Gene cloning, functional expression and characterization of a novel GH46 chitosanase from Streptomyces avermitilis (SaCsn46A)

A novel glycoside hydrolase (GH) family 46 chitosanase (SaCsn46A) from Streptomyces avermitilis was cloned and functionally expressed in Escherichia coli Rosetta (DE3) strains. SaCsn46A consists of 271 amino acids, which includes a 34-amino acids signal peptide. The protein sequence of SaCsn46A shows maximum identity (83.5%) to chitosanase from Streptomyces sp. SirexAA-E. Then the mature enzyme was puri�ed to homogeneity through Ni-chelating a�nity chromatography with a recovery yield of 78% and the molecular mass of puri�ed enzyme was estimated to be 29 kDa by SDS-PAGE. The recombinant enzyme possessed a temperature optimum of 45 o C and a pH optimum of 6.2, and it was stable at pH ranging from 4.0 to 9.0 and below 30 o C. The K m and V max values of this enzyme were 1.32 mg ∙ mL − 1 , 526.32 µM ∙ mg − 1 ∙ min − 1 , respectively (chitosan as substrate). The enzyme activity can be enhanced by Mg 2+ and especially Mn 2+ , which could enhance the activity about 3.62-fold at a 3 mM concentration. The enzyme can hydrolyze a variety of polysaccharides which linked by β-1,4-glycosidic bonds such as chitin, xylan and cellulose, but it could not hydrolyze polysaccharides linked by α-1,4-glycosidic bonds. The results of thin layer chromatography and HPLC showed that the enzyme exhibited an endo-type cleavage pattern and could hydrolyze chitosan to glucosamine (GlcN) and (GlcN) 2 . This study demonstrated that SaCsn46A is a promising enzyme to produce glucosamine and chitooligosaccharides (COS) from chitosan.


Introduction
Chitosan is a kind of linear polymer which was composed of β-1, 4-linked D-glucosamine (GlcN) units.
Chitosan is a group of biomacromolecules that is commonly prepared by the partial chemical deacetylation of chitin (34,4).As the second most abundant polymer in nature, chitin occurs in the exoskeletons of fungi, crustaceans, insects and algae.(14).
Chitooligosaccarides (COS) are the hydrolyze products of chitosan with degrees of polymerization (DP) 2-10(26).COS have many biological activities such as anti-tumor (21), immune enhancing (19), antioxidizing (13), free radical scavenging (15) and antimicrobial activity (33).GlcN has gained its popularity as nutraceutical having application in weight loss and in treatment of osteoarthritis, knee pain and back pain (9).Consequently, it is a popular compound in medicine, agriculture and rises interest in converting chitosan into COS and GlcN (24).COS and GlcN can be prepared from chitosan by a variety of methods, such as chemical, physical and enzymatic (10).Compare with other methods, enzymatic conversion to produce COS and GlcN produced has attracted extensive attention as an environmentally friendly, high e ciency and controllable and alternative to chemical and physical production.Many enzymes have been reported to hydrolyze chitosan to produce chitooligosaccharides (COS) such as chitosanase, chitinase (22), protease (20), cellulase, lipase(28).Among these enzymes, chitosanase (EC.3.2.1.132)is the most attractive because of its high activity and speci city.
Chitosanase is a kind of enzyme that hydrolyzes chitosan degradation into COS or GlcN.Various chitosanases have been identi ed from many species (17).Due to the sequences in the Carbohydrate Active Enzymes database (www.cazy.org)(16),chitosanases are categorized into glycoside hydrolases (GH) families 46 (24,26,29), 75 (34), 80 (32) and 8 (35).GH46, GH75, and GH80 currently contain exclusively chitosanases, while the GH8 family contains a few other glycoside hydrolases, such as cellulase, lichenase, and xylanase (35).It is remarkable that chitosanases from fungi are mainly distributed among the GH75 family, but chitosanases from bacteria are mainly belonging to GH46 family, a few belong to GH80 (17).S. avermitilis is a kind of aerobic bacteria and were mostly reported to produce avermectins(1) and ivermectin (3).Heggset reported a chitosanase from the GH75 family produced by S. avermitilis, which mainly produce long oligomers (DP ≥ 2) (5).However, to our knowledge, no report is available regarding on gene cloning and characterization of GH46 family chitosanase from S. avermitilis.Accordingly, in this study, a novel chitosanase designated as SaCsn46A was cloned by PCR and functionally expressed in E. coli Rosetta (DE3).The characterization of its biochemical properties suggests that SaCsn46A has high activity and pH stability and could e ciently convert colloidal chitosan into GlcN and (GlcN) 2 , which was rarely reported by other chitosanases.

DNA cloning and sequence analysis
Two primers (sacsnF, 5'-3': CGGGATCCGCACCCGTCGGCCTGGACGAC; sacsnR, 5'-3': CCCAAGCTTTCAGCCGATGTGGTAGCTGTC) were designed and used to amplify the chitosanase gene, and the signal peptide was eliminated.The PCR products were cloned into pMD18-T and then digested by the designated restrictive endonuclease and cloned into the corresponding sites (BamHI-HindIII) of the expression vector pET-28a to generate pET-SaCsn46A, the recombinant enzyme carry the His-tag and T7tag belonging to the pET-28a plasmid at the N-terminal.The signal peptide was predicted using Signal P 4.0 (http://www.cbs.dtu.dk/services/SignalP/).Homology searches in GenBank were performed using the BLAST program.The molecular mass of the protein was determined using the software DNAMAN (http://www.lynnon.com/).Multiple alignments of SaCsn46A sequence were performed with Clustal W (https://www.genome.jp/tools-bin/clustalw)and ESprint 3.0 (http://espript.ibcp.fr/ESPript/ESPript/).Phylogenetic trees were constructed using neighbor-joining algorithm in MEGA 5.0 (http://www.megasoftware.net/).Three-dimensional structure homology model of SaCsn46A was constructed by Swiss-Model (http://swissmodel.expasy.org)Expression and puri cation of SaCsn46A E. coli Rosetta (DE3) cells harboring the designated plasmid were incubated in Luria-Bertani medium containing 50 µg/mL kanamycin and grown at 37 o C to OD 600 of 0.6.The incubation of the target protein production was performed by adding IPTG to a nal concentration of 1 mM.The cultures were grown for an additional 12 h at 16 o C, and protein expression in the cells and culture supernatant were analyzed by SDS-PAGE.
All the following puri cation steps were carried out at 4 o C. To purify recombinant chitosanase, the induced culture was centrifuged for 10 min at 10000 g.The cell pellet was suspended in buffer I (0.02 M Tris-HCl, pH 8.0, 0.5 M NaCl and 10 % glycerol) and washed twice with the same buffer.The washed cell pellet was suspended in buffer I, disrupted by sonication on an ice bath, and then centrifuged for 30 min at 10000 g.The resulting supernatant was applied on a Ni-Sepharose column previously charged with Ni (II).After the column was washed with buffer II (0.02 M Tris-HCl, pH 8.0, 0.5 M NaCl, 0.02 M imidazole and 10 % glycerol), elution was performed with a 0.08 M imidazole (0.02 M Tris-HCl, pH 8.0, 0.5 M NaCl, 0.08 M imidazole and 10 % glycerol).Elution contains chitosanase activity was collected, imidazole was removed by dialysis, and the enzyme was stored at -20 o C.

Properties of puri ed SaCsn46A
Chitosanase activity was determined by 3, 5-dinitrosalicylic acid (DNS) method, as described previously (4).The content of reducing sugar was determined by measuring optical density (OD) at 520 nm using D-(+)-glucosamine as standard.The reaction mixture was incubated at 45 o C for10 min.All reactions were done in triplicate, and their mean and standard deviation values were used for analysis.One unit (U) of chitosanase activity is de ned as the amount of enzyme liberating 1 µmol of D-(+)glucosamine-equivalent reducing sugars per minute under the above conditions.
Chitosanase pro les assay for pH were determined at pH ranging from 3.0 to 8.0 at 40 o C in the following buffers: 0.05 M acetate buffer (pH 4.5-6.0)and 0.05 M phosphate buffer (pH 6.0-8.0).pH stability of chitosanase was determined by measuring the residual activities under optimal conditions (pH 6.2, 10 min) after pre-incubation at 0 o C and pH 4.0-9.0.Buffers used were 0.05 M acetate buffer (pH 4.0-6.0),0.05 M phosphate buffer (pH 6.0-8.0) and 0.05M Tris-HCl buffer (pH 9.0).Chitosanase pro les assay for temperature were determined at temperatures ranging from 20 to 90 o C at 5 intervals at optimal pH.Thermostability was conducted by measuring the residual activities under optimal conditions (pH 6.2, 45 o C) after pre-incubation at 0-60 o C and pH 7.5 for 2 h.pH stability was conducted by measuring the residual activities under optimal conditions after pre-incubation at 0 o C and pH 3.0-9.0for 2 h.A control sample with no pretreatment was taken as 100 %.
The substrate speci city of SaCsn46A was determined by DNS method.The reaction mixtures, containing 0.05 M phosphate buffer (pH 6.2), the tested polysaccharide substrates (1%) and appropriate amount of SaCsn46A, were incubated at 45 for 20 min.Control samples were treated in the same way in the absence of SaCsn46A.The amount of reducing sugar in the supernatant was determined by measuring the OD520.One unit (U) of enzyme activity was de ned as the amount of enzyme that liberated 1 µmol of reducing sugar per min under the above assay conditions.
The effects of metal ions on chitosanase activity were determined by assaying the enzyme activity at pH 6.2 and 45 o C for 10 min in the presence of various ions at different concentration.
The catalytic constant was determined by Lineweaver-Burk plot method using chitosan in ranging of 0.1-10 mg/mL at optimal pH 6.2 and 45 o C for 10 min in the presence of 3 mM Mn 2+ .

Hydrolytic properties of puri ed SaCsn46A
The analysis of the hydrolytic products of SaCsn46A was performed by analyzing the products of colloidal chitosan (1%, w/v; 90% DDA) using thin layer chromatography (TLC) method.The puri ed enzyme was incubated with colloidal chitosan at 28 o C in phosphate buffer (pH 6.2).Samples withdrawn at different times were immediately boiled for 20 min, then centrifuged for 10 min at 10000 g.The hydrolysis products were spotted onto a silica gel plate, which was developed in a solvent system which contains ethyl acetate-methanol-water-25% ammonia (5:9:1:1.5,v/v/v/v).The plate was sprayed with 0.3% ninhydrin (dissolved in 95% ethanol, w/v), followed by heating at 120 o C for 10 min.The hydrolysis products were analyzed by HPLC system (Waters 1525, Waters, USA) equipped with evaporative light scattering detector (ELSD).The GlcN and (GlcN) 2 were separated on high performance sugar column (Shodex Asahipak NH 2 P504E, Shodex, Kyoto, Japan), eluted by acetonitrile and distilled water (70/30, w/w) mixture with a ow rate of 1.0 mL•min − 1 at 30 o C.

Sequence analysis of the chitosanase
Based on Genbank database and amino acid sequence analysis, the chitosanase gene from S. avermitilis (GenBank: KUN55720.1)was selected for analysis.The gene contained 816 bp (base pair), which encoding 271 amino acids.The SaCsn46A has a 'leader peptide' in the secretion of this enzyme and a cleavage site at the N-terminus (Ala34-Ala35) which was determined by the Signal P 4.0 program.The protein sequence of SaCsn46A shows maximum identity to chitosanase from Streptomyces sp.(Genbank accession No. WP_028800608.1).
The protein sequences of SaCsn46A and other chitosanases belonging to four different GH families were selected from the GenBank database to constructed a phylogenetic tree (see Fig. 1(a)).SaCsn46A is clearly a member of GH46 family chitosanase.As can be seen from the multiple sequence alignment of GH 46 family members in Fig. 1(b), the SaCsn46A contained two key active site residues (E22 and D40), that are conserved in all GH46 family members.The residues E22 and D40 were replaced by A22 and A40, respectively and the mutants were mostly inactive (data not shown).
Homology model of SaCsn46A was built by the Swiss-Model server with the chitosanase from Streptomyces sp.SirexAA-E as template (PDB ID: 4ily.1A), which shares 83.5% identity to SaCsn46A.Two amino acid residues in the catalytic active center (E22 and D40) were illustrated with sticks in the 3Dstructure of SaCsn46A.These results suggest that SaCsn46A is a novel member of family GH46.

Heterologous expression and puri cation of the chitosanase
The SaCsn46A gene was cloned from S. avermitilis and expressed in E. coli Rosetta (DE3) successfully with a N-terminal His-tag and without signal peptide.The recombinant enzyme were puri ed to electrophoretic homogeneity by a Ni-NTA a nity column (result was shown in Fig. S1).The molecular weight of puri ed recombinant enzyme was estimated to be 29 kDa by SDS-PAGE which was about 4 kDa larger than the wild mature enzyme, corresponding to the size of T7-tag and His-tag at N-terminal of the recombinant enzyme (3.7 kDa).The molecular weight of the most reported chitosanases were ranging from 20 to 60 kDa (31,23,7,6,24), but Kim reported a chitosanase produced by Aspergillus fumigatus was 108 kDa(8) and Chen obtained a chitosanase produced by Aspergillus sp.CJ22-326 was 109 kDa (2).The molecular weight of SaCsn46A is close to a chitosanase from Streptomyces albolongus (29.6 kDa)(4).

Chitosanase activity and substrate speci city
The speci c activity of SaCsn46A was determined after puri cation step with Ni-NTA column was increased from 121.2 U•mg − 1 to 424.2 U•mg − 1 , obtaining a puri cation fold of 3.5 and recovery yield of 78% (Results are listed in Table 1).N.D., the enzyme activity was not detected.
In order to determine the substrate speci city of SaCsn46A, we tested its ability to hydrolyze different polysaccharides, and the results are listed in Table 2.We found that SaCsn46A can hydrolyze a variety of polysaccharides which were linked by β-1,4-glycosidic bonds, but it could not hydrolyze polysaccharides linked by α-1,4-glycosidic bonds such as soluble starch and dextrin.The enzyme showed a high hydrolysis activity toward colloidal chitosan, and displayed low hydrolysis activity toward powder chitosan (7.51 ± 0.72 U/mg), colloidal chitin (7.51 ± 0.93 U/mg), powder chitin (6.28 ± 0.93 U/mg), cellulose (1.95 ± 0.93 U/mg), carboxymethyl cellulose (10.14 ± 0.64 U/mg) and xylan (9.46 ± 0.93 U/mg), shows a broad of substrate spectrum.It can be seen from the experimental results that although SaCsn46A can hydrolyze a variety of polysaccharides, its catalytic capacity for cellulose is the lowest, which may be related to the solubility of cellulose and the space size of constituent monomers.The broad spectrum properties of substrate are different from the reported chitosanase belonging to family GH46, such as Csn21c from S. albolongus(4), Csn-BAC from Bacillus sp.MD-5 (29), Csn-CAP from Staphyloccus capitis (24) and BaCsn46A from Bacillus amyloliquefaciens(18).These enzymes strictly hydrolyze colloidal chitosan and have no catalytic effect on powder chitosan and other polysaccharides.

Biochemical characterization of the puri ed chitosanase
The optimal temperature for SaCsn46A was determined in different temperature ranges (20-90°C).The maximum relative chitosanase activity was observed at 45 o C (Fig. 3a), similar optimal temperature (45 o C) has been reported from Paenibacillus dendritiformis chitosanase(25), while was higher than that of Gynuella sunshinyii chitosanase ( Most reported microbial chitosanases have an optimum reaction pH in the range 4 to 8 (27).Optimal chitosanase activity was observed at pH 6.2 in 50 mM phosphate buffer (Fig. 3b), while SaScn46A was pH-sensitive (Fig. 3b), less than 30% relative activity was observed at pH value below 6 or above 7, however more than 80% relative activity was observed at pH6-7 in 50 mM phosphate buffer.
Thermostability illustrated that the SaCsn46A was stable below 30 o C (Fig. 3c).SaCsn46A has good pH stability at 4.0-9.0 (Fig. 1d), retained more than 80% of its maximal activity at pH 5.0-9.0 after incubation for 2 h, the stability characteristic of SaCsn46A was similar to Bacillus sp.chitosanase (stable at pH 3.6-9.8below 30 o C) (13).
The effects of metal ions on the activity of SaCsn46A are presented in Table 3.The enzyme activity was inhibited by Cu 2+ , Ba 2+ , Ca 2+ and Zn 2+ at both 1 mM and 2 mM concentration.The enzyme activity was inhibited by K + at 2 mM (79.92%).K + and Mg 2+ of 1mM has no effect on enzyme activity.Fe 2+ at both 1 mM and 2 mM displayed slight promoting effect on enzyme activity.Notably, the enzyme activity can be increased to 3.62-fold by Mn 2+ at 3 mM concentration, and stimulatory effect was observed when the concentration of Mn 2+ was 9 mM (Fig. 4).Most chitosanases can be enhanced activity by Mn 2+ , for example, Csn21c from S. albolongus(4), Csn-BAC from Bacillus sp.MD-5 (29) and CsnB from Bacillus sp.BY01 (30), that were enhanced to 2.0-fold, 1.79-fold and 2.57-fold, respectively, after the addition of Mn 2+ , obviously Mn 2+ shows more stimulatory effect on SaCsn46A than other chitosanases.EDTA showed a signi cant inhibitory effect on SaCsn46A, because EDTA is a chelator of divalent cations, suggesting that this chitosanase is a metalloenzyme.
The K m value (colloidal chitosan as substrate) of SaCsn46A was 1.32 mg•mL − 1 , which was lower than that of Csn21c from S. albolongus (7.4

Hydrolytic properties of chitosanase
The hydrolysis products of colloidal chitosan (1%, w/v, 90%DDA) were detected by TLC, results are listed in Fig. 5.After 7 h incubation, chitosan was complete degraded by SaCsn46A, two clear spots could be detected on the TLC plate.The mobility ratio of these spots was in good agreement with GlcN and (GlcN) 2 markers, indicated that this enzyme can hydrolyze chitosan into GlcN and (GlcN) 2 .This conclusion was followed veri ed by high performance liquid chromatography (HPLC) analysis (Fig. 4).
There is one product peak with an appearance time of 6.192 min in Fig. 4 (c), which is consistent with the appearance time of GlcN Fig. 4 (a), and the appearance time of the other product peak is 7.245 min, which is consistent with the peak time of (GlcN)2 Fig. 4 (b).The ratio of the two hydrolytic products is 1:2.8 (GlcN:(GlcN) 2 ).As shown in TLC analysis (Fig. 4), CHOS with different DPs appeared after degraded for 5 min.After 30 min incubation, (GlcN) 2 and (GlcN)

Figures
Figure 1 Bioinformatics

Supplementary Files
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Figure 3 Effects of different Mn2+ concentration on SaCsn46A Figure 4
Figure 3
3 became main products.As the reaction continued, only two visible spots were left on the TLC plate after complete degradation.Recently, most microorganisms produced chitosanases were reported to produce DP2-7 CHOS(11-13, 16-18, 24, 25, 29,  30), only Csn21c from S. albolongus was reported that can hydrolyze chitosan into GlcN and (GlcN) 2 (4), A novel chitosanase gene was cloned from S. avermitilis and expressed in E. coli Rosetta.The recombinant enzyme has its optimal enzyme activity under condition of 45 o C in 50 mM pH 6.2 phosphate buffer, and was very stable at pH range from 4.0 to 9.0 and below 30 o C. The enzyme activity can be strongly enhanced about 3.5-fold by Mn 2+ at a 3 mM concentration.The enzyme can hydrolyze a variety of β-1,4-glycosidic bonds-linked polysaccharides which shows a good application prospect.The hydrolysis products were mainly GlcN and (GlcN) 2 which demonstrated that SaCsn46A was a good potential candidate for the industrial production of GlcN and (GlcN) 2 in mild conditions. Conclusion