Continuous production of chitooligosaccharides in a column reactor by the PUF-immobilized whole cell enzymes of Mucor circinelloides IBT-83.

BACKGROUND: Chitosan oligosaccharides (COS) have great potential for applications in several fields, including agriculture, food industry or medicine. Nevertheless, the large-scale use of COS requires the development of cost-effective technologies for their production. The main objective of our investigation was to develop an effective method of enzymatic degradation of chitosan in a column reactor using Mucor circinelloides IBT-83 cells, immobilized in a polyurethane foam (PUF). These cells serve as a source of chitosanolytic enzymes. RESULTS: The study revealed that the process of freeze-drying of immobilized mycelium increases the stability of the associated enzymes during chitosan hydrolysis. The use of stabilized preparations as an active reactor bed enables the production of COS at a constant level for 16 reactor cycles (384 h in total), i.e. 216 h longer compared to non-stabilized mycelium. In the hydrolysate, oligomers ranging in structure from dimer to hexamer as well as D-glucosamine were detected. The potential application of the obtained product in agriculture has been verified. The results of phytotests have demonstrated that the introduction of COS into the soil at a concentration of 0.01 or 0.05% w/w resulted in an increase in the growth of Lepidium sativum stem and root, respectively (extensions by 38 and 44% compared to the control sample). CONCLUSIONS: The research has verified that the PUF-immobilized M. circinelloides IBT-83 mycelium, which has been stabilized through freeze-drying, is a promising biocatalyst for the environmentally friendly and efficient generation of COS. This biocatalyst has the potential to be used in fertilizers.

Investigation on anti-quorum sensing activities of chitosan AgNP's-chitosanase against MDR pathogens.

Marine bio-nanotechnology is a new promising field having high perspective in the area of biological research. In 2018 the production of crustacean shells especially from shrimp is about 54,500 tons on South East coast of India. The current study focuses on the use of extracted chitosan (Squilla shells) polymer in silver nanoparticle synthesis along with immobilized chitosanase synergistically improves the antimicrobial and quorum quenching effects against the multi drug resistant (MDR) pathogens. The main objective of the study is to synthesize the chitosan AgNPs and to immobilize the enzyme chitosanase with it and to study the anti quorum sensing (quorum quenching) activity against MDR pathogens. This study will render a new ideology to eliminate biofilm formation and suppress the pathogenicity of planktonic MDR pathogens. Since the combinations of chitosanase, as well as chitosan AgNPs, are very efficient in eliminating them.

Preparation of chitooligosaccharides with a low degree of polymerization and anti-microbial properties using the novel chitosanase AqCsn1.

Chitosanases hydrolyze chitosan into chitooligosaccharides (COSs) with various biological activities, which are widely employed in many areas including plant disease management. In this study, the novel chitosanase AqCsn1 belonging to the glycoside hydrolase family 46 (GH46) was cloned from Aquabacterium sp. A7-Y and heterologously expressed in Escherichia coli BL21 (DE3). AqCsn1 displayed the highest hydrolytic activity towards chitosan with 95% degree of deacetylation at 40 °C and pH 5.0, with a specific activity of 13.18 U/mg. Product analysis showed that AqCsn1 hydrolyzed chitosan into (GlcN)2 and (GlcN)3 as the main products, demonstrating an endo-type cleavage pattern. Evaluation of antagonistic activity showed that the hydrolysis products of AqCsn1 suppress the mycelial growth of Magnaporthe oryzae and Phytophthora sojae in a concentration-dependent manner, and the inhibition rate of P. sojae reached 39.82% at a concentration of 8 g/L. Our study demonstrates that AqCsn1 and hydrolysis products with a low degree of polymerization might have potential applications in the biological control of agricultural diseases.

Structure-based mining of a chitosanase with distinctive degradation mode and product specificity.

Chitosan derivates with varying degrees of polymerization (DP) have attracted great concern due to their excellent biological activities. Increasing the abundance of chitosanases with different degradation modes contributes to revealing their catalytic mechanisms and facilitating the production of chitosan derivates. However, the identification of endo-chitosanases capable of producing chitobiose and D-glucosamine (GlcN) from chitosan substrates has remained elusive. Herein, an endo-chitosanase (CsnCA) belonging to the GH46 family was identified based on structural analysis in phylogenetic evolution. Moreover, we demonstrate that CsnCA acts in a random endo-acting manner, producing chitosan derivatives with DP ≤ 2. The in-depth analysis of CsnCA revealed that (GlcN)3 serves as the minimal substrate, undergoing cleavage in the mode that occupies the subsites - 2 to + 1, resulting in the release of GlcN. This study succeeded in discovering a chitosanase with distinctive degradation modes, which could facilitate the mechanistic understanding of chitosanases, further empowering the production of chitosan derivates with specific DP. KEY POINTS: • Structural docking and evolutionary analysis guide to mining the chitosanase. • The endo-chitosanase exhibits a unique GlcN-producing cleavage pattern. • The cleavage direction of chitosanase to produce GlcN was identified.

Characterization of different chitosanases of Bacillus strains and their application in chitooligosaccharides production.

Chitosanases are potential candidates for chitooligosaccharides (COS) production-based industries, therefore, the discovery of chitosanases having commercial potential will remain a priority worldwide. This study aims to characterize different chitosanases of Bacillus strains for COS production. Six different indigenous Bacillus strains (B. cereus EGE-B-6.1m, B. cereus EGE-B-2.5m, B. cereus EGE-B-5.5m, B. cereus EGE-B-10.4i, B. thuringiensis EGE-B-3.5m, and B. mojavensis EGE-B-5.2i) were used to purify and characterize chitosanases. All purified chitosanases have a similar molecular weight (37 kDa) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, other characteristics such as optimum temperature and pH, kinetic parameters (Km and Vmax ), temperature, and pH stabilities were dissimilar among the strains of different Bacillus species and within the same species. Furthermore, chitosanases of all strains were able to successfully hydrolyze chitosan to COS and oligomers of the degree of polymerization 2-6 were detected with chitobiose and chitotriose as major hydrolysis products. The relative yields of COS were in a range of 19%-31% and chitosanase of B. thuringiensis EGE-B-3.5m turned out to be the best enzyme in terms of its characteristics and COS production potential with maximum relative yield (31%). Results revealed that Bacillus chitosanases could be used directly for efficient bioconversion of chitosan into COS and will be valuable for large-scale production of biologically active COS.

A Computational Biology Study on the Structure and Dynamics Determinants of Thermal Stability of the Chitosanase from Aspergillus fumigatus.

Environmentally friendly and efficient biodegradation with chitosanase for degrading chitosan to oligosaccharide has been gaining more importance. Here, we studied a chitosanase from Aspergillus fumigatus with potential for production, but does not have the ideal thermal stability. The structure predicted by the Alphafold2 model, especially the binding site and two catalytic residues, has been found to have a high similarity with the experimental structure of the chitosanase V-CSN from the same family. The effects of temperature on structure and function were studied by dynamic simulation and the results showed that the binding site had high flexibility. After heating up from 300 K to 350 K, the RMSD and RMSF of the binding site increased significantly, in particular, the downward shift of loop6 closed the binding site, resulting in the spatial hindrance of binding. The time proportions of important hydrogen bonds at the binding site decreased sharply, indicating that serious disruption of hydrogen bonds should be the main interaction factor for conformational changes. The residues contributing energetically to binding were also revealed to be in the highly flexible region, which inevitably leads to the decrease in the activity stability at high temperature. These findings provide directions for the modification of thermal stability and perspectives on the research of proteins without experimental structures.

Green-Chemical Strategies for Production of Tailor-Made Chitooligosaccharides with Enhanced Biological Activities.

Chitooligosaccharides (COSs) are b-1,4-linked homo-oligosaccharides of N-acetylglucosamine (GlcNAc) or glucosamine (GlcN), and also include hetero-oligosaccharides composed of GlcNAc and GlcN. These sugars are of practical importance because of their various biological activities, such as antimicrobial, anti-inflammatory, antioxidant and antitumor activities, as well as triggering the innate immunity in plants. The reported data on bioactivities of COSs used to contain some uncertainties or contradictions, because the experiments were conducted with poorly characterized COS mixtures. Recently, COSs have been satisfactorily characterized with respect to their structures, especially the degree of polymerization (DP) and degree of N-acetylation (DA); thus, the structure-bioactivity relationship of COSs has become more unambiguous. To date, various green-chemical strategies involving enzymatic synthesis of COSs with designed sequences and desired biological activities have been developed. The enzymatic strategies could involve transglycosylation or glycosynthase reactions using reducing end-activated sugars as the donor substrates and chitinase/chitosanase and their mutants as the biocatalysts. Site-specific chitin deacetylases were also proposed to be applicable for this purpose. Furthermore, to improve the yields of the COS products, metabolic engineering techniques could be applied. The above-mentioned approaches will provide the opportunity to produce tailor-made COSs, leading to the enhanced utilization of chitin biomass.

Exploration of the Product Specificity of chitosanase CsnMY002 and Mutants Using Molecular Dynamics Simulations.

Chitosanase CsnMY002 is a new type of enzyme isolated from Bacillus subtilis that is used to prepare chitosan oligosaccharide. Although mutants G21R and G21K could increase Chitosan yield and thus increase the commercial value of the final product, the mechanism by which this happens is not known. Herein, we used molecular dynamics simulations to explore the conformational changes in CsnMY002 wild type and mutants when they bind substrates. The binding of substrate changed the conformation of protein, stretching and deforming the active and catalytic region. Additionally, the mutants caused different binding modes and catalysis, resulting in different degrees of polymerization of the final Chitooligosaccharide degradation product. Finally, Arg37, Ile145 ~ Gly148 and Trp204 are important catalytic residues of CsnMY002. Our study provides a basis for the engineering of chitosanases.

Purification and biochemical characterization of a novel chitosanase cloned from the gene of Kitasatospora setae KM-6054 and its application in the production of chitooligosaccharides.

Chitosanase could degrade chitosan efficiently under mild conditions to prepare chitosan oligosaccharides (COSs). COS possesses versatile physiological activities and has wide application prospects in food, pharmaceutical and cosmetic fields. Herein, a new glycoside hydrolase (GH) family 46 chitosanase (CscB) was cloned from Kitasatospora setae KM-6054 and heterologously expressed in Escherichia coli. The recombinant chitosanase CscB was purified by Ni-charged magnetic beads and showed a relative molecular weight of 29.19 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). CscB showed the maximal activity (1094.21 U/mg) at pH 6.0 and 30 °C. It was revealed that CscB is a cold-adapted enzyme. CscB was determined to be an endo-type chitosanase with a polymerization degree of the final product mainly in the range of 2-4. This new cold-adapted chitosanase provides an efficient enzyme tool for clean production of COSs.

Gene cloning and molecular characterization of a thermostable chitosanase from Bacillus cereus TY24.

BACKGROUND: An important conceptual advance in health and the environment has been recognized that enzymes play a key role in the green processing industries. Of particular interest, chitosanase is beneficial for recycling the chitosan resource and producing chitosan oligosaccharides. Also, chitosan gene expression and molecular characterization will promote understanding of the biological function of bacterial chitosanase as well as explore chitosanase for utilizing chitosan resources. RESULTS: A chitosanase-producing bacterium TY24 was isolated and identified as Bacillus cereus. Moreover, the chitosanase gene was cloned and expressed in Escherichia coli. Sequence analysis reveals that the recombinant chitosanase (CHOE) belongs to the glycoside hydrolases 8 family. The purified CHOE has a molecular weight of about 48 kDa and the specific activity of 1150 U/mg. The optimal pH and temperature of CHOE were 5.5 and 65 °C, respectively. The enzyme was observed stable at the pH range of 4.5-7.5 and the temperature range of 30-65 °C. Especially, the half-life of CHOE at 65 °C was 161 min. Additionally, the activity of CHOE was remarkably enhanced in the presence of Mn2+, Cu2+, Mg2+ and K+, beside Ca2+ at 5 mM. Especially, the activity of CHOE was enhanced to more than 120% in the presence of 1% of various surfactants. CHOE exhibited the highest substrate specificity toward colloid chitosan. CONCLUSION: A bacterial chitosanase was cloned from B. cereus and successfully expressed in E. coli (BL21) DE3. The recombinant enzyme displayed good stability under acid pH and high-temperature conditions.

Insights into promiscuous chitosanases: the known and the unknown.

Chitosanase, a glycoside hydrolase (GH), catalyzes the cleavage of ß-1,4-glycosidic bonds in polysaccharides and is widely distributed in nature. Many organisms produce chitosanases, and numerous chitosanases in the GH families have been intensely studied. The reported chitosanases mainly cleaved the inter-glucosamine glycosidic bonds, while substrate specificity is not strictly unique due to the existence of bifunctional or multifunctional activity profiles. The promiscuity of chitosanases is essential for the different pathways of biomass polysaccharide conversion and understanding of the chitosanase evolutionary process. However, the reviews for this aspect are completely unknown. This review provides an overview of the promiscuous activities, also considering the substrate and product specificity of chitosanases observed to date. These contribute to important implications for the future discovery and research of promiscuous chitosanases and applications related to biomass conversion. KEY POINTS: • The promiscuity of chitosanases is reviewed for the first time. • The current review provides insights into the substrate specificity of chitosanases. • The mode-product relationship and prospect of promiscuous chitosanases are highlighted.

Biochemical characterization and cleavage pattern analysis of a novel chitosanase with cellulase activity.

Chitosanases are critical tools for the preparation of active oligosaccharides, whose composition is related to the cleavage pattern of the enzyme. Although numerous chitosanases have been characterized, the glycoside hydrolase (GH) family 5 chitosanases with other activities have rarely been investigated. Herein, a novel and second GH5 chitosanase OUC-Csngly from Streptomyces bacillaris was cloned and further characterized by expression in Escherichia coli BL21 (DE3). Interestingly, OUC-Csngly possessed dual chitosanase and cellulase activities. Molecular docking analysis showed that the C-2 group of sugar units affected the binding of the enzyme to oligosaccharides, which could result in different cleavage patterns toward chito-oligosaccharides (COSs) and cello-oligosaccharides. Further, we characterized OUC-Csngly's distinctive cleavage patterns toward two different types of oligosaccharides. Meanwhile, endo-type chitosanase OUC-Csngly generated (GlcN) - (GlcN)4 from chitosan, was significantly different from other chitosanases. To our knowledge, this is the first report to investigate the different cleavage patterns of chitosanase for COSs and cello-oligosaccharides.Key points• The molecular docking showed C-2 group of sugar units in substrate affecting the cleavage pattern.• The first chitosanase exhibited different cleavage patterns towards chito- and cello-oligosaccharides.• The groups at C-2 influence the subsite composition of the enzyme's active cleft.

Biodegradation and Prospect of Polysaccharide from Crustaceans.

Marine crustacean waste has not been fully utilized and is a rich source of chitin. Enzymatic degradation has attracted the wide attention of researchers due to its unique biocatalytic ability to protect the environment. Chitosan (CTS) and its derivative chitosan oligosaccharides (COSs) with various biological activities can be obtained by the enzymatic degradation of chitin. Many studies have shown that chitosan and its derivatives, chitosan oligosaccharides (COSs), have beneficial properties, including lipid-lowering, anti-inflammatory and antitumor activities, and have important application value in the medical treatment field, the food industry and agriculture. In this review, we describe the classification, biochemical characteristics and catalytic mechanisms of the major degrading enzymes: chitinases, chitin deacetylases (CDAs) and chitosanases. We also introduced the technology for enzymatic design and modification and proposed the current problems and development trends of enzymatic degradation of chitin polysaccharides. The discussion on the characteristics and catalytic mechanism of chitosan-degrading enzymes will help to develop new types of hydrolases by various biotechnology methods and promote their application in chitosan.

Expression and Surface Display of an Acidic Cold-Active Chitosanase in Pichia pastoris Using Multi-Copy Expression and High-Density Cultivation.

Chitosanase hydrolyzes ß-(1,4)-linked glycosidic bonds are used in chitosan chains to release oligosaccharide mixtures. Here, we cloned and expressed a cold-adapted chitosanase (CDA, Genbank: MW094131) using multi-copy expression plasmids (CDA1/2/3/4) in Pichia pastoris. We identified elevated CDA expression levels in multi-copy strains, with strain PCDA4 selected for high-density fermentation and enzyme-activity studies. The high-density fermentation approach generated a CDA yield of 20014.8 U/mL, with temperature and pH optimization experiments revealing the highest CDA activity at 20 °C and 5.0, respectively. CDA was stable at 10 °C and 20 °C. Thus, CDA could be used at low temperatures. CDA was then displayed on P. pastoris using multi-copy expression plasmids. Then, multi-copy strains were constructed and labelled as PCDA(1-3)-AGα1. Further studies showed that the expression of CDA(1-3)-AGα1 in multi-copy strains was increased, and that strain PCDA3-AGα1 was chosen for high-density fermentation and enzyme activity studies. By using a multi-copy expression and high-density fermentation approach, we observed CDA-AGα1 expression yields of 102415 U/g dry cell weight. These data showed that the displayed CDA exhibited improved thermostability and was more stable over wider temperature and pH ranges than free CDA. In addition, displayed CDA could be reused. Thus, the data showed that displaying enzymes on P. pastoris may have applications in industrial settings.

Constitutive chitosanase from Bacillus thuringiensis B-387 and its potential for preparation of antimicrobial chitooligomers.

The article proves the ability of the entomopathogenic strain B. thuringiensis var. dendrolimus B-387 to high the constitutive production (3-12.5 U/mL) of extracellular chitosanase, that was found for the first time. The enzyme was purified in 94-fold by ultrafiltration, affinity sorption and cation-exchange chromatography and characterized biochemically. The molecular mass of the chitosanase determined using SDS-PAGE is 40 kDa. Temperature and pH-optima of the enzyme are 55 °C and pH 6.5, respectively; the chitosanase was stable under 50-60 °C and pH 4-10.5. Purified chitosanase most rapidly (Vmax ~ 43 µM/mL × min, KM ~ 0.22 mg/mL, kcat ~ 4.79 × 104 s-1) hydrolyzed soluble chitosan of the deacetylation degree (DD) 85% by endo-mode, and did not degrade colloidal chitin, CM-cellulose and some other glucans. The main reaction products of the chitosan enzymolysis included chitobiose, chitotriose and chitotetraose. In addition to small chitooligosaccharides (CHOs), the studied chitosanase also generated low-molecular weight chitosan (LMWC) with average Mw in range 14-46 kDa and recovery 14-35%, depending on the enzyme/substrate ratio and incubation temperature. In some cases, the chitosan (DD 85 and 50%) oligomers prepared using crude chitosanase from B. thuringiensis B-387 indicated higher antifungal and antibacterial activities in vitro in comparison with the initial polysaccharides. The data obtained indicate the good prospect of chitosanase B-387 for the production of bioactive CHOs.

Expression and Characterization of a Novel Cold-Adapted Chitosanase from Marine Renibacterium sp. Suitable for Chitooligosaccharides Preparation.

(1) Background: Chitooligosaccharides (COS) have numerous applications due to their excellent properties. Chitosan hydrolysis using chitosanases has been proposed as an advisable method for COS preparation. Although many chitosanases from various sources have been identified, the cold-adapted ones with high stability are still rather rare but required. (2) Methods: A novel chitosanase named CsnY from marine bacterium Renibacterium sp. Y82 was expressed in Escherichia coli, following sequence analysis. Then, the characterizations of recombinant CsnY purified through Ni-NTA affinity chromatography were conducted, including effects of pH and temperature, effects of metal ions and chemicals, and final product analysis. (3) Results: The GH46 family chitosanase CsnY possessed promising thermostability at broad temperature range (0-50 °C), and with optimal activity at 40 °C and pH 6.0, especially showing relatively high activity (over 80% of its maximum activity) at low temperatures (20-30 °C), which demonstrated the cold-adapted property. Common metal ions or chemicals had no obvious effect on CsnY except Mn2+ and Co2+. Finally, CsnY was determined to be an endo-type chitosanase generating chitodisaccharides and -trisaccharides as main products, whose total concentration reached 56.74 mM within 2 h against 2% (w/v) initial chitosan substrate. (4) Conclusions: The results suggest the cold-adapted CsnY with favorable stability has desirable potential for the industrial production of COS.

An Antifungal Chitosanase from Bacillus subtilis SH21.

Bacillus subtilis SH21 was observed to produce an antifungal protein that inhibited the growth of F. solani. To purify this protein, ammonium sulfate precipitation, gel filtration chromatography, and ion-exchange chromatography were used. The purity of the purified product was 91.33% according to high-performance liquid chromatography results. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that the molecular weight of the protein is 30.72 kDa. The results of the LC-MS/MS analysis and a subsequent sequence-database search indicated that this protein was a chitosanase, and thus, we named it chitosanase SH21. Scanning and transmission electron microscopy revealed that chitosanase SH21 appeared to inhibit the growth of F. solani by causing hyphal ablation, distortion, or abnormalities, and cell-wall depression. The minimum inhibitory concentration of chitosanase SH21 against F. solani was 68 µg/mL. Subsequently, the corresponding gene was cloned and sequenced, and sequence analysis indicated an open reading frame of 831 bp. The predicted secondary structure indicated that chitosanase SH21 has a typical a-helix from the glycoside hydrolase (GH) 46 family. The tertiary structure shared 40% similarity with that of Streptomyces sp. N174. This study provides a theoretical basis for a topical cream against fungal infections in agriculture and a selection marker on fungi.

Biochemical characterization of a novel bifunctional chitosanase from Paenibacillus barengoltzii for chitooligosaccharide production.

A novel chitosanase gene, designated as PbCsn8, was cloned from Paenibacillus barengoltzii. It shared the highest identity of 73% with the glycoside hydrolase (GH) family 8 chitosanase from Bacillus thuringiensis JAM-GG01. The gene was heterologously expressed in Bacillus subtilis as an extracellular protein, and the highest chitosanase yield of 1, 108 U/mL was obtained by high-cell density fermentation in a 5-L fermentor. The recombinant chitosanase (PbCsn8) was purified to homogeneity and biochemically characterized. PbCsn8 was most active at pH 5.5 and 70 °C, respectively. It was stable in a wide pH range of 5.0-11.0 and up to 55 °C. PbCsn8 was a bifunctional enzyme, exhibiting both chitosanase and glucanase activities, with the highest specificity towards chitosan (360 U/mg), followed by barley ß-glucan (72 U/mg) and lichenan (13 U/mg). It hydrolyzed chitosan to release mainly chitooligosaccharides (COSs) with degree of polymerization (DP) 2-3, while hydrolyzed barley ß-glucan to yield mainly glucooligosaccharides with DP > 5. PbCsn8 was further applied in COS production, and the highest COS yield of 79.3% (w/w) was obtained. This is the first report on a GH family 8 chitosanase from P. barengoltzii. The high yield and remarkable hydrolysis properties may make PbCsn8 a good candidate in industrial application.

Expression of a Beauveria bassiana chitosanase (BbCSN-1) in Pichia pastoris and enzymatic analysis of the recombinant protein.

Chitosanase (EC 3.2.1.132) is an important chitosan-degrading enzyme involved in industrial applications. In this study, a chitosanase gene (BbCSN-1) from Beauveria bassiana, an insect fungal pathogen, was cloned and expressed in Pichia pastoris. The amount of BbCSN-1 in the fermentation broth of P. pastoris gradually increased after induction with methanol from one to 6 d, reaching 398 µg/ml on the 6th day. The molecular characteristics of BbCSN-1 were measured with colloidal chitosan as a substrate. The purified BbCSN-1 exhibited optimum activity at pH 5 and 30 °C and was stable at pH 2-8 and below 40 °C. The Km value of BbCSN-1 was approximately 0.8 mg/ml at 30 °C (pH 6.0). The activity of BbCSN-1 was significantly enhanced by Mn2+ but inhibited by Co2+ and Cu2+. These results indicated that BbCSN-1 from B. bassiana could be easily expressed in P. pastoris, which provided a basis for further study on its application.

Identification of a chitosanase from the marine metagenome and its molecular improvement based on evolution data.

Chitooligosaccharides have important application value in the fields of food and agriculture. Chitosanase can degrade chitosan to obtain chitooligosaccharides. The marine metagenome contains many genes related to the degradation of chitosan. However, it is difficult to mine valuable genes from large gene resources. This study proposes a method to screen chitosanases directly from the marine metagenome. Chitosanase gene chis1754 was identified from the metagenome and heterologously expressed in Escherichia coli. The optimal temperature and pH of CHIS1754 were 55 °C and 5.5, respectively. A mutant, CHIS1754T, with 15 single point mutations designed based on molecular evolution data was also expressed in E. coli. The results indicated that the thermal stability of CHIS1754T was significantly improved, as the Tm showed an increase of ~ 7.63 °C. Additionally, the kcat/Km of CHIS1754T was 4.8-fold higher than that of the wild type. This research provides new theories and foundations for the excavation, modification, and industrial application of chitosanases. KEY POINTS: A chitosanase gene, chis1754, was firstly identified from marine metagenome. A multi-site mutant was designed to improve enzyme stability and activity. The kcat/Kmof the designed mutant was 4.8-fold higher than that of the wild type.