| Chitin is a kind of natural polysaccharide composed of N-acetyl-D-glucosamine(GlcNAc)linked with ?-1,4-linkage,which are the main component of fungal cell wall,shrimp shell,crab shell and insect shell.Chitinase is a kind of glycoside hydrolase that can degrade chitin with production of chitin oligosaccharides or chitosan oligosaccharides,which are widely distributed in crustaceans,insects,plants,microbes,even some viruses.Chitinase can inhibit the growth of fungi and kill insects by damaging the cell wall and the shell.It has been reported that plants got obviously increased resistance to pathogens and insects when transferred in the chitinase gene.Moreover,it can be beneficial for environment by using chitinase to deal with the shell wastes and replace the traditional chemical method of chitin degradation.At the same time,the oligosaccharides and chitosan oligosaccharides produced by chitin degradation can be used as cosmetics,moisturizers,food additives and tumor inhibitors with the properties of hygroscopicity,antibacterial activity and tumor-suppressor activity.Accordingly,chitinase is of great interest of research and application on agriculture,industry,medicine and environment protection.Microorganism chitinase is the main field among various chitinases from different sources due to its properties of easy operation,short growth cycle and good economic benefits.Vibrio cholerae has great potential of degrading chitin because it can use chitin as the main carbon source when living in water environment.It has been reported that Vibrio cholerae produce a set of different chitin binding proteins and chitinases to degrade natural chitin efficiently by synergistic work.After the analysis,we finally chose VC1073,VCA0027 and VC1952 to research their degradation activity to chitin.What's more,we also tried to explain the catalytic mechanism of these three proteins by analyzing the crystal structure.We got the fragments of VC 1073,VCA0027 and VC 1952 without the N-terminal signal peptide since we failed to purify the full-length proteins.VC 1073,VCA0027 and VC 1952 all showed the obvious chitin degradation activity in the in vitro activity test.Then we identified the optimizing pH of these three proteins and the values turned out to be 6.0,6.0 and 5.0 respectively.By the way,the enzymes can retain>50%of activity in the pH value ranging from 5.0 to 8.5.The activity of VC1073,VCA0027 and VC 1952 would be inhibited by different metal ions and chemical reagents in different extent,while they were all strongly inhibited by Fe3+ and SDS with only<50%or<30%of activity retained respectively.We also identified the substrate specificity of VC1073,VCA0027 and VC1952,it showed that their degradation activity to colloidal-chitin is of little difference with commercial chitinase C-6137(Sigma-aldrich).An important discovery is that VC 1073 and VCA0027 showed great degradation activity to powder chitin and shrimp shell that normally were difficult to be hydrolyzed directly,which limited the utilization of chitin.Thus,VC 1073 and VCA0027 showed a prospect in the future research of chitin utilization.To identify whether the three proteins have inhibitive effect on fungi growth,we employed the Trichoderma viride and Aspergillus niger as the experimental subjects.VCA0027 and VC 1952 did not show obvious effect on the growth of Trichoderma viride and Aspergillus niger.While VC 1073 exhibited inhibition to the growth of Trichoderma viride to a certain extent,which laying the foundation for future research of the prophylaxis and treatment to pathogenic fungi of VC1073.Furthermore,we tried to get the crystal structure of these three proteins to further explain the catalytic mechanism of them.Unfortunately,we failed to get the protein crystal so there are further research need be carried out. |
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