Gene Cloning,Expression And Characterization Of A Xylanase From Microbacterium Imperiale YD-01

Xylanase belongs to the family of glycoside hydrolases.lt can hydrolyze the glycosidic bonds of the main chain of xylan.It has been widely used in food and feed,biofuel,paper and other industries and it is also the largest class of commercial enzymes currently.Xylanase genes exist in a large number of microorganisms in nature.Our laboratory has isolated a bacterium Microbacterium imperiale YD-01 that could degrade xylan,and sequenced its whole genome,in which two xylanase coding genes were predicted.The genes xyn765 and xyn1923,encoding 384 and 1352 amino acids,respectively.Comparing Xyn765 and Xyn1923 with xylanases deposited in the protein database,they have 74%and 38%amino acid identity,respectively,to their counterparts,indicating that Xyn1923 are new xylanases.These two xylanase genes were cloned and heterologous expressed in E.coli,and the activity of the recombinant proteins was determined.The results showed that Xyn1923 had xylanase activity,while Xyn765 had no xylanase activity.Then enzymatic properties of Xyn1923 were systematically studied.Subsequently,considering that xylanase is widely used in industry,it is expected that the xylanase gene xyn1923 will be expressed in Pichia pastoris.After analyzing the sequence of Xyn1923,its key active regions were predicted,including two glycosyl hydrolase domains and an endo 1,4-xylanase domain,a total of 336 amino acids,and the truncated gene encoding these domains was introduced into Pichia pastoris for expression.The activity of the crude enzyme solution was measured,and no xylanase activity was detected.The main research contents and conclusions of this study are as follows:1.Sequence alignment analysis of Microbacterium imperiale YD-01 genome predicted two xylanase genes,xyn1923 and xyn765,which have 38%and 74%identity with known xylanase enzymes.2.The designed primers were used to amplify the predicted xylanase genes xyn1923 and xyn765 by polymerase chain reaction(PCR).These two xylanase genes were cloned into p ET28a expression vector and introduced into Escherichia coli Rosetta(DE3)for heterologous expression.The enzyme activity was measured and the result showed that only Xyn1923 had xylanase activity.3.The enzymatic properties of xylanase Xyn1923 were systematically studied after expression and purification.The specific activity of the enzyme was 14.523±0.026U/mg.The optimal p H and optimal temperature of the enzyme were 7.0 and70?,respectively.The enzyme activity was relatively stable in the range of p H 6.0to 9.0 and 30-60?.1 m M of Co²⁺,Ba²⁺,Fe²⁺and Fe³⁺metal ions promoted enzyme activity,while 2 m M and 5 m M of the above metal ions inhibited enzyme activity.1 m M,2 m M and 5 m M of Mg²⁺,Ag⁺,Cu²⁺,Ca²⁺,Mn²⁺and Pb²⁺ions inhibited enzyme activity.4.Analysis of the Xyn1923 gene sequence revealed its key active functional domains,and the truncated gene encoding these functional domains was introduced into Pichia pastoris for expression,but no enzyme activity was detected.This study mainly based on the bacterium Microbacterium imperiale YD-01capable of hydrolyzing xylan.Through whole-genome sequencing and bioinformatics analysis,two candidate xylanase genes were found.Their activity was measured after heterologous expression in E.coli.Through enzymatic analysis,It was found that Xyn1923 was a weak alkaline and thermophilic xylanase.This study lays a foundation for mining new xylanase gene resources with application values from microorganisms and promotes its industrial application.