Cloning,Expression And Molecular Modification Of Chitinase

Chitooligosaccharides?CHOS?are widely used in cosmetic,medicine,food and agriculture fields because of its good water-soluble,inoxidizability and antibacterial properties.Chitinase can hydrolyze the?-1,4 glycosidic bonds of chitin to form CHOS.Compared with the traditional chemical method for chitin degradation,enzymatic method has the advantages of mild reaction,high product purity and no environmental pollution,so it has become an important tool for the production of CHOS.However,the low catalytic efficiency of chitinase by wild strains,limits its production and application.In this study,we selected Bacillus subtilis WB600 as the expression host,and the chitinase gene?chisb?from Bacillus sp.DAU101 to construct the expression vector.Finally,the expression level and catalytic efficiency of Chisb were improved by optimizing the expression elements in the expression vector,and using molecular docking,site directed mutagenesis and error-prone PCR.In addition,the concentration and substrate conversion rate of CHOS were further improved.Meanwhile,the enzymatic properties of the original enzyme and the mutants were investigated.The main contents and conclusions of the paper are as follows:?1?A recombinant expression plasmid containing the chitinase gene?chisb?from Bacillus sp.DAU101 was constructed,and successfully expressed in B.subtilis WB600.?2?The expression elements of the plasmid pP43NMK_chisb-sp-his were optimized.Through the signal peptides screening,the ribosome binding site?RBS?optimization,promoters substitution and fusing peptides at N-terminal,the enzyme activity of Chisb was improved to 58 U?mL^-1.?3?We selected the amino acids in the conserved region?DXDXE?of Chisb for site-directed mutation,and confirmed the important role of amino acids at positions D193,D195and E197 in the Chisb catalytic domain.Homologous simulation was used to construct a three-dimensional protein structure model of Chisb,and?GlcNAc?₅ was used for molecular docking with Chisb by Discovery Studio?DS?.By site-directed mutation,the only positive mutant F273W was obtained,which had a specific activity of 249.62 U?mg^-1,and it was 1.45-fold higher than the original enzyme.In order to further improve the catalytic activity of Chisb,we used the error-prone PCR and high-throughput screening technology for directed evolution.Finally,we screened two positive mutants of C43D and E336R.The specific activity of C43D and E336R were 264 U?mg^-11 and 280 U?mg^-1,respectively,which was 1.53-fold and 1.62-fold of the original enzyme.?4?The hydrolysis products analyzed by HPLC were GlcNAc,?GlcNAc?₂,?GlcNAc?₃ and?GlcNAc?₅.The CHOS concentration of C43D,E336R and F273W were 2.06 g?L^-1,2.53 g?L^-1and 2.02 g?L^-1,improved by 2.31 times,2.84 times and 2.27 times compared with the control,respectively.In addition,the substrate conversion rate of mutants C43D,E336R and F273W were 68.7%,84.3%and 67.3%,improved by 39%,54.6%and 37.6%compared with the control,respectively.?5?The optimum temperature of the original enzyme was 60oC,and the optimum pH of the original enzyme was 5.0.In addition,the optimum temperature of C43D and E336R were55oC,F273W was 60oC;the optimum pH of C43D and F273W were 5.0,E336R was 9.0.It was also found that the original enzyme had strong substrate specificity,and only had the catalytic ability on colloidal chitin and CHOS with different degrees of polymerization.Moreover,adding Mn²⁺to the reaction could significantly improve the enzyme activity of the original enzyme and its mutants.The kinetic parameters were also measured,compared with the original enzyme,the substrate affinity and the catalytic efficiency of these mutants were significantly improved.