Gene Cloning And Expression Of Chitinase From Microorganisms Isolated From Various Environments

Chitin is a biotic macromolecular polymer of a N-acetyl-D-glucosamine(NAG)linked with ?-1,4 glycosidic bonds.Chitinase catalyzes the hydrolysis of chitin into chitooligosaccharides and N-acetyl-D-glucosamine.By enzymatic degradation of shellfish waste to produce chitooligosaccharides,it can achieve high-value waste utilization and increase economic efficiency.In this study,40 microorganisms of chitinase-producing were isolated from 15 different samples.26 chitinase gene fragments were gained.Two chitinase genes were cloned,expressed and characterized.The details of results were listed as follows:(1)We collected samples from some special circumstances(such as Beihai mangroves,Dabusu salt lake etc.),and isolated 40 strains which had chitinase-produc ing capabilities.By 16s rDNA sequence analysis,isolated microorganisms of chitinase-producing were mainly belong to the genus such as Vibrio,Microbulbifer,Aeromonas.The characterization of chitinase crude enzyme showed that the optimum reaction temperature was generally between 40??60?,the optimum pH was generally between 5.0?6.0.(2)Selected strains with higher enzyme activity or specific enzymatic properties,designed degenerate primers,used PCR to aplificate chitinase gene and then by sequencing analysis,25 gene fragments were obtained.Sequence analysis showed that these genes had identities from 67%to 100%with known sequences.Among them,the highest identities of chiG31-2 genes was only 67%,it was a relatively novel gene.(3)By TAIL-PCR,we cloned two full-length chitinase genes(chiGR52-1 and chiGR52-2)from Vibrio sp.GR52.Sequence analysis showed that two genes respectively have a total length of 2553 bp and 2385 bp,each of them encoded 850 amino acids and 794 amino acids.Blastp analysis showed that the two proteins were chitinase and the highest identities in known proteins were 80%and 78%,which sources from Vibrio fluvialis.They were relatively novel enzyme proteins.(4)Chitinase genes(chiGR52-land chiGR52-2)were expressed in Escherichia coli BL21(DE3),the recombinant proteins were purified by N i-affinity chromatography.The optima reaction pH and temperature of recombinant protein rChiGR52-1 was 6.0 and 50?.And the enzyme was stable under 45? and pH5.0?10.0.By used colloidal chitin as substrate,the Vmax and Km was 4.829 ?mol/(mg·min)and 1.302 mg/mL.For rChiGR52-2,the optima reaction pH and temperature was 6.0 and 50?.By used colloidal chitin was as substrate,the Vmax and Km was 0.9606?mol/(mg-min)and 2.777 mg/mL.In this study,we isolated chitinase-producing microorganisms from various environments,and then cloned,expressed chitinase gene,studied the enzyme properties.These results may pave the way for mining new chitinase gene and the application of chitinase in industry in the future.