DEPARTMENT OF FOOD AND ENVIRONMENTAL SCIENCESFACULTY OF AGRICULTURE AND FORESTRYDOCTORAL PROGRAMME IN FOOD CHAIN AND HEALTHUNIVERSITY OF HELSINKI

Synthesis and Structural Characterization of Glucooligosaccharides and Dextran from Weissella confusa Dextransucrases

QIAO SHI

dissertationes schola doctoralis scientiae circumiectalis, alimentariae, biologicae. universitatis helsinkiensis 21/2016

21/2016

Helsinki 2016 ISSN 2342-5423 ISBN 978-951-51-2451-7

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Department of Food and Environmental SciencesFaculty of Agriculture and Forestry

University of Helsinki

Doctoral Programme in Food Chain and HealthDoctoral School in Environmental, Food and Biological Sciences

Synthesis and structural characterization ofglucooligosaccharides and dextran from

Weissella confusa dextransucrases

Qiao Shi

Doctoral Thesis

To be presented, with the permission of the Faculty of Agriculture andForestry, University of Helsinki, for public examination in Auditorium 2,

Infocenter Korona (Viikinkaari 11), on September 29th, 2016,at 12 o’clock noon.

Helsinki 2016

Custos Professor Maija TenkanenDepartment of Food and Environmental SciencesUniversity of Helsinki, Finland

Supervisor Professor Maija TenkanenDepartment of Food and Environmental SciencesUniversity of Helsinki, Finland

Reviewers Dr. Gregory L. CôtéAgricultural Research ServiceUnited States Department of Agriculture, USA

Professor Markus LinderSchool of Chemical TechnologyAalto University, Finland

Opponent Professor Henk ScholsLaboratory of Food ChemistryWageningen University, The Netherlands

Cover picture: Representation of a dextransucrase (based on Protein Data Bank no.3HZ3) and its oligosaccharide products, made by Qiao Shi.

ISBN 978-951-51-2451-7 (Paperback)

ISBN 978-951-51-2452-4 (PDF)

ISSN 2342-5423 (Print)

ISSN 2342-5431 (Online)

UnigrafiaHelsinki 2016

1

ABSTRACT

Many lactic acid bacteria are able to synthesize dextrans from sucrose bydextransucrases. Weissella confusa strains have attracted increasing attention due totheir production of texture-modifying dextrans during food fermentation. Potentialprebiotic oligosaccharides have also been produced by dextransucrases in the presenceof sucrose and acceptor sugars. However, only few W. confusa dextransucrases havebeen previously studied, and the reactions of Weissella dextransucrases to synthesizeoligosaccharides have not been investigated. In this thesis, two W. confusadextransucrases from efficient dextran-producers were studied, and their products werestructurally characterized because this information is essential for a better usability ofthese enzymes and corresponding bacteria in food and health applications.

The biochemical and kinetic properties of one of the W. confusa dextransucrases werestudied. Two activity assays were compared to determine the kinetic parameters. Asucrose radioisotope assay gave a KM of 14.7 mM and a Vmax of 8.2 µmol/(mg∙min),whereas a Nelson-Somogyi assay gave values of 13.0 mM and 19.9 µmol/(mg∙min),respectively. The dextrans from the two W. confusa dextransucrases were found by, e.g.,NMR analysis to consist of mainly α-(1→6) linkages and 3% α-(1→3) branches, ofwhich some were elongated. A high-performance size-exclusion chromatographyanalysis of the dextrans revealed high molar masses of 107‒108 g/mol.

Weissella dextransucrases were also studied with acceptor sugars for the synthesis ofglucooligosaccharides. The most efficient acceptor was maltose, followed by isomaltose,maltotriose, and nigerose, which formed series of glucooligosaccharides by the furtherelongation of intermediate acceptor products. The products derived from maltose formedtwo homologous series, with one series being predominant and the other being minor.The major maltose product series were linear isomaltooligosaccharides (IMOs) withreducing-end maltose units, as identified by multistage mass spectrometry (MSn)analysis. The minor maltose series were revealed by an NMR analysis of the isolatedminor trisaccharide product to bear a novel branched structure. These products containedan α-(1→2)-linked glucosyl residue on the reducing residue of the linear IMOs. Thesestructures have not been previously obtained by a dextransucrase. They probably formedby the attachment of a single-unit branch to linear IMOs.

For the acceptor analogs lactose and cellobiose, their main acceptor products wereidentified by NMR analysis to be branched trisaccharides, with a glucosyl residue α-(1→2) linked to the acceptor’s reducing end. Surprisingly, a side product, isomelezitose

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(6Fru-α-Glcp-sucrose), was produced when using lactose as an acceptor. The synthesisof this nonreducing trisaccharide by a dextransucrase was reported here for the first time.

Linear IMOs with a degree of polymerization ≥3.3 serve as prebiotics for their resistanceto digestion. However, industrial IMO production often leads to a high portion ofunwanted digestible sugars. The dextransucrase reaction in the presence of the efficientacceptor maltose was demonstrated here as a promising alternative synthesis process tocontrol IMO size distribution by varying the sucrose/maltose ratio. The effects ofsubstrate concentrations (0.15–1 M) and dextransucrase dosage (1–10 U/g sucrose) onthe IMO yield and profile were modeled. High sucrose (1 M) and medium maltose (0.5M) concentrations were found to be optimal for the synthesis of long-chain IMOs, with366 g/L of total IMOs attained.

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ACKNOWLEDGEMENTS

This thesis was carried out at the Department of Food and Environmental Sciences,Faculty of Agriculture and Forestry, University of Helsinki. The work was financiallysupported by the Finnish Graduate School on Applied Bioscience: Bioengineering, Food& Nutrition, Environment (ABS Graduate School), the Academy of Finland via project“Molecular and functional characterization of dextran production in Weissella spp. ‒Superior dextran producers for cereal applications (WISEDextran)” and the FinnishFood and Drink Industries' Federation (ETL).

I am deeply indebted to my supervisor Professor Maija Tenkanen. She provided me withthe great opportunity to pursue a doctoral degree in her research group and has offeredme insightful scientific guidance, advice, and encouragement throughout the years. Heropen-mindedness, curiosity, and enthusiasm towards science have inspired me. It wasalso a privilege for me to work with Docent Liisa Virkki, who introduced me to NMRspectral interpretation. I feel grateful to Professor Vieno Piironen and ResearchProfessor Kristiina Kruus for participating in my follow-up group during my thesis work.I also want to express my sincere gratitude to Dr. Gregory L. Côté and Professor MarkusLinder for their pre-examination of this thesis.

I owe my gratitude to Docent Hannu Maaheimo, Assistant Professor Kati Katina, DocentPäivi Tuomainen, Minna Juvonen, Dr. Ndegwa Henry Maina, Kaj-Roger Hurme, Dr.Sanna Koutaniemi, and Dr. Leena Pitkänen, who gave me invaluable help in their fieldsof expertise, as well as Yaxi Hou, who assisted me with laboratory work. I also thankthe project collaborators Dr. Riikka Juvonen, Ilkka Kajala, and Dr. Antti Nyyssölä fromthe VTT Technical Research Centre of Finland Ltd. and Professor Arun Goyal andShraddha Shukla from the Indian Institute of Technology Guwahati (IITG). This thesiscould not have been completed without their fruitful collaborations.

I am fortunate to have been surrounded by friendly and supportive colleagues along thejourney, including Sun-Li Chong, Taru Rautavesi, Miikka Olin, Kirsi Mikkonen,Susanna Heikkinen, Kirsti Parikka, Minna Malmberg, Laura Huikko, Paula Hirsilä,Abdul Ghafar, Suvi Alakalhunmaa, Bhawani Chamlagain, Yan Xu, and Yu Wang, toname a few. I truly appreciate the joyful time I shared with all of the nice people fromthe department, especially those in D building.

Last but not least, my warmest gratitude goes to my dearest family, Xiaoguang Shi,Wenxiu Zhu, and Ling Zou, for being my rock and having faith in me! I cannot thankthem enough!

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LIST OF ORIGINAL PUBLICATIONS

This thesis is based on the following publications:

I. Kajala I, Shi Q, Nyyssölä A, Maina NH, Hou Y, Katina K, Tenkanen M, JuvonenR. 2015. Cloning and characterization of a Weissella confusa dextransucrase andits application in high fibre baking. PLoS ONE 10:e0116418.

II. Shukla S, Shi Q, Maina NH, Juvonen M, Tenkanen M, Goyal A. 2014. Weissellaconfusa Cab3 dextransucrase: properties and in vitro synthesis of dextran andglucooligosaccharides. Carbohydr. Polym. 101:554-64.

III. Shi Q, Juvonen M, Hou Y, Kajala I, Nyyssölä A, Maina NH, Maaheimo H, VirkkiL, Tenkanen M. 2016. Lactose- and cellobiose-derived branched trisaccharidesand a sucrose-containing trisaccharide produced by acceptor reactions ofWeissella confusa dextransucrase. Food Chem. 190:226-36.

IV. Shi Q, Hou Y, Juvonen M, Tuomainen P, Kajala I, Shukla S, Goyal A, MaaheimoH, Katina K, Tenkanen M. 2016. Optimization of isomaltooligosaccharide sizedistribution by acceptor reaction of Weissella confusa dextransucrase andcharacterization of novel α-(1→2)-branched isomaltooligosaccharides. J. Agric.Food Chem. 64:3276-86.

The author’s contribution

I. Shi Q planned and performed the experiments related to dextransucrase anddextran characterization. She interpreted the results and prepared that part of themanuscript.

II. Shi Q was responsible for producing and characterizing glucooligosaccharides.She participated in planning and performing the related experiments, interpretedthe results, and prepared the manuscript, including the introduction and partsrelated to glucooligosaccharide work and most of the dextran characterization.

III. Shi Q participated in planning the study and was responsible for all of theexperimental work and result interpretation, except for the MS analysis. Sheprepared the manuscript except for the MS part and was the corresponding author.

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IV. Shi Q was co-responsible for the experimental work and results interpretationrelated to optimization. She planned and performed most of the experiments andinterpreted the results regarding novel oligosaccharides. She prepared themanuscript and was the corresponding author.

Other related publications of the author:

Juvonen R, Honkapää K, Maina NH, Shi Q, Viljanen K, Maaheimo H, Virkki L,Tenkanen M, Lantto R. 2015. The impact of fermentation with exopolysaccharideproducing lactic acid bacteria on rheological, chemical and sensory properties of pureedcarrots (Daucus carota L.). Int. J. Food Microbiol. 207:109-18.

Kajala I, Mäkelä J, Coda R, Shukla S, Shi Q, Maina NH, Juvonen R, Ekholm P, GoyalA, Tenkanen M, Katina K. 2016. Rye bran as fermentation matrix boosts in situ dextranproduction by Weissella confusa compared to wheat bran. Appl. Microbiol. Biotechnol.100:3499-510.

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ABBREVIATIONS

BIMi (i≥3) maltose-derived branched isomaltooligosaccharides ofDPs≥3

Cel3 cellobiose-derived trisaccharideCOSY homonuclear correlation spectroscopyD2O deuterium oxideDMSO dimethyl sulfoxideDNS 3,5-dinitrosalicylic acidDP degree of polymerizationDQFCOSY double-quantum-filtered COSYDSR-S dextransucrase from Leuconostoc mesenteroides NRRL B-

512FEPS exopolysaccharidesESI-IT-MSn electrospray ionization multistage ion trap mass

spectrometryGH glycoside hydrolaseGLOSs glucooligosaccharidesHePS heteropolysaccharidesHMBC heteronuclear multiple-bond correlation spectroscopyHoPS homopolysaccharidesHPAEC-PAD high-performance anion-exchange chromatography with

pulsed amperometric detectionHPSEC high-performance size-exclusion chromatographyHSQC heteronuclear single-quantum coherence spectroscopyIMOs isomaltooligosaccharidesLAB lactic acid bacteriaLac3 lactose-derived trisaccharideLIMi (i≥4) maltose-derived linear isomaltooligosaccharides of DPs≥4NMR nuclear magnetic resonanceNR3 nonreducing sucrose-containing trisaccharidePEG polyethylene glycolQ2 the coefficient of prediction in regression analysisR2 the coefficient of determination in regression analysisTLC thin-layer chromatographyTOCSY total correlation spectroscopyWcCab3-DSR dextransucrase from W. confusa Cab3WcE392-rDSR recombinant dextransucrase from W. confusa VTT E-90392

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TABLE OF CONTENTS

Abstract ........................................................................................................................... 1

Acknowledgements ......................................................................................................... 3

List of original publications ............................................................................................ 4

Abbreviations .................................................................................................................. 6

1. Introduction ................................................................................................................. 9

2. Review of the literature ............................................................................................ 11

2.1 Carbohydrates from lactic acid bacteria ............................................................. 11

2.2 Glucansucrases ................................................................................................... 12

2.2.1 Catalytic mechanism of glucansucrases ....................................................... 12

2.2.2 Production and characterization of dextransucrases ..................................... 15

2.3 Carbohydrates synthesized by dextransucrases .................................................. 16

2.3.1 Substrate and product specificity of dextransucrases ................................... 16

2.3.2 Functional oligosaccharides from dextransucrases ...................................... 19

2.3.3 Characterization of dextransucrase-synthesized carbohydrates ................... 21

2.4 Weissella spp. and Weissella dextransucrases .................................................... 23

3. Aim of the study ....................................................................................................... 26

4. Materials and methods .............................................................................................. 27

4.1 Dextransucrase preparations and commercial enzymes ..................................... 27

4.2 Biochemical characterization of WcE392-rDSR (I) ........................................... 27

4.3 Analysis of monosaccharides and oligosaccharides ........................................... 28

4.4 Synthesis, isolation, and molar mass characterization of dextrans (I and II) ..... 29

4.5 Acceptor reactions and isolation of oligosaccharides (II‒ IV) ........................... 29

4.6 Structural analysis of dextrans and oligosaccharides ......................................... 31

4.6.1 Enzymatic hydrolysis of dextrans and oligosaccharides (II and III) ............ 31

4.6.2 NMR spectroscopy analysis of dextrans and oligosaccharides (I‒IV) ......... 31

4.6.3 MSn analysis of oligosaccharides (II‒IV) ..................................................... 32

4.7 Experimental design for IMO synthesis (IV) ..................................................... 32

5. Results ....................................................................................................................... 34

5.1 Characterization of WcE392-rDSR (I) ............................................................... 34

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5.2 Characterization of Weissella dextrans (I and II) ................................................ 35

5.3 Characterization of the oligosaccharides produced by dextransucrases .............. 38

5.3.1 Acceptor selectivity (III) ............................................................................... 38

5.3.2 Principal maltose product series (II) ............................................................. 38

5.3.3 Minor maltose product series (IV) ................................................................ 41

5.3.4 Lactose- and cellobiose-derived branched trisaccharides (III) ...................... 42

5.3.5 Novel sucrose-containing trisaccharide (III) ................................................. 46

5.3.6 Applicability of MSn analysis in the characterization of dextransucrase-synthesized GLOSs (III and IV) ................................................................... 48

5.4 Optimization of IMO synthesis by response surface modeling (IV) ................... 52

6. Discussion ................................................................................................................. 57

6.1 Properties of Weissella dextransucrases and dextrans (I and II) ......................... 57

6.2 Specificity of Weissella dextransucrases in acceptor reactions ........................... 58

6.2.1 Comparison with commercial Lc. mesenteroides dextransucrase (III andIV) ................................................................................................................. 58

6.2.2 Specificity to form an α-(1→2) branch (III and IV) ..................................... 60

6.2.3 Novel sucrose-containing trisaccharide (III) ................................................. 61

6.3 Potential of dextransucrases in prebiotic GLOS synthesis .................................. 62

6.3.1 Synthesis of prebiotic IMOs from maltose (IV) ............................................ 62

6.3.2 Synthesis of other GLOSs (III) ..................................................................... 63

7. Conclusion ................................................................................................................. 65

References ..................................................................................................................... 67

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1. INTRODUCTION

The lactic acid fermentation of food has been practiced worldwide for thousands of yearsfor improved preservability and flavors. Moreover, the oligo- and polysaccharidessecreted by lactic acid bacteria (LAB) can enhance the textural and nutritional propertiesof the fermented foods (Seo DM et al., 2007; Welman 2009; Di Cagno et al., 2013; Choet al., 2014). The exopolysaccharides (EPS) produced in situ by LAB can avoid beingdeclared on food labels, because the LAB with a history of safe use have, for example,GRAS (generally recognized as safe) or QPS (qualified presumption of safety) status inthe United States and the European Union, respectively (Waldherr and Vogel 2009).Such EPS offer an attractive solution for producing additive-free foods, which areincreasingly pursued by today’s consumers. EPS-producing cultures have been appliedin fermented milks, vegetables, and fruits and in cheese and sourdoughs, where EPSexert technological advantages, such as controlling syneresis; enhancing the thickness,creaminess, and smoothness of yoghurt; and improving the dough rheology, breadvolume, and shelf life of sourdough breads (Di Cagno et al., 2006; Schwab et al., 2008;Welman 2009; Katina et al., 2009; Galle et al., 2012). In addition to sensory benefits,there is accumulated evidence on the health benefits of oligo- and polysaccharides fromLAB, including prebiotic, cholesterol-lowing, immunomodulatory, and anti-carcinogenic effects (Schwab et al., 2008; Welman 2009; Ryan et al., 2015).

In addition to the in situ synthesis of oligo- and polysaccharides in fermented foods,LAB have been used to produce α-glucans of industrial interest (Zannini et al., 2015;Miao et al., 2016). For example, dextran produced by Leuconostoc mesenteroides wasone of the first biopolymers produced on an industrial scale and has various applicationsin food, medicine, and separation technology as a food thickener, blood plasma expander,and size-exclusion chromatography matrix, respectively. (Waldherr and Vogel 2009;Zannini et al., 2015). Dextran was approved by the European Commission for use as anovel food ingredient in bakery products for improved softness, crumb texture, and loafvolume (Zannini et al., 2015). LAB produce α-glucans extracellularly via sucrose-actingα-transglucosylases, e.g., dextransucrases. These enzymes are promising biosynthetictools because, unlike Leloir type-glycosyltransferases, they do not require energy-richsugar nucleotides as glycosyl donor substrates and can readily synthesize α-glucans froma sole substrate sucrose (Monsan et al., 2010). In addition to polymers, these enzymescan also synthesize, e.g., α-glucooligosaccharides (GLOSs) in the presence of a widevariety of acceptor sugars. These enzymes of LAB have high regio- andstereospecificities compared to those of chemical approaches and have been used tosynthesize structurally diverse carbohydrates (Leemhuis et al., 2013; Vuillemin et al.,2016). For example, Leuconostoc dextransucrases have been used to produce various

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prebiotic GLOSs, e.g., isomaltooligosaccharides (IMOs) (Goffin et al., 2011; Ruiz-Matute et al., 2011; García-Cayuela et al., 2014).

Weissella spp. are LAB frequently found in plant materials and fermented foods (Fuscoet al., 2015). They have drawn attention in recent years as efficient dextran producersfor food applications. In situ-produced Weissella dextrans improve the shelf life, volume,and softness of sourdough breads and the texture of fermented vegetable foods (Katinaet al., 2009; Galle et al., 2010; Juvonen et al., 2015). Weissella spp. have an advantageover many Leuconostoc starters in that they do not lead to over-acidification in foods.To facilitate the future use of this genus and its dextransucrases, it is important tounderstand the structures and possible functions of its products. To date, only fewdextransucrases from W. confusa and W. cibaria have been characterized (Bounaix etal., 2010; Amari et al., 2012; Bejar et al., 2013; Rao and Goyal 2013), and no Weisselladextransucrase has been examined as a synthetic tool for, e.g., potential prebiotic GLOSs.In this thesis, the dextransucrases from two efficient dextran-producing W. confusastrains were studied, with one dextransucrase biochemically characterized (article I).Their dextran (articles I and II) and GLOS products (articles II‒IV) were structurallyanalyzed, focusing on GLOSs produced in the presence of various acceptor sugars. Thereaction with the acceptor maltose was explored to synthesize prebiotic IMOs (articleIV).

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2. REVIEW OF THE LITERATURE

2.1 Carbohydrates from lactic acid bacteria

Several LAB produce polysaccharides that are components of the cell wall or arereleased from the cell. The latter can be divided into capsular polysaccharides (CPS) andEPS (Zannini et al., 2015). Whereas CPS are permanently attached to the cell surface inthe form of capsules, EPS are secreted into the environment (Ruas-Madiedo et al., 2009).EPS are involved in biofilm formation and have various physiological functions, suchas in surface adhesion and the protection of bacterial cells from environmental stress(Zannini et al., 2015). EPS can be divided into two groups based on the mechanism ofbiosynthesis and the precursors required (Boels et al., 2001). One group compriseshomopolysaccharides (HoPS), namely α-glucans and β-fructans, which are synthesizedextracellularly by glucansucrases (glycoside hydrolase family 70) and fructansucrases(glycoside hydrolase family 68), respectively. Both enzymes use sucrose as a substrate,while the latter can also use, e.g., raffinose to synthesize β-fructan in the absence ofsucrose. The other group includes β-glucans (HoPS) and heteropolysaccharides (HePS).Their synthesis requires sugar nucleotide precursors and several enzymes that areinvolved in central carbon metabolism and in the synthesis of some cell-wallcomponents (Zannini et al., 2015).

Sucrose-derived HoPS are synthesized extracellularly by the action of glycansucrases ofcertain LAB, such as Leuconostoc, Lactobacillus, Streptococcus, and Weissella (Zanniniet al., 2015). One bacterium may harbor multiple glucansucrase and fructansucrasegenes (Olvera et al., 2007; Malik et al., 2009; Malang et al., 2015). When a strain secretesmultiple glucansucrases, these enzymes may act jointly to synthesize a hybrid polymerthat is different from that produced by purified enzyme (Côté and Skory 2015). Thehydrolysis of the glycosidic linkage of sucrose provides these glycansucrases with theenergy to form a new glycosidic linkage in corresponding HoPS (Zannini et al., 2015).Because the biosynthesis of these HoPS is less energy demanding than that of HePS,they can be produced in larger amounts. According to the type of glucosidic linkages, α-glucans are classified into dextrans, mutans, alternans, and reuterans (Leemhuis et al.,2013). Dextrans contain mainly α-(1→6) linkages and α-(1→2), α-(1→3), or α-(1→4)branch linkages, mutans mainly α-(1→3) linkages, reuterans mainly α-(1→4) linkages,and alternans contain alternating α-(1→6) and α-(1→3) linkages (Naessens et al., 2005;Bounaix et al., 2009). β-Fructans are categorized into levans, with mainly β-(2→6)linkages, and inulins, with mainly β-(2→1) linkages (Zannini et al., 2015). In additionto polymeric glucans and fructans, LAB can also synthesize oligosaccharides, such as

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isomaltooligosaccharides and fructooligosaccharides. The size and yield of carbohydrateproducts depend on the strain and fermentation conditions (Jeanes et al., 1954; Schwabet al., 2008; Anwar et al., 2010; Malang et al., 2015).

2.2 Glucansucrases

Because of the high interest in the carbohydrates of LAB origin for food and non-foodapplications, their synthesis machineries have been extensively explored, and a largenumber of synthesizing enzymes have been identified and characterized. Glucansucrases(EC 2.4.1.5) are produced extracellularly by LAB to synthesize α-glucans (Leemhuis etal., 2013). They are named based on the α-glucans that they synthesize, i.e.,dextransucrases, mutansucrases, alternansucrases and reuteransucrases. Althoughglucansucrases are transferases with respect to their activity, they are structurally closeto glycoside hydrolases and are classified as part of the glycoside hydrolase family 70(GH70), which contains mainly glucansucrases (Leemhuis et al., 2013). The recentlycharacterized 4,6-α-glucanotransferases, which are inactive on sucrose but convertstarch or starch hydrolysates into isomalto/maltopolysaccharides, constitute a newsubfamily of GH70 (Bai et al., 2015). Moreover, by sequencing the genomes of theproducers of original α-glucans, a cluster of branching sucrases specialized in dextrangrafting through α-(1→2) or α-(1→3) linkages have been recently discovered (Passeriniet al., 2015; Vuillemin et al., 2016). GH70 enzymes have been found almost exclusivelyin LAB according to the entries in the Carbohydrate-Active Enzymes (CAZy) database(Lombard et al., 2014). So far, the encoding genes of more than three hundred GH70enzymes have been cloned and sequenced, and the three-dimensional structures of fourtruncated enzymes have been solved (Vujicic-Zagar et al., 2010; Ito et al., 2011; Brisonet al., 2012; Pijning et al., 2012). The GH70 enzymes are mechanistically, structurally,and evolutionary closely related to GH13 (α-amylases) and GH77 (amylomaltases)enzymes. The three families containing a catalytic (β/α)8-barrel domain form the GH-Hclan (Stam et al., 2006).

2.2.1 Catalytic mechanism of glucansucrases

The catalysis of glucansucrases is proposed to be a two-step process, starting with thecleavage of the substrate sucrose and followed by the formation of a covalent β-glucosyl-enzyme intermediate and the release of a free fructose (Figure 1). In the second step, theglucosyl moiety is transferred to an acceptor, retaining the α-anomeric configuration(Leemhuis et al., 2013). Hydrolysis takes place when a water molecule acts as theacceptor. In the presence of suitable low-molar-mass molecules, e.g., maltose,

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glucansucrases catalyze so-called acceptor reactions, where the glucosyl residue fromsucrose is diverted from α-glucan formation and transferred onto the small moleculeinstead (Figure 1). Di- and oligosaccharides are formed when carbohydrates act as theacceptor molecules, whereas glucoconjugates are formed in the presence of non-carbohydrate compounds (Monsan et al., 2010).

Figure 1. Types of reactions catalyzed by dextransucrases.

The three-dimensional structures of truncated glucansucrases revealed that they exhibita U-fold shape, organized into five (A, B, C, IV, and V) domains (Vujicic-Zagar et al.,2010; Ito et al., 2011; Brison et al., 2012; Pijning et al., 2012). Except for domain C atthe base of the U-fold, other domains are formed by discontiguous N- and C-terminalsegments of the polypeptide, as illustrated by the glucansucrase of Lactobacillus reuteri180 with its N-terminal end (~740 residues) truncated (Figure 2). Domains A, B, and Cresemble family GH13 enzymes, whereas domains IV and V are unique in GH70enzymes. The catalytic domain A adopts a (β/α)8-barrel fold and harbors a catalytic triad,which is composed of two aspartates (acting separately as a nucleophile and a transition-state stabilizer) and one glutamate (acting as an acid/base catalyst) (Leemhuis et al.,2013). The acceptor binding site is shaped by residues from domains A and B.Mutagenesis studies have shown that the interplay of these residues determines how agrowing glucan chain interacts with the acceptor binding site (which hydroxyl group ofa glucosyl residue is capable of attacking the glucosyl-enzyme intermediate to form aglycosidic linkage) and thus the proportion of specific linkages in the glucan synthesized

fructose

covalent glucosyl-enzyme intermediate

H2O hydrolysis

R-OHacceptorreaction

dextransynthesis

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(Irague et al., 2013; Leemhuis et al., 2013; Meng et al., 2015a; Meng et al., 2015c).Domain V is part of the glucan binding domain consisting of N- and C-terminalsegments, while domain IV may provide a hinge that brings the glucan-bound domainV toward or away from the catalytic site to facilitate the synthesis (Leemhuis et al., 2013;Meng et al., 2015b).

Figure 2. Crystal structure of an N-terminus-truncated dextransucrase from Lactobacillus reuteri180 and schematic presentation of the U-fold shape of the polypeptide chain (adapted fromVujicic-Zagar et al., 2010).

Moulis and others (2006b) proposed a semi-processive mechanism for thepolymerization of glucans. There is a lag-phase before glucan synthesis starts, whensucrose and glucose (produced by hydrolysis) act as initiators of polymerization.Glucose is the preferred initiator when present in sufficient amounts. Elongation occursthrough the non-processive (the product is released after each elongation step of theacceptor) attachment of glucosyl moieties to the nonreducing end of the initially formedproducts. The longer oligosaccharides are better acceptors than are sucrose, fructose,and glucose, ensuring that the glucosyl moieties are used to synthesize glucans insteadof large amounts of oligosaccharides. Once a glucan chain reaches a minimal size, itmay remain attached to the enzyme, and the elongation becomes processive. The glucan-binding domain is involved in the processive mechanism, as shown by a gradual loss ofelongation efficiency (a decrease in the ratio of high-/low-molar-mass population) as thedomain is progressively deleted (Moulis et al., 2006b).

15

2.2.2 Production and characterization of dextransucrases

Among LAB, the expression of the dextransucrase-encoding gene is usually sucrose-inducible in wild-type strains of Leuconostoc spp. but constitutive in, e.g., Streptococcusspp. and some Lactobacillus strains (Naessens et al., 2005; Bounaix et al., 2010). Theconstitutive expression of the glucansucrase-encoding gene is suggested to beadvantageous in ecosystems with fluctuating sucrose concentrations, such as the oralcavity (Leemhuis et al., 2013). Dextransucrase production is affected by temperature,aeration and medium composition, and methods such as response surface methodologyhave been used to optimize these culture conditions (Purama and Goyal 2008a). Nativedextransucrases from different LAB have been purified by, e.g., salt, alcoholprecipitation, polyethylene glycol (PEG) fractionation, ultrafiltration, andchromatography (Naessens et al., 2005; Purama and Goyal 2008b). PEG fractionationcan separate dextransucrases from contaminating enzymes, such as levansucrase;however, dextran remains in the preparation (Majumder et al., 2007). Because theinduced production of dextransucrase leads to the concomitant production of dextran,which can envelop the enzyme and hinder the enzyme characterization (Moulis et al.,2006a), it is desired to produce dextransucrases by the constitutive mutants orheterologous hosts, where the expression of the dextransucrase-encoding gene does notrequire sucrose induction and therefore no dextran is produced concomitantly. Thedextran present, however, stabilizes dextransucrase, and so does PEG and Tween 80(Majumder et al., 2007). The presence of Ca2+ is essential for dextransucrase activity(Naessens et al., 2005). The Ca2+-binding site is located at the interface between domainsA and B (Figure 2). The absence of Ca2+ may lead to a compromised substrate bindingand thus a decreased activity (Vujicic-Zagar et al., 2010). Dextransucrases normallyhave a pH optimum of 5.0‒5.5 and a Michaelis-Menten constant (KM) for sucrose ofapproximately 12‒16 mM (Naessens et al., 2005).

Several assays have been used for dextransucrase activity measurement (Vettori et al.,2011). They can be divided into two groups. One group is based on the measurement ofthe fructose released, which represents sucrose consumption (from both hydrolytic andtransferase reactions). The hydrolytic activity, which is usually low, can be determinedby quantifying the free glucose released. Reducing-value assays are based on an increasein the reducing value in the reaction mixture (mostly resulting from the fructose releasedin dextran formation) and are normally used for dextransucrase activity assays, althoughthe dextran product itself and small amounts of side products, i.e., leucrose (α-D-Glcp-(1→5)-D-Frup, an acceptor product of fructose), glucose and fructose resulting fromhydrolysis, also contribute to the increased reducing value. The commonly usedreducing-value assays include the 3,5-dinitrosalicylic acid (DNS) method (Sumner and

16

Howell 1935) and the Nelson-Somogyi method (Nelson 1944; Somogyi 1945). Theother assay group is based on the quantification of dextran product by, e.g., its weight.The 14C-sucrose radioisotope method has also been used (Vettori et al., 2011; Côté andSkory 2012) and is based on the incorporation of 14C-labeled glucose from 14C-U-labeled sucrose to the growing dextran chain. The second group provides a moreaccurate measurement of dextransucrase activity (Germaine et al., 1974; Vettori et al.,2011).

2.3 Carbohydrates synthesized by dextransucrases

Glucansucrases are useful synthetic tools for various carbohydrates and glucoconjugatesbecause of their high regioselectivity in glucosyl transfer reactions, relaxed specificitytoward a variety of acceptors, and low cost of the donor substrate sucrose (Seibel et al.,2010). Moreover, glucansucrases have been used to improve the flavor, water solubility,and oxidative stability of various compounds for food, cosmetic, and therapeuticapplications (Côté et al., 2009; Kim et al., 2012; Liang et al., 2016).

2.3.1 Substrate and product specificity of dextransucrases

Although glucansucrases are highly specific towards sucrose, certain othercarbohydrates can also serve as glycosyl donors (Jung and Mayer 1981; Seto et al., 2008;Timm et al., 2013). Especially, the transfer of a different glycosyl residue than a glucosylone indicates broader applications of glucansucrases in carbohydrate bioconversion. Forexample, carbonyl-group-containing dextran was synthesized using keto-sucrose at C-2by dextransucrase (Seto et al., 2008). Similarly, a reuteransucrase was used to transferan allosyl (differs in C3 configuration from glucosyl) group to several acceptors with α-D-allopyranosyl β-D-fructofuranoside as a donor substrate (Timm et al., 2013).

The binding kinetics between Lc. mesenteroides dextransucrase and immobilizedglucose, maltose, and dextran were determined using a quartz crystal microbalance(Nihira et al., 2011; Fang et al., 2015). The dissociation constants for glucose, maltose,and dextran were 64, 35, and 18 nM, respectively, indicating an increasing order ofspecificity between the enzyme and these acceptors. The effectiveness of differentacceptors was compared for Lc. mesenteroides dextransucrases at a sucrose/acceptorratio of 1:1 (Robyt and Eklund 1983; Kothari and Goyal 2013). Among the acceptorsugars tested in both studies, maltose and, to a lesser extent, isomaltose were the bestacceptors; D-glucose, lactose, and cellobiose were moderately effective; and raffinose,D-galactose, and D-xylose were poor acceptors. The more effective acceptors can

17

generally be further glucosylated into a series of oligosaccharides and suppress dextranformation to a higher degree (Robyt and Eklund 1983; Kothari and Goyal 2013).

Dextransucrases are able to form mainly α-(1→6) linkages and, to a lesser extent, α-(1→2), α-(1→3), or α-(1→4) linkages (Leemhuis et al., 2013). It has been proposed thatthe linkage specificity depends on the orientation, in which the acceptor is guided towardthe reaction center and is therefore determined by the conformation of the acceptorbinding cleft (Leemhuis et al., 2013). Amino acid residues forming acceptor bindingsubsites affect dextransucrase specificity, as mutations of targeted residues were shownto alter the linkage composition of the product (Brison et al., 2012; Leemhuis et al., 2013;Meng et al., 2015c). Many dextransucrases catalyze the formation of more than one typeof glucosidic linkage, requiring different binding modes of an acceptor. Thus, theacceptor binding cleft needs to be wide enough to accommodate the acceptor in differentorientations. For example, the structure of a truncated dextransucrase from Lactobacillusreuteri 180, which forms both α-(1→6) and α-(1→3) linkages, shows a rather widebinding cleft. However, a unique branching sucrase derived from Leuconostoc citreumNRRL B-1299 (formerly known as Leuconostoc mesenteroides NRRL B-1299)dextransucrase only has α-(1→2) branching activity, and correspondingly, its bindingcleft is partially blocked, allowing only one acceptor binding mode (Brison et al., 2012;Leemhuis et al., 2013).

In acceptor reactions, the product pattern for a given acceptor depends on dextransucrasespecificity. Maltose is the most studied acceptor due to its high effectiveness, and thepatterns of oligosaccharide products of maltose acceptor reactions corresponded to thestructure of the dextran produced by the same strain (Côté and Leathers 2005). Forexample, the typical dextransucrases that predominantly synthesize α-(1→6) linkages,represented by the commercially used Leuconostoc mesenteroides NRRL B-512Fdextransucrase (DSR-S), produce homologous oligosaccharides in the presence ofmaltose (Robyt and Eklund 1983). The initial product, panose (α-D-Glcp-(1→6)-α-D-Glcp-(1→4)-D-Glcp), is elongated by the successive attachment of glucosyl residues tothe 6-OH of the nonreducing end glucose, forming linear isomaltooligosaccharides(IMOs), as illustrated by series A in Figure 3. For Lc. citreum NRRL B-1299dextransucrase synthesizing α-(1→2)-branched dextran, however, the maltose acceptorproducts are three homologous IMO series, with the major series constituting linearIMOs of series A (Figure 3). The two minor series (marked B and C in Figure 3) haveα-(1→6)-linked main chains with a single-unit branch α-(1→2) attached to the terminaland the penultimate α-(1→6)-linked residue of the nonreducing end, respectively (Dolset al., 1998).

18

Figure 3. Oligosaccharide series (named A, B, and C) produced in the maltose acceptor reactionof dextransucrases. Series A are produced by Lc. mesenteroides NRRL B-512F and Lc. citreumNRRL B-1299 dextransucrases, whereas series B and C are only produced by B-1299dextransucrase (adapted from Dols et al., 1998).

In addition to this thesis work, GLOS products from different acceptors (Table 1) haveonly been characterized for DSR-S from Lc. mesenteroides B-512F (Yamauchi andOhwada 1969; Robyt 1995; Demuth et al., 2002; Côté et al., 2009; Díez-Municio et al.,2012a; Barea-Alvarez et al., 2014; Côté et al., 2014). The type of linkage that is formedis a function of the acceptor structure. Surprisingly, α-(1→2)-linkage is formed onlactose and cellobiose, even though this enzyme is known to synthesize α-(1→6) and α-(1→3) linkages in dextran. In addition to 2Glc-α-D-Glcp-lactose, an unknowntrisaccharide has been reported for the lactose acceptor reaction (Díez-Municio et al.,2012b). Controversial results have been reported regarding the main cellobiose product.Instead of 21-α-D-Glcp-cellobiose, the enzyme from a dextransucrase-hyper-producingLc. mesenteroides B-512F constitutive mutant was reported to produce 22-α-D-Glcp-cellobiose (Kim et al., 2010). However, both the wild-type and mutant enzymes formeda minor trisaccharide: linear 62-α-D-Glcp-cellobiose (Argüello Morales et al., 2001; Kimet al., 2010).

19

Table 1. Acceptor products formed by DSR-S from Lc. mesenteroides NRRL B-512F.Acceptor First acceptor producta ReferenceMaltose (α-D-Glcp-(1→4)-D-Glcp) Panose* Robyt 1995

Isomaltose (α-D-Glcp-(1→6)-D-Glcp) Isomaltotriose* "

Nigerose (α-D-Glcp-(1→3)-D-Glcp) 62-α-D-Glcp-nigerose* "Methyl α-D-glucopyranoside Methyl α-isomaltoside* "D-Glucose Isomaltose* "Lactose (β-D-Galp-(1→4)-D-Glcp) 2Glc-α-D-Glcp-lactose "Cellobiose (β-D-Glcp-(1→4)-D-Glcp) 21-α-D-Glcp-cellobiose* "D-Fructose Leucrose

(α-D-Glcp-(1→5)-D-Frup)Isomaltulose(α-D-Glcp-(1→6)-D-Fruf)

"

D-Galactose α-D-Glcp-(1→1)-β-D-Galf "Maltotriose 63-α-D-Glcp-maltotriose*

61-α-D-Glcp-maltotriose"

Maltotetraose 64-α-D-Glcp-maltotetraose*61-α-D-Glcp-maltotetraose

"

Gentiobiose (β-D-Glcp-(1→6)-D-Glcp) 62-α-D-Glcp-gentiobiose Yamauchi and Ohwada 1969D-Mannose α-D-Glcp-(1→6)-D-Manp

α-D-Glcp-(1→1)-β-D-Manp

Côté et al., 2014Robyt 1995

Raffinose(α-D-Galp-(1→6)-α-D-Glcp-(1→2)-β-D-Fruf)

4Gal-α-D-Glcp-raffinose Côté et al., 2009

Isomaltulose (α-D-Glcp-(1→6)-D-Fruf) 62-α-D-Glcp-isomaltulose* Barea-Alvarez et al., 2014

Lactulose (β-D-Galp-(1→4)-D-Fruf) β-D-Galp-(1→4)-β-D-Fruf-(2→1)-α-D-Glcp

Díez-Municio et al., 2012b

Maltulose (α-D-Glcp-(1→4)-D-Fruf) α-D-Glcp-(1→4)-β-D-Fruf-(2→1)-α-D-Glcp

Demuth et al., 2002

a The starred product is the first member of a homologous series with α-isomalto-oligosaccharides attached to the acceptor.

2.3.2 Functional oligosaccharides from dextransucrases

Glucansucrase-catalyzed synthesis provides access to a large range of bioactiveglucooligosaccharides (GLOSs), particularly non-digestible oligosaccharides withprebiotic effects (Monsan et al., 2010). The use of prebiotics is gaining popularitybecause, compared to probiotics, they are resistant to the aggressive gastro-intestinalenvironment and cheaper and easier to incorporate into the diet (Goffin et al., 2011; Al-

20

Sheraji et al., 2013). Prebiotics are currently defined as the dietary components thatselectively stimulate the growth and/or activity(ies) of one or a limited number ofmicrobial genus(era)/species in the gut microbiota and thus confer health benefits to thehost (Roberfroid et al., 2010). The acceptor reactions of glucansucrases, especiallydextransucrases, from Leuconostoc spp. have been used to synthesize functional GLOSs(Chung and Day 2002; Seo DM et al., 2007; Holt et al., 2008; Goffin et al., 2011; Kothariand Goyal 2015). The GLOSs derived from maltose, lactose, lactulose, and cellobioseby DSR-S from Lc. mesenteroides NRRL B-512F have shown prebiotic potential in vitroand for maltose products also in vivo (Argüello Morales et al., 2001; Ruiz-Matute et al.,2011; Díez-Municio et al., 2012b; García-Cayuela et al., 2014). GLOS products ofLeuconostoc dextransucrases have also shown other health benefits, e.g., preventing theinstallation of type 2 diabetes, preventing dental caries, and inhibiting cancer cells (Kimet al., 2010; Monsan et al., 2010; Kothari and Goyal 2015).

Among the GLOSs from dextransucrases, maltose-derived isomaltooligosaccharides(IMOs) represent the most commercialized prebiotic products. In particular, the IMOscontaining α-(1→2)-linkages from Lc. citreum NRRL B-1299 dextransucrase aremarketed as prebiotics for their low digestibility and produced on an industrial scale of40 metric tons/year (Plou et al., 2002). The linear IMOs with higher degrees ofpolymerization (DPs) also demonstrated prebiotic effects in humans and animals (Goffinet al., 2011; Yen et al., 2011; Ketabi et al., 2011). In fact, IMOs of DP 3.3 were fermentedin the large intestine of rats, while IMOs of DP 2.7 were mainly digested and absorbedin the small intestine, without reaching the large intestine (Iwaya et al., 2012). However,commercial IMOs usually contain a high proportion of digestible carbohydrates, whichdecreases their functionality as prebiotics (Hu et al., 2013). The industrial IMOproduction includes the enzymatic hydrolysis of starch into high maltose syrup, followedby the action of α-transglucosidase, which catalyzes the transfer of the nonreducingglucosyl residue of maltose to the 6-OH group of glucose (released by hydrolysis), thenonreducing glucosyl unit of maltose or any α-glucooligosaccharide present in thesolution (Goffin et al., 2011). Isomaltose, panose and isomaltotriose are the maincomponents in the IMOs thus produced. The dextransucrase acceptor reaction holdspromise for the size-controlled synthesis of IMOs by varying concentrations of sucrose(donor) and maltose (acceptor). With an increase in the sucrose/maltose ratio, the DP ofboth the major and the longest products increased (Su and Robyt 1993; Lee et al., 1997;Lee et al., 2008). However, detailed studies are needed to select the optimal synthesisconditions for IMOs of higher DP.

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2.3.3 Characterization of dextransucrase-synthesized carbohydrates

Knowledge of the structure of dextransucrase-synthesized products is essential for theapplication of enzymes and the dextran and oligosaccharide products. Typical strategiesfor the structural characterization of dextrans and oligosaccharides are presented inFigure 4. Extracellular water-soluble glucans can simply be purified by the solvent(normally ethanol) precipitation of the cell-free culture and dialysis against water whenno severe interfering compound, e.g., protein is present (Ruas-Madiedo and de losReyes-Gavilán 2005). The molar mass of dextrans can be determined by, e.g.,ultracentrifugation, high-performance size-exclusion chromatography (HPSEC), andasymmetric flow field-flow fractionation (AsFlFFF) (Irague et al., 2012; Maina et al.,2014). HPSEC is widely used to analyze the molar mass distribution based on theseparation of molecules by hydrodynamic radius (Pitkänen 2011). Weight-averagemolar mass (Mw) can be determined absolutely by HPSEC using universal calibration(applying refractive index and viscosity detectors) or static light scattering detection. Awide range of Mw (106‒109 g/mol) has been reported for native dextrans; however,discrepancy can arise from the different analysis methods applied (Maina 2012). DMSOwas reported to be the solvent of choice for dextran Mw determination by HPSEC, asaggregate formation was less prevalent in DMSO than in aqueous solution. Moreover,the degradation of dextrans due to shear forces during HPSEC analysis was notsignificant in DMSO (Maina et al., 2014). High-molar-mass dextrans with low-branchedstructures have applications in food industries as hydrocolloids (Lacaze et al., 2007;Waldherr and Vogel 2009).

The monosaccharide compositions of polysaccharides can be analyzed after totaldepolymerization by acid methanolysis or acid hydrolysis (Leemhuis et al., 2013). Themonomers released are analyzed by gas chromatography (GC) or high-performanceliquid chromatography (HPLC). High-performance anion-exchange chromatographywith pulsed amperometric detection (HPAEC-PAD) is one of the most frequently usedtechniques for mono- and oligosaccharide analysis. It exempts derivatization and affordssensitive detection. Carbohydrates are readily separated by HPAEC under alkalineconditions, where retention increases with decreasing pKa (Corradini et al., 2012). Forstructural elucidation, large α-glucans often need to be degraded into the constitutingoligosaccharides by partial acid hydrolysis or specific enzymes. The fragments are thenanalyzed by, e.g., thin-layer chromatography, HPLC, or NMR spectroscopy, and thestructure and abundance of these building blocks are used to build a composite polymerstructure (Remaud-Slmeon et al., 1994; van Leeuwen et al., 2008b; Vettori et al., 2012).Enzyme-aided methods are useful for fingerprinting the type of branch linkages indifferent dextrans (Sawai et al., 1978; Maina 2012). One method involves the specific

22

hydrolysis of dextran with a mixture of dextranase and α-glucosidase. Thechromatographic profiles of enzyme-resistant oligosaccharides vary depending on thebranch points of dextran and can therefore be used as structural markers (Maina 2012).

Figure 4. Commonly used strategies and techniques for the structural analysis of products fromglucansucrases.

The position of linkages between monosaccharide units in oligo- and polysaccharide canbe determined by methylation analysis. In this method, all of the free hydroxyl groupsin a carbohydrate are tagged by methylation prior to hydrolysis, after which themonomers are reduced and derivatized for gas chromatography-mass spectrometry (GC-MS) analysis (Naessens et al., 2005). Nuclear magnetic resonance (NMR) spectroscopyis the most powerful technique for the structure elucidation of oligosaccharides and hasbeen intensively used for characterizing glucansucrase-synthesized carbohydrates (Côtéand Skory 2012; Díez-Municio et al., 2012a; Barea-Alvarez et al., 2014). It can provideall of the information about the type of constituent monosaccharides, including ring sizeand anomeric configuration, and the sequence of glycosidic linkages (Bubb 2003).Combinations of two-dimensional 1H and 13C techniques have been developed, such ashomonuclear (1H-1H) COSY and TOCSY experiments for proton chemical shifts of eachresidue and heteronuclear (1H-13C) HSQC and HMBC experiments for substitutioninformation (Bubb 2003). The primary structures of α-glucans have been characterizedbased on the 1H NMR structural-reporter-group concept (van Leeuwen et al., 2008a; vanLeeuwen et al., 2008b).

Glucansucrase or LAB

Glucan OligosaccharidesPartial hydrolysis

Profiling by e.g.HPAEC-PAD

Mw determinationby e.g. HPSEC

Monosaccharidecomposition

Linkage ratio bymethylation

Detailed informationby NMR spectroscopy

Linkage sequenceby ESI-MSn

Isolatedoligosaccharides

23

Multistage mass spectrometry (MSn) has proven valuable in the sequential analysis ofthe glycosidic linkages in oligosaccharides (Usui et al., 2009; Mischnick 2012; Mainaet al., 2013). Matrix-assisted laser desorption/ionization (MALDI) and electrosprayionization (ESI) are the main ionization techniques used, with the latter coupling MSwith liquid chromatography (Harvey 2012). In negative ion MSn, C-ions (Figure 5) arepredominantly produced due to glycosidic cleavage, whereas isomeric C- and Y-ionsare produced in positive ion MSn. Negative ion MSn is preferably used for the structuralanalysis of an underivatized linear oligosaccharide (Maina et al., 2013). In each MSn

stage, the cross-ring cleavages at the reducing end residue of the isolated C-ion arediagnostic of the reducing end linkage. The linkage sequence of an unknownoligosaccharide can be deduced by comparing its fragmentation behaviors in MSn

against a library of model compounds (Mischnick 2012). Unlike NMR spectroscopyanalysis, which requires purified carbohydrates, MS coupled with liquidchromatography provides an integrated strategy for the rapid characterization ofoligosaccharides in mixtures (Wuhrer et al., 2009).

Figure 5. Nomenclature of fragment ions of cellotriose according to Domon and Costello (1988).

2.4 Weissella spp. and Weissella dextransucrases

The genus Weissella was proposed in 1993 after the reclassification of someLeuconostoc-like bacteria (Collins et al., 1993). Weissella are Gram-positive, catalase-negative, non-endospore-forming, coccoid or rod-shaped heterofermentative LAB fromthe family Leuconostocaceae (Fusco et al., 2015). Dextran production is a phenotypiccriterion for the identification of bacteria in this genus. Weissella strains have been foundin a wide range of habitats, such as plants and vegetables and the gastrointestinal tractof animals and humans (Fusco et al., 2015). Some strains of W. confusa and W. cibariaspecies have been suggested to be probiotics (Ayeni et al., 2011; Lee et al., 2012).Although W. confusa has rarely been described as a human opportunistic pathogen,infections usually occur in patients with immunosuppression or underlying diseases(Fusco et al., 2015).

24

Weissella spp. are among the most frequently occurring LAB in raw and spontaneouslyfermented cereals, vegetables, and fruits. They have adapted to the specific plant matrixand usually lead to desired and predictable changes in the shelf life, nutritional andsensory properties of the matrix (Di Cagno et al., 2013). In a study to modify the textureof pureed carrots by fermentation with EPS-producing LAB, dextran-producing W.confusa VTT E-90392, which was initially isolated from a sour carrot mash, conferredthe highest viscosity and desirable sensory profile (Juvonen et al., 2015). The highlynutritious barley malt extract was fermented by W. cibaria MG1, resulting in afunctional alcohol-free beverage with desirable textural properties (Zannini et al., 2013).

Sourdough is a cocktail of LAB and yeasts originating from wheat or rye flour and isused traditionally as a leavening agent in baking (Di Cagno et al., 2006). As commonsourdough isolates, the closely related W. confusa and W. cibaria are promising instarting sourdoughs (Di Cagno et al., 2006; Katina et al., 2009; Galle et al., 2010). Inwheat sourdough, W. confusa VTT E-90392 synthesized a high yield of dextran, whereasthe commercial dextran-producing Leuconostoc mesenteroides NRRL B-512F formedalmost none under the same conditions. Unlike many Leuconostoc spp., Weissella spp.produce dextran without extensive acidification, as they do not convert fructose intomannitol, with concomitant acetate production (Galle et al., 2010). Therefore, thetechnological benefits of Weissella dextran are not overridden by high acidity insourdough bread baking. The in situ-produced dextran improved the moisture retention,dough rheology, bread volume and crumb softness and retarded staling (Di Cagno et al.,2006; Schwab et al., 2008; Katina et al., 2009; Galle et al., 2010; Galle et al., 2012).Moreover, W. confusa and W. cibaria also produced a significant amount of IMOs insourdoughs and thus provided extra health benefits (Schwab et al., 2008; Katina et al.,2009; Galle et al., 2010). The amounts of IMOs and dextran in a W. confusa VTT E-90392 sourdough were estimated to be 4.0% and 1.8% (both dry weight), respectively,produced from 10% (dry weight) added sucrose (Maina 2012). The supplementation ofsucrose for Weissella dextran production also leads to free fructose production insourdough (5% fructose produced from 10% added sucrose), which does not comply tothe general diet advised to reduce sugar consumption. However, fructose leads to a lowerblood glucose increase compared to sucrose or glucose (European Food Safety Authority(EFSA) 2011) and thus has a relatively small health implication.

Dextransucrases from Weissella spp. may also find various uses, e.g., to modify foodstructure without concomitant acidification or for safety considerations compared towhole-cell fermentation. The crude dextransucrase from pear-derived Weissella sp.TN610 induced the solidification of sucrose-supplemented milk and was suggested as apromising, safe food additive (Bejar et al., 2013). Several W. cibaria and W. confusa

25

isolates from sourdoughs were found to produce constitutive soluble dextransucrases, asthe enzyme activities were always higher when they grew on glucose than on sucrose(Bounaix et al., 2010). However, for W. confusa Cab3 isolated from fermented cabbage,a strain related to this thesis work, dextransucrase production was boosted with sucrose(Shukla and Goyal 2011). In addition to this thesis, the biochemical and kineticproperties of dextransucrases from two other Weissella strains have been recentlystudied, with one being a native enzyme and the other being a recombinant one (Amariet al., 2012; Rao and Goyal 2013). The gene encoding the latter (of strain LBAE C39-2)was cloned and expressed in E. coli. An amino acid sequence alignment indicated thatWeissella dextransucrases form a distinct phylogenetic group within glucansucrases(Amari et al., 2012). The two enzymes exhibited similar molar masses (approximately180 kDa), pH optima (pH 5.4), thermal stabilities (inactivated rapidly above 35 °C), andMichaelis-Menten kinetic parameters (KM and Vmax values of 8.6–13 mM and 20–27.5µmol/(mg∙min), respectively), which were also comparable to the properties reportedfor Leuconostoc dextransucrases (Naessens et al., 2005; Côté and Skory 2012). Of note,the activity and thus the kinetic values of both enzymes were measured by conventionalreducing-value assays.

Dextrans from Weissella spp. share highly similar structures, normally consisting ofpredominantly α-(1→6) linkages and only a few α-(1→3) branch linkages (<5%)(Bounaix et al., 2009; Maina et al., 2011; Ahmed et al., 2012; Bejar et al., 2013).Weissella dextrans show high molar masses of >106 g/mol (Ahmed et al., 2012; Mainaet al., 2014). In fact, the technological benefits of dextrans in sourdoughs are attributedto their low degree of branching and high molar mass (Lacaze et al., 2007). Other thanthe GLOSs formed in the presence of maltose (Katina et al., 2009), the acceptor productsof Weissella dextransucrases have not yet been investigated. Weissella spp. may form,simultaneously with dextrans, byproducts from, e.g., lactose acceptor reaction whenendogenous sugars are present in food matrices. Furthermore, the enzymes may providea new opportunity for the biosynthesis of GLOSs of industrial interest via acceptorreactions.

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3. AIM OF THE STUDY

The overall aim of the thesis was to study the function and applicability of Weissella

dextransucrases, especially in the synthesis of glucooligosaccharides. Dextransucrases

from two efficient dextran-producing W. confusa strains were studied, namely, a native

enzyme from W. confusa Cab3 (WcCab3-DSR) and a recombinant one from W. confusa

VTT E-90392 (WcE392-rDSR), as no W. confusa dextransucrase had been studied in

detail by the time that this work started.

The specific objectives were to:

· study the biochemical and kinetic properties of WcE392-rDSR and compare a

reducing-value assay with a 14C-sucrose radioisotope assay to determine these

parameters (article I)

· structurally characterize the dextrans synthesized by the enzymes (articles I and

II)

· explore the selectivity of Weissella dextransucrase towards various acceptor

sugars and structurally characterize the selected GLOSs (articles II‒IV)

· apply Weissella dextransucrase for the synthesis of prebiotic IMOs (article IV)

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4. MATERIALS AND METHODS

4.1 Dextransucrase preparations and commercial enzymes

Sources of Weissella dextransucrases and other enzymes used in this work are presentedin Table 2. The Weissella preparations are described in articles I and II. Briefly, the geneencoding dextransucrase of W. confusa VTT E-90392 was cloned into Lactococcuslactis NZ9800. The resultant recombinant enzyme WcE392-rDSR was purified bynickel affinity chromatography or partially purified (the crude form) by ultrafiltering theculture supernatant (article I). W. confusa Cab3 was previously isolated from fermentedcabbage, and WcCab3-DSR was partially purified by PEG fractionation (article II).

Table 2. Dextransucrase preparations and commercial enzymes used in the study.Type Name Source ArticleDextran-sucrase

Purified recombinant dextransucrase fromW. confusa E-90392 (WcE392-rDSR)

VTT Technical ResearchCentre of Finland Ltd.

I

A crude preparation of WcE392-rDSR VTT I, III, IVNative dextransucrase from W. confusaCab3 (WcCab3-DSR)

Indian Institute ofTechnology Guwahati

II, IV

Commercial dextransucrase fromLeuconostoc mesenteroides

Sigma-Aldrich, MO IV

Hydrolyticenzyme

Chaetomium erraticum dextranase Sigma-Aldrich IIAspergillus niger α-glucosidase(E-TRNGL)

Megazyme, Ireland II, III

Bacillus stearothermophilus α-glucosidase (E-TSAGL)

Megazyme II

A. niger β-galactosidase Megazyme IIIAgrobacterium sp. β-glucosidase Megazyme IIIYeast invertase (β-fructosidase) Megazyme III

4.2 Biochemical characterization of WcE392-rDSR (I)

The protein concentration of the WcE392-rDSR preparation was determined by the DCProtein Assay Kit (Bio-Rad, CA) with bovine serum albumin as the standard. Theactivity of WcE392-rDSR was assayed by the 14C-sucrose radioisotope method and theNelson-Somogyi method (Nelson 1944; Somogyi 1945; Côté and Skory 2012). Oneactivity unit was defined as the amount of enzyme that catalyzed the incorporation of 1

28

µmol of D-glucose into dextran and the formation of 1 µmol of reducing sugar in 1 min,respectively.

The Nelson-Somogyi method was used to determine the pH optimum, the effect oftemperature on the activity, and the thermal stability of WcE392-rDSR. Kineticparameters were determined by both the 14C-sucrose radioisotope and Nelson-Somogyimethods, with the sucrose concentration ranging from 3 to 146 mM. The initial velocitydata were analyzed by nonlinear regression using GraphPad Prism 6 (GraphPad, CA).

4.3 Analysis of monosaccharides and oligosaccharides

HPAEC-PAD was used to analyze mono- and oligosaccharides. Prior to analysis, thesamples were filtered through a 10 kDa Amicon Ultra-0.5 centrifugal filter (Millipore,MA) or a 0.45 μm membrane (Pall Corporation, NY). The instrument (unless otherwisespecified) was equipped with a CarboPac PA-100 column (250 × 4 mm, i.d, Dionex,CA), a Decade detector (Antec Leyden, The Netherlands), a Waters 717 autosampler,and two Waters 515 pumps. For a qualitative analysis of the oligosaccharide profiles,the elution was performed with 100 mM NaOH (15 min), followed by a gradient to 120mM Na-acetate in 100 mM NaOH (20 min) with a flow rate of 1 ml/min.

For the quantification of mono- and oligosaccharides, the same system was run byanother elution method, starting with 75 mM of NaOH (8 min) and followed by agradient to 100 mM of Na-acetate in 75 mM of NaOH (27 min) with a flow rate of 1ml/min. In the case of the co-elution of lactose and sucrose, another HPAEC-PADsystem was used in article III. Glucose, fructose, galactose, maltose, sucrose, lactose,cellobiose (Merck, Germany), leucrose (Santa Cruz Biotechnology, CA), isomaltose,isomaltotriose, panose (TCI Europe nv, Belgium) and kojibiose (Carbosynth, UK) wereused as standards. Panose was also used as a standard for the quantification of total IMOproducts (by integrating the total peak area) and individual IMOs of DP4‒6 in article IV,as no reference compounds were available. Maltooligosaccharides of DP3‒6 (Sigma–Aldrich, MO) were used in article IV to study the effect of DP on the elution time anddetector response factor for members of a homologous series. Oligosaccharides inisolated fractions in article III were also separated by thin-layer chromatography (TLC)(Côté and Leathers 2005) to confirm that the peaks detected by HPAEC-PAD consistedof single compounds.

29

The total carbohydrate concentration in oligosaccharide and dextran solutions (inarticles III and IV) was determined by the phenol-sulfuric acid method (Dubois et al.,1956).

4.4 Synthesis, isolation, and molar mass characterization of dextrans (Iand II)

Dextrans were synthesized in vitro by incubating 1.06 U/ml (Nelson-Somogyi unitsunless otherwise specified) of WcE392-rDSR or WcCab3-DSR in 20 mM Na-acetatebuffer, pH 5.4, containing 2 mM CaCl2 and 146 mM sucrose at 35 °C for 24 h. After theaddition of two or three volumes of ethanol, the solution was centrifuged at 10,000 gand 4 °C for 15 min. The re-dissolved dextran pellet was precipitated and re-dissolvedagain. Dextrans were produced on agars by the native host W. confusa VTT E-90392 orthe heterologous host L. lactis and extracted according to Maina et al. (2008). Thedextrans were freeze-dried for characterization. The selective enzymatic hydrolysis andNMR spectroscopy analysis of dextrans are described in following sections.

The molar mass of dextrans was determined with a high-performance size-exclusionchromatography (HPSEC) multidetector system (Maina et al., 2014). Dextran solutionsof 2.0 mg/mL were prepared in dimethylsulfoxide (DMSO) with 10 mM LiBr. Thesolutions were kept at room temperature for at least four days for dissolution and thenfiltered using a 0.45 μm membrane filter before analysis. The system was equipped withtwo analytical columns (exclusion limit >2 × 106) and a guard column. The HPSEC datawere processed with the Viscotek OmniSEC 4.5 software. A dη/dc value of 72 µL/g fordextran in DMSO-based eluent was used (Basedow and Ebert 1979).

4.5 Acceptor reactions and isolation of oligosaccharides (II‒ IV)

To compare the specificity of WcE392-rDSR, WcCab3-DSR, and the commercial Lc.mesenteroides dextransucrase, maltose acceptor reactions were carried out under similarconditions (Table 3). WcE392-rDSR was also tested with several other di- andtrisaccharides as acceptors (Table 3). The reactions were conducted at 30 °C in 0.2 mlof a 20 mM Na-acetate buffer (pH 5.4) containing 2 mM of CaCl2 for 24 h. The enzymeaction was terminated by incubation in a boiling water bath for 10 min. Asucrose:acceptor molar ratio of 4 was used, except for the lactose reaction, where asecond major product was promoted with an increasing sucrose:lactose ratio. In articleIV, panose and a mixture of maltose trisaccharide products of a total carbohydrate

30

concentration of 1 mM were studied as acceptors in reactions using 5 mM of sucroseand 5.5 U/g sucrose of WcE392-rDSR at room temperature for 24 h.

Table 3. Acceptor information and the initial concentrations of sucrose (Suc) and the acceptor(Acc) in WcE392-rDSR-catalyzed (10 U/g of sucrose) reactions.Acceptor Structure Supplier Acc

(mM)Suc:Accmolar ratio

Maltose α-D-Glcp-(1→4)-D-Glcp Merck, Germany 150 4Lactose β-D-Galp-(1→4)-D-Glcp " 150 6.7Cellobiose β-D-Glcp-(1→4)-D-Glcp " 50 4Laminaribiose β-D-Glcp-(1→3)-D-Glcp Megazyme, Ireland 50 4Nigerose α-D-Glcp-(1→3)-D-Glcp Sigma-Aldrich, MO 50 4Melibiose α-D-Galp-(1→6)-D-Glcp " 50 4Isomaltose α-D-Glcp-(1→6)-D-Glcp TCI Europe nv, Belgium 25 4Maltotriose α-D-Glcp-(1→4)-α-D-

Glcp-(1→4)-D-GlcpSigma-Aldrich 50 4

Forkosea α-D-Glcp-(1→4)-[α-D-Glcp-(1→6)]-D-Glcp

Carbosynth, UK 50 4

a Name according to Hansen et al. (2008).

A common series of oligosaccharides were synthesized by WcE392-rDSR and WcCab3-DSR in the presence of maltose, as exhibited by their similar HPAEC-PAD profiles.Thus, only the products from WcCab3-DSR were taken for structural characterization.The synthesis was carried out in 100 ml using 292 mM sucrose, 146 mM maltose, and aWcCab3-DSR dosage of 10 U/g sucrose. Moreover, to characterize the two mainproducts (named Lac3 and NR3) observed in the WcE392-rDSR lactose acceptorreaction, the synthesis was carried out in 50 ml. The major product of the cellobioseacceptor reaction (named Cel3) was also structurally characterized for comparison withthe product derived from analogous lactose. The cellobiose acceptor reaction was scaledup to 50 ml.

After the reaction was stopped, two or three volumes of ethanol (Etax A, AltiaCorporation, Finland) were added to the reaction mixture, which was then centrifugedto remove polymeric dextran. The supernatant containing fructose, residual acceptor,and acceptor products was concentrated using a rotatory vacuum evaporator (HeidolphLaborota 4001 Digital, Sigma-Aldrich, MO). Prior to injection into a Biogel P2 column(5 × 95 cm; Bio-Rad, Hercules, CA), the concentrate was filtered through a 0.45 μmmembrane filter. Water was the eluent at a flow rate ≤0.7 ml/min. The oligosaccharides

31

in the collected fractions were analyzed by HPAEC-PAD. The fractions containing thehighest proportion of products of interest were used for a subsequent structural analysis.

4.6 Structural analysis of dextrans and oligosaccharides

4.6.1 Enzymatic hydrolysis of dextrans and oligosaccharides (II and III)

Dextransucrase products were selectively digested with specific enzymes for structuralinformation. The hydrolysis reactions are presented in Table 4. After enzymeinactivation in a boiling water bath for 10 min, the digests were analyzed by HPAEC-PAD and compared to controls without enzyme treatment.

Table 4. Enzymatic hydrolysis conditions for dextran and oligosaccharide characterization. Analyte Enzyme dosagesa Conditionb

Dextran 0.6 U/mg of C. erraticum dextranase and0.06 U/mg of A. niger α-glucosidase

30 °C, 48 h

Maltose-derivedproduct series

0.3 U/mg of C. erraticum dextranase or0.04 U/mg of B. stearothermophilus α-glucosidase

"

Lactose-derivedtrisaccharide (Lac3)

25 U/mg of A. niger α-glucosidase or50 U/mg of A. niger β-galactosidase

overnight at30 °C

Cellobiose-derivedtrisaccharide (Cel3)

25 U/mg of A. niger α-glucosidase or75 U/mg of Agrobacterium sp. β-glucosidase

"

Nonreducingtrisaccharide (NR3)

25 U/mg of A. niger α-glucosidase,200 U/mg of dextranase,or 100 U/mg of yeast invertase

"

a Enzyme dosages reference the amount of dextran, total carbohydrate in dextran-removedmaltose acceptor reaction mixture, or total carbohydrate in isolated product fractions for Lac3,Cel3 and NR3.b Reactions were performed in Na-acetate or Na-citrate buffer at 20‒50 mM and pH 5.0‒5.5.

4.6.2 NMR spectroscopy analysis of dextrans and oligosaccharides (I‒IV)

NMR spectra were recorded with a 600 MHz Bruker Avance III NMR spectrometer(Bruker BioSpin, Germany). The dextran (10 mg/ml) and isolated trisaccharides (>0.3mg/ml) were exchanged with D2O (MagniSolv, Merck, Germany) and placed in NMRtubes (Wilmad NMR tubes, 5 mm, ultra-imperial grade, Sigma-Aldrich, MO). The 1Hand the corresponding 2D (multiplicity-edited HSQC, DQFCOSY, TOCSY, and HMBC)NMR experiments were performed at a temperature at which the residual water signaldid not affect the signals of the analyte. Topspin v.3.2 (Bruker, Germany) software was

32

used to process and analyze the NMR spectra. The chemical shifts of 1H (δH) and 13C(δC) were referenced to acetone (δH 2.225 and δC 31.55, respectively).

4.6.3 MSn analysis of oligosaccharides (II‒IV)

Electrospray ionization multistage ion trap mass spectrometry (ESI-IT-MSn) was usedfor the molar mass determination and characterization of the oligosaccharides isolatedby gel filtration. The analysis was carried out in negative or positive mode ([M+Cl]- and[M+Li]+, respectively) using the Agilent XCT Plus Ion Trap mass spectrometer with anelectrospray ion source (Agilent Technologies, CA) or a Bruker Esquire LC quadrupoleion trap mass spectrometer with an electrospray ion source (Bruker Daltonik GmbH,Germany). A 1–10 µl sample (~0.5 mg/ml) was diluted in 200–400 µl of methanol-water-formic acid (50:49:1, v:v:v), and 0.5–1 µl of 10 mg/ml ammonium chloride orlithium acetate was added for adduct ion formation. The samples were introduced to theion source with a direct infusion syringe pump at a flow rate of 5 µl/min. MS spectrawere processed with LC/MSD Trap Software (Bruker Daltonik GmbH). Cellotriose(Megazyme) and panose (TCI Europe nv) were used as model compounds for thecharacterization of Cel3 and BIM3 (the minor trisaccharide obtained in the maltoseacceptor reaction), respectively.

The maltose and lactose acceptor reactions were further carried out using [UL-13C6glc]sucrose (Omicron Biochemicals Inc. IN) so that the GLOS products were 13C6glc-labeledfor ESI-IT-MSn analysis. The maltose reaction was conducted by incubating 0.3 U ofcommercial Lc. mesenteroides dextransucrase in 0.5 ml of 20 mM Na-acetate buffer, pH5.4, containing 0.1 M 13C6glc sucrose and 0.2 M maltose at 30 °C for 48 h. The GLOSproducts were isolated by gel filtration. According to TLC analysis, the fractioncontaining solely DP5 was selected for MSn analysis as [M+Cl]- and [M+Li]+ ions.13C6glc sucrose was also used to synthesize the novel trisaccharides (Lac3 and NR3)identified in the lactose reaction mixture. The product mixture obtained was analyzedby MSn to characterize the fragmentation patterns of the two labeled products (articleIII).

4.7 Experimental design for IMO synthesis (IV)

The concentrations of sucrose, maltose, and WcE392-rDSR were selected as threeindependent variables in the experimental design. Preliminary experiments were carriedout to select the following ranges of factors: sucrose (0.15–1 M), maltose (0.15–1 M),and dextransucrase in relation to sucrose concentration (1–10 U/g sucrose). An

33

experimental series consisting of 18 experiments was designed to model the effects ofthe factors on IMO synthesis. The reactions were conducted in 20 mM Na-acetate buffer,pH 5.4, containing 2 mM CaCl2 at 30 °C for 24 h.

The yields of individual IMOs of DP3‒6, the yield of total IMOs, and the percent ofconsumed maltose were each modelled by a quadratic regression model. Modde 9.0(Umetrics AB, Sweden) was used to analyze the results by multiple linear regression(MLR) or partial least squares (PLS) method and to generate contour plots. The validityof the regression models was evaluated by the coefficient of determination (R2) and thecoefficient of prediction (Q2).

34

5. RESULTS

The dextransucrases from two efficient dextran-producing Weissella confusa strains (W.confusa Cab3 isolated at IITG and W. confusa E-90392 from VTT Culture Collection)were the subjects of this study. In the beginning, the dextransucrase from native W.confusa E-90392 was produced and partially purified by PEG fractionation. However,the yield was low and subsequent purification attempts failed to remove theconcomitantly produced dextran from the enzyme preparation (unpublished). Becausethe presence of dextran is known to affect the enzyme characteristics, dextran-freeenzyme was obtained by cloning and expressing the dextransucrase gene in Lactococcuslactis with a nisin-inducible expression system (others’ work). The recombinant W.confusa E-90392 dextransucrase (WcE392-rDSR) was used in biochemical and kineticstudies. Moreover, the dextransucrase from the other strain, W. confusa Cab3 (WcCab3-DSR), was obtained with better yield by PEG fractionation and was used to synthesizedextran and GLOSs in parallel with WcE392-rDSR to compare their product specificity.

5.1 Characterization of WcE392-rDSR (I)

The specific activity of the nickel-affinity-purified WcE392-rDSR was 7.2 ± 0.3 U/mgand 18.9 ± 0.6 U/mg, as determined by the 14C-sucrose radioisotope assay and theNelson-Somogyi assay, respectively. According to the HPAEC-PAD analysis of thereaction mixture after sucrose depletion, glucose and leucrose accounted for only 2%and 5% (w/w) of the total mono- and disaccharides. Fructose represented the remaining93%, indicating that the transferase reaction synthesizing dextran comprises the mainproportion of the WcE392-rDSR activity.

The Nelson-Somogyi assay was used to determine the effects of pH and temperature onthe activity. The optimal pH for WcE392-rDSR activity was observed at pH 5.4 in 20 MNa-acetate buffer (Figure 6A). The rate of dextran synthesis increased with highertemperatures up to 35 °C, after which it decreased (Figure 6A), as shown in the activityassay (15 min) conducted at different temperatures. The thermal stability of WcE392-rDSR was studied at 25, 35 and 40 °C in the presence or absence of glycerol up to 24 h(Figure 6B). The enzyme stability decreased sharply when the temperature increasedfrom 35 °C (half-life of 26.3 h) to 40 °C (half-life of 7.4 h). Glycerol enhanced thestability at each of the three temperatures; for example, it increased the enzyme half-lifeat 35 °C from 26.3 to 37.5 h.

35

WcE392-rDSR exhibited Michaelis-Menten kinetics with a KM of 14.7 mM, a Vmax of8.2 µmol/(mg∙min) and a kcat of 23.2 1/s, determined using the sucrose radioisotope assay.The sucrose radioisotope assay and the Nelson-Somogyi assay gave similar KM values,whereas values approximately 2.5 times higher were obtained for Vmax, kcat and kcat/KM

with the Nelson-Somogyi method (Table 5).

Table 5. Specific activities and kinetics of WcE392-rDSR (Table 5 in article I).Assay Activity

(U/mg)KM

(mM)Vmax

[µmol/(mg∙min)]kcat

(1/s)kcat/KM

[1/(M∙s)]

14C-Sucroseradioisotope

7.2 ± 0.3 14.7 ± 2.3 8.2 ± 0.4 23.2 ± 1.1 (1.6 ± 0.3) × 103

Nelson-Somogyi 18.9 ± 0.6 13.0 ± 1.1 19.9 ± 0.6 56.4 ± 1.7 (4.3 ± 0.5) × 103

Figure 6. (A) Effects of pH and temperature on WcE392-rDSR activity. The effect of pH wasassayed in 20 mM Na-acetate buffer (pH 4‒6) or in 20 mM Tris-HCl buffer (pH 6.5‒7.0), andthe effect of temperature in 20 mM Na-acetate buffer, pH 5.4. Error bars represent the standarderrors of the mean (n=3). (B) The thermal stability of WcE392-rDSR in the presence and absenceof 0.5% (v/v) glycerol after incubation for 1, 2, 5 and 24 h. (Adapted from Figures 3 and 4 inarticle I.)

5.2 Characterization of Weissella dextrans (I and II)

The dextrans synthesized in vitro by the two Weissella dextransucrases were isolated byethanol precipitation and lyophilized for enzyme-aided branch-type fingerprinting,NMR spectroscopy and HPSEC analysis. For the fingerprinting of branch type, the

A B

36

dextrans were hydrolyzed with a mixture of dextranase and α-glucosidase. The resultingresistant oligosaccharides exhibited a similar HPAEC-PAD profile (Figure 7A forWcCab3-DSR dextran, whereas that for WcE392-rDSR dextran is not shown) to thoseof dextran extracted from W. confusa VTT E-90392 culture (Maina et al., 2011). Basedon the structure identified earlier for the in vivo-synthesized dextran (Maina et al., 2011),the enzymatically produced dextrans were shown to contain α-(1→3)-linked branches,with some branches elongated.

WcCab3-DSR dextran (Figure 7B) had a similar 1H NMR spectrum to that of thedextrans produced by WcE392-rDSR and its heterologous host L. lactis (Figure S3 inarticle I). Based on the relative intensities of the anomeric proton signals at 4.97 and5.32 ppm, the ratio of α-(1→6) to α-(1→3) linkages was 97:3. The remaining protonresonances at 3.97, 3.91, 3.77, 3.73, 3.58, and 3.52 ppm were assigned to H-6b, H-5, H-6a, H-3, H-2, and H-4 of the α-(1→6)-linked glucopyranosyl residues, respectively. Inthe edited HSQC NMR spectrum of WcCab3-DSR dextran (Figure 7C), a cross peak of4.18→71.6 ppm was assigned to H-5→C-5 of the elongated branch point residue [-(1→6)-α-D-Glcp-(1→3)-] (Maina et al., 2011), confirming the presence of brancheswith more than one glucosyl unit.

The dextrans that were enzymatically synthesized and those produced in vivo by nativeW. confusa VTT E-90392 and heterologous host L. lactis were analyzed by HPSEC. Themolar masses were obtained using DMSO with 10 mM LiBr as the solvent becausedextrans are prone to aggregation in aqueous solutions (Maina et al., 2014). One majorpeak was observed with both refractive index (RI) and light scattering detectors (HPSECchromatograms not shown). The molar masses of dextrans were in the range of 1.8 to8.5 × 107 g/mol.

37

Figure 7. (A) HPAEC-PAD chromatograms showing resistant oligosaccharides from WcCab3-DSR dextran after dextranase and α-glucosidase treatment. 1H (B) and edited HSQC (C) NMRspectra of WcCab3-DSR dextran. The spectra were recorded at 600 MHz in D2O at 50 °C. Peaksin the NMR spectra were referenced to internal acetone (δH 2.225 ppm and δC 31.55 ppm).(Adapted from Figures 3 and 4 in article II.)

38

5.3 Characterization of the oligosaccharides produced bydextransucrases

5.3.1 Acceptor selectivity (III)

The action of WcE392-rDSR on several glucose-containing di- and trisaccharides wasevaluated. In all of the reactions, a large amount of fructose was released, and dextranwas produced. Judging by the product peak area in the HPAEC-PAD chromatograms(Figure S1 in article III), the more effective acceptors among those tested were maltose,isomaltose, maltotriose, and nigerose. A series of products were detected in thesereaction mixtures, indicating that the acceptor products could be further elongated.Lactose and cellobiose showed moderate effectiveness (section 5.3.4), whereasmelibiose, laminaribiose, and forkose were poor acceptors (data not shown) forWcE392-rDSR.

5.3.2 Principal maltose product series (II)

The HPAEC-PAD profile of the glucooligosaccharides (GLOSs) produced in thepresence of maltose was similar between WcCab3-DSR, WcE392-rDSR, and thecommercial Lc. mesenteroides dextransucrase. According to the HPAEC-PADchromatogram of WcCab3-DSR products (Figure 8A, those of WcE392-rDSR and Lc.mesenteroides dextransucrase are not shown), a number of GLOSs were produced, withsome being predominant and others being minor. After treatment with endo-actingdextranase, most GLOSs eluting after panose were degraded into their building blocks,i.e., glucose, isomaltose, isomaltotriose, and panose (Figure 8B), indicating that theyhave consecutive α-(1→6) linked glucosyl residues attached to the nonreducing end ofthe maltose and therefore belong to isomaltooligosaccharides (IMOs). As expected, theGLOSs were resistant to the hydrolysis of an α-glucosidase exo-acting on α-(1→4)linkages from the nonreducing end (article II).

The GLOSs that were produced in the WcCab3-DSR maltose reaction were isolatedaccording to DP by gel filtration (Figures 8C‒F). A HPAEC-PAD and MS analysis ofthe fractions showed that for each principal IMO product, there was a minor product ofthe same DP. The minor products eluted earlier than did the corresponding majorproducts (Figures 8C‒F) and seemed to constitute another series. The HPAEC retentiontimes of the two product series were plotted versus their DP (Figure S4 in article IV).The plot for the minor series exhibited a similar slope to that of the principal IMOs butdistinct from that of the α-(1→4)-linked maltooligosaccharides. For GLOSs growing viathe same linkage, the slope (representing the peak interval between adjacent members

39

in the series) appeared to depend on the type of linkage. The HPAEC mobility resultindicated that the two series observed in the maltose reaction mixture were IMOs. Thiswas confirmed by the susceptibility of higher DP products of both series to dextranase(Figure 8B). Because a minor DP4 product coeluted with panose in HPAEC, thedextranase hydrolysate was further fractionated by gel filtration (data not shown). AnHPAEC-PAD analysis of the isolated DP4 fractions showed that the minor DP4remained after dextranase hydrolysis. The members in principal series, supposedlylinear, were named after panose, LIM4 (DP4), LIM5 (DP5), and so on, while the minorIMOs were suspected to be branched and named BIM3 (DP3), BIM4 (DP4), and so on.

Figure 8. HPAEC-PAD profiles of (A and B) acceptor products synthesized by WcCab3-DSRfrom 5% (w/v) maltose and 10% (w/v) sucrose: (A) unhydrolyzed; (B) hydrolyzed by dextranase;and (C‒F) selected fractions of isolated products by gel filtration. The m/z of DP3‒5 as [M+Cl]-

ions are shown. The principal product series are named Pan and LIM4‒6 and the minor seriesBIM3‒6. Labeled peaks are: Glc, glucose; Fru, fructose; IM2, isomaltose; IM3, isomaltotriose;Mal, maltose; and Pan, panose. (Adapted from Figure 4 and Figure 2 in articles I and IV,respectively.)

To confirm the proposed structures for the principal series, negative ion MSn was usedto analyze isolated principal products of DP3‒5 ([M+Cl]- ions of m/z at 539, 701, and863, respectively). The diagnostic fragment ions at m/z 425 and 383 (loss of 78 and 120Da, respectively) and m/z 281, 251, and 221 (loss of 60, 90 and 120 Da, respectively) in

Glc

Fru

IM2

IM3

A

B

DP4‒DP12Mal

Pan

BIM4

LIM4

BIM5

LIM5

BIM6

LIM6

BIM3

Pan

C

D

E

Fm/z 539

m/z 701

m/z 863

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 min

40

the MS2 spectrum of DP3 (not shown) confirmed the α-(1→4) linkage at its reducingend and the α-(1→6) linkage at its nonreducing end, i.e., panose (Maina et al., 2013).Similarly, in the MS2 spectrum of the [M+Cl]- ion of DP4 (Figure 9A), fragment ions atm/z 587 and 545 (loss of 78 Da and 120 Da, respectively) indicated the presence of anα-(1→4) linkage at the reducing end. The ions at m/z 443, 413, and 383 (loss of 60, 90,and 120 Da, respectively) in the MS3 spectrum, starting with the ion at m/z 503, indicatedthe subsequent α-(1→6) linkage (Figure 9B). The α-(1→6) linkage at the nonreducingend was confirmed with the MS3 spectrum of the ion at m/z 341 (Figure 9C), whichcontained fragment ions at m/z 281, 251, and 221 (loss of 60, 90, and 120 Da,respectively). The MSn spectra of DP5 showed a similar trend (Figure 3S in article I):an α-(1→4) linkage at its reducing end and the remaining linkages being α-(1→6). Thesefindings confirmed that the principal maltose products are homologous IMOs thatcontain maltose at the reducing end.

Figure 9. MS2 (A) and MS3 (B and C) spectra of the [M+Cl]- ion of the principal maltose productof DP4 (LIM4). The MS3 spectra in B and C are for the fragment ions at m/z 503 and 341,respectively. (Adapted from Figure 5 in article I.)

C2341

0,4A3383

0,3A3413

0,2A3443

B3485

C3503

2,4A4545

0,2A4-H2O587

[M-H]-665

[M+Cl]-701

0.0

0.2

0.4

0.6

0.8

1.0

6x10Intens.

179281 323

383

413

443

485

503

0

2000

4000

6000

8000

B1161

C1179

0,4A2221

0,3A2251

0,2A2281

B2323

C2341

0

1000

2000

3000

4000

5000

100 200 300 400 500 600 700 m/z

A DP4 -MS2

B -MS3 m/z 503

C -MS3 m/z 341

41

5.3.3 Minor maltose product series (IV)

Because BIM3 of the minor series eluted slightly earlier than did panose in gel filtration,few fractions collected before the elution of panose contained enriched BIM3 and wereselected for NMR spectroscopy and ESI-IT-MSn (section 5.3.6) analysis. The structureof BIM3 was determined by a set of NMR experiments using a fraction containing anapproximately equimolar mixture of panose and BIM3. Pure panose was analyzed inparallel so that the signals from panose could be subtracted from those of the mixture,and the remaining signals were interpreted. Compared to the 1D 1H spectrum of panose,five doublets in the anomeric proton region were found for BIM3 (Figure 10). Theintegrals of the anomeric protons indicated that the trisaccharide was an anomericmixture, with the resonances of one nonreducing residue affected by mutarotationequilibrium. The proton signals of each spin system were assigned using TOCSY andDQFCOSY spectra (Figure S5 in article IV). The signals at δH 5.45 (d J1-2 3.4 Hz) andδH 4.81 (d J1-2 8.0 Hz) were assigned to the α and β anomeric protons of the reducingglucopyranosyl residue, respectively. The signal at δH 5.49 (d J1-2 3.5 Hz) correspondedto the anomeric proton of the α-(1→4)-linked glucopyranosyl residue in maltose. Theother two anomeric signals at δH 5.10 (d J1-2 3.6 Hz) and δH 5.41 (d J1-2 3.9 Hz) wereassigned to a terminal α-(1→2)-linked glucopyranosyl residue in α and β anomers,respectively (Figure 10).

Figure 10. 1D 1H NMR spectra of (A) pure panose and (B) a mixture containing equimolaramounts of panose and BIM3. The spectra were measured at 600 MHz in D2O at 11 °C. Thestructure of BIM3 is illustrated. (Adapted from Figure 4 in article IV.)

42

The downfield shift of C2 signals of the reducing residue in the edited HSQC spectrum(Figure 4C and Table S3 in article IV) indicated that the third glucosyl residue wasattached to the C2 of maltose-reducing end, forming a branched structure. Directevidence for the α-(1→2) linkage was observed in the HMBC (Figure 4D in article IV)correlation peaks: between GlcI-C2 (δC 76.76) and GlcIII-H1 (δH 5.10) and betweenGlcIII-C1 (δC 97.41) and GlcI-H2 (δH 3.67) for the α-anomer and between GlcI-C2 (δC

79.52) and GlcIII-H1(δH 5.41) and between GlcIII-C1 (δC 98.83) and GlcI-H2 (δH 3.41)for the β-anomer. Thus, BIM3 was identified as α-D-Glcp-(1→2)-[α-D-Glcp-(1→4)]-D-Glcp, also known as centose.

To understand the structures and synthesis mechanism of the higher homologues of theminor series, BIM3 (in a fraction containing an equal molarity of BIM3 and panose) wastested as an acceptor. The HPAEC-PAD analysis before and after the reaction showedthat BIM3 did not act as an acceptor (data not shown). Thus, the higher DP minorproducts contained a single-unit α-(1→2)-branch at the reducing end, and additional α-(1→6)-linked residues in the main chain, as the α-(1→2)-linked branch was not able toelongate further. Moreover, the minor series were proposed to be synthesized throughdirect branch formation on an elongated linear IMO rather than through the elongationof the main chain after branch formation. This mechanism was supported by the productpattern observed using panose as a sole acceptor. A trace of BIM5 was formed (FigureS6 in article IV) most probably by branch formation on LIM4. The branch may beformed on panose as well, although the resulting BIM4 could not be directly detected inthe HPAEC analysis (as it was not well separated from panose).

5.3.4 Lactose- and cellobiose-derived branched trisaccharides (III)

The major products observed in the reaction mixtures of lactose and cellobiose (Figure11A and 11B) were selected for structural analysis to determine whether the same typeof linkage was formed on the two analogs by Weissella dextransucrase. The productsderived from lactose and cellobiose were referred to as Lac3 and Cel3, respectively, asthey were later found to be trisaccharides. Moreover, in the lactose reactions withvarying sucrose and enzyme concentrations (Figure S2 in article III), a second productwas promoted when both concentrations increased. This product was also selected forcharacterization and named NR3 (later found to be a nonreducing trisaccharide, Figure11A). The two products from the WcE392-rDSR lactose reaction mixture and one fromthe cellobiose reaction were isolated by gel filtration (Figure 11C). The selected Lac3and Cel3 fractions contained negligible amounts of impurities, whereas the NR3 fractioncontained a minor portion of Lac3, in addition to other impurities. The three product

43

fractions were subjected to an ESI-IT-MS2 and NMR spectroscopy analysis andenzymatic hydrolysis.

Figure 11. HPAEC-PAD profiles of (A and B) acceptor product mixtures of WcE392-rDSR (10U/g sucrose) with (A) 150 mM lactose and 1 M sucrose and (B) 50 mM cellobiose and 200 mMsucrose and those of (C) selected product fractions isolated by gel filtration in order of elution.The insets show the zoomed-in chromatograms of the product areas. Labeled peaks are: Glc,glucose; Fru, fructose; Leu, leucrose; Lac, lactose; Cel, cellobiose. The peak DP3# waspresumed to be NR3. (Modified from Figure 1 in article III.)

C

Isolated from lactoseproduct mixture

Isolated from cellobioseproduct mixture

Fraction Lac3

Fraction NR3

Fraction Cel3

5.00 10.00 15.00 20.00 25.00 30.00 35.00 min

Cel

Cel3

DP3#

Lac3

NR3

44

The three isolated products were preliminarily analyzed by negative ion MS2. The threeproducts ([M+Cl]- ions) exhibited an m/z value of 539 and were therefore trisaccharides.The MS2 spectra of [M+Cl]- ions of Lac3 and Cel3 were similar (Figure 12A), indicatingsimilar structures and thus fragmentation patterns. The relative abundance of thefragment ions resulting from the cross-ring cleavage of the reducing-end residue wasdifferent from that of cellotriose (data not shown) and other linear oligosaccharidescontaining a β-(1→4)-linked reducing-end hexopyranose (Maina et al., 2013). Thissuggests that Lac3 and Cel3 probably have a rare branched structure. The absence ofcross-ring fragment ions in the MS2 spectrum of the [M+Cl]- ion of NR3 (Figure 12A,the ion at m/z 425 probably resulted from Lac3 present in the fraction) indicated thatNR3 is possibly a nonreducing trisaccharide.

The structures of Lac3 and Cel3 were determined by a set of NMR experiments. Theiranomeric proton regions in 1D 1H spectra (Figure 12B) were similar. Both showed sixanomeric proton signals representing two sets of resonances due to the effect ofmutarotation equilibrium on the two other residues. For example, the signals of Cel3 atδH 5.44 (d J1-2 3.5 Hz) and δH 4.81 (d J1-2 8.0 Hz) corresponded to α- and β-anomericprotons of the reducing glucopyranosyl residue, respectively. The signals at δH 4.52 (dJ1-2 7.8 Hz) and δH 4.51 (d J1-2 7.9 Hz) corresponded to the anomeric proton of the β-(1→4)-linked glucopyranosyl residue. The last two anomeric signals at δH 5.10 and δH

5.37 (both d J1-2 3.8 Hz) were from an α-(1→2)-linked glucopyranosyl residue. Evidencefor α-(1→2)-linkage in Cel3 was observed in the HMBC (Figure S3 in article III)correlation peaks: between GlcI-C2 (δC 76.84) and GlcIII-H1 (δH 5.10) and betweenGlcIII-C1 (δC 97.70) and GlcI-H2 (δH 3.67) for the α-anomer and between GlcI-C2 (δC

79.60) and GlcIII-H1(δH 5.37) and between GlcIII-C1 (δC 99.20) and GlcI-H2 (δH 3.41)for the β-anomer. Thus, Cel3 was identified as α-D-Glcp-(1→2)-[β-D-Glcp-(1→4)]-D-Glcp (21-α-Glcp-cellobiose). Similarly, Lac3 was identified as α-D-Glcp-(1→2)-[β-D-Galp-(1→4)]-D-Glcp (2Glc-α-Glcp-lactose). Thus, both Lac3 and Cel3 contain aglucosyl residue that is α-(1→2) linked to the reducing end of the acceptor disaccharide(lactose and cellobiose, respectively).

45

Figure 12. MS2 spectra of [M+Cl]- ions (A) and 1D 1H NMR spectra (B) of isolated Cel3, Lac3, and NR3. The NMR spectra were measured at 600 MHz inD2O at 16 ºC. The β-anomeric signals of the reducing-end residue of Lac3 and Cel3 were partially suppressed with the residual water signal. (Adapted fromFigures 2 and S6‒8 in article III.)

α-GlcIII(α)α-GlcIII(β) β-GlcII(α)

β-GlcIα-GlcI

β-GlcII(β)

α-GlcII(α)

α-GlcII(β) β-Gal(α)

β-GlcIα-GlcI

β-Gal(β)

α-GlcI α-GlcII

Lac3

NR3

B

Glc II Glc I

Glc III

Cel3179

221 263 341

425

467485

503

539

0

2

4

6

6x10

[M-H]-

[M+Cl]-Hex

Intens.

425

503

539467

485

[M-H] -

[M+Cl] -

179

221263 341

383

0.5

1.0

1.5

6x10

Hex

425

503

539

1

2

37x10

[M-H]-

[M+Cl] -

100 150 200 250 300 350 400 450 500 m/z

A

Glc II

Gal Glc I

Glc II

Fru

Glc I

46

Figure 13. HPAEC-PAD profiles of isolated product fractions before (dashed lines) and after(solid lines) specific enzymatic digestion. The labeled peaks were identified based on theirretention time against standards: Glc, glucose; Fru, fructose; Lac, lactose; Cel, cellobiose; Gal,galactose; Suc, sucrose; and Koj, kojibiose. (Adapted from Figure 3 in article III.)

The product fractions of Lac3 and Cel3 were selectively digested with specific enzymesand then analyzed by HPAEC-PAD (Figure 13). The α-(1→2) linkage was hydrolyzedby α-glucosidase, generating glucose and the corresponding acceptor (lactose andcellobiose, respectively). The β-(1→4) linkage in Lac3 was hydrolyzed by β-galactosidase, leading to the formation of galactose and kojibiose (α-D-Glcp-(1→2)-D-Glcp), as expected. The glucose that formed concomitantly was due to the side reactionof the β-galactosidase preparation on the α-(1→2)-linked glucosyl residue, asmanifested by the reaction of the β-galactosidase preparation on Cel3 and pure kojibiose(data not shown). Although Cel3 was not very susceptible to hydrolysis by the β-glucosidase used, a small amount of it was converted to glucose and kojibiose. Theabove results further support the notion that Lac3 and Cel3 contain a glucosyl unit thatis α-(1→2) linked to the reducing end of their acceptors.

5.3.5 Novel sucrose-containing trisaccharide (III)

Under the conditions selected for the lactose acceptor reaction, a second product denotedby NR3 (Figure 11A) was obtained. The 1D 1H spectrum of the isolated NR3 (Figure12B) was different from that of Lac3 and Cel3. Apart from the signals from theimpurities (mainly Lac3) in isolated NR3, there were two anomeric protons at δH 5.42(d J1-2 3.9 Hz) and δH 4.96 (d J1-2 3.7 Hz), both with an α configuration (Figure 12B).The presence of only two anomeric protons for a trisaccharide indicated that one of its

15.00 25.00 35.00 45.00

Cel3

Lac3NR3

Lac3

min

Cel3

Lac3

GlcLac

Suc Fru

Glc

GlcCel

Gal Koj

KojGlc

Glc

Fraction Lac3

Fraction NR3

Fraction Cel3

Fraction Lac3 + β-galactosidase

Fraction Cel3 + β-glucosidase

+ α-glucosidase

47

residues has a quaternary anomeric carbon and no corresponding anomeric proton.Because sucrose was the only sugar in the reaction system with this feature, NR3 wasinferred to contain sucrose, which was supported by comparing its 13C assignments withthose of sucrose (Table S3 in article III). The third residue exhibited resonances typicalof a terminal α-(1→6)-linked α-glucopyranosyl unit, which was found attached to thefructofuranosyl residue of sucrose, as manifested by the downfield shift offructofuranosyl C6 signal in the edited HSQC. The linkage was also confirmed bycorrelation peaks in the HMBC spectrum (Figure S5 in article III) between Fru-C2 (δC

104.71) and GlcI-H1 (δH 5.42), Fru-C6 (δC 70.08) and GlcII-H1 (δH 4.96), and GlcII-C1(δC 99.58) and Fru-H6 (δH 4.06 and 3.76). NR3 was consequently identified as an α-D-Glcp-(1→2)-[α-D-Glcp-(1→6)]-β-D-Fruf (6Fru-α-Glcp-sucrose / 2Fru-α-Glcp-isomaltulose), also called isomelezitose.

NR3 was hydrolyzed to glucose, fructose, and sucrose by α-glucosidase (Figure 13). Therelease of glucose and sucrose is in line with the proposed structure, whereas fructoseseems to result from the further hydrolysis of sucrose. NR3 was resistant to dextranase(data not shown), which requires two consecutive α-(1→6)-linked glucosyl units for itsfunction (Maina et al., 2011). NR3 was not hydrolyzed by invertase, whereas raffinose(α-D-Galp-(1→6)-α-D-Glcp-(1→2)-β-D-Fruf) was hydrolyzed to fructose andmelibiose (α-D-Galp-(1→6)-D-Glcp) (data not shown). This agrees with the structureof NR3 having a glucosyl unit, which is α-(1→6) linked to the fructosyl residue, whereasthe galactosyl unit in raffinose is α-(1→6) linked to the glucosyl residue of sucrose.

In the product mixture of cellobiose acceptor reaction, NR3, if present, could not bedirectly detected by HPAEC-PAD due to its coelution with cellobiose. However, theanalysis of the product fractions isolated by gel filtration (Figure 11C) showed thepresence of an unknown product, probably a trisaccharide according to its elutionbehavior in gel filtration. This product was presumed to be NR3 based on its HPAECretention time. Moreover, a product with a similar HPAEC retention time to NR3 wasfound in the product mixtures of all the tested acceptors except for maltose andisomaltose (Figure S1 in article III and others unpublished). However, higher dilutionwas used for the product mixtures of the two effective acceptors and thus the possiblyformed NR3 could not be detected. These observations indicated the formation of NR3as a side product from endogenous sugars in dextransucrase-catalyzed reactions.

48

5.3.6 Applicability of MSn analysis in the characterization ofdextransucrase-synthesized GLOSs (III and IV)13C6glc sucrose (6 carbon atoms in the glucosyl residue are 13C-labeled, while those inthe fructosyl residue are unlabeled) was used for the synthesis of GLOSs, the MSn

analysis of which provided information on the monosaccharide composition of unknownGLOSs. In the beginning of the characterization of the minor products in the maltoseacceptor reaction, they were first suspected to be fructose-containing GLOSs, asdextransucrases are known to use fructose and sucrose as acceptors (Moulis et al., 2006b;Seo E et al., 2007). The reaction was thus carried out with 13C6glc sucrose, as fructose-containing GLOSs (with one unlabeled residue and the remaining residues labeled) canbe differentiated from the major maltose-based GLOSs (with two unlabeled residues).Therefore, if the minor series contain fructose, their m/z will be 6 plus that of the majormaltose-based product of the same DP. However, when analyzing the two products ofDP5 isolated from the maltose reaction mixture, only a single m/z signal was detectedthat corresponded to a pentaose with 2 unlabeled residues (data not shown). As the minorDP5 product was present in an appreciable amount in the isolated DP5 mixture (Figure8D), the result indicated that the minor series probably also contained maltose ratherthan fructose. Unfortunately, the negative and positive MSn spectra of the labeled DP5product mixture (data not shown) did not show any diagnostic fragment ions other thanthose known to be formed from the major LIM5, which was later explained by thefragmentation patterns of the minor products.

The use of 13C6glc sucrose was also useful in the MSn characterization of the two novelstructures (Lac3 and NR3) from lactose acceptor reaction after their structuralelucidation by NMR spectroscopy analysis. Attempts to characterize unlabeled Lac3were not successful due to the presence of isomeric impurity hypothesized to be afructose-containing linear structure, the presence of which was later confirmed (articleIII). To solve this problem, synthesis was carried out again with 13C6glc sucrose. Becausethe m/z of resultant Lac3 and NR3 differed by 6, which enabled their separatefragmentation in MSn, the reaction mixture was directly analyzed without fractionation.The GLOSs composition and their sugar components were reflected in the MS spectrumof the labeled product mixture (Figure 14). Notably, the GLOSs containing twounlabeled hexose units (lactose-derived) were found in only DP3 and 4 and were notfurther elongated. Because of the addition of a labeled glucosyl branch in Lac3, thedifferentiation of the two nonreducing end residues was made possible, enabling athorough MSn characterization of its fragmentation behavior (not within the scope ofthis thesis). In short, the combination of both positive and negative ionization techniqueswas beneficial for the analysis of rare oligosaccharides containing a 2,4-O-disubstituted

49

reducing end residue. The negative ion MS2 spectra of Lac3 and Cel3 showed an intensesignal corresponding to a loss of 60 Da+H2O. In positive ion MS2 spectra, the cross-ringions resulting from a loss of <162 Da were not observed (others’ results in article III).These results provide reference MSn spectral data for such rare structures.

Figure 14. MS spectrum ([M+Cl]-) of the 13C6glc-labeled oligosaccharide mixture from the

WcE392-rDSR lactose reaction. In the spectra, the number of * shows that of the 13C6glc-labeled

residues in the compound. Solid circles symbolize the labeled glucosyl residues transferred fromsucrose, open circles represent unlabeled residues in the acceptor lactose, and triangles areunlabeled fructosyl residues. The inset shows the HPAEC-PAD chromatogram of the samemixture. (Adapted from Figure S9 in article III.)

The MSn fragmentation behavior of Lac3 was useful for the subsequent preliminarycharacterization of BIM3 by MSn. A mixture of panose and BIM3 with a ratio of ~7:3was used for MSn analysis. It was analyzed in parallel with pure panose; thus, thedifference in their fragment ion profiles indicated the behavior of BIM3 in the mixture.The fragmentation pattern of panose (Maina et al., 2013) is illustrated in Figure 15I. Inthe negative MS2 spectrum of the [M+Cl]- ions in the mixture (Figure 15II-A), thepresence of BIM3 resulted in an increase in cross-ring fragment ions, such as m/z 425,263, and 221, compared to pure panose (Figure 15I-A). The profile of the fragment ionsoriginating from BIM3 resembled that from Lac3 and Cel3 (paper III), which containeda 2,4-disubstituted reducing residue.

In positive ionization, the MS2 spectrum of the [M+Li]+ ion of panose was generallysimilar to that of the mixture with BIM3 (Figure 15I-B and II-B, respectively). However,the fragment ions at m/z 391 and 451 in the spectrum of the mixture were lower inintensity compared to those in the spectrum of panose, indicating a lack of such fragmentions from BIM3. Similarly, cross-ring cleavage ions that formed by the loss of <162 Da(corresponding to ions at m/z 391 and 451) in the MS2 spectrum of the [M+Li]+ ion werealso absent from Lac3 (article III). When the Y2 and C2 ions at m/z 349 were furtherfragmented at the MS3 stage, a marked increase in cross-ring fragment ion at m/z 229

50

was observed in the spectrum of the mixture (Figure 15II-C) compared to thecorresponding spectrum of panose (Figure 15I-C). The cross-ring fragment ion at m/z229 (loss of 120 Da) was previously reported to be diagnostic of a (1→2) linkage (articleIII). The above evidence indicates that BIM3 contains a (1→2)-linked hexopyranosylresidue on the reducing residue of maltose. The analysis of BIM3 based on negative andpositive MSn spectral data of the reference compound Lac3 demonstrated the potentialof MSn analysis in the structural characterization of rare oligosaccharides, even inimpure samples.

51

Figure 15. MSn spectra of (I) pure panose and (II) a fraction containing panose and BIM3 at aratio of ~7:3. (A) Negative ion MS2 of [M+Cl]- m/z 539, (B) positive ion MS2 of [M+Li]+ m/z511, and (C) positive ion MS3 for fragment ions at m/z 349. The fragmentation pathways ofpanose are illustrated in section I. The downward arrows show the intensity changes of thediagnostic fragment ions due to the presence of BIM3 (Figure 3 in article IV).

52

5.4 Optimization of IMO synthesis by response surface modeling (IV)

The IMO product profile (mainly principal linear IMOs) was systematically studied asa function of sucrose and maltose concentrations and WcE392-rDSR dosage. Thepreliminary experiments showed that dextran synthesis was largely suppressed in theexperimental region (Table S1 in article IV), within which a central composite designwas carried out (Table 6). The yield of individual IMOs of DP3‒6 and the yield of totalIMOs and the percent of consumed maltose were examined as responses after 24 h ofreaction (Table 6). The branched minor products were not quantified; however, thecontent of BIM3 was affected in a similar way as was the content of panose (data notshown). According to a carbohydrate analysis of the product mixtures (Table S2 inarticle IV and Table 6), the substrates consumed and the products formed were roughlybalanced. The IMO profile varied between the experimental points, and the fourreplicates at the center point of the design showed good reproducibility (Table 6 andFigure 16). A quadratic equation was used to model each response by excludingirrelevant terms to obtain the highest coefficients of determination (R2) and prediction(Q2) possible. The relatively high R2 and Q2 values (Table 7) indicated that the modelsfit well with the experimental data and could be used to predict the behavior of responseswithin the experimental region.

53

Table 6. The central composite experimental design and response data for modeling the effectsof reaction conditions on IMO synthesis by WcE392-rDSR (Table 1 in article IV).

Run Maltose(mol/L)

Sucrose(mol/L)

Enzyme(U/gsucrose)

Panose(g/L)

LIM4(g/L)

LIM5(g/L)

LIM6(g/L)

TotalIMOsa

(g/L)

Consumedmaltose (%)

1 0.15 0.15 1 10.39b 17.24 7.69 1.46 45.69 36.712 1 0.15 1 19.33 5.03 - - 25.89 20.553 0.15 1 1 3.89 22.33 39.67 31.35 162.84 86.254 1 1 1 120.10 105.46 31.20 3.90 287.16 38.155 0.15 0.15 10 13.45 22.90 15.63 4.50 70.01 37.036 1 0.15 10 47.87 23.38 2.58 - 82.98 28.507 0.15 1 10 3.86 13.18 25.98 31.19 204.43 94.198 1 1 10 120.55 138.99 80.15 19.00 408.68 50.199 0.15 0.575 5.5 5.96 19.30 33.83 27.96 154.46 84.2010 1 0.575 5.5 91.66 98.77 37.78 5.92 265.03 40.3711 0.575 0.15 5.5 37.91 31.57 6.58 0.64 89.98 23.0212 0.575 1 5.5 45.70 98.17 97.11 46.89 365.89 64.7413 0.575 0.575 1 48.53 79.17 40.06 8.82 199.51 39.7714 0.575 0.575 10 43.40 62.11 45.95 14.28 191.76 59.1715 0.575 0.575 5.5 47.62 79.08 56.26 17.00 229.36 49.8416 0.575 0.575 5.5 49.39 83.00 55.05 15.82 240.15 46.6917 0.575 0.575 5.5 47.50 81.13 58.39 18.23 236.31 49.9618 0.575 0.575 5.5 49.00 84.61 58.82 17.83 243.86 49.26

a Total IMOs represents all of the IMO products, including DP>6 members of principal linearseries and members of minor branched series.b Samples were analyzed twice, and the average values are presented. A hyphen (-) denotes thatthe value was below the lower limit of quantification.

54

Figure 16. HPAEC-PAD profiles of acceptor product mixtures in experimental design. Theconditions of each run are shown in Table 6. Labeled peaks are: Fru, fructose; Suc, sucrose; Mal,maltose; and Pan and LIM4−6, the major IMO products in the DP range of 3–6. Thechromatograms are under the same intensity scale, and the samples were diluted by the sameamount. (Figure S2 in article IV.)

Run 8

Run 12

Run 7

Run 1

Run 5

Run 11

Run 4

Run 10

Run 6

Run 2

Run 13

Run 3

Run 9

Run 15−18

Run 14

FruSuc

Mal Pan LIM4

LIM5 LIM6

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 min

55

Table 7. Effects of factors expressed as their corresponding coefficients obtained by modelingfor IMO yield (with respect to DP) and maltose consumption in WcE392-rDSR-catalyzedmaltose acceptor reaction (Table 2 in article IV).

Responses Regression equationsa Coefficients ofdetermination (R2)and prediction (Q2)

Panose (g/L) 44.78 + 36.20M + 16.52S + 2.69E + 23.69M∙S R2=0.95, Q2=0.89LIM4 (g/L) 78.46 + 27.67M + 27.80S + 3.13E - 20.26M2

- 14.43S2 + 27.58M∙SR2=0.95, Q2=0.82

LIM5 (g/L) 55.09 + 2.22M + 18.58S + 3.97E - 10.09M2

- 7.49E2 + 4.90M∙S + 4.22M∙ER2=0.85, Q2=0.60

LIM6 (g/L) 18.79 - 5.19M + 9.64S + 1.80E - 4.32E2

- 2.48M∙S + 0.88S∙ER2=0.85, Q2=0.68

Total IMOs (g/L) 236.07 + 42.46M + 111.45S + 23.68E - 27.90M2

- 45.86E2 + 41.93M∙S + 14.09M∙ER2=0.97, Q2=0.87

Consumedmaltose (%)

50.16 - 16.06M + 18.77S + 4.77E + 8.99M2

- 9.42S2 - 8.43M∙SR2=0.97, Q2=0.90

a M, initial maltose concentration (mol/L); S, initial sucrose concentration (mol/L); E, WcE392-rDSR concentration (U/g sucrose).

For each response, three two-dimensional contour plots with maltose and sucroseconcentrations as the two axes (Figure S3 in article IV) were used to illustrate the models,and each plot corresponded to a different enzyme dosage. Because the enzyme dosagein relation to the sucrose concentration had a minor effect on the responses, contour plotsat an enzyme dosage of 5.5 U/g sucrose were chosen to represent the effects of the twomain variables in Figure 17. The yield of each IMO of DP3−6 and the total IMOs, aswell as the percent of consumed maltose, increased by increasing the initial sucroseconcentration, whereas the initial maltose concentration exhibited varying effects on theproduction of each IMO (Figure 17). The maximal production of panose, LIM4, andtotal IMOs was attained when maltose and sucrose were both at the highestconcentrations tested. The production of LIM5 and LIM6, however, was favored at themedium and lowest maltose concentrations, respectively, while maintaining the highestsucrose concentration. The maltose utilization ratio was influenced positively by sucroseinput and negatively by maltose input. Aiming for a good yield of total IMOs and aproduct profile towards higher DP, run 12 employing a medium enzyme load (5.5 U/gsucrose) and medium maltose (0.575 M) and the highest sucrose (1 M) concentrationswas considered close to the optimum. The run led to sucrose depletion and a 65%

56

consumption of maltose. A yield of 366 g/L of total IMOs was attained, with a weightedaverage DP of >4.5 (taking into account the amounts of DP3−6, not higher DP).

Figure 17. Contour plots showing the influence of the initial sucrose and maltose concentrationsin the WcE392-rDSR acceptor reaction on the yields (g/L) of (A) panose, (B) LIM4, (C) LIM5,(D) LIM6, (E) total IMOs, and (F) the consumed maltose (%). The reactions were conductedwith an enzyme dosage of 5.5 U/g sucrose for 24 h (Figure 1 in article IV).

57

6. DISCUSSION

6.1 Properties of Weissella dextransucrases and dextrans (I and II)

Biochemical characterization showed that WcE392-rDSR was affected similarly to otherreported Weissella dextransucrases by pH and temperature (Kang et al., 2009; Shuklaand Goyal 2011; Amari et al., 2012; Bejar et al., 2013; Rao and Goyal 2013). It shouldbe pointed out that in the study of the effect of pH on WcE392-rDSR activity, the Tris-HCl buffer used for pH 6.5‒7.0 contained Tris, which may have inhibited the enzymeactivity (Côté and Robyt 1982). Thus, the decrease of activity in this pH range may haveresulted from the inhibiting effect of Tris, in addition to that of pH per se. However, forthe purpose of determining the pH optimum, the experiments were not repeated withanother buffer of pH 6.5‒7.0, given that the enzyme activity at pH 6.0 already decreaseddrastically in Na-acetate buffer. The quick inactivation above 40 °C and the stabilizingeffect of glycerol were observed for both WcCab3-DSR (others’ results in article II) andWcE392-rDSR. It should be noted that WcCab3-DSR was produced by sucroseinduction and partially purified by PEG fractionation and thus was bound to dextran(Majumder et al., 2007), whereas WcE392-rDSR was produced in glucose-containingmedium and devoid of bound dextran. The half-lives of WcCab3-DSR and WcE392-rDSR of similar protein concentrations at ~30 °C without any additive were 10.3 h and26.3 h, respectively, despite the fact that the former was dextran bound and dextran isknown to stabilize dextransucrase. In a recent study of the recombinant dextransucrasefrom W. confusa Cab3, the addition of dextran could increase the enzyme activity by atmost 14% (Shukla et al., 2016). The stabilizing and activating effect of dextran onWeissella dextransucrases thus seems to be minor.

The kinetics for a recombinant dextransucrase from W. confusa LBAE C39-2 and anative one from W. cibaria JAG8 have been reported to follow Michaelis-Mentenkinetics, with KM and Vmax values of 8.6 mM and 20 µmol/(mg∙min), respectively, forthe former, and 13 mM and 27.5 µmol/(mg∙min), respectively, for the latter (Amari etal., 2012; Rao and Goyal 2013). The kinetics parameters of the C39-2 dextransucrasewere determined by a 3,5-dinitrosalicylic acid (DNS) assay (Amari et al., 2012), whichhas been suggested to overestimate the dextransucrase activity due to the significantover-oxidation of dextran and other carbohydrates in the sample, thus giving invalidenzyme assay and kinetics results (Vettori et al., 2011; McIntyre et al., 2013). Accordingto Vettori et al. (2011), the DNS method generates a 5.8-fold higher activity than the14C-sucrose radioisotope method. However, it is not known how accurate is the Nelson-Somogyi assay, the other commonly used reducing-value assay for dextransucrase

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activity and kinetics measurements. It was thus compared with the 14C-sucroseradioisotope assay in this study. The Nelson-Somogyi method gave an approximately2.5-fold higher activity and Vmax than did the 14C-sucrose radioisotope method, while theKM values were similar regardless of the method used. Because small amounts of glucoseand leucrose formed as side products, the higher values produced by the Nelson-Somogyi method seemed to be due to the oxidation of several residues down the dextranchain other than the reducing residue. As determined by Nelson-Somogyi method, thekinetic values for WcE392-rDSR (KM of 13.0 mM and Vmax of 19.9 µmol/(mg∙min))were similar to those of W. cibaria JAG8 dextransucrase (KM of 13 mM and Vmax of 27.5µmol/(mg∙min)) (Rao and Goyal 2013). The WcE392-rDSR KM obtained by the 14C-sucrose radioisotope method (14.7 mM) was comparable to that of a glucansucrase fromLeuconostoc mesenteroides NRRL B-1118 (12 mM) using the same method (Côté andSkory 2012). In this thesis, the Vmax (8.2 µmol/(mg∙min)) determined by the 14C-sucroseradioisotope assay was reported for the first time for a glucansucrase.

The dextran produced in vitro by WcE392-rDSR exhibited high similarity to thatproduced in vivo by native and heterologous hosts as well as that by the other enzymeWcCab3-DSR, as characterized by 1H NMR spectroscopy and HPSEC. The Weisselladextrans showed an α-(1→6) to α-(1→3) linkage ratio of 97:3, being slightly lessbranched than the dextran from Lc. mesenteroides NRRL B-512F (with a ratio of 96:4),which was analyzed comparatively (Maina et al., 2008; Bounaix et al., 2009). The molarmasses reported for Weissella dextrans ranged between 106 and 107 g/mol (Amari et al.,2012; Maina et al., 2014). Similarly high molar masses (107‒108 g/mol) were obtainedin this study (articles I and II) by HPSEC. Significantly high molar masses (108 g/mol)were reported for dextrans synthesized in vitro with Lc. mesenteroides B-512Fdextransucrase mutants applying the asymmetrical flow field flow fractionationtechnique (Irague et al., 2012). The high molar mass and low branching make Weisselladextrans attractive hydrocolloids for food applications, such as in sourdough baking(Lacaze et al., 2007).

6.2 Specificity of Weissella dextransucrases in acceptor reactions

6.2.1 Comparison with commercial Lc. mesenteroides dextransucrase (IIIand IV)

Before this thesis work, the dextransucrase acceptor reactions for GLOS synthesis wereonly investigated for DSR-S from Lc. mesenteroides B-512F. WcE392-rDSR wassimilar to DSR-S in its selectivity towards the acceptors tested (in decreasing order):

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maltose, isomaltose, nigerose, lactose, cellobiose, and melibiose (Robyt and Eklund1983). The unusual acceptors tested in this thesis work, laminaribiose and forkose,showed poor effectiveness for WcE392-rDSR. Moreover, the product patterns ofWcE392-rDSR were also similar to those of DSR-S based on the product profiles frommaltose, isomaltose, maltotriose, nigerose, lactose, and cellobiose (Robyt and Eklund1983; Fu and Robyt 1990).

In the presence of lactose or cellobiose, both WcE392-rDSR and DSR-S form mainly atrisaccharide with a glucosyl residue α-(1→2) linked to the acceptor’s reducing end(Robyt 1995). In their maltose acceptor reaction, a principal series of linear IMOs aresynthesized from further elongation on panose. Moreover, a number of previouslyunknown minor products were also observed in the reactions of both enzymes. In thisthesis, the minor trisaccharide from WcE392-rDSR was identified as centose with aglucosyl residue α-(1→2) linked to the maltose-reducing end. However, a trace amountof forkose (α-D-Glcp-(1→4)-[α-D-Glcp-(1→6)]-D-Glcp) has been reported to beformed by DSR-S from Lc. mesenteroides B-512F (Fu and Robyt 1990). To determinewhether the minor trisaccharide derived from maltose varied with the enzyme source,the commercial Lc. mesenteroides enzyme (results not shown) was compared toWcE392-rDSR and WcCab3-DSR in maltose reaction. Their product profiles weresimilar in HPAEC-PAD analysis, showing that the ability to synthesize centose (insteadof forkose) and branched IMOs from maltose is shared by these dextransucrases. Thefinding was consistent with the use of maltose acceptor product patterns to classifyglucansucrases of different specificities (Côté and Leathers 2005). For dextransucrasesproducing typical dextrans (with predominantly α-(1→6) linkages and a low degree ofα-(1→3) branching), their GLOS products from maltose showed a unique pattern,although this was based on only principal IMO series detected in TLC analysis (Côtéand Leathers 2005). This thesis demonstrates that the dextransucrases highly specific forα-(1→6) linkage formation also produce the same minor IMO series.

The shared specificity between the two Weissella dextransucrases is consistent with theclose resemblance in their sequences (93% amino acid sequence identity; GenBank:AKE50934 and AHU88292). The amino acid substitutions are unlikely to cause drasticdifferences in their catalytic properties (others’ results in article I). According to thelatest constructed phylogenetic tree of GH70 enzymes (Vuillemin et al., 2016),WcE392-rDSR and W. confusa LBAE C39-2 dextransucrase (with 100% amino acididentity to WcCab3-DSR) cluster together and form a branch with a subgroup of two W.cibaria dextransucrases and a Lactobacillus fermentum dextransucrase. Although DSR-S from Lc. mesenteroides NRRL B-512F is separated from Weissella dextransucrases,and its sequence (GenBank: AAD10952) bears only 52% amino acid similarity to the

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two Weissella dextransucrases studied, they seem to belong to a phylogenetic group oftypical dextransucrases producing predominantly α-(1→6) linkages. This correspondsto the similar selectivity and specificity observed for the three dextransucrases.

6.2.2 Specificity to form an α-(1→2) branch (III and IV)

A typical dextransucrase DSR-S was shown to form an α-(1→2)-branched trisaccharidefrom β-(1→4)-linked lactose or cellobiose but form an α-(1→6) linkage on thenonreducing end of α-linked acceptors (Robyt 1995). However, this thesis reports forthe first time that α-linked maltose can be α-(1→2) branched as well and form centosevia typical dextransucrases. The higher DP homologues were proposed to be IMOscarrying a single-unit α-(1→2) branch at the reducing end. This correlates with an earlierobservation that the α-(1→2) branch added to a maltose derivative, whose C-6 of thenonreducing end was blocked, could not grow further (Bhattacharjee and Mayer 1993).Such branched products have not been previously described from the acceptor reactionof an enzyme of GH family 70. Dextransucrase from Lc. citreum NRRL B-1299 and itstruncated form GBD-CD2 (a branching sucrase) are able to form α-(1→2) branches onα-(1→6)-linked residues in GLOSs (Dols et al., 1998; Brison et al., 2013). Nevertheless,in the α-(1→2)-branched IMOs formed by typical dextransucrases, the branch is locatedon the reducing-end residue.

Based on the observation that centose did not serve as an acceptor, which ruled out thepossibility of main chain elongation after branch formation, the higher DP homologueswere proposed to be formed through direct branch formation on an elongated linear IMO.In this thesis, the dextransucrases have been shown to form an α-(1→2) branch not onlyon lactose and cellobiose but also on maltose, panose and higher linear IMOs. Thus,these acceptor sugars interact with the enzymes so that the C-2 of the reducing residuecan be oriented toward the active site and be linked to the C-1 of the glucosyl-enzymeintermediate. The resultant branched GLOSs, however, were expectedly no longeraccommodated in the enzyme acceptor site to form a regular α-(1→6) linkage at thenonreducing end. This complies with the fact that the branched trisaccharides derivedfrom maltose (this study) and cellobiose (Ruiz-Matute et al., 2011) do not elongatefurther. GLOSs of only DP3 and DP4 were observed to contain lactose under thesynthesis with 13C6glc-labeled sucrose (Figure 14). Although other lactose-derivedGLOSs were too low in amount to be characterized in this and previous studies (Díez-Municio et al., 2012b), a portion of lactose is expected to be elongated linearly via α-(1→6) linkages, explaining the presence of the 13C6glc-labeled lactose-containing DP4product. In the isolation of Lac3 or Cel3 by gel filtration, they were found coelutingclosely with a small amount of another product (not shown), which may be the linear

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trisaccharide with an α-(1→6)-linked glucosyl residue. It is interesting to note thatalthough typical dextransucrases form α-(1→3) branches in dextran, no α-(1→3) linkagehas been found in GLOSs. The recognition of α-(1→2)-branch linkage specificity shedslight on a new interaction mode between acceptors and the enzyme active site. Enzymestructure-guided mutations may alter dextransucrase specificity to produce a higherportion of α-(1→2)-branched GLOSs for novel applications.

6.2.3 Novel sucrose-containing trisaccharide (III)

In the WcE392-rDSR acceptor reaction in the presence of lactose, the production ofsucrose-containing isomelezitose (NR3) by a dextransucrase was described for the firsttime. It was independent from the exogenous acceptor and was promoted by higherconcentrations of sucrose and dextransucrase. Previously, an unknown trisaccharideproduct was noticed in the lactose reaction mixture of Lc. mesenteroides B-512Fdextransucrase, which may have been isomelezitose (Díez-Municio et al., 2012b). Thisproduct may have also been formed in the presence of cellobiose and other acceptorstested. However, the reaction rate for isomelezitose formation is much lower comparedto that for effective acceptors to form IMO.

Isomelezitose was previously obtained from the fructose acceptor reaction of analternansucrase, where two possible pathways were proposed for its synthesis (Côté etal., 2008). One involves glucosyl transfer to the 2-OH of the reducing residue ofisomaltulose (α-D-Glcp-(1→6)-D-Fruf, an acceptor product of fructose), resulting in theformation of a sucrose-type (β-D-Fruf-(2→1)-α-D-Glcp) linkage. The other pathwayinvolves the transfer of a glucosyl residue to the 6-OH of the fructofuranosyl residue ofanother sucrose molecule. In this thesis, isomelezitose was hypothesized to be formedby the first pathway i.e., as an acceptor product of isomaltulose. Some supportiveevidence has been reported. First, a sucrose-type linkage is formed on lactulose (β-D-Galp-(1→4)-D-Fruf) and maltulose (α-D-Glcp-(1→4)-D-Fruf) by Lc. mesenteroidesdextransucrase (Demuth et al., 2002; Díez-Municio et al., 2012a), indicating that asimilar sucrose-type linkage could be formed on isomaltulose. Second, a majorimbalance existed between the consumption of isomaltulose and the yield ofisomaltulose-derived IMOs according to the data published on the isomaltulose acceptorreaction of Lc. mesenteroides dextransucrase (Barea-Alvarez et al., 2014), indicatingthat other compounds may have also been produced from isomaltulose. Third, if sucroseacts as an acceptor, a glucosyl residue is added to the sucrose-glucosyl moiety (Mouliset al., 2006b; Dobruchowska et al., 2013) and not on the fructosyl moiety to formisomelezitose. To test the hypothesis, an acceptor reaction with isomaltulose and 13C6glc-labeled sucrose can be carried out. According to the hypothesis, the two glucosyl

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residues in the isomelezitose thus synthesized can be differentiated in MSn analysis. Byreference to the fragmentation pattern of isomelezitose reported in article III, one shouldbe able to prove its origin from isomaltulose.

Other sucrose analogs, β-D-Galp-(1→4)-β-D-Fruf-(2→1)-α-D-Glcp (lactulosucrose)and α-D-Glcp-(1→4)-β-D-Fruf-(2→1)-α-D-Glcp, derived from lactulose and maltulose,respectively, have been considered donor substrates for dextransucrase after theconsumption of sucrose (Demuth et al., 2002; Díez-Municio et al., 2012a). Additionalstudy is needed to test the possible capacity of isomelezitose to act as a donor substrate.Because isomelezitose derives from endogenous sugars in acceptor reactions of bothalternansucrase (Côté et al., 2008) and dextransucrase, it may widely occur as a sideproduct among glucansucrases.

6.3 Potential of dextransucrases in prebiotic GLOS synthesis

6.3.1 Synthesis of prebiotic IMOs from maltose (IV)

The dextransucrase maltose acceptor reaction represents a promising synthetic pathwayfor prebiotic IMOs because of the cost-effectiveness and the abundance of the substratesused (sucrose and maltose), as well as its ability to control the size distribution of IMOs(Su and Robyt 1993; Lee et al., 2008). Compared to classical IMO production via afungal α-transglucosidase on high-maltose syrup, which preferentially produces low-DPIMOs (Goffin et al., 2011; Hu et al., 2013), the dextransucrase-catalyzed reaction can betailored for the synthesis of higher-DP IMOs. A slight increase in DP (from 2.7 to 3.3)endowed IMOs with non-digestibility for their function as prebiotics (Iwaya et al., 2012).Moreover, in the presence of the effective acceptor maltose, dextran synthesis can belargely inhibited to maximize sucrose utilization for IMO synthesis (Su and Robyt 1993;Rabelo et al., 2009).

In this thesis, the effects of reaction factors (concentrations of sucrose, maltose, anddextransucrase) on the IMO product profile were studied for the first time byoptimization design. A high sucrose concentration was favorable for IMO yield. Enzymedosage showed a small effect on the yield and profile of IMOs over a prolonged reactiontime for maximal sucrose conversion. Maltose input, however, had varying effects onthe production of IMO components. It promoted the production of lower DP whileinhibiting that of DP6. This indicates that maltose has a higher affinity to dextransucrasethan do the higher-DP IMOs. Thus, only when maltose concentration was low, did theintermediate IMO products have a better chance to act as acceptors. The total IMO yield

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peaked when the sucrose and maltose inputs were at their highest. This was alsoobserved in a previous study with Lc. mesenteroides NRRL B-512F dextransucrase, inwhich the IMO size distribution and maltose consumption were unfortunately notreported (Rabelo et al., 2009). However, this thesis showed that for a good yield ofhigher-DP IMOs, medium maltose (~0.5 M) and high sucrose (1 M) concentrations wereoptimal and also resulted in a better maltose utilization.

In vitro fermentation by human fecal bacteria showed that linear maltose-based IMOswith DP5−7 had a relatively high selectivity toward beneficial bacteria compared tothose with a lower DP (Sanz et al., 2006). The higher IMOs also persist longer in thecolon and can reach the most distal regions, where most chronic intestinal disordersoriginate (Sanz et al., 2006). In addition, dextransucrases can simultaneously synthesizeminor α-(1→2)-branched IMOs, which may have enhanced resistance to gastrointestinaldigestion and selectivity toward probiotic bacteria (Sanz et al., 2005). Thus, thedextransucrase-catalyzed maltose acceptor reaction represents a promising syntheticpathway for prebiotic IMOs. Dextransucrase from Lc. mesenteroides B-512F wassuccessfully immobilized for the repetitive production of IMOs (Tanriseven and Doğan2002). Several purification methods have been developed to remove unwantedmonosaccharides (e.g., glucose and fructose) and disaccharides (e.g., maltose andsucrose) from IMO products, including adsorption separation and selective fermentationby yeasts or bacteria (Crittenden and Playne 2002; Yoon et al., 2003; Pan and Lee 2005;Goffin et al., 2011). Beneficial IMOs can also be produced in situ by bacterial cellsduring food fermentation (Katina et al., 2009; Cho et al., 2014).

6.3.2 Synthesis of other GLOSs (III)

Leuconostoc dextransucrases have been used to synthesize GLOSs from a variety ofacceptors, such as lactose, cellobiose, isomaltulose, lactulose, and gentiobiose. Theseproducts have been demonstrated in vitro as potential prebiotics (Ruiz-Matute et al.,2011; Coelho et al., 2014; Díez-Municio et al., 2014; Barea-Alvarez et al., 2014; García-Cayuela et al., 2014; Kothari and Goyal 2015). Although these acceptors are lesseffective than maltose, a reasonable yield of acceptor products can be achieved byincreasing the substrate concentrations and the ratio of acceptor to sucrose, which divertsmore sucrose for use in the acceptor reaction at the cost of dextran and leucroseproduction (Díez-Municio et al., 2012b). In the Lc. mesenteroides B-512Fdextransucrase-catalyzed synthesis of 2Glc-α-Glcp-lactose (Lac3) using concentratedsucrose and lactose both at 0.88 M, the trisaccharide yield was 130 g/L correspondingto a consumption of approximately 36% of the initial lactose (Díez-Municio et al.,2012b). In this thesis, the lactose product yield was limited by the low lactose input

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concentration of 0.15 M. The highest Lac3 yield was achieved (with a 27% lactoseconsumption) when the sucrose and dextransucrase concentrations were at their highest,which resulted in the concomitant formation of a large amount of dextran. Under theseconditions for lactose acceptor reaction, a novel side product, isomelezitose (NR3), wasidentified and is a potential nutraceutical (Görl et al., 2012).

Dextransucrase-synthesized 2Glc-α-Glcp-lactose has found use as a precursor for thesynthesis of kojibiose, a bioactive disaccharide with low availability due to difficultiesin its isolation or synthesis. After the initial lactose acceptor reaction, the sugars presentin the mixture (fructose, glucose, galactose, and sucrose) were removed by yeastSaccharomyces cerevisiae treatment. The remaining lactose and 2Glc-α-Glcp-lactosewere then hydrolyzed by Kluyveromyces lactis β-galactosidase without removing theyeast cells. Kojibiose was obtained with good yield: 38% in weight with respect to theinitial amount of lactose (Díez-Municio et al., 2014). Dextransucrase acceptor reactionsmay provide affordable synthesis processes for a variety of rare di- and oligosaccharides,thus expanding their applications.

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7. CONCLUSION

In this thesis, two W. confusa dextransucrases (mostly WcE392-rDSR and partlyWcCab3-DSR) were studied for their catalytic properties and dextran- and GLOS-synthesizing functionalities. Focus was given to the structural characterization of theirGLOS products because the information on dextransucrase-synthesized GLOSs waspreviously only available for a commercial Lc. mesenteroides dextransucrase.

The effects of pH and temperature on the WcE392-rDSR activity were determined. Theenzyme lost activity quickly above 40 °C and was stabilized by glycerol. It showedMichaelis-Menten kinetics with a KM of 14.7 mM and a Vmax of 8.2 µmol/(mg∙min) asdetermined by the sucrose radioisotope assay. A similar KM value and a 2.5-fold higherVmax were obtained by the reducing-value Nelson-Somogyi assay. Dextrans from theWeissella dextransucrases consisted of 97% α-(1→6) linkages and 3% α-(1→3) linkages,which resembled that from the commercial Lc. mesenteroides B-512F dextransucrase.The dextrans obtained showed high molar masses in the range of 107‒108 g/mol inHPSEC analysis.

The selectivity of WcE392-rDSR towards several acceptors and the acceptor productpatterns were similar to those of Lc. mesenteroides B-512F dextransucrase. The mostefficient acceptor maltose was tested with Weissella dextransucrases and the Lc.mesenteroides dextransucrase, all exhibiting a similar product profile in HPAEC-PADanalysis. The GLOS products were isolated according to DP for structuralcharacterization. Interestingly, in addition to a series of principal products, there seemedto be another series of minor products. The principal products were identified as linearmaltose-based IMOs, which corresponded to the predominant α-(1→6) linkages indextran products from typical dextransucrases. The minor products were confirmed toform another homologous series, where a single glucosyl residue was α-(1→2) linked tothe reducing residue of linear IMOs. Such branched IMOs have not been reported for aglucansucrase. The branch was proposed to be added to each member of the linear IMOs.Unlike maltose, WcE392-rDSR produced dominantly branched trisaccharides withanalogous lactose and cellobiose and with a glucosyl residue α-(1→2) linked to theacceptor’s reducing end. Moreover, the side product isomelezitose was proven for thefirst time to be formed by a dextransucrase. This nonreducing trisaccharide wasapparently synthesized from the fructose acceptor product isomaltulose. An MSn

analysis of the 13C6glc-labeled GLOS products was proven useful in, e.g., distinguishingfructose-containing side products from products derived from exogenous acceptors.

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To apply WcE392-rDSR for the synthesis of higher-DP linear IMOs for better prebioticproperties, the effects of sucrose, maltose, and dextransucrase concentrations on theIMO product profile were studied here for the first time. According to response surfacemodelling, the yields of individual IMOs of DP3−6 were all maximized with the highestsucrose input (1 M) but varying maltose inputs (0.15−1 M). The optimal maltoseconcentration for DP3−4, DP5 and DP6 was at its highest, medium and lowest levels,respectively. The dextransucrase dosage (1–10 U/g sucrose) had a smaller effect on theseresponses.

Overall, this study examined the catalytic properties of Weissella dextransucrases andthe structures of their dextran and GLOS products. Several GLOSs from Weisselladextransucrases were identified, which enhanced the understanding of the catalyticmechanism of these enzymes and more generally of typical dextransucrases producingpredominantly α-(1→6) linkages, such as the commercial one from Lc. mesenteroidesB-512F. The study also demonstrated the usability of Weissella dextransucrases insynthesizing dextran and oligosaccharides for food use.

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