Identification,Modification And Application Potential Of A Novel Chitosanase With High Activity

Chitosan has great potential application value in many fields such as medicine,industry and agriculture due to its good biocompatibility,biodegradability,non-toxicity and many other unique properties.Compared with chitosan,chitosan oligosaccharides with low polymerization degree,which are resulted from the hydrolysisof chitosan,have better water solubility and higher biological activity.Therefore,the preparation and application of chitosan oligosaccharides have become a research hotspot in many fields.At present,chitosanase has become the research focus of enzymatic preparation of chitosan oligosaccharides in industrial production,but the identified chitosanases often have problems such as low enzyme activity,low expression,and poor stability,which seriously restrict their application in industrial production.In this study,a chitosanase gene CSnⅡ was found from the metagenome of marine mud,and was cloned,expressed,and investigated the basic enzymatic properties;on this basis,CSnⅡwas truncated,and the soluble expression,specific enzyme activity and stability of the modified enzyme CSnⅡ-TB were significantly improved;then,the shake flask fermentation conditions of CSnⅡ-TB were optimized;finally,the conditions for the preparation of chitosan oligosaccharides by using CSnⅡ-TB degrading chitosan were preliminarily investigated,which laid the foundation for the application of this enzyme.The results are as follows:1.Cloning,expression and analysis of basic enzymatic properties of CSnⅡ:the molecular mass of CSnⅡ is 31.4 kDa,and it is mainly expressed in the form of inclusion bodies;after renaturation of inclusion bodies,the optimal reaction temperature and buffer of CSnⅡ are 45℃ and pH 5.6 0.2 M NaAc-HAc buffer solution,Mn2+can significantly promote the enzymatic activity,2.5 mM Mn2+ can increase the enzylatic activity of CSnⅡ up to 2 times,while Fe3+and Cu2+can strongly inhibit the enzymatic activity;under the optimal reaction conditions,the specific enzyme activity of CSnⅡwas 799.08±39.80 IU/mg.In addition,when analyzing the recombinant expression of CSnⅡ by SDS-PAGE,it was observed that there was a protein band with a high watersoluble expression and a lower molecular weight(-28 kDa)below the full-length CSnⅡprotein band,which was presumed to be the product of CSnⅡ digested by endogenous enzyme during its expression,suggesting that the water-soluble expression of CSnⅡcan be improved by truncation modification.2.Truncation modification and basic enzymatic properties of CSnⅡ:first,the water-soluble fragment of about 28 kDa produced during the expression of CSnⅡ was purified and analyzed by protein spectrum,which proved that the protein was the product of CSnⅡ missing 35 amino acids from the N-terminus,and based on the result,the truncated form(CSnⅡ-TB)of CSnⅡ was obtained through cloning and expression.The molecular weight of CSnII-TB is 28.3 kDa,and has good water-soluble expression;the optimal reaction temperature and buffer of CSnⅡ-TB are 45℃ and 0.2 M NaAcHAc buffer(pH 5.6);similar to CSnⅡ,Mn2+ has the strongest stimulation to CSnⅡ-TB,2.5 mM Mn2+can increase the enzymatic activity of CSnⅡ-TB up to 2.5 times,while Fe3+and Cu2+strongly inhibit the activity of CSnⅡ-TB;under the optimal reaction conditions,the specific enzyme activity of CSnⅡ-TB reaches 1744.98±75.93 IU/mg,which is nearly 2.2 times of CSnⅡ before modification;the substrate action mode and final product analysis showed that the final product of CSnⅡ-TB degrading chitosan was mainly DP2-4 oligosaccharides,and the 2-N-Acetylation of GlcN residues inhibited the enzymatic degradation of the glycan chain.3.Preliminary determination of CSnⅡ-TB shake flask fermentation conditions:For the large-scale fermentation production of CSnⅡ-TB in the future,we explored and optimized the carbon/nitrogen sources and fermentation conditions required for the shake flask fermentation of this enzyme.The experimental results showed that the optimal medium composition for CSnⅡ-TB shake flask fermentation was 1%glycerol,2.5%tryptone,0.2%K2HPO4+0.1%KH2PO4 buffer system and 0.07%MgSO4;the induction expression conditions were initial pH 6.5,induction expression at 25℃,3%inoculum volum,and bottle volume of 50 mL/250 mL.Under these fermentation conditions,the enzyme activity of CSnⅡ-TB fermentation broth reached 651.12±19.59 U/mL,which was 2.32 times of enzymatic activity obtained from the conventional fermentation in LB medium,and thus had the potential for further large-scale fermentation production and application.4.Preliminary study on the application potential of CSnII-TB in the enzymatic preparation of chitosan oligosaccharides:through preliminary optimization of enzymatic hydrolysis conditions such as chitosan concentration,enzyme dosage,and reaction time,it is basically determined that when the chitosan concentration is 10%,and the mass ratio of enzyme to substrate is 1:70,000,the reaction time is 8 hours,the polymerization degree of chitosan oligosaccharides produced by the enzymatic digestion is di-to heptasaccharides,of which the tetra-to hexasaccharides considered to have high biological activity are the main components,indicating that the enzyme has great application potential.In summary,the novel chitosanase CSnⅡ-TB obtained in this study has high enzyme activity,easy recombinant expression,good stability,and low polymerization degree of the product,and its reaction conditions are suitable for the large-scale production of low polymerization chitosan oligosaccharides with high added value,and thus has a good application prospect.