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Progress on Chemistry and Application of Chitin and Its ..., Volume XIV, 2009 89

ENZYMATIC PREPARATIONS FROM Mucor MOULDS AND THEIR APPLICATION IN OLIGOAMINOSACCHARIDES PRODUCTION

Katarzyna Struszczyk, Mirosława Szczęsna-Antczak, Marta Walczak, Emilia Pomianowska,

Justyna Wojciechowska, Tadeusz Antczak

Institute of Technical Biochemistry, Technical University of Lodz (TUL)

ul. Stefanowskiego 4/10, 90-924 Łódź, PolandE-mail: [email protected]

AbstractA method of chitosan hydrolysis catalyzed by intracellular chitosanolytic enzymes produced by strains of Mucor circinelloides and Mucor racemosus was developed within the scope of presented study. Hydrolysis of the biopolymer was carried out with the use of: I) mycelial preparation of chitosanolytic enzymes, II) preparation of partially purified enzymes and III) mycelium immobilized in a polyurethane carrier (used in the system of continuous hydrolysis of biopolymer). All these enzymatic preparations extremely effectively catalyzed hydrolysis of various chitosan preparations. They converted chitosan to products having different molecular weight, such as low-molecular weight chitosan, chitooligosaccharides and D-glucosamine. Furthermore, these preparations are relatively inexpensive and highly stable during long-term storage.

Key words: chitosan, chitosanase, chitooligosaccharides, low-molecular weight chitosan.

Progress on Chemistry and Application of Chitin and Its ..., Volume XIV, 200990

K. Struszczyk, M. Szczęsna-Antczak, M. Walczak, E. Pomianowska, J. Wojciechowska, T. Antczak

1. IntroductionChitosan is an N-deacetylated derivative of chitin - one of most abundant natural

polysaccharides. This biopolymer has found numerous applications in various fields, such as: production of food and cosmetics, medicine, agriculture, biotechnology and environmental protection. However, its high molecular weight, high viscosity and poor solubility render chitosan difficult to use in industrial-scale.

Chitooligosaccharides (CHOS, with an average molecular weight below 3.9 kDa) and the so-called low molecular weight chitosan (LMWC, with an average molecular weight ranging between 3.9 kDa and 20 kDa) obtained through chemical, physical or enzymatic degradation of the biopolymer, have many interesting properties. They are biodegradable and non-toxic. These chitosan derivatives display numerous biological activities: antibacterial, antifungal, antiviral, antitumor and antioxidant, stimulate immune system, exert fat and high blood pressure lowering and hypocholesteromic effects [1 - 2]. Products with the low molecular weigh are very well soluble at neutral pH which paved way to their industrial application. It was found that CHOS with a relatively high degree of polymerization (especially those with six residues or more) as well as LMWC were more biologically active than the high molecular weight chitosan and oligomers with low DP [1 - 4]. Acid hydrolysis of chitosan generates less oligomers with high DP and more D-glucosamine than its enzymatic digestion. An advantage of the latter method consists in an intended degree of polymerization and N-deacetylation of hydrolysis products and these two parameters determine the potential application and biological activity of chitosan digests. Enzyme-mediated chitosan degradation is also easier to control and environmentally friendly but its commercial use is limited due to high prices and scarcity of specific enzymes, such as: chitosanase and chitinase. Currently, many of non-specific enzymes have been effectively used in chitosan hydrolysis. These enzymes were as follows: lysozyme, cellulase, hemicellulase, lipase, papain, pectinase, pepsin and pronase [5 - 11]. Enzymatic processes of chitosan oligomers production were carried out in traditional batch reactors containing free enzymes, in column reactors with immobilized enzymes, in ultrafiltration membrane reactors and in other reactor systems which were combinations of aforementioned ones [2].

Three different chitosanolytic preparations from Mucor circinelloides and Mucor racemosus have been obtained within the scope of research conducted in 2005 - 2008 at the Institute of Technical Biochemistry of Technical University of Lodz [12-14]. Application of these enzymatic preparations in the process of chitosan degradation yielded functional, biologically active products with a wide range of polymerization degree (so-called low-molecular weight chitosan, chitooligosaccharides and also D-glucosamine).

2. Materials and methods2. 1. Microorganisms and culture conditions

The strains of Mucor circinelloides and Mucor racemosus from the pure culture collection of the Institute of Technical Biochemistry of TUL were cultivated with agitation


Enzymatic Preparations from Mucor Moulds and Their Application in Oligoaminosaccharides Production

at 180 r.p.m. for 72 h at 30 °0C in culture medium containing corn steep liquor (3.7% w/v) and olive oil (2.7% v/v). The initial pH of the medium was 4.7.

2.2. Methods of obtaining enzymatic preparations from Mucor myceliumPreparation I – mycelial preparation of chitosanolytic enzymes. Mycelium of

Mucor was harvested by filtration, carefully washed with water, defatted with acetone and air-dried at room temperature [12]. Preparation II - preparation of partially purified enzymes was obtained through a two-step procedure comprising chromatography of proteins extracted from Mucor mycelium on CNBr-Sepharose 4B with covalently linked bacitracin, followed by molecular sieving on Sephadex G-100, as was described in [14]. Preparation III - the mycelium immobilized in polyurethane carriers (PU). The Mucor strains were cultivated under conditions described in section 2.1. Medium before sterilization was supplemented with one of the following polyurethane foams: type A – the foam produced by Corpura B.V., Netherlands, with pore diameter varying between 170 and 220 mm, diced into 1.0 × 1.0 × 0.5 cm cubes, amount of this carrier in the culture medium was – 2.7% (w/v); type B – the foam produced by Eurofoam, Poland, with pore diameter of ~ 1.96 mm, cut into 1.0 × 1.5 × 0.2 cm cubes and used as a carrier in the culture medium (in amount of 1.3% (w/v)). The mycelia grown in polyurethane carriers were defatted, dehydrated, dried and used in continuous processes of chitosan hydrolysis carried out in a column reactor.

2.3. Determination of enzymatic activityThe chitosanolytic activity (endo-chitosanolytic activity Aendo-CH) of enzymatic

preparations was determined on the basis of the decrease in a viscosity average molecular weight (Mvh) of chitosan and was expressed as an amount of enzyme necessary to decrease Mvh of chitosan by 1 kDa in 1 minute under the conditions described below [1 unit = 1 kDa min-1]. Reaction mixtures for preparation I (pH 5.5) contained 2% chitosan solution in 2% acetic acid (1 ml), 2 M CH3COONa (1 ml) and enzyme preparation (10 mg). Chitosan hydrolysis was carried out at 37 °C for 120 min and was terminated by 5 min incubation in a boiling water bath. Controls (with the same composition as the samples) were first incubated for 5 min in the boiling water bath to inactivate enzymes and then for 120 min at 37 °C. Reaction mixtures for preparation II (pH 5.5) contained 2% chitosan solution in 2% acetic acid (1 ml), 1 M CH3COONa (0.85 ml) and enzyme solution (0.15 ml). Chitosan hydrolysis was carried out at 37 °C for 60 min and was terminated by 5 min incubation in a boiling water bath. Controls (with the same composition as the samples) were incubated for 5 min in the boiling water bath to inactivate enzymes and then for 60 min at 37 °C.

2.4. Analytical methodsThe viscosity average molecular weight (Mvh) of chitosan and its digestion

products was determined by the viscometric method using one of the following solutions: 1) 0.1 M sodium chloride, 0.2 M acetic acid and 4.0 M urea (for chitosan with Mvh between 113 kDa and 492 kDa), 2) 0.30 M sodium chloride and 0.33 M acetic acid (for chitosan with Mvh between 13 kDa and 135 kDa) and calculated according to the Mark-Houwink’s equation [h = kMvha] with 1) k = 8.93 × 10-4 and a = 0.71 or 2) k = 3.41 × 10-3 and a = 1.02, respectively [15]. The viscosity measurements were conducted at 25.0 ± 0.1 °C using an Ubbelohde’s viscometer (Schott GmbH, type 53110/I).



The concentration of reducing sugars (RSC) released from chitosan was determined by Somogyi-Nelson method [16] using D-glucosamine as standard.

The gel permeation chromatography (GPC) of chitosan and its digestion products was done using Hewlett Packard 1050 system equipped with two PL aquagel-OH Mixed columns and RI HP 1047 detector. The eluent was acetate buffer, pH 4.3. The temperature of columns was 30.0 ± 0.1 °C and the eluent flow rate was 0.8 ml min-1. The standards used to calibrate the column were: polyethylene glycol and polyethylene oxide.

Products released from chitosan were analyzed by thin layer chromatography (TLC) on Silica G 60 plates using n-propanol/30% ammonia water (2:1 v/v) as the developing solvent. Aminosugars were visualized by 0.1 % (w/v) nihydrin in 99 % ethanol solvent spray.

3. Results and discussion 3.1. Characterization of Mucor enzymatic preparations

Our studies [11, 12] revealed that defatted and air-dried mycelia of Mucor moulds (preparation I) displayed mainly the activity of endo-chitosanase capable of cleaving b-1,4-glycosidic bonds in chitosan. Their exo-chitosanolytic activity was minor. The A endo-CH of M. circinelloides preparations was 429 ± 42 unit/g and was higher than the activity of M. racemosus preparations (343.5 ± 43 unit/g). The optimum conditions for biopolymer hydrolysis by preparations obtained from M. circinelloides mycelium were: the temperature of 37 °C and pH 5.4 – 5.6. These chitosanolytic preparations were relatively stable below 60 °C and at pH between 4.0 and 8.0. Preparations from the second strain were optimally active at 37 °C and in pH range of 5.0 – 5.5. They were stable below 60 °C and at pH between 4.0 and 7.5. The endo-hydrolases bounded to M. circinelloides mycelium preferred chitosan with the low DD (these enzymes hydrolyzed also colloidal chitin and NaCMC) whereas the highly deacetylated biopolymer was the preferred substrate of M. racemosus enzymes. These mycelial enzymatic preparations retained 85% of their initial chitosanolytic activity for 60 days at 24 °C. After one year of storage their activity dropped by 37% (for M. circinelloides enzymes) and by 51% (for M. racemosus enzymes). The higher chitosanolytic activity and storage-stability of M. circinelloides crude preparations renders them more suitable for industrial-scale applications in comparison to preparations from the second Mucor strain. Characteristics of M. circinelloides and M. racemosus preparations are shown in Table 1.

The endo-acting chitosanase was extracted from defatted mycelium of M. circinelloides with a non-ionic detergent Triton X-100 and purified through a two-step procedure comprising chromatography on CNBr-Sepharose 4B with covalently linked bacitracin and gel filtration on Sephadex G-100 [14]. Partially purified enzyme (preparation II) was optimally active at 37 °C and pH of 5.5 - 6.0 and activated by Ca+2, Mn+2 and Mg+2 ions. It preferred chitosan with a high degree of deacetylation as a substrate. However, this preparation showed no activity for colloidal chitin, Na-CMC and starch. Its



SDS-PAGE analysis showed two protein bands with molecular mass of 38 and 43 kDa [14]. Characterization of partially purified enzymes of M. circinelloides is shown in Table 2.

Table 1. Characteristics of M. circinelloides and M. racemosus chitosanolytic mycelial prepara-tions.

ParameterMycelial enzymatic preparation

M. circinelloides M. racemosus

Aendo-CH, unit/g 429 ± 42 343.5 ± 43

Optimum temperature 1) 37 °COptimum pH 2) 5.4 – 5.6 5.0 –5.5

Substrate specificity 3) Preferred chitosan with low DD; colloidal chitin; NaCMC

Preferred chitosan with high DD; colloidal chitin

Thermostability 4) Stable below 60 °CpH –stability 5) 4.0 – 8.0 4.0 – 7.5

Storage stability, days6) 60 85% activity is retained 85% activity is retained360 63% activity is retained 49% activity is retained

1) The temperature optimum of chitosanolytic enzymes was determined by measuring relative activity (section 2.3.) at pH 5.5 over a temperature range between 5 °C and 60 °C; 2) The effect of pH on activity of endo-hydrolases was estimated on the basis of assays carried out at 37 °C and over pH range between 3.5 and 8.0; 3) For determination of substrate specificity of Mucors chitosanolytic enzymes, various substrates such as chitosan with Mvh ranging between 121 kDa and 421 kDa and DD ranging between 66% and 96%, colloidal chitin and NaCMC were used; 4) Thermostability of chitosanolytic enzymes was evaluated by their incubation for 30 min at temperature varying between 5 and 100 °C followed by residual activity assays under standard conditions (section 2.3.); 5) The pH stability of the enzymes was determined by measuring residual activity (section 2.3.) after their pre-incubation (for 60 min at 4 °C) in one of the following buffers: 0.1 M acetate buffer (pH 3.6 – 5.6), 0.1 M phosphate buffer (pH 5.7 - 8.0), 0.1 M Tris-HCl buffer (pH 8.0 - 9.0); 6) Stability of enzymatic preparations was estimated by measuring residual activity (section 2.3.) after their storage at 24 °C for 2 to 360 days.

Results of gel permeation chromatography of products of chitosan hydrolysis catalyzed by M. circinelloides preparations are shown in Figure 1. After 2 h digestion (under optimum conditions) catalyzed by preparations of partially purified enzymes (Aendo-CH of approximately 18.5 unit ml-1) or after 4 h digestion with mycelial enzymatic preparation (Aendo-CH of approximately 382 unit g-1) the hydrolysates contained mainly low molecular weight chitosan (molecular mass of 5 kDa – 50 kDa) and 4 - 5% of oligoaminosaccharides with molecular mass below 5 kDa.

When the time of hydrolysis catalyzed by crude enzymatic preparation was longer (24 – 48 hours) the amount of products with the low degree of polymerization (even monomers) was increased. However, the partially purified enzyme released only oligosaccharides with the high DP.

Both enzymatic preparations were also extremely effective in hydrolysis of different chitosan preparations [13 - 14]. They are relatively inexpensive and characterized



by the high long-term storage stability. Therefore their application in the large scale can be cost effective. °

Table 2. Characterization of partially purified enzymes from M. circinelloides

Parameter Preparation of partially purified enzymes of M. circinelloides

Optimum temperature 1) 37 °COptimum pH 2) 5.5 – 6.0Thermostability 3) Stable below 50 °C pH –stability 4) 4.5 – 7.5Activators 5) Ca2+, Mn2+and Mg2+ ionsInhibitors 5) Hg2+, Cu2+ and Ag2+ ions; Tween 20, Tween 80, Triton X-100, EDTA, SDSPreferred chitosan with 6) high DDStability during storage 7) Retain of 90% of initial activityMolecular masses 8), kDa 38 and 43

1) The temperature optimum of chitosanolytic enzymes was determined by measuring relative activity (section 2.3.) at pH 5.5 over a temperature range between 5 °C and 60 °C ; 2) The effect of pH on activity of chitosanolytic enzymes (endo-hydrolases) was estimated on the basis of assays carried out at 37 °C over pH range between 3.0 and 8.0; 3) Thermostability of chitosanolytic enzymes was evaluated by their incubation for 30 min at temperature varying between 5 and 100 °C followed by residual activity assays under standard conditions (section 2.3.); 4) The pH stability of the purified enzymes was determined by measuring residual activity (section 2.3.) after pre-incubation (for 60 min at 4 °C) in 0.1 M citrate buffer (pH 3.0 - 6.0) or in 0.1 M phosphate buffer (pH 6.0 - 8.0); 5) The effect of various metals ions (Ag2+, Ba2+, Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Hg2+, Mg2+, Mn2+, Pb2+, Sn2+, Zn2+ - 1 mM) and other chemicals (Tween 20, Tween 80, Triton X-100, EDTA, SDS – 1 % w/v) on the activity of Mucor chitosanolytic enzymes was investigated by their pre-incubation with these compounds for 30 min at 37 °C followed by measuring residual activity under standard conditions (section 2.3.); 6) For determination of substrate specificity of M. circinelloides proteins, various substrates such as chitosan Mvh ranging between 121 kDa and 421 kDa, DD ranging between 66% and 96%), colloidal chitin, sodium carboxymethyl cellulose and starch were used; 7) Stability of enzymes was estimated by measuring residual activity (section 2.3.) during their storage at –20 °C for 30 days; 8) The molecular mass of the separated proteins was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli [17] using 12.5% polyacrylamide gel.

3.2. Continuous production of chitosan oligomersThe continuous hydrolysis of chitosan was conducted in a column reactor packed

with mycelia of M. circinelloides or M. racemosus immobilized in polyurethane (PU) carriers (preparations type III). Two kinds of PU foams (with different pore dimensions) were used for immobilization (section 2.2.). Figure 2 presents SEM images of these preparations.

The schematic diagram of the column reactor system used for chitosan hydrolysis (consisting of substrate and product tanks, peristaltic pumps, UF membrane and column reactor packed with immobilized mycelium) is presented in Figure 3. Changes in reducing sugars concentration (RSC) and in Mvh of chitosan during its continuous hydrolysis by M. circinelloides (column I) and M. racemosus (column II) enzymes immobilized in PU carriers are presented in Figures 4 - 7.



Figure 1 (and table below). Molecular weight distribution of chitosan (sample 1) and enzymati-cally degraded chitosan (samples 2 and 3).

No SampleFraction content [% w/w)]

Molecular mass (x 103) [Da]<5 5-50 50-100 100-200 200-400 400-800 >800

1. Undigested chitosan 1 32 24 23 13 6 1

2. Chitosan degradation products released by mycelial enzymatic preparation 1) 4 55 23 13 1 0 0

3. Chitosan degradation products released by partially purified enzymes 2), 3) 5 69 19 6 1 0 0

The preparation of M. circinelloides enzymes that was used in the experiment displayed the endo-chitosanolytic activity of 1) 382 unit g-1 and 2) 18.4 unit ml-1. Chitosan digestion was carried out for 1) 240 min and 2) 120 min. 3) Reaction mixture (pH 5.5) contained 2% chitosan solution in 2% acetic acid (1 ml), 1 M CH3COONa (0.70 ml) and enzyme solution (0.30 ml). Other details on reaction mixture composition and conditions of chitosan hydrolysis are described in section 2.3.

Figure 2. SEM image of M. circinelloides mycelium immobilized in PU foam: (a) pore diameter of 170 - 220 mm, produced by Corpura B.V., Netherlands; (b) pore diameter of ~ 1.96 mm, produced by Eurofoam, Poland.

a) b)



Figure 3. Schematic diagram of a column reactor used for continuous production of oligoami-nosaccharides; 1) Substrate feed tank (solution of chitosan); 2) Column containing immobilized mycelium of Mucor; 3 and 6) Pumps; 4) Product feed tank I; 5) UF membrane; 7) Product feed tank II

Figure 4. Column I. Dynamics of continuous process of chitosan hydrolysis by M. circinelloides mycelium immobilized in PU carrier with pore diameter of 170 – 220 mm. Samples of chitosan degradation products were taken from product feed tank I (see Figure 3) and changes in viscosity average molecular weight of chitosan (Mvη) and reducing sugars concentration (RSC) were determined. Explanation: column dimensions – 2.5 × 18.5 cm, flow rate of chitosan solution (0.5% chitosan acetate, pH 5.5) – 1 cm3 cm-2 h-1, mass of biocatalyst – 12 g (PU containing approximately 7 g of dried mycelium), temperature of chitosan hydrolysis of 24 °C.



Figure 5. Column II. Dynamics of continuous process of chitosan hydrolysis by M. racemosus mycelium immobilized in PU carrier with pore diameter of ~1.96 mmSamples of chitosan degradation products were taken from product feed tank I (see Figure 3) and changes in viscosity average molecular weight of chitosan (Mvη) and reducing sugars concentration (RSC) were determined. Explanation: column dimension – 2.5 × 18.5 cm, flow rate of chitosan solution (0.5% chitosan acetate, pH 5.5) – 1 cm3 cm-2 h-1, mass of biocatalyst - 3 g (PU containing approximately 1.6 g of dried mycelium), temperature of chitosan hydrolysis of 24 °C.

Figure 6. TLC profiles of eluates from the column I used for chitosan oligomers production. Samples of chitosan degradation products were taken from product feed tank II (see Figure 3). Products dialysable through UF membrane with 5 kDa cut off were analyzed by TLC. Lane S: standards of GlcN and its oligomers from (GlcN)2 to (GlcN)5 , Lanes 1 – 18: mixtures of chitooligosaccharides obtained as described under Figure 4.

Figure 7. TLC profiles of eluates from the column II used for chitosan oligomers production. Samples of chitosan degradation products were taken from product feed tank II (see Figure 3). Products dialysable through UF membrane with 5 kDa cut off were analyzed by TLC. Lane S: standards of GlcN and its oligomers from (GlcN)2 to (GlcN)5 Lanes 1 – 16: mixtures of chitooligosaccharides obtained as described under Figure 5.



The mycelia of Mucor moulds immobilized in PU carriers are extremely effective in continuous hydrolysis of chitosan. The TLC profiles (Figures 6 and 7) of eluates from the column I and column II (which were taken from the feed tank II and filtered through UF membrane with 5 kDa cut off) provided evidence of the presence of D-glucosamine and its oligomers – from dimer to pentamer. It indicates that Mucor mycelium contains not only endo-chitosanases but also exo-chitosanases that liberate D-glucosamine from nonreducing ends of chitosan chains. However, the activity of the latter enzymes is relatively low and therefore the complete hydrolysis of chitosan to D-glucosamine catalyzed by preparations of Mucor mycelium (preparation I) is very long and requires the high E:S ratio.

It is to note that in contrast to the immobilized enzyme preparations, the powdered Mucor mycelia could not be used for packing the columns because they were immediately plugged. Only columns packed with Mucor mycelia immobilized in PU carriers can be used for continuous chitosan hydrolysis. DP of reaction products can be tailored by regulation of process temperature and the flow rate and concentration of chitosan solution.

Operational stability of immobilized, mycelial preparations of chitosanolytic enzymes in column I and column II implies that there were 2 phases of function of these columns (Fig. 4 and 5). In the first phase (corresponding to first 14 and 6 days for column I and II, respectively) the activity of chitosanases decreased gradually by approximately 1-2.5% per day due to their gradual inactivation. Our results indicate that the exo-chitosanases of both Mucor strains have the lower operational stability than the endo-chitosanases. After 6 days of the process the Aexo-CH was decreased by 14 and 13% while Aendo-CH was lowered by 3 and 11% for M. circinelloides (column I) and M. racemosus (column I), respectively. Time of duration of phase I depends mainly on the portion of enzyme preparation in the column (the more enzyme preparation, the longer phase I). On completion of phase I the hydrolysis of chitosan is considerably slowed down (despite the constant rate of substrate flow through the column of 1 cm3cm-2h-1) because of much faster inactivation of the enzymes (by approximately 10 % per day). We think that it was caused by the leakage of proteins from the mycelium because some proteins were detected in the eluate from the column (data were not shown).

The activity of endo- and exo-chitosanases contained in immobilized Mucor preparations packed into column I and II was decreased by 50% after 16 and 10 days, respectively. This difference resulted most probably from different doses of these preparations used for packing the columns (column I and II: 7 g and 1.6 g of dried mycelium, respectively) but it also could be caused by different stability of chitosanases from M. circinelloides and M. racemosus.

Because the operating stability is a crucial parameter for large scale applications (in particular for continuous processes) we intend to improve it through stabilization of enzymes and elimination of enzyme leakage from columns.



4. ConclusionsEnzymatic preparations from M. circinelloides and M. racemosus mycelia

are characterized by high chitosanolytic activity, high stability during their storage and low production costs. Their application in processes (batch or continuous) of chitosan degradation yields products with different polymerization degrees: chitooligosaccharides, so-called low-molecular weight chitosan and D-glucosamine. They can be successfully used in many branches of industry.

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AcknowledgmentThis work was partially supported by the Polish Ministry of Science and Higher Education, research project No. N507 181 32/2133.

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