Bioresource Technology 102 (2011) 1715–1721

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Bioresource Technology

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Biochemical characterization of an intracellular 6G-fructofuranosidasefrom Xanthophyllomyces dendrorhous and its use in production ofneo-fructooligosaccharides (neo-FOSs)

Jing Chen, Xiaoming Chen, Xueming Xu, Yawei Ning, Zhengyu Jin ⇑, Yaoqi Tian ⇑⇑The State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China

a r t i c l e i n f o a b s t r a c t

Article history:Received 11 April 2010Received in revised form 7 August 2010Accepted 10 August 2010Available online 13 August 2010

Keywords:Intracellular 6G-fructofuranosidasesXanthophyllomyces dendrorhous 269Enzyme characterizationNeo-fructooligosaccharides

0960-8524/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.biortech.2010.08.033

⇑ Corresponding author. Tel./fax: +86 510 8591392⇑⇑ Corresponding author.

E-mail addresses: jinlab2008@yahoo.com (Z. Jin), y

An intracellular 6G-fructofuranosidase (endo-type enzyme) extracted from Xanthophyllomyces dendror-hous 269 efficiently hydrolyzes fructosyl-b-(2?1)-linked sucrose to produce neo-kestose as a main trans-glycosylation product. The enzyme with a molecular weight of 33 kDa was purified by DEAE-52 cellulosechromatography. Thirty-fivefold purification and a 13.4% enzyme activity recovery were achieved. Opti-mum enzyme activity occurred at pH 6.4 and 45 �C and the enzyme was stable at pH 4–7 and at 45 �C.Using sucrose as a substrate, the Km and Vmax values were, respectively, 511 mmol/l and 233 lmol/(min mg) for transfer activity and 62 mmol/l and 164 lmol/(min mg) for hydrolytic activity. Under opti-mum conditions, a maximum concentration (73.9 g/l) of neo-fructooligosaccharides catalyzed by theendo-enzyme was obtained. These findings suggest that the purified endo-enzyme exhibits a high trans-fructosylation activity and it has potential for the industrial production of neo-FOSs.

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1. Introduction

Fructo-oligosaccharides (FOSs) are of nutritional interest be-cause they can improve growth of bifidobacteria and potentiallyinhibit pathogenic microorganisms (Nadeau, 2000). FOSs are com-posed of 1-kestose (GF2), nystose (GF3), and 1F-fructofuranosyl(GF4) in which fructosyl units are linked at b-2,1 positions of su-crose. Neo-fructooligosaccharides (neo-FOSs) with b-2, 6 linkagebetween two fructose units (6F-FOSs: 6-kestose) or between fruc-tose and a glucosyl group (6G-FOSs: neo-kestose, neo-nystose, andneo-fructofuranosylnystose) have a structure different from that ofFOSs (Kilian et al., 1996; Hayashi et al., 2000) and might have bet-ter prebiotic properties and chemical stability compared to FOSs(Kilian et al., 2002; Marx et al., 2000; Lim et al., 2007).

FOSs are commercially produced using either microbial fruc-tosyltransferases (EC 2.4.1.9) or b-fructofuranosidases (EC3.2.1.26). b-fructofuranosidases from Saccharomyces cerevisiae(Taussig and Carlson, 1983; Reddy and Maley, 1990, 1996), Schizo-saccharomyces pombe (Moreno et al., 1990), Pichia anomala (Rodri-guez et al., 1995), Candida utilis (Chávez et al., 1998), Arxulaadeninivorans (Böer et al., 2004), and Schwanniomyces occidentalis(Álvaro-Benito et al., 2007) have been shown to release D-glucoseand D-fructose from sucrose and to catalyze the synthesis of

ll rights reserved.

2.

qtian@yahoo.com (Y. Tian).

short-chain fructooligosaccharides (FOSs). However, neo-6G-FOSshave not been widely explored, probably because they are not pro-duced by fructofuranosidases in microorganisms (Álvaro-Benitoet al., 2007; Ghazi et al., 2007) or they represent only a minor bio-synthetic product (Farine et al., 2001).

The basidiomycetous yeast Xanthophyllomyces dendrorhous (X.dendrorhous, formerly Phaffia rhodozyma) is known to produce anextracellular b-fructofuranosidase (Linde et al., 2009). In the pres-ent study we reported that it also produces an intracellular 6G-fructofuranosidase (endo-6G-FFase). The enzyme was purifiedfrom X. dendrorhous 269 and optimal pH and temperature, stabilityat different temperatures and pH, kinetic parameters and produc-tion of neo-FOSs were studied.

2. Methods

2.1. Materials

X. dendrorhous 269 was obtained from School of Food Scienceand Technology of Jiangnan University (Wuxi, China). The neo-kes-tose (P98%) was produced in our laboratory (Su et al., 2009).DEAE-52 cellulose was purchased from Sigma Chemical Co. (St.Louis, MO, USA). An electrophoresis calibration kit for proteinmolecular weight determination was purchased from Xibasi(Shanghai, China). All other chemicals and reagents used were ofanalytical grade unless other stated.

1716 J. Chen et al. / Bioresource Technology 102 (2011) 1715–1721

2.2. Microorganism and culture conditions

X. dendrorhous 269 was maintained at 4 �C on YEPS agar med-ium including (g/l): sucrose, 10; yeast extract, 3; peptone, 5; andagar, 18. A different medium was used for growth of an inoculumand for enzyme production. The seed culture medium consisted of(g/l), sucrose, 30; yeast extract, 3; peptone, 5. The medium for en-zyme production contained (g/l) sucrose, 30; yeast extract, 3; pep-tone, 5; MgSO4�7H2O, 0.5. The pH was adjusted with 0.2 mol/lsodium phosphate buffer (pH 8.0) to 6.0 before the sterilization.The inoculum and enzyme production cultures were grown at20 �C with shaking at 220 rpm for 48 and 40 h, respectively.

2.3. Extraction of X. dendrorhous intracellular enzymes

Cells were harvested by centrifugation (8000g, 10 min) andwashed twice with deionized water. The mycelium (20 g) was sus-pended in 50 ml of deionized water and disrupted with a cell dis-ruption system (Constant Systerm, UK) at 4 �C. The workingpressure and power were 30 Kpsi (2000 bar) and 0.75 kW, respec-tively. The lysate was centrifuged at 13,000g for 20 min and thesupernatant was used as crude enzymatic extract.

2.4. Protein isolation and purification

The 6G-fructofuranosidase extracted in a dialysis bag was con-centrated via a PEG 20000 powder. The enzyme solution (50 ml)was dialyzed against 20 mmol/l sodium phosphate (pH 7.0) (BufferA) overnight at 4 �C and purified via a DEAE-52 cellulose chroma-tography column (2 � 20 cm), previously equilibrated with thesame buffer. The protein was eluted with a step gradient of NaClfrom 0 to 0.5 mol/l in the same buffer at a flow rate of 1 ml/min.Absorbance was determined at 280 nm. Four milliliters of eachfraction were collected and assayed for enzyme activity on sucrose.The fractions with transfructosylation activity were eluted by aNaCl solution (0.13 mol/l), dialyzed in 20 mmol/l sodium acetatewith pH 5.0 (Buffer B), and applied to a DEAE-52 cellulose chroma-tography column equilibrated with the same buffer. Protein waseluted with a 0–0.2 mol/l NaCl gradient. The effluents containinginvertase activity were collected and concentrated using a PEG20000 powder. The purified enzyme was stored at �70 �C.

2.5. Determination of enzymatic activities

Enzyme activity was determined in 0.5 ml of 40% sucrose in0.2 mol/l sodium phosphate buffer (pH 6.0) and 0.5 ml enzymesolution at 40 �C for 60 min and stopped by heating in a boilingbath water for 10 min. A control sample was incubated for10 min in boiling water to inactivate the enzyme and incubatedat the same conditions as above.

One unit of intracellular 6G-fructofuranosidase was defined asthe amount of enzyme that produced 1 lmol of neo-kestose permin under below assay conditions.

The analysis of the reaction products were performed by HPLC(LC-20A, Shimadzu, Japan) as described previously (Hang et al.,1995). The Waters Sugarpak1 Column (6.5 mm � 300 mm) wasmaintained at 85 �C and ultrapure water was used as the mobilephase at a flow rate of 0.5 ml/min. A 10 ll sample was injectedand the detection was performed by a refractive index detector.

2.6. Measurement of protein concentration

Protein measurement was carried out according to the proce-dure described by Bradford (1976). Bovine serum albumin wasthe standard.

2.7. Protein purity determination and molecular weight estimation

The purity and molecular weight of the purified enzyme weredetermined by SDS–PAGE as described by Laemmli (1970) in a ver-tical slab gel with 12% polyacrylamide gel containing 0.1% SDS andTris–glycine buffer containing 0.1% SDS (pH 8.3) at a constant cur-rent of 15 mA. Rabbit phosphorylase b (97 kDa), bovine serumalbumin (66 kDa), rabbit actin (43 kDa), bovine carbonic anhydrase(31 kDa), trypsin inhibitor (20.1 kDa) and hen egg white lysozyme(14.4 kDa) were used as standards for the molecular weight deter-mination. Proteins were stained with Coomassie Brilliant Blue G-250.

2.8. Effect of pH on the activity and stability of the endo-6G-FFase

The effect of pH on activity of the endo-6G-FFase was deter-mined by assaying enzyme activity at different pHs ranging from2.6 to 8.0 at 40 �C for 60 min, using 0.1 mol/l sodium acetate buffer(pH 2.6–5.4) and 0.2 mol/l sodium phosphate buffer (pH 6.0–8.0).

The pH stability of the enzyme was estimated by measuring theresidual activity after protein pre-incubation at pH 2.6–8.0 for60 min at 4 �C.

2.9. Effect of temperature on the enzyme activity and stability

The optimal temperature for the enzyme activity was deter-mined by incubating the enzyme-substrate mixtures at varioustemperatures (10–70 �C) in 0.2 mol/l phosphate buffer (pH 6.0)for 60 min. Thermal stability of endo-6G-FFase was assayed interms of residual activity after incubation of the enzyme at differ-ent temperatures (20–70 �C) for 1 h.

2.10. Effect of metal ions and ethylene diamine tetraacetic acid (EDTA)and sodium dodecyl sulfate (SDS) on endo-6G-FFase activity

The enzyme was incubated with 2 mM of KCl, MgSO4�7H2O,CaCl2, MnCl2�4H2O, Al(NO3)3�9H2O, ZnCl2, LiCl�H2O, BaCl2�2H2O,AgCl, EDTA, and SDS in 50 mM sodium phosphate buffer (pH 6.4)at 4 �C for 30 min followed by measuring the residual activity un-der the standard conditions described in Section 2.5.

2.11. Determination of kinetic parameters

The initial reaction rate of the endo-6G-FFase activity wasdetermined at optimum conditions (pH 6.4, 45 �C) and various su-crose concentrations (0.029–1.749 mol/l, 10–600 g/l). The reac-tions were terminated by heating in boiling water for 10 min andthe products were analyzed by the HPLC to determine the contentsof fructose and glucose (Hang et al., 1995). One unit of hydrolyticactivity was defined as catalyzing the release of 1 lmol fructoseper minute, whereas one unit of transferase activity was definedas catalyzing the transfer of 1 lmol of fructose per minute.

2.12. Production of neo-FOSs

The reaction mixture (4 ml) consisted of 2 ml sucrose solution(2 mmol/l Al3+) with 0.2 mol/l sodium phosphate buffer (pH 6.4)and 2 ml enzyme solution. The enzyme activity in the mixturewas 1.0 U/ml and then the mixture was incubated at 45 �C for8 h in an orbital shaker at 220 rpm. The reaction product was di-luted to tenfold with deionized water and terminated at 100 �Cfor 10 min to inactivate the enzyme. Samples were centrifuged at13,000g for 5 min, filtered through 0.45 lm Durapore1 membrane(Millipore), and analyzed by HPLC. Each experiment was repeatedthree times.

J. Chen et al. / Bioresource Technology 102 (2011) 1715–1721 1717

3. Results and discussion

3.1. Purification and identification of endo-6G-FFase

After DEAE-52 cellulose ion-exchange chromatography in Fig. 1,the endo-6G-FFase was purified 35-fold with an ultimate specificactivity of 239 U/mg and the overall yield of 13.4% (Table 1). Onlyone band corresponding to a molecular mass of 33 kDa was ob-served by SDS–PAGE (Fig. 2). This molecular weight was lowerthan that of extracellular fructofuranosidases from X. dendrorhous(molecular weight of 160 kDa that was derived from a 66 kDaunglycosylated monomer) reported previously (Linde et al.,2009). b-Fructofuranosidases (invertases) described in a numberof microorganisms were dimeric or multimeric enzymes that hadan average molecular weight of 60–65 kDa in the nonglyco-

0 5 10 15 20 25 300.0

0.1

0.2

0.3

0.4

0.5

Fraction num

OD

at 2

80 n

m

a

0.13 M NaCl

0 5 10 15 200.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

Fraction num

OD

at 2

80 n

m

b

Fig. 1. Elution profiles of intracellular 6G-fructofuranosidases from X. dendrorhous. (a) Io52 cellulose chromatography column (pH 7.0); (b) DEAE-52 cellulose column chromato

Table 1Purification of the intracellular 6G-fructofuranosidases from X. dendrorhous.

Purification step Total protein (mg) Total activity (U)

Intracellular fraction 31.49 217.02DEAE-52 cellulose, pH 7.0 2.10 39.02DEAE-52 cellulose, pH 5.0 0.12 29.15

sylated-monomeric forms, including S. cerevisiae (Taussig and Carl-son, 1983), Bacillus macerans (Park et al., 2001), P. anomala(Rodriguez et al., 1995), S. pombe (Moreno et al., 1990), and C. utilis(Chávez et al., 1997). However, one from S. occidentalis had anapproximate molecular weight of 85 kDa (Álvaro-Benito et al.,2007) and another from Rhodotorula glutinis had around 47 kDa(Rubio et al., 2002). It was thus suggested that the intracellular en-zyme from X. dendrorhous we purified in this work had lowermolecular weight.

3.2. Characterization of X. dendrorhous endo-6G-FFase

3.2.1. Effect of pH on activity and stabilityThe purified endo-6G-FFase from X. dendrorhous was active

from pH 6.0 to 7.0 (Fig. 3a) and showed maximum activity at pH

0.00

0.25

0.50

0.75

1.00

35 40 45 50

OD at 280 nmFFase activity (U/ml)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

ber

NaC

l gra

dien

t (0

- 0.

5M)

FFas

e ac

tivity

(U

/ml)

0.0

0.1

0.2

0.3

0.4

25 30 35

OD at 280 nm FFase activity (U/ml)

ber

0.0

0.5

1.0

1.5

2.0

2.5

3.0

FFas

e ac

tivity

(U

/ml)

NaC

l gra

dien

t (0

- 0.

2M)

n-exchange chromatography of the crude fructofuranosidases preparation of DEAE-graphy (pH 5.0) profile of invertase.

Specific activity (U/mg) Purification degree [fold] Yield (%)

6.89 1 10018.87 3 18.3

238.64 35 13.4

4 3 2 1

[kDa]

97

66

43

31

20

14

Fig. 2. SDS–PAGE (12%) of intracellular 6G-fructofuranosidases from X. dendrorhous.Lane 1: crude extract; lane 2: Proteins purified by DEAE-52 cellulose chromatog-raphy column (pH 7.0); lane 3: Proteins purified by DEAE-52 cellulose chromatog-raphy column (pH 5.0); lane 4: Molecular mass markers.

Table 2Effects of metal ions and other chemicals on the activity of endo-6G-FFase from X. dendrorhous.

Compound Concentration (mmol/l)

Relative activity(%)

None 100K+ 2 96Mg2+ 2 97Ca2+ 2 129Zn2+ 2 86Li+ 2 131Mn2+ 2 131Ba2+ 2 129Al3+ 2 142EDTA 2 137SDS 2 65

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.80

40

80

120

160

200

240

Sucrose concentration (mol/l)

Act

ivity

(µm

ol/(

min

·mg)

prot

ein)

Hydrolytic activityTransfer activity

Fig. 4. Kinetic behaviors of intracellular 6G-fructofuranosidases from X. dendror-hous. Reactions were carried out in 0.2 M sodium phosphate buffer (pH 6.4) at 45 �C.

1718 J. Chen et al. / Bioresource Technology 102 (2011) 1715–1721

6.4. This value was almost the same as the optimal pH of the extra-cellular enzyme from X. dendrorhous (pH 5.0–6.5) (Linde et al.,2009). In addition, it was only a slightly higher than that found

pH

Rel

ativ

e ac

tivity

(%

) a

pH

Rel

ativ

e ac

tivity

(%

)

b

Temperature (°C)

c

Temperature (°C)

Rel

ativ

e ac

tivity

(%

) R

elat

ive

activ

ity (

%)

d

Fig. 3. Effects of pH and temperature on the activity and stability of X. dendrorhous endo-6G-FFase. (a) Enzyme activity plotted to pH; (b) Enzyme stability plotted to pH; (c)Enzyme activity plotted to temperature; (d) Enzyme stability plotted to temperature.

J. Chen et al. / Bioresource Technology 102 (2011) 1715–1721 1719

for S. occidentalis (pH 5.5) (Álvaro-Benito et al., 2007). Endo-6G-FFase was stable between pH 4.0–7.0 at 45 �C and retained 85–97% of relative activity (Fig. 3b).

0 1 2 3 40

25

50

75

100

125

150

175

200

225

Con

cent

ratio

n g/

l

Reaction t

b

0 1 2 3 4

10

20

30

40

50

60

70

80

neo-

FOSs

(g/

l)

Reaction tim

c

0.0 2.5 5.0

0

25

50

75

100

125

150

175

200

1/6.0

1

2

Det

ecto

r re

spon

se (

mV

)

Retention

a

Fig. 5. (a) Reaction of sucrose (3 h) with the endo-6G-FFase from X. dendrorhous. Peaks: 1of fructooligosaccharides production catalyzed by the intracellular 6G- fructofuranosida

3.2.2. Effect of temperature on activity and stabilityThe optimum temperature for enzyme activity at pH 6.4 was

45 �C (Fig. 3c), which was in accordance with the optimum temper-

5 6 7 8 9ime (h)

Tetrasaccharides Neokestose Sucrose Glucose Fructose

5 6 7 8 9e (h)

7.5 10.0 min4.00

4.25

4.50

4.75

5.00

5.25

5.50

5.75

6.00

MPa

14

2/6.7053/7.858

4/9.761

5/11.767

3

4

5

time (min)

, tetrasaccharides; 2, neo-kestose; 3, sucrose; 4, glucose; 5, fructose; (b) Time courseses from X. dendrorhous.; (c) Formation of total neo-FOSs.

1720 J. Chen et al. / Bioresource Technology 102 (2011) 1715–1721

ature of other yeast invertases such as S. occidentalis (45–55 �C)(Álvaro-Benito et al., 2007). The thermal stability experimentsdetermined that endo-6G-FFase was relatively stable below 45 �Cwhile a sharp decrease in activity was observed above 50 �C(Fig. 3d). This result is different from that observed for the extracel-lular b-fructofuranosidase from X. dendrorhous which exhibited anoptimum temperature of 65–70 �C and only minor inactivation ofthe enzyme (10%) was detected after 4 days at 40–50 �C (Lindeet al., 2009).

3.2.3. Effect of metal ions and other reagents on activity of enzymeAs shown in Table 2, K+ and Mg2+ had no obvious effect on the

activity of endo-6G-FFase. However, Ca2+, Li+, Mn2+, Ba2+ and Al3+

caused a 1.25–1.4-fold increase and a slight inhibition was inducedby Zn2+. EDTA increased the enzyme activity whereas SDS signifi-cantly reduced its activity. These results indicated that the activityof purified endo-6G-FFase from X. dendrorhous was significantly in-creased by Al3+.

3.3. Determination of kinetic parameters

The kinetic parameters (Km and Vmax) for both hydrolysis andtransfer reactions were shown in Fig. 4. It was possible to use theMichaellis–Menten model (Eq. (1)) for transfer activity but thismodel did not fit well the experimental data for hydrolytic activitythat had a substrate inhibition (Eq. (2)).

V ¼ Vmax

1þ Km½S�

ð1Þ

V ¼ Vmax

1þ Km½S� þ

½S�KSI

ð2Þ

½S�opt ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiKmKSI

pð3Þ

KSI, the inhibitor constant, related to the inhibitory effect of the sub-strate; [S]opt, the optimum sucrose concentration.

For transfer activity, the Km and Vmax values were 511 mmol/land 233 lmol/(min mg). The Km was slightly lower than that ofthe Aspergillus aculeatus fructosyltransferase (535 mmol/l) (Ghaziet al., 2007). Regarding the hydrolytic reaction, the Km and Vmax

values were 62 mmol/l and 164 lmol/(min�mg). The KSI value forhydrolysis activity was 877 mmol/l (Eq. (3)). Other invertases frommicroorganisms like Candida sp. (Hernalsteens and Maugeri, 2008)and Aspergillus niger (L’Hocine et al., 2000) also had kinetics ex-plained by the Michaellis–Menten model (with or without sub-strate inhibition).

3.4. Production of neo-FOSs

Pure endo-6G-FFase from X. dendrorhous was used for the pro-duction of neo-FOSs, and analysis of the reaction products by HPLCshowed that the intracellular enzyme had transfructosylatingactivity. More glucose than fructose was detected (Fig. 5a). Basedon its chromatographic mobility, the compounds correspondingto peaks 2 and 3 were identified as tetrasaccharides and neo-kes-tose (6G-FOS series), respectively (Fig. 5a). At the point of maxi-mum neo-kestose production (3 h), the reaction mixturecontained 9.0 g/l tetrasaccharide, 64.9 g/l neo-kestose, 18.4 g/l su-crose, 55.8 g/l glucose, and 25.3 g/l fructose (Fig. 5b). A sucroseconversion of 90% was achieved (Fig. 5c), and 42.6% (w/w)(73.9 g/l) of neo-FOSs was obtained in the mixture after 7 h. Thisrate and level of production were higher than that previous re-ported by Linde et al. (2009) who obtained 65.9 g/l neo-FOSs(15.8% w/w in the sugar composition) with intracellular b-fructofu-ranosidase X. dendrorhous ATCC MYA-131 after 48 h using 410 g/l

sucrose as substrate. A lower neo-FOSs yield was also observedin the process with enzymes from intact immobilized P. citrinumcells, in which 49.4 g/l of neo-FOSs (8.2% [w/w] in the sugar com-position) was obtained (Lim et al., 2005).

4. Conclusions

Purified intracellular 6G-fructofuranosidase from the basidio-mycetous yeast X. dendrorhous efficiently catalyzes and hydrolyzeshigh concentration sucrose to produce neo-FOSs. The enzymepreparation exhibited higher activity and thus reduced time forneo-FOSs. This work therefore is capable of understanding of theunusual behaviors of the X. dendrorhous endo-6G-FFase and willprovide useful data for the production of neo-FOSs.

Acknowledgement

This study was financially supported by Excellent Doctoral Cul-tivation Foundation of Jiangnan University, New Century ExcellentTalents in University (No. NCET-07-0379), Graduate Student Inno-vation Project of Jiangsu Province (No. CX09B_177Z), NationalNatural Science Foundation of China (No. 20976070), Nature Sci-ence Foundation of Jiangsu Province (Nos. BK2008003 andBK2009069), and Project of State Key Laboratory of Food Scienceand Technology, Jiangnan University (SKLF-MB-200804).

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