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3 Huihui Sun1, Xiangzhao Mao2, Na Guo2, Ling Zhao1, Rong Cao*1, Qi Liu1
1
4 Department of Food Engineering and Nutrition, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery
2
6 College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China
10
12 Address: Department of Food Engineering and Nutrition, Yellow Sea Fisheries Research Institute, Chinese Academy of
14 Tel.: +86-532-85830760
15 Email: caorong@ysfri.ac.cn
16
17 ABSTRACT: In the process of genome mining for novel chitosanases by phylogeny-based enzymatic product specificity
18 prediction, a gene named Csn-PD from Paenibacillus dendritiformis was discovered. The enzyme was classified as a
19 member of the GH46 family of glycoside hydrolase based on sequence alignment, and it was functionally expressed in
20 Escherichia coli BL21 (DE3). The recombinant chitosanase was purified, and its molecular weight was estimated to be
21 31 kDa by SDS-PAGE. Csn-PD displayed maximal activity toward colloidal chitosan at pH 7.0 and 45 °C, respectively.
22 A combination of thin-layer chromatography and electrospray ionization-mass spectrometry results showed that Csn-PD
23 exhibited an endo-type cleavage pattern and hydrolyzed chitosan to yield (GlcN)2 as the major product. The unique
24 enzymatic properties of this chitosanase may make it a good candidate for (GlcN)2 production.
26
27 ■ INTRODUCTION
28 Chitosan, a linear polysaccharide composed of β-1,4 linked D-glucosamine (GlcN), is a totally or partially
29 deacetylated form of chitin, which is the second most abundant polysaccharide on earth1,2. Chitin can be commercially
30 obtained from the exoskeletons (shells) of crustacea, such as shrimp and crab, which are continuously generated in nature
31 and thus an almost unlimited renewable resource3. Chitosans have been reported to support a broad range of potential
32 applications in agriculture, biotechnology, medicine and environmental treatment4-8. However, the commercial use of
33 chitosan is restricted because of its poor solubility and high viscosity in neutral or basic aqueous media9-11. Therefore,
34 there is considerable interest in converting chitosan to chitooligosaccharides (COSs), which are water-soluble; these even
35 possess additional biological properties such as antimicrobial, antitumor, and immuno-enhancing activities12-15.
36 Chemical and physical methods can be used to produce COSs16,17. However, there are many problems with both
37 processes, such as requirements for high temperature and pressure, production of secondary compounds, high separation
38 cost, and low yields of COSs. In contrast, chitosan may be biologically converted to COSs under ambient and
39 environmentally friendly conditions. Chitosanases (EC 3.2.1.132) are enzymes that can hydrolyze chitosan into COSs
40 and glucosamine and are found in many kinds of organisms, including bacteria, fungi, and plants18.
41 Chitosanases can be grouped into six glycosyl hydrolase (GH) families, i.e. GH5, GH7, GH8, GH46, GH75 and GH80,
42 based on their amino acid sequences rather than their substrate specificities19. Families GH46, GH75, and GH80 are
43 believed to be more substrate specific (limited to chitosan), while chitosanases from families GH5 and GH8 also tend to
44 possess other glycoside hydrolase activities such as cellulose and licheninase20. Most chitosanases have been shown to be
45 the endo-type enzyme, which randomly cleave glycosidic bonds of chitosan to produce a mixture of oligosaccharides
46 with various degrees of polymerization (DP)18,21,22. There is to date no successful method to obtain COSs with a single
47 DP directly with one endo-chitosanase. Costly and time-consuming separation of COSs with different DPs, such as size
48 exclusion chromatography and ultrafiltration, is commonly employed, but is only practical on a small scale23,24. However,
49 the biological functionalities of COSs have been shown to be strongly dependent on their DPs25-27. Thus, the production
50 of defined oligomers has generated significant interest both for fundamental structure-function relationship research and
52 As far as we know, there have been several works that described the existence of exo-β-D-glucosaminidases
53 (belonging to GH2 and not classified into the chitosanase families listed above), that can hydrolyze chitosan to generate
54 glucosamine (GlcN)28-32. In another study, the endo-type chitosanase from Bacillus circulans MH-K1 was converted into
55 an exo-type chitosanase by inserting two surface loops; this produced (GlcN)2 as the dominant product, but, unfortunately,
56 the catalytic rate of the mutant was only 3% that of the wild-type chitosanase22.
57 In the present study, phylogeny-based enzymatic product specificity prediction (PEPSP) was introduced for the
58 efficient discovery of chitosanases to produce COSs with different DPs. A novel chitosanase, Csn-PD, belonging to
59 family GH46, was discovered from Paenibacillus dendritiformis. It was cloned and overexpressed in Escherichia coli
60 BL21 (DE3). The substrate specificity of Csn-PD and its stability as a function of temperature and pH were investigated.
62 Materials. All the three chitosanase genes extracted from the GenBank database, including Csn-PD from
63 Paenibacillus dendritiformis (WP_006679998.1), Csn-CAP from Staphylococcus capitis (OAN23142), and Csn-But from
64 Butyrivibrio sp. MC2013 (WP_026508362), were synthesized by Talen-bio Scientific (Shanghai) Co., Ltd. and inserted
65 into plasmid pET28a(+). The resulting plasmids were respectively transformed into E. coli BL21 (DE3) for expression of
66 the chitosanase. Chitosans with a degree of deacetylation (DDA) of 85% and 95% were purchased from Sigma Chemical
67 Co. (St. Louis, MO, USA). COSs with DP 2–6 were purchased from Qingdao BZ Oligo Biotech Co., Ltd. (Qingdao,
68 China). Other chemicals and reagents used were of analytical grade and purchased from local markets.
69 Database mining and sequence analysis. Searching for novel chitosanase gene sequences was performed using the
70 BLASTP program
71 (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins&PROGRAM=blastp&BLAST_PROGRAMSblastp&PAGE_TY
73 (BAA01474) was chosen as the identifier to detect potential chitosanases to produce COSs with different DPs33. This
74 chitosanase has been studied extensively, and its product ((GlcN)1–(GlcN)5) was completely different from those of other
75 reported chitosanases22. Chitosanase sequences showing 20–80% amino acid identity were extracted from the GenBank
76 database. Chitosanases with experimentally defined product specificity belonging to family GH46 were chosen as a
77 priority to refine the phylogenetic analysis. Sequence alignments were conducted using Clustal W34. Phylogenetic
78 relationships among chitosanolytic enzymes were analyzed through a neighbor-joining method packaged in MEGA 6.035.
79 Expression of the three chitosanases genes in E. coli. Recombinant E. coli BL21 (DE3) cells for chitosanases
80 expression, including Csn-PD, Csn-CAP, and Csn-But, were cultivated in Luria-Bertani medium containing kanamycin
81 (50 µg/mL) at 37 °C in a shaker (200 rpm). When the OD600 of the culture reached 0.6, isopropyl-β-D-thio-
82 galactopyranoside was added to a final concentration of 1 mM. The induced cultures were further incubated for 16 h at
83 30 °C. Then, cells were harvested by centrifugation (10,000 × g, 10 min), washed twice with 0.85% NaCl solution, and
84 stored at −20°C.
85 Purification of Csn-PD. The obtained cell pellets were resuspended in sodium phosphate buffer (50 mM, pH 7.0).
86 Cell disruption was performed by sonication, and the debris was removed by centrifugation (10,000 × g, 30 min). The
87 resulting crude extract was loaded onto a Ni-NTA column (1 mL; Qiagen, Hilden, Germany) at a flow rate of 1 mL/min.
88 The column was equilibrated with buffer A (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0). Then the
89 column was washed with 20 mM imidazole in buffer A. Fractions containing the target protein were eluted with 200 mM
90 imidazole in buffer A. The eluted protein was desalted and concentrated by ultrafiltration. The crude extract and the
91 purified protein were analyzed by SDS-PAGE. Protein concentration was determined using the Bradford method. All
93 Enzyme assay. Standard assays were performed in a reaction mixture (1 mL) containing sodium phosphate buffer (50
94 mM, pH 7.0), colloidal chitosan (1%, w/v, DDA of 85%), and an appropriate amount of chitosanase. The mixture was
95 incubated at 30 °C for 10 min and then the reaction was quenched in boiling water for 10 min. The reducing sugars
96 released were determined by the DNS method with minor modification36. All the experiments were performed in
97 triplicate. One unit (U) of the enzyme activity was defined as the amount of enzyme that produced 1 µmol of reducing
98 sugar per min under the above assay conditions using GlcN as the standard.
99 Characterization of purified Csn-PD. The optimum temperature for Csn-PD activity was determined by incubating
100 the enzyme with colloidal chitosan (1%, w/v, DDA of 85%) at 20–60 °C in 50 mM phosphate buffer (pH 7.0). The
101 thermostability of Csn-PD was determined by measuring the residual activity after the enzyme was incubated at different
102 temperatures (20–60 °C) for 30 min. The optimum pH for Csn-PD activity was determined at pH 4.0 to 8.0 (citrate buffer
103 pH 4.0 to 6.0, phosphate buffer pH 6.0 to 8.0). The residual chitosanase activity was also measured after the enzyme was
105 Substrate specificity of Csn-PD was determined at 30 °C for 30 min in 50 mM phosphate buffer (pH 7.0). The tested
106 polysaccharide substrates (1%, w/v) included colloidal chitosan, chitosan with DDAs of 85% and 95% respectively,
108 Hydrolytic properties of the purified Csn-PD. The hydrolytic properties of Csn-PD were investigated using
109 colloidal chitosan and COSs (DP 2–6) by analyzing the hydrolysis products by thin-layer chromatography (TLC) and
110 electrospray ionization-mass spectrometry (ESI-MS). The reaction mixture was centrifuged at 10,000 × g for 10 min,
111 then the supernatant was spotted onto a silica gel plate (Merck, Darmstadt, Germany), which was developed in a solvent
112 system containing propanol–25% ammonia–water (8:3:1, v/v/v). The oligosaccharide spots were visualized by spraying
113 0.1% ninhydrin reagent (dissolved in ethanol), followed by heating at 105 °C for 10 min. ESI-MS studies were performed
114 in positive-ion mode and conducted by Shanghai Micronmr Infor Technology Co., Ltd. Samples were analyzed by
115 WATERS UPLC-MS SQD2 scanning with a ratio of mass to charge in the range of 50–2000 (m/z). In all ESI-MS
116 experiments, the scan mode was used under a capillary needle at 3 kV and the ion source temperature at 150 °C.
118 Discovery and identification of chitosanase Csn-PD by PEPSP. Based on the GenBank database mining and amino
119 acid sequence analysis, a total of 16 proteins, annotated as chitosanases, were chosen for analysis of their products
120 toward chitosan. Among them, 13 chitosanases, belonging to GH46, have been experimentally determined to produce
121 COSs with different DPs (Figure 1). Multiple sequence alignment (Figure 1A) showed that the catalytic domain of the
122 three novel chitosanases contained two key active site residues (Glu and Asp), which are conserved in all members of
123 GH46. These results indicated that chitosanases Csn-But, Csn-CAP, and Csn-PD were novel members of chitosanolytic
125 Based on the phylogenetic analysis (Figure 1B), the 13 chitosanases that have been studied previously clustered into
126 five groups. For example, the main products of the first six chitosanases are (GlcN)2 and (GlcN)337-42. The next four
127 chitosanases produce mixtures of (GlcN)2–643-46. The products of the chitosanases from Burkholderia gladioli, Bacillus
128 circulans MH-K1, and Bacillus coagulans include (GlcN)2–(GlcN)4, (GlcN)1–(GlcN)5, and (GlcN)3–(GlcN)5,
129 respectively22,47,48. The three novel chitosanases are distributed in different groups. Chitosanases Csn-But and Csn-CAP
130 clustered into the first group which can produce (GlcN)2 and (GlcN)3. However, chitosanase Csn-PD did not belong to
132 To verify the accuracy of this prediction method and to confirm the product of the three novel chitosanases, the three
133 chitosanase genes Csn-PD, Csn-CAP, and Csn-But, were respectively expressed in E. coli. All three enzymes were active
134 toward colloidal chitosan. The results of TLC showed that the final products of chitosanases Csn-But and Csn-CAP were
135 (GlcN)2 and (GlcN)3 (Figure S1), which was in accordance with the prediction. Csn-PD produced only (GlcN)2. These
136 results not only confirmed the accuracy of the PEPSP method, but also identified a novel chitosanase Csn-PD that could
137 produce COS with a single DP. Csn-PD was thus selected for further study.
138 Purification of chitosanase Csn-PD. The chitosanase Csn-PD from P. dendritiformis was successfully expressed in E.
139 coli as an active enzyme. Using the 6×His affinity tag, Csn-PD was purified to electrophoretic homogeneity by nickel
140 affinity chromatography. The purified Csn-PD was separated as a single protein band of approximately 31 kDa by SDS-
141 PAGE (Figure 2). The molecular mass is lower than those of the chitosanases from Janthinobacterium sp. 4239 (33
142 kDa)41 and Bacillus subtilis (36 kDa)46, but higher than those of most other reported bacterial chitosanases, e.g., from B.
143 circulans MH-K1 (29 kDa)33 and Amycolatopsis sp. CsO-2 (27 kDa)37. The specific activity of purified Csn-PD was 76.4
144 U/mg toward chitosan with DDA of 85%, which was much higher than the activity of CSN from Penicillium sp. D-118.
145 Characterization of the purified Csn-PD. The optimum reaction temperature of purified Csn-PD was at 45 °C, and
146 there was a decrease to 31.8% of the maximal activity at 60 °C (Figure 3A). Csn-PD could maintain >80% of the initial
147 activity after incubation at 20–50 °C. However, the residual activity of Csn-PD decreased sharply when it was incubated
148 at 60 °C for 30 min (relative activity 10%) (Figure 3B). The purified Csn-PD was very pH-sensitive, with the highest
149 activity at pH 7.0 in 50 mM phosphate buffer, some activity at acidic pH values, but inactivation at pH 8.0 (Figure 3C).
150 The enzyme was stable at pH 6.0–7.0 (retained >90% of the maximal activity after incubation) but showed a dramatic
151 decrease in activity after incubation at pH <6.0 and >7.0 (Figure 3D).
152 The specificity of the purified Csn-PD for chitin and chitosans with different DDAs is presented in Table 1. The
153 enzyme showed higher hydrolysis of chitosan and colloidal chitosan with DDA 95% than DDA 85%. However, it was
154 not active toward colloidal chitin or CMC. Csn-PD thus displayed strict substrate specificity, in accordance with most
156 Hydrolytic properties of purified Csn-PD. The hydrolytic properties of Csn-PD toward colloidal chitosan were
157 investigated in detail. On incubation of colloidal chitosan with the purified recombinant enzyme at 30 °C for 1 h (Figure
158 4), Csn-PD initially hydrolyzed the substrate to yield mainly (GlcN)2 and (GlcN)3 (0–0.5 h). (GlcN)3 was further
159 converted to (GlcN)2 with extension of the reaction time (0.5–1 h), but no GlcN was observed. ESI-MS analysis also
160 confirmed that (GlcN)2 was the dominant product. As shown in Figure 5A, apart from (GlcN)2, no other oligomers,
161 including GlcN, was detected, which indicated that (GlcN)2 was not hydrolyzed any more. Meanwhile, it was
162 demonstrated from Figure 5B that the products derived from hydrolysis of colloidal chitosan by Csn-PD were only
163 (GlcN)2. These results suggested that Csn-PD exhibited a random catalytic mode of action toward chitosan with a high
164 ability to form (GlcN)2. This property is similar to most chitosanases from microbes that belong to family GH46, which
166 COSs (DP 2–6) were also used as substrates to test the hydrolytic properties of Csn-PD. As shown in Figure 6, the
167 enzyme mainly hydrolyzed these COSs to (GlcN)2 and (GlcN)3 in the initial stage, but (GlcN)2 was not hydrolyzed at all.
168 (GlcN)3 was completely converted to (GlcN)2 in 4 h, with no GlcN production. This result implied that (GlcN)3
169 molecules must have combined then been hydrolyzed to produce (GlcN)2, rather than (GlcN)3 being cleaved to (GlcN)2
170 and GlcN, which was confirmed by the hydrolysis of (GlcN)5. (GlcN)5 was hydrolyzed to mainly yield equivalent
171 amounts of (GlcN)2 and (GlcN)3 in the initial stage (0–1 h), and the released (GlcN)3 was then further converted to
172 (GlcN)2 within 4 h. (GlcN)4 and (GlcN)6 were converted to (GlcN)2 in much less time than (GlcN)3 and (GlcN)5.
173 As far as we know, Csn-PD is the first reported chitosanase that exhibits an endo-type pattern and produces (GlcN)2 as
174 the dominant product. A mutant of B. circulans MH-K1 chitosanase functions as an exo-type enzyme with (GlcN)2 as the
175 dominant product22. Strangely, these two chitosanases share only 72% identity in amino acid sequence. Thus, the PEPSP
176 method we used. This method discovered a novel enzyme based on its product specificity toward chitosan, rather than the
177 sequence identity. Therefore, despite the low identity among these chitosanases, they were successfully clustered.
178 In this study, a novel chitosanase, named Csn-PD, was discovered from Paenibacillus dendritiformis by PEPSP, and
179 was successfully expressed in E. coli BL21 (DE3). The recombinant chitosanase was purified and biochemically
180 characterized. It was most active at pH 7.0 and 45 °C. Csn-PD converted chitosan to, mainly, (GlcN)2, via an endo-type
181 mechanism. The excellent biochemical properties may make this enzyme a good candidate for (GlcN)2 production. More
182 broadly, this work shows that there is every possibility to screen diverse chitosanases for different product specificities.
183 Moreover, before the characterization of an enzyme, use of the PEPSP approach can save large amounts of experimental
184 labor, time, and cost. Better catalysts will be developed in this way for efficient production of pure COSs with specific
185 DP.
186 ■ ABBREVIATIONS
187 PEPSP: phylogeny-based enzymatic product specificity prediction; COSs: chitooligosaccharides; GH: glycosyl hydrolase;
188 DP: degree of polymerization; DDA: degree of deacetylation; CMC: carboxylmethyl cellulose; TLC: thin-layer
190 ■ FUNDING
191 This work was supported by the China Postdoctoral Science Foundation (funded project No. 2017M612379), and the
193 Notes
196 Figure S1. TLC analysis of hydrolysis products of Csn-CAP and Csn-But.
197 ■ REFERENCES
198 (1) Shinya, S.; Fukamizo, T. Interaction between chitosan and its related enzymes: A review. Int. J. Biol. Macromol.
200 (2) Younes, I.; Rinaudo, M. Chitin and chitosan preparation from marine sources. Structure, properties and applications.
202 (3) Su, C. X.; Wang, D. M.; Yao, L. M.; Yu, Z. L. Purification, characterization, and gene cloning of a chitosanase from
203 Bacillus species strain S65. J. Agric. Food Chem. 2006, 54(12), 4208-14.
204 (4) Alishahi, A.; Aïder, M. Applications of chitosan in the seafood industry and aquaculture: A review. Food Bioprocess
206 (5) Jayakumar, R.; Menon, D.; Manzoor, K.; Nair, S. V.; Tamura, H. Biomedical applications of chitin and chitosan
207 based nanomaterials-a short review. Carbohydr. Polym. 2010, 82, 227-232.
208 (6) Antonia, M.; Ana I, R. M.; Nieves, C.; Cecilia, G.; Gabriela, I. Enzymatic generation of chitooligosaccharides from
209 chitosan using soluble and immobilized glycosyltransferase (branchzyme). J. Agric. Food Chem. 2013, 61, 10360-10367.
210 (7) Aranaz, I.; Mengíbar, M.; Harris, R.; Paños, I.; Miralles, B.; Acosta, N.; Galed, G.; Heras, A. Functional
211 characterization of chitin and chitosan. Curr. Chem. Biol. 2009, 3, 203-230.
212 (8) Xia, W. S.; Liu, P.; Zhang, J. L.; Chen, J. Biological activities of chitosan and chitooligosaccharides. Food
214 (9) Park, P. J.; Je, J. Y.; Kim, S. K. Free radical scavenging activity of chitooligosaccharides by electron spin resonance
216 (10) Maria I, Q. V.; Berit B, A.; John, R.; Morten, S.; Vincent G H, E.; Robert W, H. Adherence
217 inhibition of enteropathogenic Escherichia coli by chitooligosaccharides with specific degrees of acetylation and
219 (11) Aam, B. B.; Heggset, E. B.; Norberg, A. L.; Sørlie, M.; Varum, K. M.; Eijsink, V. G. H. Production of
220 chitooligosaccharides and their potential applications in medicine. Mar. Drugs. 2010, 8, 1482-1517.
221 (12) Benhabiles, M. S.; Salah, R.; Lounici, H.; Drouiche, N.; Goosen, M. F. A.; Mameri, N. Antibacterial activity of
222 chitin, chitosan and its oligomers prepared from shrimp shell waste. Food Hydrocolloids. 2012, 29, 48-56.
223 (13) Wu, H.; Aam, B. B.; Wang, W.; Norberg, A. L.; Sørlie, M.; Eijsink, V. G. H.; Du, Y. Inhibition of angiogenesis by
224 chitooligosaccharides with specific degrees of acetylation and polymerization. Carbohydr. Polym. 2012, 89, 511-518.
225 (14) Yan, Y. L.; Hu, Y.; Simpson, D. J.; Michael G, G. Enzymatic synthesis and purification of galactosylated chitosan
226 oligosaccharides reducing adhesion of enterotoxigenic Escherichia coli K88. J. Agric. Food Chem. 2017, 65(25).
227 (15) Hrynets, Y.; Ndagijimana, M.; Betti, M. Studies on the formation of Maillard and caramelization products from
229 (16) Mourya, V. K.; Inamdar, N. N.; Choudhari, Y. M. Chitooligosaccharides: synthesis, characterization and
231 (17) Cabrera, J. C.; Van Cutsem, P. Preparation of chitooligosaccharides with degree of polymerization higher than 6 by
232 acid or enzymatic degradation of chitosan. Biochem. Eng. J. 2005, 25, 165-172.
233 (18) Zhu, X. F.; Tan, H. Q.; Zhu, C.; Liao, L.; Zhang, X. Q.; Wu, M. Cloning and overexpression of a new chitosanase
234 gene from Penicillium sp. D-1. AMB Express, 2012, 2(1), 13.
235 (19) Cantarel, B. L.; Coutinho, P. M.; Rancurel, C.; Bernard, T.; Lombard, V.; Henrissat, B. The carbohydrate-active
236 enzymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res. 2009, 37, D233-D238.
237 (20) Bueren, A. L. V.; Ghinet, M. G.; Gregg, K.; Fleury, A.; Brzezinski, R.; Boraston1, A. B. The structural basis of
238 substrate recognition in an exo-β-D-glucosaminidase involved in chitosan hydrolysis. J. Mol. Biol. 2009, 385, 131-139.
239 (21) Thadathil, N.; Velappan, S. P. Recent developments in chitosanase research and its biotechnological applications: A
241 (22) Yao, Y. Y.; Shrestha, K. L.; Wu, Y. J.; Tasi, H. J.; Chen, C. C.; Yang, J. M.; Ando, A.; Cheng, C. Y.; Li, Y. K.
242 Structural simulation and protein engineering to convert an endo-chitosanase to an exo-chitosanase. Protein Eng., Des.
244 (23) Le, D. F.; Bazinet, L.; Furtos, A.; Venne, K.; Brunet, S.; Alexandru, M. M. Separation of chitosan oligomers by
246 (24) Shee, F. L. T.; Arul, J.; Brunet, S.; Bazinet, L. Chitosan solubilization by bipolar membrane electroacidification:
248 (25) Naqvi, S.; Moerschbacher, B. M. The cell factory approach toward biotechnological production of high-value
249 chitosan oligomers and their derivatives: an update. Crit. Rev. Biotechnol. 2017, 37(1), 11-25.
250 (26) Das, S. N.; Madhuprakash, J.; Sarma, P. V.; Purushotham, P.; Suma, K.; Manjeet, K.; Rambabu, S.; Gueddari, N. E.;
251 Moerschbacher, M. M.; Podile, A. R. Biotechnological approaches for field applications of chitooligosaccharides (COS)
252 to induce innate immunity in plants. Crit. Rev. Biotechnol. 2015, 35(1), 29-43.
253 (27) Kohlhoff, M.; Niehues, A.; Wattjes, J.; Bénéteau, J.; Cord-Landwehr, S.; Gueddari, N. E. E.; Bernard, F.; Rivera-
254 Rodriguez, G. R.; Moerschbacher, B. M. Chitinosanase: A fungal chitosan hydrolyzing enzyme with a new and unusually
256 (28) Cote, N.; Fleury, A.; Dumont-Blanchette, E.; Fukamizo, T.; Mitsutomi, M.; Brzezinski, R. Two exo-β-D-
257 glucosaminidases/exochitosanases from actinomycetes define a new subfamily within family 2 of glycoside hydrolases.
259 (29) Nanjo, F.; Katsumi, R.; Sakai, K. Purification and characterization of an exo-β-D-glucosaminidase, a novel type of
260 enzyme, from Nocardia orientalis. J. Biol. Chem. 1990, 265, 10088-10094.
261 (30) Nogawa, M.; Takahashi, H.; Kashiwagi, A.; Ohshima, K.; Okada, H.; Morikawa, Y. Purification and
262 characterization of exo-β-D-glucosaminidase from a cellulolytic fungus, Trichoderma reesei PC-3-7. Appl. Environ.
264 (31) Fukamizo, T.; Fleury, A.; Côté, N.; Mitsutomi, M.; Brzezinski, R. Exo-β-D-glucosaminidase from Amycolatopsis
265 orientalis: catalytic residues, sugar recognition specificity, kinetics, and synergism. Glycobiology. 2006, 16, 1064-1072.
266 (32) Ike, M.; Isami, K.; Tanabe, Y.; Nogawa, M.; Ogasawara, W.; Okada, H.; Morikawa, Y. Cloning and heterologous
267 expression of the exo-β-D-glucosaminidase-encoding gene (gls93) from a filamentous fungus, Trichoderma reesei PC-3-
269 (33) Ando, A.; Noguchi, K.; Yanagi, M.; Shinoyama, H.; Kagawa, Y.; Hirata, H.; Yabuki, M.; Fujii, T. Primary structure
270 of chitosanase produced by Bacillus circulans MH-K1. J. Gen. Appl. Microbiol. 1992, 38(2), 135-144.
271 (34) Larkin, M. A.; Blackshields, G.; Brown, N. P.; Chenna, R.; McGettigan, P. A.; McWilliam, H.; Valentin, F.;
272 Wallace, I. M.; Wilm, A.; Lopez, R.; et al. Clustal W and Clustal X version 2.0. Bioinformatics. 2007, 23, 2947-2948.
273 (35) Tamura, K.; Stecher, G.; Peterson, D.; Filipski, A.; Kumar, S. MEGA6: Molecular evolutionary genetics analysis
275 (36) Miller, F. M.; Gamson, R. M.; Kramer, D. N. A study of the physical and chemical properties of the esters of
277 (37) Masson, J. Y.; Boucher, I.; Neugebauer, W. A.; . A new chitosanase gene from a Nocardioides sp. is a third member
278 of glycosyl hydrolase family 46. Microbiology. 1995, 141 (10), 2629-2635.
279 (38) Okajima, S.; Ando, A.; Shinoyama, H.; Fujii, T. Purification and characterization of an extracellular chitosanase
280 produced by Amycolatopsis sp. CsO-2. J. Ferment. Bioeng. 1994, 77(6), 617-620.
281 (39) Boucher, I.; Dupuy, A.; Vidal, P.; Neugebauer, W. A.; Brzezinski, R. Purification and characterization of a
282 chitosanase from Streptomyces N174. Appl. Microbiol. Biotechnol. 1992, 38, 188-193.
283 (40) Takasuka, T. E.; Bianchetti, C. M.; Tobimatsu, Y.; Bergeman, L. F.; Ralph, J.; Fox, B. G. Structure-guided analysis
284 of catalytic specificity of the abundantly secreted chitosanase SACTE_5457 from Streptomyces sp. SirexAA-E. Proteins:
286 (41) Johnsen, M. G.; Hansen, O. C.; Stougaard, P. Isolation, characterization and heterologous expression of a novel
287 chitosanase from Janthinobacterium sp. strain 4239. Microb. Cell Fact. 2010, 9(1), 5.
288 (42) Lyu, Q.; Wang, S.; Xu, W.; Han, B.; Liu, W.; David, N. M. J.; Liu, W. Structural insights into the substrate-binding
290 (43) Lee, Y. S.; Yoo, J. S.; Chung, S. Y.; Lee, Y. C.; Cho, Y. S.; Choi, Y. L. Cloning, purification, and characterization
291 of chitosanase from Bacillus sp. DAU101. Appl. Microbiol. Biotechnol. 2006, 73(1):113-121.
292 (44) Yoon, H. G.; Kim, H. Y.; Lim, Y. H.; Kim, H. K.; Shin, D. H.; Hong, B. S.; Cho, H. Y. Thermostable chitosanase
293 from Bacillus sp. strain CK4: cloning and expression of the gene and characterization of the enzyme. Appl. Environ.
295 (45) Li, H.; Fei, Z.; Gong, J. S.; Yang, T.; Xu, Z. H.; Shi, J. S. Screening and characterization of a highly active
296 chitosanase based on metagenomic technology. J. Mol. Catal. B: Enzym. 2015, 111, 29-35.
297 (46) Chiang, C. L.; Chang, C. T.; Sung, H. Y. Purification and properties of chitosanase from a mutant of Bacillus
299 (47) Shimosaka, M.; Fukumori, Y.; Zhang, X. Y.; He, N. J.; Kodaira, R.; Okazaki, M. Molecular cloning and
300 characterization of a chitosanase from the chitosanolytic bacterium Burkholderia gladioli strain CHB101.
302 (48) Yoon, H. G.; Lee, K. H.; Kim, H. Y.; Kim, H. K.; Shin, D. H.; Hong, B. S.; Cho, H. Y. Gene cloning and
303 biochemical analysis of thermostable chitosanase (TCH-2) from Bacillus coagulans CK108. Biosci. Biotechnol. Biochem.
306 Figure 1. Bioinformatic analysis of Csn-PD. (A) Multiple amino acid sequence alignment of Csn-PD and other
307 chitosanases belonging to family GH46. The typical catalytic sites (Glu and Asp) were emphasized with triangles. (B)
308 Neighbor-joining phylogenetic tree. Phylogenetic analysis was carried out using MEGA 6.0 software. The novel
309 chitosanases are in black frames. Groups with different product specificities are in red frames.
310 Figure 2. SDS-PAGE analysis of Csn-PD. Lane M, protein marker; Lane 1, whole cell lysate of recombinant Csn-PD;
312 Figure 3. The Characterization of Csn-PD. (A) Effect of temperature on enzyme activity; (B) Effect of temperature on
313 enzyme stability. (C) Effect of pH on enzyme activity; (D) Effect of pH on enzyme stability.
315 Figure 5. ESI-MS analyses of products derived from hydrolysis of (GlcN)2 and colloidal chitosan by Csn-PD. A: ESI-
316 MS analyses of products derived from hydrolysis of (GlcN)2 by Csn-PD. The (GlcN)2 was used as a control. (GlcN)2 (m/z
317 341 and +K+ m/z 379). B: ESI-MS analyses of products derived from hydrolysis of colloidal chitosan by Csn-PD.
320
321 Tables
Chitosan 85 100
Chitosan 95 128.4
Colloidal chitin 0 0
CMC 0 0
323 Figures
324 Figure 1.
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326 Figure 2.
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328 Figure 3.
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331 Figure 4.
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333 Figure 5.
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336 Figure 6.
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TOC Graphic
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