Breeding And Enzymatic Properties Of High Yield Chitinase-producing Strain

Chitin,a linear polysaccharide of ?-l,4-linked N-acetyl-D-glucosamine?GlcNAc?,is the second abundant natural resource next to the cellulose.As a member of the glycosyl hydrolase family,chitinase can catalyze chitin into N-acetyl-D-glucosamine or chitosan oligosaccharide.Currently,chitinase and chitosan oligosaccharides are applied in various fields,such as industry,agriculture and biomedicine.However,large-scale applications are greatly limited by the low yield and low activity of chitinase by microoragnisms.In current study,gene cloning and expression,site directed mutagenesis were used to improve the yield and activity of the recombinant chitinase.The recombinant chitinase was purified and characterized,and the products were analyzed.The main results are as follows:1.In this paper,the chitinase gene GF21-2 from Serratia marcescens GF-21 was cloned into pPICZa-C named pPICZa-C-GF2]-2 and was successfully expressed in Pichia pastoris.The activity of recombinant chitinase rchiGF21-2 was 2.1 U/mL.2.The recombinant chitinase rchiGF21-2 was purified by Ni-NTA affinity chromatography and SDS-PAGE showed a monomer with a molecular mass of approximate 55 kDa and a specific activity of 3.84 U/mg.The optimum catalytic pH was 6.0 and it was stable in the pH range from 5.0 to 11.0.The enzyme showed the highest activity at 55? and retained its most activity after incubation below 45?.The half-life of rchiGF21-2 was 349.2 minutes at 45? and was only 1.9 minutes at 60?.Ag+,Cat+,Mn²⁺,Co²⁺,Hg+ and SDS showed different inhibitory effects on the enzyme,while Hg+ and SDS can inhibit it totally.The recombinant chitinase rchiGF21-2 could specifically hydrolyze colloidal chitin and the kinetic parameter Km,Vma,and kcat were 8.68 g/L,3.88 tmol/?mL·min?and 9.22 s-1 respect:ively.3.The three-dimensional structure of rchiGF21-2 was predicted by the method of homologous modeling and optimized by Chiron server.The text results of PROCHECK,ERRAT,Verify-3D and PROSA showed that the model was reasonable.The semi-flexible docking of molecules was carried out by Autodock Tools and the optimal docking conformation was analyzed by Autodock tools analyze and pymol software.The mutation sites?F12A?P14A,F191 A,S341A and G406A?were determined referring to the literature reports.The activity of mutant G406A increased by 12.5%compared with the original enzyme.4.Chitin oligosaccharides were prepared by enzymatic method.The optimum enzymatic hydrolysis conditions contain 45?,pH6.0,8 h,1.0%substrate concentration and 10 U enzyme.The hydrolysis level was 72%under the optimum conditions.It showed that the recombinant enzyme rchiGF21-2 could specifically hydrolyze colloidal chitin into?G1cNAc?2 as the major product and was verified to be an exo-chitinase through the analysis of TLC?HPLC?ESI-MS.