Expression And Purification Of Thermophilic Xylanase Gene In Pichia Pastoris And Study On Enzymatic Properties

Hemicellulose is an important component of plant cell walls,in the nature of carbohydrate quantity after semi fiber,is a kind of important renewable energy,which is an important component of xylanhemicellulose.But the structure of xylan degration process is very complex,it needs severalenzymes assist in action,including xylanase could be the hydrolysis of xylaninto xylooligo saccharides of various length and a small amount of xylose.Therefore,xylanase could be hemicellulose degraded into small molecular substances can be used,improving the utilization efficiency,is therestriction enzyme degradation of hemicellulose.Xylanase is widely used in the field of industrial production.However,the xylanase activity is low,unstable factors limit its application in industry.In recent years,improve xylanase activity and stability has become the world's hot spots,including construction of genetic engineering bacteria is one of the ways.Xylanase has a wide application prospect in the industry,but it is easy to cause the inactivation of enzymes due to the high temperature in the environment of industrial production,and the industrial demand of the heat resistance and the stability of the enzyme become higher and higher.Heat resistant enzymes produced by thermophiles have broad prospect in solving enzyme inactivation.Thermostable enzyme isolated from hot springs in the strains in the lab and cloned thermostable xylanase gene,combination of molecular biology and gene engineering technology,the thermostable xylanase was cloned into Pichia pastoris to construct the engineering bacteria which can adapt to industrial environment and produce thermostable xylanases.The conclusions are as follows:The sequencing results showed that,by using pGEX-4T-2-XynF and pGEX-4T-2-XynR synthesis of xylanase gene is an open reading frame of 1204bp,encoding 407 amino acids.Signal peptide of gene xylanase XynA is between twenty-eighth and twenty-ninth amino acids.In the process of protein synthesis,the signal peptide is a signal of synthesis extracellular proteins,however,because the signal pPIC9K carrier itself with the synthesis of extracellular proteins signal,so using pPIC9K-XynF and pPIC9K-XynR synthesis of xylanase gene fragment of approximately 1080bp.Because the pPIC9K carrier size is about 9300bp,the recombinant plasmid of pPIC9K-XynA size is about 10320bp.After linearization by clicking into Pichia pastoris,through the medium of MD and PCR can be screened for positive clones.By sequence comparison,similarity in Pichia pastoris expression of xylanase gene and expression in Escherichia coli of xylanase is about 93%.Pichia pastoris AOX1 gene promoter can be used to regulate the expression of exogenous gene,methanol is the induction of alcohol oxidase promoter of AOX1 gene,through the analysis of enzyme producing conditions,at a temperature of 30?,pH 4,inducer concentration is 1%,the induction time of 72h enzyme activity reached the maximum.Because the xylanase produced by the Escherichia coli expression system is intracellular enzyme,xylanase is released from the cell through ultrasonic wall breaking process the purification process,then use the GST purification column to obtain purified xylanase.While the xylanase produced by the Pichia pastoris expression system is extracellular enzyme,purification process is simpler than Escherichia coli.Only through ammonium sulfate,G-25 and G-75 and other steps to obtain the purified xylanase,and the G-25 and G-75 can be reused.In summary,the Pichia pastoris expression system of xylanase has more widely application value than Escherichia coli expression system of xylanase.Xylanase enzyme spectrum image show that size of xylanase expressed in Escherichia coli is about 48 KD,and that expressed in Pichia pastoris is about 55KD.This is because of the presence of glycosylation proteins during expression in Pichia pastoris.Through the research on the enzymology properties found,Pichia pastoris expression of xylanase activity than Escherichia coli expression of xylanaseactivity.In addition,whole using Pichia pastoris expression of xylanase's resistance to the external environment than coli expression of xylanase strong.This may be due to expression in Pichia pastoris process-modification of protein.