Cellulases Mining From Reticulitermes Perilucifugus Gut Based On Proteomics

As clean and renewable biomass energy,lignocellulose has the largest reserves on the earth.Making full use of the lignocellulose is effective to relieve the energy crisis and environmental pollution from fossil fuels.With a efficiency lignocellulose degradation enzymes system consists of endogenous enzymes and enzymes produced by intestinal symbiotic,termite has the most efficient ablity to degrade lignocellulose,and it is considered as the most efficient natural mechanism of lignocellulose degradation in the nature.The research object of this study is Reticulitermes perilucifugus,isotope labeling relative and absolute quantitative(i TRAQ)technology was used to study the kinds of protein in termites and the expression difference of pertein between promesenteron with hindgut,analysis the main functions and the metabolic pathways of the differential protein,explore the enzymatic mechanism of the lignocellulose degradation in ternites gut.Using reverse transcription PCR and RACE technology to clone cellulase of Reticulitermes perilucifugus,two new ?-glucosidase were heterologous expressed in pichia expression system,and their crude enzyme activities were detect after inducing.Resultes are followed:1)Proreins in the gut of R.perilucifugus were identificated and quantitative analysised by i TRAQ technology,1925 proteins were identificated in the whole gut and 411 different proteins between promesenteron with hindgut was found through quantitative analysis,including 222 up-regulation proteins and 189 down-regulation proteins.Through GO annotation function and KEGG pathway analysis,the differential proteins mainly involved in binding,catalytic activity functions and mainly involved in the Ribosome,Carbon metabolism and other metabolic pathways.2)29 kinds of lignocellulose degradation enzymes was identified in the gut of R.perilucifugus,analyzed the distribution of lignocellulolytic enzymes in termites gut based on quantitative information,speculated that the termite digestive process of lignocellulose: lignocellulose food was lapped in the foregut and endogenous EGs and BGs mixed into the food at the same time.Symbiotic protists in hindgut endocytosed wood particles into their cell and secreted CBHs into the food vacuoles to continue to digest the lignocellulose.In the enteric cavity of hindgut,BG enzyme hydrolysis of oligosaccharides is more than that in foregut and midgut.Hemicellulase and lignin enzymes are distributed in the whole gut,the hemicellulases acted on the corresponding hemicellulose component,which causes the hemicellulose to crack to make it easier for cellulose to combine with cellulose.Lignin enaymes protected digestive enzymes and gut symbionts from xenobiotic/oxidative stress created during lignocellulose degradation.Lignin can not be degraded and excreted in faeces.3)Using reverse transcription PCR and RACE method amplified 5 kinds of cellulose genes from the gut of R.perilucifugus,inclouding 2 kinds of cellobiohydrolase(NO.1 and 2)from symbiotic protists,1 kind of endogenous endoglucanase(NO.9)and 2 kings of endogenous Beta-glucosidase(NO.7 and 8).The protein and gene sequences of 5 kinds of cloned cellulose wre aligned and analysised,the most similar gene in Gene Bank Database of NO.7 and 8 protein is JN565079,with the similarity around 70% in nucleotide sequence and 80% in protein amino acid sequence.Both the above two enzymes endogenous beta-glucosidase can be seen as two new beta-glucoside enzyme.4)Two new endogenous ?-glucosidase from the gut of R.perilucifugus were heterologous expressed in pichia expression system,and their crude enzyme activities were detect after inducing.The crude enzyme activities of two transformations of two kinds of ?-glucosidases were detected,the crude enzyme activities of NO.7 protein,intracellular is 4.43 U and extracellular is 7.47 U;the crude enzyme activities of NO.8 protein,intracellular is 5.93 U and extracellular is 9.00 U.The crude enzyme activities of NO.8 is higher than that of NO.7,and both the crude enzyme activities of extracellular is higher than that of intracellular.Through proteome research and cellulase of minning from the gut of R.perilucifugus,this study further clarify the process of the lignocellulose degradation in termites gut,and enriched the number of termites cellulases in database,provide theoretical basis and data support to build a new and efficient system of lignocellulose degradation technologies.