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Purification and characterization of two types of chitosanase from Aspergillus sp. CJ22-326
Food Research International 38 (2005) 315–322 www.elsevier.com/locate/foodres
Purification and characterization of two types of chitosanase from Aspergillus sp. CJ22-326 XiaoÕe Chen a
c
a,b
, Wenshui Xia
a,*
, Xiaobin Yu
c
School of Food Science, Southern Yangtze University, Wuxi 214036, PR China b Zhejiang Ocean University, Zhoushan 316004, PR China School of Biotechnology, Southern Yangtze University, Wuxi 214036, PR China Received 14 January 2004; accepted 6 April 2004
Abstract Aspergillus CJ22-326, a fungi strain capable of utilizing chitosan as a carbon source, was isolated from soil samples. Two types of chitosanase (ChiA and ChiB) produced from the culture supernatant of Aspergillus CJ22-326 were purified to an apparent homogeneity identified by SDS–PAGE through ammonium sulfate precipitation, CM-Sepharose FF chromatography, and Sephacryl S-200 gel filtration. Molecular weights of the enzymes were 109 kDa (ChiA) and 29 kDa (ChiB). Optimum pH values and temperature of ChiA were 4.0 and 50 C, respectively, those of ChiB were 6.0 and 65 C. The enzyme activities of ChiA and ChiB were increased by about 0.5-fold and 1.5-fold, respectively, by the addition of 1 mM Mn2+. However, 2.5 mM Ag+, Hg2+ and Fe3+ strongly inhibited ChiA and ChiB activities. Viscosimetric assay and analysis of reaction products of these enzymes, using chitosan as a substrate, by TLC indicated endo- and exo-type cleavage of chitosan by ChiB and ChiA, respectively. ChiB catalysed the hydrolysis of glucosamine (GlcN) oligomers larger than pentamer, and chitosan with a low degree of acetylation (0–30%), and formed chitotriose with chitohexaose as the major products. ChiA released a single glucosamine residue from chitosan and glucosamine oligomers. Both of the activities of ChiA and ChiB increased with the degree of deacetylation of chitosan. The enzyme ChiB had a useful reactivity and a high specific activity for producing functional chitooligosaccharides with high degree of polymerization. 2004 Elsevier Ltd. All rights reserved. Keywords: Chitosanase; Chitooligosaccharides; Aspergillus; Chitosan; Endo-type; Exo-type
1. Introduction Chitosan that have different degrees of deacetylation (DDA) can be readily obtained by N-deacetylating chitin that is extracted from an abundant natural source, shrimp and crab shells. It has high safety and peculiar physical properties because of its high cation content. Its biological properties, like compatibility and antimicrobial activity, are remarked. Actually, chitosan is applied widely to health foods such as for the prevention and treatment of hyperuricemia, and as an antimicrobial *
Corresponding author. Tel.: +86510 5869455. E-mail address: [email protected] (W. Xia).
0963-9969/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2004.04.012
agent, preservative agent and edible film. But there is doubt about their level of absorption in the human intestine, and their high molecular weights and highly viscous nature may restrict their uses in in vivo systems. Recently much attention has been paid to converting chitosan to safe and functional chitooligosacchrides, because chitooligosaccharides with high degrees of polymerization (DP), especially those with six residues or more, show strong physiological activities, such as antitumor activities (Suzuki et al., 1986; Suzuki, Matsumoto, Tsukada, Aizawa, & Suzuki, 1989), immuno-enchancing effects (Suzuki, 1996), enhancing protective effects against infection with pathogens in mice (Tokoro, Kobayashi, Tatekawa, Suzuki, & Suzuki,
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1989; Yamada, Shibuya, Kodama, & Akatsuka, 1993), antifungal activity (Hirano & Nagao, 1989; Kendra, Christian & Hadwiger, 1989) and antimicrobial activity (Jeon, Park, & Kim, 2001; Savard, Beaulieu, Boucher, & Champagne, 2002; Uchida, Izume, & Ohtakara, 1989). Chitooligosaccharide functions have led to progressively increased utilization in the food and pharmaceutical fields for human health. Chitooligosaccharides can be made by hydrolyzing chitosan with chitosanase. Chitosanase (EC 3.2.1.132) catalyses the hydrolysis of the glycosidic bonds of chitosan, and has been found in a variety of microorganisms, including bacteria (Akiyama et al., 1999; Chiang, Chang, & Sung, 2003; Omumasaba, Yoshida, Sekiguchi, Kariya, & Ogawa, 2000; Yoon et al., 2000), actinomycetes (Boucher, Dupuy, Vidal, Neugebauer, & Brzezinski, 1992; Sakai, Katsumi, Isobe, & Nanjo, 1991), and fungi (Shimosaka, Nagawa, Ohno, & Okazaki, 1991; Zhang et al., 2000). Chitosanases from individual organisms differ in their hydrolytic action pattern. However, most chitosanases from the isolated microorganisms intend to make dimers, trimers and tetramers rather than oligomers above DP 4, so the utility of these enzymes is not good. To obtain a novel chitosanase which could be used for large-scale production of chitosan oligomers above DP 4, we screened various types of microorganisms. An Aspergillus CJ22-326 produced high chitosanase activities when grown on medium with wheat bran and chitosan as carbon source. In this paper, we describe purification and characterization of two different chitosanolytic enzymes, and one of the endo-type chitosanase ChiB is potentially valuable for industrial applications for producing functional chitooligosaccharides.
2. Materials and methods 2.1. Strain and culture conditions The strain used in this study is one of the fungal strains isolated from marine soil in China. The culture medium composed of 1.0% soluble chitosan, 2.0% wheat bran, 0.2% (NH4)2SO4, 0.2% KH2PO4, and 0.05% MgSO4 per liter (pH 5.6). The cultivation was carried out in a 500 ml baffle flask with 150 ml of the culture medium at 30 C for 96 h with agitation at 150 rpm. 2.2. Chemicals and substrates CM-Sepharose FF, Sephacryl S-200 and Phenyl CL4B were from Pharmacia. Chitosan and chitin were purchased from the local suppliers in China. Glucosamine (GlcN), chitobiose (GlcN)2, chitotriose (GlcN)3, chitotetraose (GlcN)4, chitopentose (GlcN)5 chitohexaose (GlcN)6 were purchased from Seikagaku Co. All other reagents were of analytical grade.
2.3. Enzyme purification Ammonium sulfate precipitation – After cultivation, the cells were removed from the medium by centrifugation at 4000 rpm for 30 min. Solid ammonium sulfate was added to the culture filtrate to 80% saturation. After standing overnight, the precipitate was collected by centrifugation and dissolved in 50 ml of 20 mM sodium acetate buffer, pH 5.6 (buffer A). The enzyme solution was dialyzed against the same buffer. Chromatography on CM-Sepharose FF – The dialyzed solution was put on a CM-Sepharose FF column (2.6 · 50 cm) that had been equilibrated with 20 mM sodium acetate buffer. The column was washed with the same 500 ml buffer, and then eluted with a 600 ml linear gradient of 0–1.0 M NaCl in buffer A, at a flow rate of 30 ml/h, and 5 ml fractions were collected. The two active fractions were pooled and concentrated separately by ultrafiltration. Gel filtration on Sephacryl S-200 – Fractions from each peak were pooled separately and put on a Sephacryl S-200 column (2.0 · 100 cm) equilibrated with buffer A containing 0.1 M NaCl (buffer B). The proteins were eluted with buffer B at a flow rate of 12 ml/h, and the resulting active fractions were collected and used as the purified enzyme preparations throughout this study. All purification steps were performed at 4 C. 2.4. Enzyme assay Unless indicated otherwise, chitosan with a DDA of 83% was used as the substrate in the chitoanase assay. The incubation mixture contained 1 ml of 0.5% soluble chitosan and 1 ml of diluted enzyme solution (pH 5.6). The incubation was carried out at 37 C for 15 min, with shaking. The amount of reducing sugar in the supernatant was measured using the modified dinitrosalicyclic acid (DNS) method (Miller, 1959). One enzyme unit was defined as the amount of enzyme required to produce 1 lmol of reducing sugar as glucosamine per min. 2.5. Protein assay and electrophoresis Protein was determined by the method of Lowry, Rosebrough, Farr, and Randall (1951), using bovine serum albumin as the standard. During purification, protein was estimated by measuring absorbance at 280 nm, with bovine serum albumin as the standard. Polyacrylamide gel electrophoresis (SDS–PAGE) was used to determine protein purity and the molecular mass of the purified enzyme under denaturing conditions using a 12% acrylamide gel, as described by Laemmli (1970). Protein was stained by Coomassie blue.
X. Chen et al. / Food Research International 38 (2005) 315–322
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2.6. Enzyme characterization The effect of pH on the enzyme activity was determined by changing the pH of the reaction mixtures using 200 mM sodium acetate (pH 3.6–5.8) and 200 mM sodium phosphate (pH 5.8–8.0). The effect of temperature on the enzyme activity was determined at pH 5.6, in the range 30–80 C. The effects of metallic ions and some enzyme inhibitors on ChiA activity were determined after preincubation at 37 C for 30 min. 2.7. Viscosimetric assay and analysis of hydrolysis products Viscosimetric assay was performed by the method of Ohtakara (1988). The end products of the enzymatic hydrolysis of chitosan were analyzed by thin-layer chromatography (TLC) on Silica G 60 plates using n-propanol/30% ammonia (3:1, v/v) as the developing solvent (Sakai et al., 1991). Sugars were visualized by ninhydrin spray.
Fig. 2. SDS–PAGE of the purified ChiA and ChiB. The purified enzyme ChiA (lane 1), standard molecular mass markers (lane 2), the purified enzyme ChiB (lane 3), protein was stained with Coomassie brilliant blue R-250.
Sephacryl S-200 columns in the positions predicted for proteins with their molecular weights, indicating that they were monomeric. The purification procedure yielded a 4.01-fold purified ChiA with 7.4% recovery and 11.34-fold purified ChiB with 37.0% recovery. The final specific activity of ChiA and ChiB were 6.46 and 18.26 u/mg using 83% DDA, respectively. It should be noted that the chitosanase assay reflects total enzymatic activity; individual chitosanase activities A and B cannot be distinguished in the crude extract. These purification steps from culture broth are summarized in Table 1.
3. Results 3.1. Purification of chitosanases Crude enzyme solution prepared by salting out with ammonium sulfate was applied to cation exchange chromatography on CM-Sepharose FF. As shown in Fig. 1 two chitosanase peaks of A (minor fraction) and B (major fraction) were separated. The chitosanolytic proteins that eluted in peaks A and peaks B were designated ChiA and ChiB, respectively. Both of these chitosanases were purified by successive Sephacryl S200 gel chromatography to homogeneity as judged by SDS–PAGE (Fig. 2). The molecular masses of ChiA and ChiB were estimated to be 109 and 29 kDa, respectively. Both of these chitosanases eluted from
3.2. Effects of pH and temperature on chitosanases The effects of pH and temperature on ChiA and ChiB were investigated. Fig. 3 shows the effects of pH on ChiA and ChiB activity and enzyme stability.
3.0
B
35
25 1.5
20
A
15
1.0 10
0.75
0.50
NacCL(mol/l)
30 2.0
Chitosanase activity(u/ml)
2.5
OD(280nm)
1.00
40
OD(280nm) Chitosanase activity
0.25
0.5 5 0.0
0 0
100
200
300
400
0.00
500
Elution volume(ml) Fig. 1. Elution profile of chitosanase on CM-Sepharose FF column chromatography.
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Table 1 Purification of chitosanase ChiA and ChiB from Aspergillus CJ22-326 Step
Total activity (u)
Total protein (mg)
Specific activity (u/mg)
Crude extract 0–80% (NH4)2SO4
1134 804
703 169
1.61 4.75
100 70.8
1 2.95
ChiA CM-Sepharose FF Sephacryl S-200
116 84
21 13
5.52 6.46
10.2 7.4
3.42 4.01
ChiB CM-Sepharose FF Sephacryl S-200
580 420
42 23
13.8 18.26
51.1 37.0
8.57 11.34
Relative activity /%
Relative activity /%
The effects of metal ions on chitosanases are presented in Table 2. Hg2+, Ag+ and Fe3+ (2.5 mM) inhibited the activities of ChiA and ChiB about 90%. 1 mM Mn2+ remarkably increased the activities of ChiA and ChiB to 150% and 250%, respectively. This result was similar to those for other chitosanlytic enzymes. ChiA was not significantly affected by other metal ions. However, the activities of ChiB were also inhibited by Cd2+, Cu2+, Pb2+ and Zn2+.
Chi A
3
4
5
6
7
8
9
Purification folds
3.3. Effects of metal ions on chitosanases
Optimum pHs were about 4.0 and 6.0, respectively. ChiA was stable in the pH range of 3.5–7.5 and ChiB was stable in the pH range of 4.5–8.0. Fig. 4 shows the optimum temperatures of ChiA and ChiB were 50 and 65 C, respectively. ChiB was stable at a temperature lower than 75 C, but ChiA was stable at temperature lower than 60 C, and dramatically inactivated by incubation at 70 C in pH 5.6 for 60 min. Both enzymes did not lose their activities during storage in sodium acetate buffer (pH 5.6) at 4 C for 6 months.
100 90 80 70 60 50 40 30 20 10 0
Yield (%)
Chi B
100 90 80 70 60 50 40 30 20 10 0 3
10
4
5
6
7
8
9
10
pH
pH
-
- Enzyme activity;
- -.Relative stability
Chi A
100 90 80 70 60 50 40 30 20 10 0
Relative activity/%
Relative activity/%
Fig. 3. Effect of pH on the activity and stability of enzyme ChiA and ChiB.
30
40
50
60
-
70
80
Chi B
100 90 80 70 60 50 40 30 20 10 0 30
40
50
60
-Enzyme activity; - -.Relative stability
Fig. 4. Effect of temperature on the activity and stability of enzymes ChiA and ChiB.
70
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319
Table 2 Effect of metal ions on the activity of ChiA and ChiB Ion ChiA Relative activity ChiB Relative activity
Control
Cu2+
Hg2+
Cd2+
Mn2+
Sn2+
100
108
15
93
150
84
100
20
3
22
250
97
3.4. Substrate specificity The activities of the two chitosanases with various substrates are summarized in Table 3. ChiA degraded chitosan with a DDA of 83% to 95% effectively, and exhibited low activity with chitosan having DDA of 65%. On the other hand, the most susceptible chitosan for ChiB had DDA of 72–96%. Neither ChiA nor ChiB hydrolyzed colloidal chitin, or CMC at all. 3.5. Viscosimetric assay To characterize the mode of action of both enzymes, a decreasing rate in viscosity of 95% deacetylated chitosan was investigated during enzymatic hydrolysis. ChiB reduced the viscosity of chitosan solution at an early stage of reaction, while the ChiA reduced the viscosity to a much lower extent when the same amount of enzyme was used, as judged by the values obtained in Table 3 Substrate specificity of chitosanases ChiA and ChiB Substrate (0.5%)
Relative activity (%) ChiA
ChiB
Chitosan (95% DDA) Chitosan (83% DDA) Chitosan (72% DDA) Chitosan (65% DDA) Colloidal chitin CMC
100.0 91.2 83.5 72.7 0 0
100.0 80.6 65.2 44.1 0 0
Ba2+
Pb2+
Zn2+
Ca2+
Co2+
Mg2+
Ag+
Fe3+
97
90
85
101
86
89
13
8
102
60
67
98
80
91
15
12
the standard reducing sugar assay (Fig. 5). This result indicates that the ChiB would probably hydrolyze chitosan in an endo-type fashion in contrast with an exo-type cleavage pattern for ChiA. 3.6. Analysis of the reaction products The hydrolysates of chitosan (DDA 95%) by the purified ChiA and ChiB were analyzed by thin-layer chromatography (TLC) (Fig. 6). A change in the hydrolysis products was observed during incubation with the ChiB at 50 C for 2 h. At appropriate intervals during an early stage, chitosan was hydrolyzed to chitosan oligomers larger than tetramer and a little trimer (Fig. 6, lanes 1–3). After a prolonged reaction (2 h), the amount of (GlcN)5 and (GlcN)6 in the hydrolysate decreased, while (GlcN)3 and (GlcN)4 levels increased (lane 4). The shorter oligomer of GlcN increased as digestion time increased. It is suggested that the mode of action of the enzyme is the endo-type. On the other hand, ChiA gave only GlcN as a final product after a
Specific viscosity
1 0.8 0.6 0.4 0.2 0 0
2 4 6 Reaction time(min) -
- 0.1u of Chi B; -
8
10
- 0.1u of Chi A
Fig. 5. Reduction in viscosity of chitosan colution with ChiA and ChiB. The reaction mixture (7 ml) contained 0.05% chitosan as the substrate. The flow time of the mixture was measured at appropriate times. Specific viscosity = (the flow time of the reaction mixture/flow time of distilled water).
Fig. 6. Analysis of enzymatic hydrolysates by TLC. The substrate used was 95% deacetylated chitosan (lanes 1–5). The enzymes used were purified ChiB (lanes 1–4) and ChiA (lane 5). At different time intervals, 1 ml of reaction mixture were taken out and heated for 10 min in a boiling water bath. Lanes S, standard (GlcN) to (GlcN)6; lanes 1–4, ChiB hydrolysates after 10 min, 20 min, 30 min and 2 h, respectively; lane 5, ChiA hydrolysates after 1 h.
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Fig. 7. TLC of chitooligosaccharides after treatment of ChiA (a) and ChiB (b). Lanes: 1 and 4, unhydrolyzed substrate (GlcN)2 and (GlcN)3, respectively; 2 and 3, digestion of (GlcN)2 and (GlcN)3with ChiA for 24 h at 37, respectively; 5–8, digestion of (GlcN)3 to (GlcN)6 with ChiB for 24 h at 37, respectively; S, standard (GlcN) to (GlcN)6.
prolonged reaction, suggesting an exo-type cleavage manner to release a single GlcN residue from chitosan. ChiB could not hydrolyze either (GlcN)2, (GlcN)3, or (GlcN)4 to any extent, (GlcN)5 was hydrolyzed to produce a mixture of (GlcN)2 and (GlcN)3 slowly, (GlcN)6 was hydrolyzed to (GlcN)3 (Fig. 7(b)). From the results of a viscosimetric assay and TLC analysis of hydrolysates, ChiB catalyzes an endo-type cleavage of chitosan, and should be designated as chitosanase. On the other hand, ChiA gave only GlcN as a final product after a prolonged reaction (Fig. 7(a)), suggesting an exo-type cleavage manner to release a single GlcN residue from the substrate (GlcN)2 and (GlcN)3.
4. Discussion This paper described the purification and characterization of chitosanase from an Aspergillus mutant. It is
noteworthy that the level of chitosanlytic activity of Aspergillus CJ22-326 (3.61 u/ml of culture fluid) was fairly high as compared to Aspergillus oryzae IAM2660 (0.05 u/ml of culture fluid) and F. solani (0.0015 u/ml) (Zhang et al., 2000). So, Aspergillus CJ22-326 was selected as a strong producer of chitosanolytic enzymes, and two enzymes, ChiA (exo-type) and ChiB (endo-type), were purified to homogeneity from the culture fluid. The molecular masses of ChiA and ChiB were 108 and 29 kDa, respectively. Most microbial chitosanases have been characterized as endo-type chitosanases, and exo-type b-GlcNase were reported from a limited case. To our knowledge, exo-type chitosanase called b-GlcNase has been purified from only five microorganisms, that is the actinomycete Nocardia orientalis (Nanjo, Katsumi, & Sakai, 1990), the fungus Trichoderma reesei PC-3-7 (Nogawa et al., 1998), A. fumigatus KH-94 (Kim, Shon, & Lee, 1998), A. oryzae IAM2660 (Zhang et al., 2000) and Aspergillus CJ22-326 (this study). All the reported molecular weights of b-GlcNase were in the range of 97–135 kDa, while endo-type chitosanase is about 20–50 kDa. The molecular weight of ChiA and ChiB were in the range of reported values. It is possible that chitosanolytic enzymes are widely distributed in Aspergillus sp. (Cheng & Li, 2000), and are composed of two different types (endo- and exotypes). Some properties of Aspergillus chitosanases are summarized in Table 4. Almost all of the chitosanase from Aspergillus remarkably increased activity with Mn2+, were inhibited by Hg2+, and the optimal pH and temperature values for endo-chitosanase activity were in the ranges of pH 5.5–6.5 and 60–80 C, respectively, and for exo-chitosanase activity were in the ranges of pH 4.0–5.5 and 50–60 C. For most chitosanases, a sharp drop in activity was observed at pH values higher than 6.5, coinciding with precipitation of the substrate chitosan. The chitosanase of various species were found to be stable up to 50 C (Somashekar & Joseph, 1996), and there is no exception of ChiA and ChiB.
Table 4 Some properties of chitosanase from Aspergillus strains Strain
Aspergillus CJ22-326
A. fumigatus KH-94 A. oryaze IAM2660 Aspergillus sp.Y 2K a b
Endo-type chitosanase. Exo-type chitosanase.
Molecular weight (kDa) 29a 109b 22.5a 108b 40a 135b 25a
Optimum pH
Optimum temperature (C)
6.0
60–65
4.0 5.5 4.5–5.5 5.5 5.5 6.5
50–55 70–80 50–60 50 50 65–70
pI
Metal-ion activators
Metal-ion inhibitors
Major end products (DP)
Mn2+
3,4,5,6 1 2,3,4 1 1 3,4,5
7.3 4.8
Mn2+, Cu2+ Mn2+ Mn2++
Hg2+, Ag+, Fe3+, Cd2+, Cu2 Hg2+, Ag+, Fe3+ Hg2+, Cu2+ Hg2+, Cu2+
8.4
Mn2+, Cu2+
Hg2+, Cd2+
X. Chen et al. / Food Research International 38 (2005) 315–322
In many respects, the Aspergillus CJ22-326 chitosanase, described in this report, resemble that of A. fumigatus KH-94 (Kim et al., 1998). Both chitosanases are inducible by the substrate chitosan, and have endoand exo-types, and exhibit the same mechanism of chitosan hydrolysis, degraded highly-deacetylated chitosan with higher reaction velocity (Table 3), they do not attack chitin and CMC and they have similar molecular mass. The pI of ChiA and ChiB were not determined, but can be deduced as being near to neutral from its behavior in cation exchange chromatography, while the pI of chitosanase I (endo-type) and chitosanase II (exo-type) of A. fumigatus KH-94 was about 7.3 and 4.8, respectively, thus they are different from that of A. fumigatus KH-94. We tentatively identified the strain as a member of the genus Aspergillus (Raper & Fennel, 1965) but not same with A. fumigatus according to the shape of the spores (data not shown). To produce chitooligosaccharides, endo-type chitosanase is needed. ChiB is the best candidate for producing high DP chitooligosaccharides according to the major end products as shown in Table 4. The ChiB first hydrolyzed chitosan to high DP oligomers and these oligomers were then hydrolyzed into chitotriose, chitotetraose, chitopentaose and chitohexaose as the final products (Fig. 6). The action pattern of this enzyme is distinguished from most known chitosanases from actinomycetes, such as Streptomyces sp. N174, which is one of the most efficient producers of chitosanase described so far in the literature, they normally produce chitobiose and chitotrios as their major products (Boucher et al., 1992), because of the substrate of chitosan oligomers other than (GlcN)5 and (GlcN)6, (GlcN)4 were also digested by the chitosanase from Streptomyces sp. N174. It is disadvantageous that almost all of chitosanase from bacteria, such as Bacillus subtilis IMR-NK1 (Chiang et al., 2003), B. subtilis KH1 (Omumasaba et al., 2000) and actinomycetes can hydrolyze (GlcN)4 to (GlcN)2, but endo-type chitosanase from Aspergillus do not hydrolyze substrate (GlcN)4. ChiB of Aspergillus CJ22-326 hydrolyzed substrate (GlcN)5 to (GlcN)2 and (GlcN)3 very slowly compared with (GlcN)6. This leads to only a trace amount of chitobiose being produced in enzymatic degradation of chitosan. This trait is highly desirable for large-scale production of functional oligosaccharides, with trimers through hexamers as the major products. The functional properties of chitooligosaccharides have clearly revealed their high dependency on DP. It should be noted that enzymatic hydrolysis course and product distribution are subject to more facile control, in order to efficiently get oligomers of above DP 4, we can control the hydrolysis time. As shown in Fig. 6, 30 min is enough, or the bioreacter method by Jeon et al. (2001) would be applicable in future experiments.
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Acknowledgement This work was supported by the National Natural Science Foundation of China (NO.20271023).
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Tokoro, A., Kobayashi, M., Tatekawa, N., Suzuki, S., & Suzuki, M. (1989). Protective effect of N-acetyl chitohexaose on listeria monocytogens infection in mice. Microbiology and Immunology, 33, 357–367. Uchida, Y., Izume, M., & Ohtakara, A. (1989). Preparation of chitosan oligomers with purified chitosanase and its application. In G. Skjak Braek, T. Anthonsen, & P. Sandford (Eds.), Chitin and chitosan (pp. 373–382). London: Elsevier. Yamada, A., Shibuya, N., Kodama, O., & Akatsuka, T. (1993). Induction of phytoalexin formation in suspension-cultured rice cells by N-acetyl chitooligosacchrides. Bioscience, Biotechnology, and Biochemistry, 57, 405–409. Yoon, H. G., Kim, H. Y., Lim, Y. H., Kim, H. K., Shin, D. H., Hong, B. S., et al. (2000). Thermostable chitosanase from Bacillus sp. strain CK4: cloning and expression of the gene and characterization of the enzyme. Applied and Environmental Microbiology, 66, 3727–3734. Zhang, X. Y., Dai, A. L., Zhang, X. K., Kuroiwa, K., Kodaira, R., Shimosaka, M., et al. (2000). Purification and characterization of chitosanase and exo-b-D -glucosaminidase from a koji mold. Aspergillus oryzae IAM2660. Bioscience Biotechnology and Biochemistry, 64, 1896–1902.
Purification and characterization of two types of chitosanase from Aspergillus sp. CJ22-326 XiaoÕe Chen a
c
a,b
, Wenshui Xia
a,*
, Xiaobin Yu
c
School of Food Science, Southern Yangtze University, Wuxi 214036, PR China b Zhejiang Ocean University, Zhoushan 316004, PR China School of Biotechnology, Southern Yangtze University, Wuxi 214036, PR China Received 14 January 2004; accepted 6 April 2004
Abstract Aspergillus CJ22-326, a fungi strain capable of utilizing chitosan as a carbon source, was isolated from soil samples. Two types of chitosanase (ChiA and ChiB) produced from the culture supernatant of Aspergillus CJ22-326 were purified to an apparent homogeneity identified by SDS–PAGE through ammonium sulfate precipitation, CM-Sepharose FF chromatography, and Sephacryl S-200 gel filtration. Molecular weights of the enzymes were 109 kDa (ChiA) and 29 kDa (ChiB). Optimum pH values and temperature of ChiA were 4.0 and 50 C, respectively, those of ChiB were 6.0 and 65 C. The enzyme activities of ChiA and ChiB were increased by about 0.5-fold and 1.5-fold, respectively, by the addition of 1 mM Mn2+. However, 2.5 mM Ag+, Hg2+ and Fe3+ strongly inhibited ChiA and ChiB activities. Viscosimetric assay and analysis of reaction products of these enzymes, using chitosan as a substrate, by TLC indicated endo- and exo-type cleavage of chitosan by ChiB and ChiA, respectively. ChiB catalysed the hydrolysis of glucosamine (GlcN) oligomers larger than pentamer, and chitosan with a low degree of acetylation (0–30%), and formed chitotriose with chitohexaose as the major products. ChiA released a single glucosamine residue from chitosan and glucosamine oligomers. Both of the activities of ChiA and ChiB increased with the degree of deacetylation of chitosan. The enzyme ChiB had a useful reactivity and a high specific activity for producing functional chitooligosaccharides with high degree of polymerization. 2004 Elsevier Ltd. All rights reserved. Keywords: Chitosanase; Chitooligosaccharides; Aspergillus; Chitosan; Endo-type; Exo-type
1. Introduction Chitosan that have different degrees of deacetylation (DDA) can be readily obtained by N-deacetylating chitin that is extracted from an abundant natural source, shrimp and crab shells. It has high safety and peculiar physical properties because of its high cation content. Its biological properties, like compatibility and antimicrobial activity, are remarked. Actually, chitosan is applied widely to health foods such as for the prevention and treatment of hyperuricemia, and as an antimicrobial *
Corresponding author. Tel.: +86510 5869455. E-mail address: [email protected] (W. Xia).
0963-9969/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2004.04.012
agent, preservative agent and edible film. But there is doubt about their level of absorption in the human intestine, and their high molecular weights and highly viscous nature may restrict their uses in in vivo systems. Recently much attention has been paid to converting chitosan to safe and functional chitooligosacchrides, because chitooligosaccharides with high degrees of polymerization (DP), especially those with six residues or more, show strong physiological activities, such as antitumor activities (Suzuki et al., 1986; Suzuki, Matsumoto, Tsukada, Aizawa, & Suzuki, 1989), immuno-enchancing effects (Suzuki, 1996), enhancing protective effects against infection with pathogens in mice (Tokoro, Kobayashi, Tatekawa, Suzuki, & Suzuki,
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1989; Yamada, Shibuya, Kodama, & Akatsuka, 1993), antifungal activity (Hirano & Nagao, 1989; Kendra, Christian & Hadwiger, 1989) and antimicrobial activity (Jeon, Park, & Kim, 2001; Savard, Beaulieu, Boucher, & Champagne, 2002; Uchida, Izume, & Ohtakara, 1989). Chitooligosaccharide functions have led to progressively increased utilization in the food and pharmaceutical fields for human health. Chitooligosaccharides can be made by hydrolyzing chitosan with chitosanase. Chitosanase (EC 3.2.1.132) catalyses the hydrolysis of the glycosidic bonds of chitosan, and has been found in a variety of microorganisms, including bacteria (Akiyama et al., 1999; Chiang, Chang, & Sung, 2003; Omumasaba, Yoshida, Sekiguchi, Kariya, & Ogawa, 2000; Yoon et al., 2000), actinomycetes (Boucher, Dupuy, Vidal, Neugebauer, & Brzezinski, 1992; Sakai, Katsumi, Isobe, & Nanjo, 1991), and fungi (Shimosaka, Nagawa, Ohno, & Okazaki, 1991; Zhang et al., 2000). Chitosanases from individual organisms differ in their hydrolytic action pattern. However, most chitosanases from the isolated microorganisms intend to make dimers, trimers and tetramers rather than oligomers above DP 4, so the utility of these enzymes is not good. To obtain a novel chitosanase which could be used for large-scale production of chitosan oligomers above DP 4, we screened various types of microorganisms. An Aspergillus CJ22-326 produced high chitosanase activities when grown on medium with wheat bran and chitosan as carbon source. In this paper, we describe purification and characterization of two different chitosanolytic enzymes, and one of the endo-type chitosanase ChiB is potentially valuable for industrial applications for producing functional chitooligosaccharides.
2. Materials and methods 2.1. Strain and culture conditions The strain used in this study is one of the fungal strains isolated from marine soil in China. The culture medium composed of 1.0% soluble chitosan, 2.0% wheat bran, 0.2% (NH4)2SO4, 0.2% KH2PO4, and 0.05% MgSO4 per liter (pH 5.6). The cultivation was carried out in a 500 ml baffle flask with 150 ml of the culture medium at 30 C for 96 h with agitation at 150 rpm. 2.2. Chemicals and substrates CM-Sepharose FF, Sephacryl S-200 and Phenyl CL4B were from Pharmacia. Chitosan and chitin were purchased from the local suppliers in China. Glucosamine (GlcN), chitobiose (GlcN)2, chitotriose (GlcN)3, chitotetraose (GlcN)4, chitopentose (GlcN)5 chitohexaose (GlcN)6 were purchased from Seikagaku Co. All other reagents were of analytical grade.
2.3. Enzyme purification Ammonium sulfate precipitation – After cultivation, the cells were removed from the medium by centrifugation at 4000 rpm for 30 min. Solid ammonium sulfate was added to the culture filtrate to 80% saturation. After standing overnight, the precipitate was collected by centrifugation and dissolved in 50 ml of 20 mM sodium acetate buffer, pH 5.6 (buffer A). The enzyme solution was dialyzed against the same buffer. Chromatography on CM-Sepharose FF – The dialyzed solution was put on a CM-Sepharose FF column (2.6 · 50 cm) that had been equilibrated with 20 mM sodium acetate buffer. The column was washed with the same 500 ml buffer, and then eluted with a 600 ml linear gradient of 0–1.0 M NaCl in buffer A, at a flow rate of 30 ml/h, and 5 ml fractions were collected. The two active fractions were pooled and concentrated separately by ultrafiltration. Gel filtration on Sephacryl S-200 – Fractions from each peak were pooled separately and put on a Sephacryl S-200 column (2.0 · 100 cm) equilibrated with buffer A containing 0.1 M NaCl (buffer B). The proteins were eluted with buffer B at a flow rate of 12 ml/h, and the resulting active fractions were collected and used as the purified enzyme preparations throughout this study. All purification steps were performed at 4 C. 2.4. Enzyme assay Unless indicated otherwise, chitosan with a DDA of 83% was used as the substrate in the chitoanase assay. The incubation mixture contained 1 ml of 0.5% soluble chitosan and 1 ml of diluted enzyme solution (pH 5.6). The incubation was carried out at 37 C for 15 min, with shaking. The amount of reducing sugar in the supernatant was measured using the modified dinitrosalicyclic acid (DNS) method (Miller, 1959). One enzyme unit was defined as the amount of enzyme required to produce 1 lmol of reducing sugar as glucosamine per min. 2.5. Protein assay and electrophoresis Protein was determined by the method of Lowry, Rosebrough, Farr, and Randall (1951), using bovine serum albumin as the standard. During purification, protein was estimated by measuring absorbance at 280 nm, with bovine serum albumin as the standard. Polyacrylamide gel electrophoresis (SDS–PAGE) was used to determine protein purity and the molecular mass of the purified enzyme under denaturing conditions using a 12% acrylamide gel, as described by Laemmli (1970). Protein was stained by Coomassie blue.
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2.6. Enzyme characterization The effect of pH on the enzyme activity was determined by changing the pH of the reaction mixtures using 200 mM sodium acetate (pH 3.6–5.8) and 200 mM sodium phosphate (pH 5.8–8.0). The effect of temperature on the enzyme activity was determined at pH 5.6, in the range 30–80 C. The effects of metallic ions and some enzyme inhibitors on ChiA activity were determined after preincubation at 37 C for 30 min. 2.7. Viscosimetric assay and analysis of hydrolysis products Viscosimetric assay was performed by the method of Ohtakara (1988). The end products of the enzymatic hydrolysis of chitosan were analyzed by thin-layer chromatography (TLC) on Silica G 60 plates using n-propanol/30% ammonia (3:1, v/v) as the developing solvent (Sakai et al., 1991). Sugars were visualized by ninhydrin spray.
Fig. 2. SDS–PAGE of the purified ChiA and ChiB. The purified enzyme ChiA (lane 1), standard molecular mass markers (lane 2), the purified enzyme ChiB (lane 3), protein was stained with Coomassie brilliant blue R-250.
Sephacryl S-200 columns in the positions predicted for proteins with their molecular weights, indicating that they were monomeric. The purification procedure yielded a 4.01-fold purified ChiA with 7.4% recovery and 11.34-fold purified ChiB with 37.0% recovery. The final specific activity of ChiA and ChiB were 6.46 and 18.26 u/mg using 83% DDA, respectively. It should be noted that the chitosanase assay reflects total enzymatic activity; individual chitosanase activities A and B cannot be distinguished in the crude extract. These purification steps from culture broth are summarized in Table 1.
3. Results 3.1. Purification of chitosanases Crude enzyme solution prepared by salting out with ammonium sulfate was applied to cation exchange chromatography on CM-Sepharose FF. As shown in Fig. 1 two chitosanase peaks of A (minor fraction) and B (major fraction) were separated. The chitosanolytic proteins that eluted in peaks A and peaks B were designated ChiA and ChiB, respectively. Both of these chitosanases were purified by successive Sephacryl S200 gel chromatography to homogeneity as judged by SDS–PAGE (Fig. 2). The molecular masses of ChiA and ChiB were estimated to be 109 and 29 kDa, respectively. Both of these chitosanases eluted from
3.2. Effects of pH and temperature on chitosanases The effects of pH and temperature on ChiA and ChiB were investigated. Fig. 3 shows the effects of pH on ChiA and ChiB activity and enzyme stability.
3.0
B
35
25 1.5
20
A
15
1.0 10
0.75
0.50
NacCL(mol/l)
30 2.0
Chitosanase activity(u/ml)
2.5
OD(280nm)
1.00
40
OD(280nm) Chitosanase activity
0.25
0.5 5 0.0
0 0
100
200
300
400
0.00
500
Elution volume(ml) Fig. 1. Elution profile of chitosanase on CM-Sepharose FF column chromatography.
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Table 1 Purification of chitosanase ChiA and ChiB from Aspergillus CJ22-326 Step
Total activity (u)
Total protein (mg)
Specific activity (u/mg)
Crude extract 0–80% (NH4)2SO4
1134 804
703 169
1.61 4.75
100 70.8
1 2.95
ChiA CM-Sepharose FF Sephacryl S-200
116 84
21 13
5.52 6.46
10.2 7.4
3.42 4.01
ChiB CM-Sepharose FF Sephacryl S-200
580 420
42 23
13.8 18.26
51.1 37.0
8.57 11.34
Relative activity /%
Relative activity /%
The effects of metal ions on chitosanases are presented in Table 2. Hg2+, Ag+ and Fe3+ (2.5 mM) inhibited the activities of ChiA and ChiB about 90%. 1 mM Mn2+ remarkably increased the activities of ChiA and ChiB to 150% and 250%, respectively. This result was similar to those for other chitosanlytic enzymes. ChiA was not significantly affected by other metal ions. However, the activities of ChiB were also inhibited by Cd2+, Cu2+, Pb2+ and Zn2+.
Chi A
3
4
5
6
7
8
9
Purification folds
3.3. Effects of metal ions on chitosanases
Optimum pHs were about 4.0 and 6.0, respectively. ChiA was stable in the pH range of 3.5–7.5 and ChiB was stable in the pH range of 4.5–8.0. Fig. 4 shows the optimum temperatures of ChiA and ChiB were 50 and 65 C, respectively. ChiB was stable at a temperature lower than 75 C, but ChiA was stable at temperature lower than 60 C, and dramatically inactivated by incubation at 70 C in pH 5.6 for 60 min. Both enzymes did not lose their activities during storage in sodium acetate buffer (pH 5.6) at 4 C for 6 months.
100 90 80 70 60 50 40 30 20 10 0
Yield (%)
Chi B
100 90 80 70 60 50 40 30 20 10 0 3
10
4
5
6
7
8
9
10
pH
pH
-
- Enzyme activity;
- -.Relative stability
Chi A
100 90 80 70 60 50 40 30 20 10 0
Relative activity/%
Relative activity/%
Fig. 3. Effect of pH on the activity and stability of enzyme ChiA and ChiB.
30
40
50
60
-
70
80
Chi B
100 90 80 70 60 50 40 30 20 10 0 30
40
50
60
-Enzyme activity; - -.Relative stability
Fig. 4. Effect of temperature on the activity and stability of enzymes ChiA and ChiB.
70
80
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319
Table 2 Effect of metal ions on the activity of ChiA and ChiB Ion ChiA Relative activity ChiB Relative activity
Control
Cu2+
Hg2+
Cd2+
Mn2+
Sn2+
100
108
15
93
150
84
100
20
3
22
250
97
3.4. Substrate specificity The activities of the two chitosanases with various substrates are summarized in Table 3. ChiA degraded chitosan with a DDA of 83% to 95% effectively, and exhibited low activity with chitosan having DDA of 65%. On the other hand, the most susceptible chitosan for ChiB had DDA of 72–96%. Neither ChiA nor ChiB hydrolyzed colloidal chitin, or CMC at all. 3.5. Viscosimetric assay To characterize the mode of action of both enzymes, a decreasing rate in viscosity of 95% deacetylated chitosan was investigated during enzymatic hydrolysis. ChiB reduced the viscosity of chitosan solution at an early stage of reaction, while the ChiA reduced the viscosity to a much lower extent when the same amount of enzyme was used, as judged by the values obtained in Table 3 Substrate specificity of chitosanases ChiA and ChiB Substrate (0.5%)
Relative activity (%) ChiA
ChiB
Chitosan (95% DDA) Chitosan (83% DDA) Chitosan (72% DDA) Chitosan (65% DDA) Colloidal chitin CMC
100.0 91.2 83.5 72.7 0 0
100.0 80.6 65.2 44.1 0 0
Ba2+
Pb2+
Zn2+
Ca2+
Co2+
Mg2+
Ag+
Fe3+
97
90
85
101
86
89
13
8
102
60
67
98
80
91
15
12
the standard reducing sugar assay (Fig. 5). This result indicates that the ChiB would probably hydrolyze chitosan in an endo-type fashion in contrast with an exo-type cleavage pattern for ChiA. 3.6. Analysis of the reaction products The hydrolysates of chitosan (DDA 95%) by the purified ChiA and ChiB were analyzed by thin-layer chromatography (TLC) (Fig. 6). A change in the hydrolysis products was observed during incubation with the ChiB at 50 C for 2 h. At appropriate intervals during an early stage, chitosan was hydrolyzed to chitosan oligomers larger than tetramer and a little trimer (Fig. 6, lanes 1–3). After a prolonged reaction (2 h), the amount of (GlcN)5 and (GlcN)6 in the hydrolysate decreased, while (GlcN)3 and (GlcN)4 levels increased (lane 4). The shorter oligomer of GlcN increased as digestion time increased. It is suggested that the mode of action of the enzyme is the endo-type. On the other hand, ChiA gave only GlcN as a final product after a
Specific viscosity
1 0.8 0.6 0.4 0.2 0 0
2 4 6 Reaction time(min) -
- 0.1u of Chi B; -
8
10
- 0.1u of Chi A
Fig. 5. Reduction in viscosity of chitosan colution with ChiA and ChiB. The reaction mixture (7 ml) contained 0.05% chitosan as the substrate. The flow time of the mixture was measured at appropriate times. Specific viscosity = (the flow time of the reaction mixture/flow time of distilled water).
Fig. 6. Analysis of enzymatic hydrolysates by TLC. The substrate used was 95% deacetylated chitosan (lanes 1–5). The enzymes used were purified ChiB (lanes 1–4) and ChiA (lane 5). At different time intervals, 1 ml of reaction mixture were taken out and heated for 10 min in a boiling water bath. Lanes S, standard (GlcN) to (GlcN)6; lanes 1–4, ChiB hydrolysates after 10 min, 20 min, 30 min and 2 h, respectively; lane 5, ChiA hydrolysates after 1 h.
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Fig. 7. TLC of chitooligosaccharides after treatment of ChiA (a) and ChiB (b). Lanes: 1 and 4, unhydrolyzed substrate (GlcN)2 and (GlcN)3, respectively; 2 and 3, digestion of (GlcN)2 and (GlcN)3with ChiA for 24 h at 37, respectively; 5–8, digestion of (GlcN)3 to (GlcN)6 with ChiB for 24 h at 37, respectively; S, standard (GlcN) to (GlcN)6.
prolonged reaction, suggesting an exo-type cleavage manner to release a single GlcN residue from chitosan. ChiB could not hydrolyze either (GlcN)2, (GlcN)3, or (GlcN)4 to any extent, (GlcN)5 was hydrolyzed to produce a mixture of (GlcN)2 and (GlcN)3 slowly, (GlcN)6 was hydrolyzed to (GlcN)3 (Fig. 7(b)). From the results of a viscosimetric assay and TLC analysis of hydrolysates, ChiB catalyzes an endo-type cleavage of chitosan, and should be designated as chitosanase. On the other hand, ChiA gave only GlcN as a final product after a prolonged reaction (Fig. 7(a)), suggesting an exo-type cleavage manner to release a single GlcN residue from the substrate (GlcN)2 and (GlcN)3.
4. Discussion This paper described the purification and characterization of chitosanase from an Aspergillus mutant. It is
noteworthy that the level of chitosanlytic activity of Aspergillus CJ22-326 (3.61 u/ml of culture fluid) was fairly high as compared to Aspergillus oryzae IAM2660 (0.05 u/ml of culture fluid) and F. solani (0.0015 u/ml) (Zhang et al., 2000). So, Aspergillus CJ22-326 was selected as a strong producer of chitosanolytic enzymes, and two enzymes, ChiA (exo-type) and ChiB (endo-type), were purified to homogeneity from the culture fluid. The molecular masses of ChiA and ChiB were 108 and 29 kDa, respectively. Most microbial chitosanases have been characterized as endo-type chitosanases, and exo-type b-GlcNase were reported from a limited case. To our knowledge, exo-type chitosanase called b-GlcNase has been purified from only five microorganisms, that is the actinomycete Nocardia orientalis (Nanjo, Katsumi, & Sakai, 1990), the fungus Trichoderma reesei PC-3-7 (Nogawa et al., 1998), A. fumigatus KH-94 (Kim, Shon, & Lee, 1998), A. oryzae IAM2660 (Zhang et al., 2000) and Aspergillus CJ22-326 (this study). All the reported molecular weights of b-GlcNase were in the range of 97–135 kDa, while endo-type chitosanase is about 20–50 kDa. The molecular weight of ChiA and ChiB were in the range of reported values. It is possible that chitosanolytic enzymes are widely distributed in Aspergillus sp. (Cheng & Li, 2000), and are composed of two different types (endo- and exotypes). Some properties of Aspergillus chitosanases are summarized in Table 4. Almost all of the chitosanase from Aspergillus remarkably increased activity with Mn2+, were inhibited by Hg2+, and the optimal pH and temperature values for endo-chitosanase activity were in the ranges of pH 5.5–6.5 and 60–80 C, respectively, and for exo-chitosanase activity were in the ranges of pH 4.0–5.5 and 50–60 C. For most chitosanases, a sharp drop in activity was observed at pH values higher than 6.5, coinciding with precipitation of the substrate chitosan. The chitosanase of various species were found to be stable up to 50 C (Somashekar & Joseph, 1996), and there is no exception of ChiA and ChiB.
Table 4 Some properties of chitosanase from Aspergillus strains Strain
Aspergillus CJ22-326
A. fumigatus KH-94 A. oryaze IAM2660 Aspergillus sp.Y 2K a b
Endo-type chitosanase. Exo-type chitosanase.
Molecular weight (kDa) 29a 109b 22.5a 108b 40a 135b 25a
Optimum pH
Optimum temperature (C)
6.0
60–65
4.0 5.5 4.5–5.5 5.5 5.5 6.5
50–55 70–80 50–60 50 50 65–70
pI
Metal-ion activators
Metal-ion inhibitors
Major end products (DP)
Mn2+
3,4,5,6 1 2,3,4 1 1 3,4,5
7.3 4.8
Mn2+, Cu2+ Mn2+ Mn2++
Hg2+, Ag+, Fe3+, Cd2+, Cu2 Hg2+, Ag+, Fe3+ Hg2+, Cu2+ Hg2+, Cu2+
8.4
Mn2+, Cu2+
Hg2+, Cd2+
X. Chen et al. / Food Research International 38 (2005) 315–322
In many respects, the Aspergillus CJ22-326 chitosanase, described in this report, resemble that of A. fumigatus KH-94 (Kim et al., 1998). Both chitosanases are inducible by the substrate chitosan, and have endoand exo-types, and exhibit the same mechanism of chitosan hydrolysis, degraded highly-deacetylated chitosan with higher reaction velocity (Table 3), they do not attack chitin and CMC and they have similar molecular mass. The pI of ChiA and ChiB were not determined, but can be deduced as being near to neutral from its behavior in cation exchange chromatography, while the pI of chitosanase I (endo-type) and chitosanase II (exo-type) of A. fumigatus KH-94 was about 7.3 and 4.8, respectively, thus they are different from that of A. fumigatus KH-94. We tentatively identified the strain as a member of the genus Aspergillus (Raper & Fennel, 1965) but not same with A. fumigatus according to the shape of the spores (data not shown). To produce chitooligosaccharides, endo-type chitosanase is needed. ChiB is the best candidate for producing high DP chitooligosaccharides according to the major end products as shown in Table 4. The ChiB first hydrolyzed chitosan to high DP oligomers and these oligomers were then hydrolyzed into chitotriose, chitotetraose, chitopentaose and chitohexaose as the final products (Fig. 6). The action pattern of this enzyme is distinguished from most known chitosanases from actinomycetes, such as Streptomyces sp. N174, which is one of the most efficient producers of chitosanase described so far in the literature, they normally produce chitobiose and chitotrios as their major products (Boucher et al., 1992), because of the substrate of chitosan oligomers other than (GlcN)5 and (GlcN)6, (GlcN)4 were also digested by the chitosanase from Streptomyces sp. N174. It is disadvantageous that almost all of chitosanase from bacteria, such as Bacillus subtilis IMR-NK1 (Chiang et al., 2003), B. subtilis KH1 (Omumasaba et al., 2000) and actinomycetes can hydrolyze (GlcN)4 to (GlcN)2, but endo-type chitosanase from Aspergillus do not hydrolyze substrate (GlcN)4. ChiB of Aspergillus CJ22-326 hydrolyzed substrate (GlcN)5 to (GlcN)2 and (GlcN)3 very slowly compared with (GlcN)6. This leads to only a trace amount of chitobiose being produced in enzymatic degradation of chitosan. This trait is highly desirable for large-scale production of functional oligosaccharides, with trimers through hexamers as the major products. The functional properties of chitooligosaccharides have clearly revealed their high dependency on DP. It should be noted that enzymatic hydrolysis course and product distribution are subject to more facile control, in order to efficiently get oligomers of above DP 4, we can control the hydrolysis time. As shown in Fig. 6, 30 min is enough, or the bioreacter method by Jeon et al. (2001) would be applicable in future experiments.
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Acknowledgement This work was supported by the National Natural Science Foundation of China (NO.20271023).
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