Preparation Of Xylanase Polyclonal Antibody And Its Application In Saccharomyces Cerevisiaeexpression System

Xylanase is a class of hydrolases that can degrade xylan into xylose and xylooligosaccharides.Xylanase is of great value in many aspects such as food research and development,feed production,industrial papermaking and environmental protection.Since the production of naturally occurring xylanase cannot meet the application requirements,more and more scholars have used molecular biology to construct xylanase engineering bacteria.However,in the course of the research,the detection of xylanase protein only stayed at the detection level of enzyme activity,lacking efficient and intuitive xylanase protein quantification method.In this study,by preparing a xylanase antibody,it is expected that the antibody can be used for preliminary quantification of the xylanase protein in the bacterium,andexplore the relationship between gene copy number,protein expression and enzyme activity in different copy number xylanase Saccharomyces cerevisiae engineering bacteria.In this study,the xylanase gene derived from Aspergillus niger CICC2462 is prokaryotically expressed in E.coli BL21,and the purified recombinant xylanase protein is used as antigen to prepare polyclonal antibody,quantitative analysis of xylanase protein is performed on the constructed random multicopy xylanase S.cerevisiae engineering strain using the prepared xylanase polyclonal antibodye,xploring the relationship between xylanase gene copy number,protein expression and enzyme activity.In order to provide technical support for the production of enzyme preparations and detection of enzyme preparation proteins and strain modification.The research results are as follows:1.The recombinant expression vector pET28a-xynB is constructed and expressed in E.coli BL21.The induction temperature is 37?,the induction time is 7 h,the shaking speed is 180 r/min,and the final concentration of IPTG is 100?g/mL.Excellent expression conditions,the amount of recombinant xylanase protein is 12.8 times that of the original strain under this condition.2.Purified recombinant xylanase protein is used as antigen to prepare xylanase polyclonal antibody,and polyclonal antibody with titer greater than 1:10000 and capable of specifically identifying xylanase is obtained.3.Construction of random multi-copy xylanase S.cerevisiae engineering bacteria,obtaining 1,2,3,5,7,8,10 and 11 copies of 8 different xylanase gene copy numbers of S.cerevisiae engineering bacteria positive transformants.4.Xylanase polyclonal antibody quantitative analysis of xylanase protein in bacteria and fermentation broth in multicopy xylanase Saccharomyces cerevisiae expression system,the relationship between the xylanase gene copy number,the expression level of the xylanase protein,and the xylanase enzyme activity is obtained.In the bacteria,the amount of xylanase protein increases as the copy number increases;In the fermentation broth,when the xylanase gene copy number is 1-8 copies,the enzyme activity is gradually increased,the 8 copy enzyme activity is up to 326 U/mL,and when the xylanase gene copy number is greater than 8,the fermentation is performed.The amount of xylanase protein in the liquid decreased,the activity of xylanase decreased,the enzyme activity decreased to 263 U/mL at 10 copies,and 248 U/mL at 11 copies,which was 30%less than that at 8 copies.