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 3D-structure of SaCsn46A. These results suggest that SaCsn46A is a novel member of family GH46.
Heterologous expression and purification 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 purified to electrophoretic homogeneity by a Ni-NTA affinity column (result was shown in Fig. S1). The molecular weight of purified 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 specificity
The specific activity of SaCsn46A was determined after purification step with Ni-NTA column was increased from 121.2 U∙mg− 1 to 424.2 U∙mg− 1, obtaining a purification fold of 3.5 and recovery yield of 78% (Results are listed in Table 1).
Table 1
Effects of different subtracts on SaCsn46A
Subtracts | Relative activity (%) |
Colloidal chitosan | 100 ± 1.67 |
Power chitosan | 1.77 ± 0.12 |
Colloidal chitin | 1.77 ± 0.22 |
Power chitin | 1.48 ± 0.17 |
Cellulose | 0.46 ± 0.09 |
Carboxymethyl cellulose | 2.39 ± 0.15 |
Xylan | 2.23 ± 0.17 |
Soluble starch | N.D. |
Dextrin | N.D. |
N.D., the enzyme activity was not detected. |
In order to determine the substrate specificity 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.
Table 2
Effects of different ions on SaCsn46A
Ions | Relative activity (%) | |
| 1 mM | 2 mM |
Control | 100 ± 1.89 | 100 ± 2.18 |
Cu2+ | 95.32 ± 2.45 | 46.76 ± 2.34 |
Fe2+ | 103.20 ± 2.04 | 107.25 ± 1.51 |
K+ | 100.67 ± 1.88 | 79.92 ± 2.52 |
Mg2+ | 103.73 ± 1.98 | 127.85 ± 1.74 |
Ba2+ | 92.79 ± 1.94 | 76.94 ± 2.43 |
Ca2+ | 87.85 ± 1.59 | 63.47 ± 1.89 |
Zn2+ | 89.05 ± 2.62 | 48.70 ± 2.09 |
EDTA | 34.56 ± 3.28 | 21.61 ± 2.79 |
Biochemical characterization of the purified chitosanase
The optimal temperature for SaCsn46A was determined in different temperature ranges (20–90°C). The maximum relative chitosanase activity was observed at 45 oC (Fig. 3a), similar optimal temperature (45 oC) has been reported from Paenibacillus dendritiformis chitosanase(25), while was higher than that of Gynuella sunshinyii chitosanase (30 oC)(17), Bacillus sp. BY01 chitosanase (35 oC)(30), Bacillus sp. MD-5 chitosanase (40 oC)(29) and S. capitis chitosanase (40 oC)(24), and lower than Bacillus sp. chitosanase (60 oC)(13), B. amyloliquefaciens chitosanase (55 oC)(12) and S. albolongus chitosanase (50 oC)(4). 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 oC (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 3.6–9.8 below 30 oC)(13).
The effects of metal ions on the activity of SaCsn46A are presented in Table 3. The enzyme activity was inhibited by Cu2+, Ba2+, Ca2+ and Zn2+ at both 1 mM and 2 mM concentration. The enzyme activity was inhibited by K+ at 2 mM (79.92%). K+ and Mg2+of 1mM has no effect on enzyme activity. Fe2+ 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 Mn2+ at 3 mM concentration, and stimulatory effect was observed when the concentration of Mn2+ was 9 mM (Fig. 4). Most chitosanases can be enhanced activity by Mn2+, 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 Mn2+, obviously Mn2+ shows more stimulatory effect on SaCsn46A than other chitosanases. EDTA showed a significant inhibitory effect on SaCsn46A, because EDTA is a chelator of divalent cations, suggesting that this chitosanase is a metalloenzyme.
The Km 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 mg∙mL− 1)(4), BaCsn46A from B. amyloliquefaciens (2.8 mg∙mL− 1)(18), GsCsn46A from G. sunshinyii (1.97 mg∙mL− 1)(17). Notably, this value is very low, indicating a high affinity with the substrate. The Vmax value of SaCsn46A was determined that was 526.32 µM∙mg− 1∙min− 1,which was higher than that of Csn21c from S. albolongus (263.1 µM∙mg− 1∙min− 1) and GsCsn46A from G. sunshinyii (358.65 µM∙mg− 1∙min− 1)(17), while was much lower than BaCsn46A from B. amyloliquefaciens (7142.9 µM∙mg− 1∙min− 1)(18).
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 verified 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)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), however, the hydrolysis behavior of SaCsn46A is completely different from that of Csn21c. When Csn21c hydrolyzes chitosan, GlcN was existed at the initial stage of the hydrolysis process, however, SaCsn46A hydrolyzes chitosan, higher DP products appear first, then gradually degrade into GlcN and (GlcN)2, that indicate SaCsn46A is an endo-type enzyme catalyzing the cleavage of β-1,4-glycosidic linkage. Although this hydrolysis characteristic of SaCsn46A is similar to other reported GH46 family chitosanases, hydrolysates of SaCsn46A have lower degree of polymerization.