Study On The Functional Genes Of Xylanase From Actinomycetes From Tengchong Hot Springs

Xylanase has important applications in animal feed,paper industry,food processing and biotransformation,so it has attracted much attention.Xylanase widely exists in bacteria,archaea,fungi and animals.However,the protein subunit of fungal xylanase is more complex than that of bacterial xylanase,and its thermal stability is worse than that of bacterial xylanase.In the process of practical application,it often encounters the challenges of special conditions such as high temperature,high pressure and extreme acid-base.At present,the industrial application of xylanase mostly comes from bacteria,but due to the limitation of its own evolution,the xylanase produced by microorganisms can not meet these extreme conditions in the industrial application environment.Therefore,people turn to the use of Metagenomics and genomes to excavate new thermophilic xylanase functional genes from the special high-temperature environment of hot spring.In this experiment,two new xylanase genes named Xyn19B and Xyn21A were cloned from Actinomadura amylolytica YIM 77502^Tfrom genomes;A new xylanase gene named xyndrty1 was obtained from Tengchong hot springs Metagenomics.The total length of Xyn19B gene was 861 bp,and the total length of Xyn21A gene was 1296bp.Through informatics analysis,Xyn19B belongs to xylanase family 11(GH11),and Xyn21A belongs to xylanase family 10(GH10).The total length of xyndrty1 gene was1221 bp,which belongs to xylanase family 10(GH10)by informatics analysis.Three xylanase genes were transferred into E.coli for high-efficiency expression,purified by Ni-NTA nucleophilic chromatography column and detected by sds-paeg,and the enzymatic properties of recombinant xylanase were studied.The results show that:(1)The catalytic activity of recombinant xylanase Xyn19B on Corncob xylan(12U/mg).The recombinant xylanase Xyn19B showed good thermal stability at the optimum temperature of 55?and p H 6.Metal ions and inhibitors had no effect on the activity of the enzyme.(2)The catalytic activity of recombinant xylanase Xyn19B on Corncobt xylan(7.5U/mg).The optimum reaction temperature of Xyn21A is 65?.The optimum reaction p H is 5.6,and the activity is relatively high at high temperature of 55-70?.Metal ions K⁺,Mg²⁺,Fe³⁺,Ca²⁺,Ba²⁺,Cu²⁺,Zn²⁺and Al³⁺all have certain inhibitory effects on the enzyme,and the inhibitory effect of inhibitor SDS is obvious.(3)The catalytic activity of recombinant xylanase Xyn DRTY1 on birch xylan(21.2±3U/mg)and oat xylan(8.2±0.3U/mg).The optimum p H value is 6.0 and the optimum temperature is 65?.After incubation at 60?for 20 min and 120 min,the stability exceeded 140%and 110%of the relative enzyme activity,respectively.In p H3.0-7.0,the relative activity remains above 80%,and the thermal stability and p H stability are good.The above research shows that the new xylanase genes derived from hot spring microorganism and hot spring macrogenome are thermophilic xylanase,indicating that hot spring is an important resource bank of thermophilic enzymes.This study provides an enzyme resource reserve for the production,processing and application of thermophilic xylanase.The above research shows that xylanase(Xyn19B and Xyn21A)and metage nomics xylanase(xyndrty1)from pure cultured microorganisms in hot spring are thermophilic xylanases,which shows that hot spring microorganisms are an impo rtant source of thermophilic xylanase.This study provides an important enzyme r esource reserve for the production and application of thermophilic xylanase,and has certain guiding significance for the mining of thermophilic xylanase and othe r thermophilic enzymes.