Screening Of Bacteria Producing High-efficiency Xylanase And Cloning And Expression Of Xylanase Gene

At present,xylanase is widely used in various industrial production.However,the application of enzymes is also affected by some factors,such as low enzyme activity,poor acid and alkali resistance,poor thermal stability.In this paper,we mainly screened xylanase producing alkali-resistant strains and obtained alkaline xylanase with high enzyme activity by genetic engineering method.The research is mainly carried out from the following aspects:1.The tobacco straw wastes were mainly used as raw materials to screen out strains capable of degrading xylanase efficiently,and then purified.Four strains capable of degrading xylanase were screened out.Through the transparent circle method of Congo red staining,it was found that 2 strains（No.1 and No.4）had significantly larger transparent circles.The results showed that these two strains had the ability to degrade xylan efficiently.Morphological observation and molecular biological identification were carried out,and the experimental results showed that:The first strain was Bacillus stratericus（SG-1）,and the fourth strain was Bacillus safensis（SG-4）.Through the DNS enzyme activity test,the specific situation of the two strains’xylanase degradation ability was further explored,that is,the optimal screening of the culture conditions for xylanase production of highly efficient xylanase degrading bacteria（SG-1 and SG-4）was mainly explored from the growth environment of the microbial strains.Single factor variable method was used to carry out experiments from fermentation seven days,different temperature（℃）,different initial p H,different carbon sources,different nitrogen sources and other aspects.The results showed that the optimal screening conditions for xylanase production by strains SG-1 and SG-4 were basically the same,but the enzyme activity was different.The results were as follows:the optimal fermentation time of strains SG-1 and SG-4was three days;The optimum temperature is 37℃.The best source of C was xylan.The best N source is peptone.The optimum initial p H was 11.0.Under the optimum conditions,the maximum enzyme activity of strain SG-4 was 640.71 U/m L.In other words,strain SG-4 had high alkalinity tolerance.2.The cloned endoxylanase gene Xyn43 was analyzed by BLAST.The xylanase gene has a full length of 1538 bp and a 512 amino acid sequence.According to informatics analysis,the xylanase belongs to the endoxylanase of the xylanase family GH43.The theoretical isoelectric point of the enzyme is 7.77,the molecular weight is54 k Da,and the protein structure is relatively stable.It belongs to the hydrophilic protein and phosphoric acid.16 chemical sites3.The recombinant xylanase gene Xyn43 can be transferred into E.coli（E.coli Top10）for high-efficiency expression.However,its expression was affected by different inducer concentration and induction time.It was found that the expression effect was better when the inducer concentration was 0.2 mmol/L and the induction time was 6 h.Finally,Ni-NTA nucleophilic chromatography column was used for purification and SDS-PAEG detection.A single protein band can be obtained,and the results were consistent with what we expected after analysis,which proved that the expression was successful.By studying the enzymatic properties of recombinant xylanase,it was found that the enzyme activity of recombinant xylanase was better when the temperature was 40℃and the p H was between 4-8.Under the influence of metal ion Cu²⁺,it has a certain promotion effect,while under the influence of Ba²⁺,it has a certain inhibition effect.