Expression and Biochemical Characterization of a Novel Marine Chitosanase from Streptomyces niveus Suitable for Preparation of Chitobiose

It is known that bioactivities of chitooligosaccharide (COS) are closely related to the degree of polymerization (DP); therefore, it is essential to prepare COS with controllable DP, such as chitobiose showing high antioxidant and antihyperlipidemia activities. In this study, BLAST, sequence alignment and phylogenetic analysis of characterized glycoside hydrolase (GH) 46 endo-chitosanases revealed that a chitosanase Sn1-CSN from Streptomyces niveus was different from others. Sn1-CSN was overexpressed in E. coli, purified and characterized in detail. It showed the highest activity at pH 6.0 and exhibited superior stability between pH 4.0 and pH 11.0. Sn1-CSN displayed the highest activity at 50 °C and was fairly stable at ≤45 °C. Its apparent kinetic parameters against chitosan (DDA: degree of deacetylation, >94%) were determined, with Km and kcat values of 1.8 mg/mL and 88.3 s−1, respectively. Cu2+ enhanced the activity of Sn1-CSN by 54.2%, whereas Fe3+ inhibited activity by 15.1%. Hydrolysis products of chitosan (DDA > 94%) by Sn1-CSN were mainly composed of chitobiose (87.3%), whereas partially acetylated chitosan with DDA 69% was mainly converted into partially acetylated COS with DP 2-13. This endo-chitosanase has great potential to be used for the preparation of chitobiose and partially acetylated COS with different DPs.


Introduction
Chitin is a β-1,4-linked polysaccharide of N-acetylglucosamine (GlcNAc, A) and is the second-largest natural organic, renewable resource. It is widely distributed in living organisms, including insects, arthropod shells, shrimps, crabs, and some higher plants and fungal cell walls [1]. Chitosan, which is the deacetylated product of chitin, is a linear polysaccharide, consisting of N-acetylglucosamine and glucosamine (GlcN, D) linked by β-1,4-glucosidic bonds [2], and possesses diverse biological activities such as antifungal activity, antibacterial activity, antiviral activity, etc. However, its application and commercial development are greatly restricted by its large molecular weight and poor water solubility [3]. By comparison, COS has a lower molecular weight and viscosity and COS is the degradation product of chitosan and is the only natural oligosac with a positive charge. COS can be prepared by chemical, physical, and enzymatic dation [6]. Chemical methods mainly include oxidative and acidic degradation. Ho these methods have the disadvantages such as violent reaction conditions, unwan products, poor selectivity, high monosaccharide content, uncontrollability, poor ducibility, and serious environmental pollution [7]. Physical methods mainly invol todegradation, ultrasonic and microwave degradation. Compared with chemical dation, physical methods can be operated under mild conditions, with good contr ity and simple operation, but they are difficult to obtain biologically active oligom zymatic approaches refer to enzymatic degradation of chitosan, which is advant over the other two approaches in mild reaction conditions, high yields, good sele controllability and reproducibility, and beneficial to the environment. They hav widely used for COS production [6].
Mar. Drugs 2021, 19, 300 3 of 20 The bioactivities of COS depend closely on its structure and physicochemical properties. The DP, degree of deacetylation (DDA), charge distribution, and the oligomer structure pattern have an important influence on its bioactivities. In particular, the DP of COS is a very important factor in the study of structure-function relationship of COS. Chen et al. reported that the oral bioavailability of chitobiose was higher than that of chitotriose at all doses (30,100, and 300 mg/kg) examined [18]. In addition, it was found that chitobiose effectively protected rats from hepatic injury induced by carbon tetrachloride and showed the most active antihyperlipidemia effect [19,20]. Moreover, it has been reported that the antibacterial activity required COS with a DP of at least 5, and the inhibitory effects improved with increasing DPs of COS [21]. Furthermore, chitohexaose and chitoheptaose exhibited the most effective activities in alleviating chilling stress to wheat seedlings [22]. COS with DDA 50% was the most effective in alleviating salt stress to wheat seedlings, indicating that the activity of COS was closely related to its DDAs too [23].
Therefore, the identification and development of new chitosanases to produce COS with controllable DP and DDA are essential. Until now, few marine chitosanases have been characterized in detail and used for COS production [15]. In this study, one new GH 46 chitosanase Sn1-CSN (GenBank: AQU65829.1) from marine microorganism Streptomyces niveus was found by subjecting a well-characterized chitosanase from Streptomyces sp. N174 (CSN-174) to a BLAST search (Figure 2A), and it showed the highest sequence identity (75.6%) to the reported SACTE_5457 chitosanase from Streptomyces sp. SirexAA-E, implying that Sn1-CSN might have different enzymological properties from those characterized GH 46 chitosanases [24]. It was overexpressed and purified, and its biological properties, kinetic parameters, and degradation products of chitosan (DDA > 94%) and partially acetylated chitosan (DDA 69%) were investigated. In addition, structure modelling of Sn1-CSN with docked chitobiose and chitotriose was performed.

Sequence Alignment and Phylogenetic Analysis of Sn1-CSN with Characterized GH 46 Chitosanases
Though many GH 46 chitosanases have been identified and characterized, until now, few marine GH 46 chitosanases have been investigated [15,25]. One GH 46 chitosanase (GenBank: AQU65829.1) from Streptomyces niveus in deep-sea sediment was found by subjecting the chitosanase CSN-174 from Streptomyces sp. N174 (GenBank: AAA19865.1) to a BLAST search. The chitosanase from Streptomyces niveus was named Sn1-CSN in the current study.
The GH 46 family chitosanases can be further grouped into five subclasses: Cluster A-E, among which Cluster C chitosanases have been found exclusively in viruses, whereas Cluster E groups together proteins for which no enzymatic activity has been reported so far [9]. Therefore, these two subclasses were not included for multiple sequence alignment and phylogenetic analysis ( Figure 2). Sn1-CSN exhibited the highest sequence identity (75.6%) to SACTE_5457 (GenBank AEN13266.1) from Streptomyces sp. SirexAA-E, suggesting that Sn1-CSN is different from those characterized GH 46 chitosanases at least in amino acid sequence ( Figure S1, Supplementary Materials) [24]. Sn1-CSN shares the same branch with SACTE_5457 and ScCsnA from Streptomyces coelicolor A3(2) (GenBank No. CAB61194.1) and belongs to Cluster A (Figure 2A). Two conserved glutamic acid (Glu40) and aspartic acid (Asp58) residues were considered as the catalytic residues in Sn1-CSN ( Figure 2B) [24]. SACTE_5457 could not degrade chitin but can hydrolyze chitosans having different DDAs (75->90%) with markedly different k cat /K M values [24]. Based on the above analysis, it was presumed that Sn1-CSN might have some different enzymological properties from those reported GH 46 chitosanases. Therefore, Sn1-CSN was chosen for further investigation.
The modeled structure of Sn1-CSN based on homologous enzymes (PDB ID: 4ILY from Streptomyces sp. SirexAA-E; PDB ID: 1CHK from Streptomyces N174) was obtained by the I-TASSER server. Five top-ranking 3D models were generated. Each model was validated based on confidence score (C-score), template modeling score (TM-score), the root-mean-Mar. Drugs 2021, 19, 300 4 of 20 square deviation (RMSD), and cluster density. In general, models with C-score > −1.5 have a correct fold. Model 1 had the highest C-scorevalue (1.31), reflecting a model of better quality (TM-score = 0.90 ± 0.06 and RMSD = 3.2 ± 2.3 Å) ( Figure S1, Supplementary Materials). Similar to homologous GH 46 chitosanases, the modeled three-dimensional structure of Sn1-CSN was composed of two α-helical domains connected by a bent α-helix (residues 126-151). The smaller one of the two domains, designated here as the N-terminal domain, is composed of residues 46-123, while the larger domain, named as the C-terminal domain, includes residues 1-45 and 152-255 [9,16].
In order to insight into the possible interaction mode of Sn1-CSN with chitosan, its binding interactions with chitobiose and chitotriose were done by molecular docking. The TotalScores of binding of Sn1-CSN with chitobiose and chitotriose were 6.08 and 7.12, respectively, indicating that the binding interaction of Sn1-CSN with chitobiose is weaker than that with chitotriose. Structural analyses showed that chitobiose binds with Sn1-CSN by electrostatic interaction with Glu215 and hydrogen bonding with Arg60, Ile67, Asp75, Val166, and Met167, whereas chitotriose interacts with Sn1-CSN by electrostatic interaction with Asp75 and hydrogen bonding with Ile67, Gly68, Thr73, Asp75, Tyr140, Val166, Met167, Gly169, and Asp170 ( Figure 3). Therefore, more hydrogen bonds are involved in the interaction of Sn1-CSN with chitotriose, leading to stronger binding interaction between Sn1-CSN and chitotriose than that between Sn1-CSN and chitobiose. Some involved residues are highly conserved among GH 46 chitosanases [9,26].  [41], and Streptomyces sp. SirexAA-E (SACTE_5457, GenBank No. AEN13266.1) [24]. (B) Multiple sequence alignment of some GH 46 chitosanases. Putative conserved residues involved in catalysis were marked with red dots above. Phylogenetic, sequence analysis, and classification of GH46 chitosanases [9]. (A) Phylogenetic analysis and classification of GH46 chitosanases. The maximum likelihood method was used to construct the phylogenetic tree. The homologous sequences were found using BLAST at NCBI. Sequences included in the alignment were derived from Bacillus amyloliquefaciens YX01 (BaCsn46A, GenBank No. AFJ63438.1) [14], Bacillus sp.  . The modeled structure of Sn1-CSN was displayed in the ribbon mode, and the ligands, chitobiose and chitotriose, and amino acids participating in hydrogen bonding were shown as a ball-and-stick model. Residues 1-45, 46-123, 152-255 corresponding to domains were colored blue, pink, and orange, respectively, while the bent α-helix consisting of residues 124-151 was highlighted in green.

Overexpression and Purification of Sn1-CSN in E. coli
The gene encoding Sn1-CSN was cloned into pET-22b and overexpressed in E. coli BL21(DE3). The overexpressed protein was purified by nickel-chelating affinity chromatography (the predicted molecular weight based on the amino acid sequence: 29.8 kDa) ( Figure S2, Supplementary Materials). The yield of Sn1-CSN was about 30 mg per liter LB medium.

Determination of pH Optima and pH Stability of Sn1-CSN
As shown in Figure 4, no obvious activities were detected below pH 4.0 and above pH 9.0 (data not shown). Sn1-CSN showed the highest activity at pH 6.0 and retained . The modeled structure of Sn1-CSN was displayed in the ribbon mode, and the ligands, chitobiose and chitotriose, and amino acids participating in hydrogen bonding were shown as a ball-and-stick model. Residues 1-45, 46-123, 152-255 corresponding to domains were colored blue, pink, and orange, respectively, while the bent α-helix consisting of residues 124-151 was highlighted in green.

Overexpression and Purification of Sn1-CSN in E. coli
The gene encoding Sn1-CSN was cloned into pET-22b and overexpressed in E. coli BL21(DE3). The overexpressed protein was purified by nickel-chelating affinity chromatography (the predicted molecular weight based on the amino acid sequence: 29.8 kDa) ( Figure  S2, Supplementary Materials). The yield of Sn1-CSN was about 30 mg per liter LB medium.

Determination of pH Optima and pH Stability of Sn1-CSN
As shown in Figure 4, no obvious activities were detected below pH 4.0 and above pH 9.0 (data not shown). Sn1-CSN showed the highest activity at pH 6.0 and retained residual activity above 80% between pH 5.0 and pH 7.5. It maintained 74.1% and 77% of residual activity at pH 4.5 and pH 8.0, respectively.
The pH stability of Sn1-CSN was investigated after being preincubated for a fixed time over the pH range of 4.0-11.0 ( Figure 5). Sn1-CSN was stable over the pH range of 4.0-11.0, retaining > 90.0% original activity after 120 h. Therefore, Sn1-CSN was very stable between pH 4.0 and pH 11.0 and exhibited superior pH stability over a wide pH range.

Determination of Temperature Optima and Thermal Stability of Sn1-CSN
The optimal temperature of Sn1-CSN was determined ( Figure 6). It showed the highest activity at 50 °C and kept >70% of residual activity between 45 °C and 55 °C and retained less than 55% of residual activity at 60 °C and below 40 °C. and 120 h, respectively. The residual activities were then measured immediately under standard conditions. The initial activity at optimal pH (6.0) was taken as 100%, and the percentages of the residual activities at different time points and pH values against the one at optimal pH (6.0) and 37 • C were calculated. The mean values ± standard deviations of three replicates were shown.

Determination of Temperature Optima and Thermal Stability of Sn1-CSN
The optimal temperature of Sn1-CSN was determined ( Figure 6). It showed the highest activity at 50 • C and kept >70% of residual activity between 45 • C and 55 • C and retained less than 55% of residual activity at 60 • C and below 40 • C. Figure 5. pH stability of Sn1-CSN. The pH stability assay was investigated by first preincubating Sn1-CSN in Britton-Robinson buffer at different pH values (pH 4.0-11.0) at 4 °C for 1 h, 5 h, 24 h, and 120 h, respectively. The residual activities were then measured immediately under standard conditions. The initial activity at optimal pH (6.0) was taken as 100%, and the percentages of the residual activities at different time points and pH values against the one at optimal pH (6.0) and 37 °C were calculated. The mean values ± standard deviations of three replicates were shown.
The thermal stability of Sn1-CSN was investigated after being preincubated for a fixed period at pH 6.0, and 30 • C, 35 • C, 40 • C, 45 • C, and 50 • C, respectively (Figure 7). Sn1-CSN was very stable below 35 • C and lost 9.6-14.4% of original activity after incubated at 40 • C and 45 • C for 2 h. It was completely inactivated at 50 • C after 1 h. Therefore, Sn1-CSN was a thermolabile chitosanase and was fairly stable below 45 • C.

Determination of Kinetic Parameters of Sn1-CSN
The kinetic parameters of recombinant Sn1-CSN against chitosan were determined by substrate hydrolysis from 0.4 to 2.5% (w/v) for 15 min. The deduced kinetic values were apparent parameters since saturation was not achieved even when high chitosan (DDA > 94%) concentrations were used (Figure 8). The apparent K m , k cat , and V max values of Sn1-CSN for chitosan were 1.8 ± 0.3 mg/mL, 88.3 ± 7.6 s −1 , and 1800 ± 145 µmol/min/mg, respectively.

Effects of Metal Ions on Enzyme Activity
The effects of metal ions on Sn1-CSN activity were examined, too (Figure 9). Cu upregulated the activity of Sn1-CSN by 54.2%, while Fe 3+ decreased the activity of Sn CSN by 15.1%. Other metal ions did not show obvious influence on the catalytic activi of Sn1-CSN.

Effects of Metal Ions on Enzyme Activity
The effects of metal ions on Sn1-CSN activity were examined, too (Figure 9). Cu 2+ upregulated the activity of Sn1-CSN by 54.2%, while Fe 3+ decreased the activity of Sn1-CSN by 15.1%. Other metal ions did not show obvious influence on the catalytic activity of Sn1-CSN.

Identification of Hydrolysis Products of Chitosan Catalyzed by Sn1-CSN
Thin-layer chromatography (TLC) analysis of soluble sugars released from chitosan (DDA > 94%) by Sn1-CSN indicated that chitobiose was predominantly produced at the stage of initial 30 min and accumulated as hydrolysis continued, and less chitotriose was also generated ( Figure 10). No monomer glucosamine (GlcN, D) was observed even after 24 h, suggesting that it was an endo-acting enzyme. The HPLC results further confirmed the above results, with chitobiose (DP2), chitotriose (DP3), and chitotetraose (DP4) accounting for 87.3%, 11.3%, and 1.4%, respectively (hydrolysis time of 4 h) ( Figure 11). The formation of chitobiose, chitotriose, and chitotetraose were also proved by MS (Figure 12).

Analysis of Hydrolysis Products of Partially Acetylated Chitosan Catalyzed by Sn1-CSN
Since it has been found degree and position of acetylation of COS had important fects on biological activities of COS, partially acetylated chitooligosaccharides were a prepared from partially acetylated chitosan by Sn1-CSN. TLC results of partially ace lated chitosan (DDA 69%) were very similar to those of chitosan (DDA > 94%): chitobio chitotriose, and chitotetraose were detected, and chitobiose was still the main degradati product ( Figure S3, Supplementary Materials), so were the HPLC results (Data n shown). However, MALDI-TOF MS analysis indicated that COSs with different DPs (D 2-13) and different compositions of N-acetylglucosamine and glucosamine were clea observed ( Figure 13). Therefore, Sn1-CSN can be used to produce partially acetylated C (paCOS).

Analysis of Hydrolysis Products of Partially Acetylated Chitosan Catalyzed by Sn1-CSN
Since it has been found degree and position of acetylation of COS had important effects on biological activities of COS, partially acetylated chitooligosaccharides were also prepared from partially acetylated chitosan by Sn1-CSN. TLC results of partially acetylated chitosan (DDA 69%) were very similar to those of chitosan (DDA > 94%): chitobiose, chitotriose, and chitotetraose were detected, and chitobiose was still the main degradation product ( Figure S3, Supplementary Materials), so were the HPLC results (Data not shown). However, MALDI-TOF MS analysis indicated that COSs with different DPs (DP 2-13) and different compositions of N-acetylglucosamine and glucosamine were clearly observed ( Figure 13). Therefore, Sn1-CSN can be used to produce partially acetylated COS (paCOS). It has been reported that paCOS also possess numerous bioactivities and have already been implemented in different applications [42]. However, few chitosanases have It has been reported that paCOS also possess numerous bioactivities and have already been implemented in different applications [42]. However, few chitosanases have been used to produce them [43,44]. Therefore, Sn1-CSN might become a useful tool enzyme for the production of paCOS.

Bacterial Strains, Plasmids, and Media
E. coli DH5α was used for routine DNA transformation and plasmid isolation. E. coli BL21(DE3) was utilized for chitosanase overexpression. E. coli strains were routinely grown in Luria-Bertani broth at 37 • C with aeration or on LB supplemented with 1.5% (w/v) agar. Ampicillin (100 µg/mL) was added when required.
To insight into the possible mode of action of Sn1-CSN with chitosan, its binding interactions with chitobiose and chitotriose were mimicked with the Surflex-Dock method in SYBYL-X software, respectively (Tripos Inc., St. Louis, MO, USA). For molecular docking, the hydrogen atoms were added, and the atomic charges were calculated with the MMFF94 method for protein and the Gasteiger-Huckel method for chitobiose and chitotriose. The binding sites of chitosanase with chitobiose and chitotriose were found with Automatic mode in Protomol generation, in which the Threshold and Bloat were set as 0.5 and 10 Å, respectively. The default values were used for all other parameters. To reduce the impact of initial ligand conformation on the docking results, three starting conformations of each ligand, which were obtained according to energy minimization with Max Iterations set to 0, 100, and 1000 were used. The highest TotalScore of each ligand was used to compare their binding interaction, and its conformation was used for structural analysis.

DNA Manipulation
Molecular cloning was done by following the standard protocol [47].The plasmid for the synthesized chitosanase gene cloned into the vector PUC57 at the restriction sites of BamHI (5 -terminal) and XhoI (3 -terminal) was digested with BamHI and XhoI and re-cloned into the vector pET-22b(+) digested with the same restriction enzymes. The final construct pET-22b-Sn1-CSN was confirmed by DNA sequencing.

Protein Over-Expression and Purification
The expression construct pET-22b-Sn1-CSN was overexpressed in E. coli BL21(DE3) in the presence of isopropyl-β-D-thiogalactopyranoside (IPTG) [47]. A single colony was cultured in 50 mL LB containing ampicillin (100 µg/mL) at 220 rpm and 37 • C overnight. The overnight culture was used to inoculate 100 mL LB medium with 100 µg/mL ampicillin. When OD 600nm was about 0.6, protein expression was induced at 16 • C with different IPTG concentrations and induction time. Finally protein was expressed under optimized conditions (0.1 mM IPTG, 16 • C for 12 h). The cells were harvested by centrifugation at 3950 g and 4 • C for 20 min.
All purification procedures were carried out at 4 • C. The cell pellet was suspended in 20 mL buffer A (50 mM Tris-HCl, pH 8.0, 0.5M NaCl), and 1 mM phenylmethanesulfonylfluoride (PMSF) was used as the protease inhibitor. The cell suspension was homogenized by sonication at 50% amplitude (working time 6 min). The crude protein solution was obtained by spinning down at 12,000 g and 4 • C for 20 min.
Nickel chelating resin (5 mL) was equilibrated with buffer A. The crude protein was loaded onto the column, which was washed with buffer A containing 20-500 mM imidazole in sequence. The enzyme purity was checked by SDS-PAGE. The fractions containing Sn1-CSN were combined and dialyzed against buffer A. The protein concentration was determined by the Bradford method using bovine serum alumin as a standard [48].

Enzyme Assay
All enzymatic assays were performed in triplicate. Chitosanase activity was quantified by measuring the amount of released reducing sugars from chitosan through the 3,5dinitrosalicylic acid (DNS) method [49]. D-Glucosamine was used as a standard. The assay mixture (1 mL) was composed of 0.5% (w/v) chitosan and enzyme solution in 50 mM Britton-Robinson buffer (pH 5.5), and enzymatic reactions were done at 37 • C for 15 min. One unit (U) of chitosanase activity was defined as the amount of chitosanase required to release 1 µmol of reducing sugar per min under standard assay conditions, and specific activity was defined as units mg −1 chitosanase.

Determination of Optimal pH and pH Stability
The optimal pH of Sn1-CSN was carried out in 50 mM Britton-Robinson buffer at 37 • C and pH values between 2.0 and 11.0, and all enzymatic assays under different conditions were incubated for 15 min. Specific activities were calculated as above.
The pH stability assay was evaluated by first preincubating Sn1-CSN in 50 mM Britton-Robinson buffer at different pH values (pH 4.0-11.0) at 4 • C for 1 h, 5 h, 24 h, and 120 h, respectively. The residual activities were analyzed immediately under standard conditions (optimal pH, 37 • C for 15 min). The original activity at optimal pH (6.0) was considered as 100%, and the percentage of the residual activity at different time points and pH values against the original one at optimal pH (6.0) was calculated.

Determination of Optimal Temperature and Thermal Stability
The optimal temperature was determined in 50 mM Britton-Robinson buffer (pH 6.0) between 30 and 60 • C, and all enzymatic reactions at different temperatures were incubated for 15 min.
To determine the thermal stability of Sn1-CSN, it was pre-incubated for different intervals (15 min-2 h) at pH 6.0, and 30 • C, 35 • C, 40 • C, 45 • C, 50 • C, and 55 • C, respectively. and. The residual activities were measured under standard conditions (optimal pH, 37 • C for 15 min) after all samples were chilled on ice for at least 15 min. The original activity at pH 6.0 considered as 100%, and the percentage of the residual activity at different time points and temperatures against the initial one was calculated.

Determination of Kinetic Parameters
Kinetic parameters were measured under initial rate conditions through non-linear regression analysis of the Michaelis-Menten equation [50,51]. The initial rates of Sn1-CSN were analyzed at 37 • C in the presence of chitosan ranging from 0.1 to 1.5% (w/v) in a 50 mM Britton-Robinson buffer (pH 6.0). The released reducing sugars were quantified as above after being incubated for 15 min.

Effects of Metal Ions on Enzyme Activity
The impact of some metal ions on the catalytic activity of Sn1-CSN was investigated in the presence of 1 mM of various metal ions (Pb(CH 3 COO) 2 , NiSO 4 , CuSO 4 , BaCl 2 , ZnSO 4 , CoCl 2 , CaCl 2 , MgCl 2 , Fe 2 (SO) 3 , and Li 2 SO 4 ). Since phosphate in Britton-Robinson buffer might affect the assay, it was replaced with 100 mM sodium acetate (pH 6.0). The reaction in the absence of metal ions was used as the control, which was taken as 100%. The percentage of the activity in the presence of different metal ions against the control was determined.

Hydrolysis of Partially Acetylated Chitosan by Sn1-CSN and Characterization
Partially acetylated chitosan was prepared according to the published procedure [52], and DDA was determined by 1 H NMR following the standard procedure [53]. The hydrolysis procedure of partially acetylated chitosan and characterization of degradation products was carried out as above.

Conclusions
In summary, multiple sequence alignment and phylogenetic analysis of Sn1-CSN from Streptomyces niveus and homologous GH 46 endo-chitosanases suggested that Sn1-CSN is a new GH 46 endo-chitosanase. Sn1-CSN was overexpressed in E. coli and characterized in detail. It showed the highest activity at pH 6.0 and 50 • C. It was very stable between pH 4.0 and pH 11.0. Sn1-CSN exhibited high stability at ≤45 • C and was unstable above 50 • C. The apparent K m and k cat values of Sn1-Csn were 1.8 mg/mL and 88.3 s −1 , respectively. Cu 2+ showed some stimulatory effects on Sn1-CSN, while Sn1-CSN was inhibited by Fe 3+ . The structural characteristics of GH 46 endo-chitosanases were demonstrated by structure modelling of Sn1-CSN and molecular docking. The main hydrolysis product of chitosan (degree of deacetylation, DDA > 94) by Sn1-CSN was chitobiose (87.3%), whereas degradation products of partially acetylated chitosan (DDA 69%) were very complicated with DP 2-15. This endo-chitosanase could be potentially for the production of chitobiose and partially acetylated COS.