Biochemical Characterization And Inhibitor Screening Of Chitin Deacetylases 7 From Bombyx Mori

Chitin deacetylase(EC 3.5.1.41,CDA)is an enzyme of great significance involved in chitin modification.It could hydrolyze the acetyl group of N-acetyl-D-glucosamine,the basic structural unit of chitin,to generate chitosan and release acetic acid.CDAs are widely distributed in the cuticle,trachea,midgut and other chitin-rich organism of insects,and interference with the expression of CDAs will result in abnormal development of insects.Therefore,CDAs are potential pest control targets,and the study of inhibitors toward CDAs can develop green biological pesticides.However,only fungal and bacterial CDA inhibitors have been studied,and no insect CDAs inhibitors have been reported to date.The structure of insect CDAs protein are less similar to that of fungal and bacterial CDA.Previous studies have limited significance for the development of insect CDAs inhibitors.In this study,we focused on BmCDA7,a CDA from the midgut of Bombyx mori,and several assays were carried out for the purpose of inhibitor development:(1)Firstly,recombinant protein BmCDA7 expressed in Pichia pastoris and purified by metal chelate chromatography followed with ion exchange chromatography from the fermentation broth to obtain high purity proteins;(2)Secondly,the catalytic activity of recombinant protein BmCDA7 was analyzed qualitatively through polyacrylamide gel electrophoresis(PAGE)combined with Calcofluor White M2R(CFW)staining.The activity staining results revealed that the purified BmCDA7 protein in gel showed a clear white fluorescence band under the UV-transilluminator,indicating that the recombinant protein BmCDA7 could deacetylate glycol chitin substrate presented in gel in vitro;(3)Thirdly,the catalytic activity and biochemical properties of recombinant protein BmCDA7 against a variety of substrates was quantitatively determined based on the determination of acetic acid release.When glycol chitin was used as a substrate,BmCDA7 revealed a broad optimum reaction temperature ranged from 50 °C to 65 °C and also exhibited high activity over a wide pH range 7.0–11.0.In addition,BmCDA7 exhibited high stability below 60 °C and under the alkaline conditions.BmCDA7 is extremely sensitive to the acidic conditions.BmCDA7 was less active when incubated with the substrate at low pH,and its enzyme activity reduced significantly within the acidic range.BmCDA7 showed activity of 2.9926 and 0.4270 ?mol/min/?mol against glycol chitin and colloidal chitin,respectively;(4)Finally,we verified the inhibitory effect of 23 compounds on the enzymatic activity of BmCDA7.The results showed that two of the compounds,ZINC33253064 and ZINC33253026,have inhibitory effects on BmCDA7 activity,and the inhibitory rates were(31.01±2.17)% and(25.53±1.62)%,respectively.In conclusion,based on the fine separation and purification of recombinant BmCDA7 followed by the research on its biochemical properties and inhibitors,we have had a better knowledge of the catalytic properties of chitin deacetylase in silkworm and this work also assisted in the development of specific biopesticide for pest control.