Sequence Analysis And Cloning And Expression Of Chitinase From Streptomyces Diastaticus CS1801

Chitinases are a class of glycosidic bond hydrolases that catalyze the formation of chitin from β-1,4 glycosidic bonds and specifically degrade them into chitooligosaccharides,chitosan and N-acetylglucosamine.The product of chitin degradation by chitinase,chitooligosaccharide,is widely used in cosmetics,pharmaceuticals,food and pesticides because of its good water solubility,antioxidant and antibacterial properties.Compared with the traditional chemical degradation of chitin,the enzymatic production of chitin oligosaccharides by chitinase has become the development direction of chitin oligosaccharide preparation with mild conditions and little environmental pollution.However,the low catalytic efficiency of chitinases produced by wild bacteria and the availability of only a few mucilaginous Serratia marcescens,Streptomyces and Trichoderma for industrial production limit their production and application.In this study,E.coli BL21(DE3)was used as the expression host to obtain a highly active recombinant chitinase with a short culture cycle and high catalytic efficiency.The chitinase PROKKA01070 gene from Streptomyces diastaticus CS1801 was analyzed by bioinformatics to predict the properties and structure of chitinase;the highly active recombinant chitinase was obtained by constructing an expression vector to transform E.coli BL21(DE3)and induce expression;the key amino acid residues that play a catalytic role in the structure of chitinase were analyzed and identified.At the same time,the enzymatic properties of the gibberellinase were investigated to provide a theoretical basis for the application of gibberellinase engineering bacteria in industrial production.The main results of the study are as follows:(1)Bioinformatics analysis of chitinases.The gene sequence of chitinase was analyzed using bioinformatics database and software.The analysis of physicochemical properties showed that the enzyme encodes 354 amino acids,with a molecular weight of 38 kDa and a theoretical isoelectric point pI of 4.24.The enzyme was judged to be a stable protein by stability index and a hydrophilic protein with a hydrophilic mean value of-0.363.The analysis of transmembrane domain and signal peptide showed that the enzyme did not exercise transmembrane function and no signal peptide existed;secondary and tertiary structure prediction showed that the secondary structure of the chitinase consisted of 21.75%helix,67.23%loop and 11.02%strand,and the tertiary structure consisted of α-helical inner barrel andβ-folded outer barrel;amino acid sequence comparison of the catalytic domain of the chitinase revealed that The amino acid sequences of the catalytic domain of the enzyme were compared,and it was found that the enzyme had the typical 18-family conserved regions "DxxDxDxE" and "SxGG" in the catalytic domain,and the enzyme belonged to the 18-family glycoside hydrolases.(2)Clonal expression of chitinase from Streptomyces diastaticus CS1801.By PCR amplification and TA cloning of the chitinase PROKKA01070 gene,a chitinase DNA fragment of approximately 1065 bp in size and the cloning vector ChiA/pMD19-T were obtained;by constructing the expression vector and prokaryotic expression,the recombinant plasmid ChiA/pET-32a(+)and recombinant engineered bacteria ChiA/pET-32a(+)/BL21(DE3);induced expression,SDS-PAGE analysis and enzyme activity assay of recombinant E.coli ChiA/pET-32a(+)/BL21(DE3)to obtain soluble chitinase of size 38 kDa with an enzyme activity of 0.132 U·mL-1,which is 32%higher than the original enzyme activity(0.1 U·mL-1);targeted mutation of the active amino acids in the catalytic domain of chitinase to verify The amino acid residue sites of the catalytic activity of the enzyme were D128,D130 and E132.(3)Study of the enzymatic properties of recombinant chitinase.The optimum reaction temperature of recombinant chitinase was 50℃ and the optimum reaction pH was 7.0;studies on the thermal and pH stability of recombinant chitinase found that recombinant chitinase was most stable at 25℃ and pH 5.0 buffer;metal ions and chemical reagents had effects on the activity of recombinant chitinase,Zn2+and SDS had strong inhibitory effects on the enzyme at three concentrations,Na+ and Li+at any concentration,Fe3+ and Mg2+ and Ca2+ at 1 mM,had a promotion effect on the recombinant enzyme;the study on the substrate specificity of recombinant chitinase found that the recombinant chitinase had the strongest ability to decompose colloidal chitin at 100%,and the ability to decompose powder chitin,shrimp shell powder and crab shell powder was very weak at 9.8%,6.5%and 5.8%,respectively,and could not decompose Chitosan and carboxymethyl cellulose sodium;Km was 0.281 mg·mL-1,Vmax was 0.536 μM·mL-1·min-1,and catalytic efficiency was 21.2 mL·s-1·mg-1 when colloidal chitin was used as substrate.