Screening Of Straw-degrading Bacteria As Well As Enzyme Activity Analysis And Identification Of Gene Function

Straw resource is abundant and renewable biomass resource in the world,which is rich in carbon,nitrogen,phosphorus,potassium and mineral elements.It plays an important role in improving soil cultivation and increasing organic matter content.However,being formed by cellulose,hemicellulose,lignin and other intertwining dense structures,the straw is not easily degraded and utilized,.Environmental protection and effective biodegradation methods have attracted much attention in recent years.The main components in straw can be degraded by cellulase,hemicellulase and ligninase of microorganism.These enzymes play a key role in improving the comprehensive utilization of straw.Three strains of straw-decomposing bacteria were screened from saline-alkali soil samples and straw degradation and fermentation conditions,straw degradability,enzyme activity characteristics,and key enzyme genes were cloned and functionally analyzed.The main results are as follows:1.By testing and comparing the enzymes' activity of endoglucanase,?-glucosidase,xylanase,LiP,MnP,Lac in six soil samples,three strains of straw-decomposing bacteria,designated yj1,yj2 and yj3 were screened from saline-alkali soil,pine soil,poplar soil samples.The screened strains yj1,yj2 and yj3 were identified as Pseudomonas(G-),Bacillus(G+)and Brevibacterium(G+)respectively by Gram stain and 16 S rDNA.It shows that the three straw-degrading bacteria have obtained a certain alkali resistance and have a strong ability to degradecorn stalks.The straw degradation rate of yj2 was the highest.2.A variety of cellulose degrading enzymes are required for straw degradation.Under the CMCNa as a carbon source,the highest values of EG,BG and xylanase the activities for yj2 were 37.34 U,266.89 U,and 516.08 U,respectively.But no activity of xylanase were found for yj1 and yj3.The activities of EG,BG for yj1 and yj2 were 2.34U?2.23 U.Under the straw as a carbon source,all of the six enzymes in strain yj2 have higher activity than yj1?yj3 and collaboratively complete straw degradation.The strains yj1 and yj3 have no xylanase activity and mainly depend on LiP?MnP?Lac to degradate of straw.Therefore,it is concluded that the degradation effect of strain yj2 is mainly played by xylanase.3.Under the CMCNa and straw as carbon sources,the expression levels of EG,BG and xylanase in 24 h for yj2 were higher than 12 h,and this change trend was consistent with the enzyme activity.It indicated that the straw degradation process was induced and the enzyme activity increased with the increase of gene expression.4.The analysis of enzyme activity in the three strains showed that xylanase plays an important role in degradating straw.The gene was cloned and named xylanase,which was 642 bp.The similarity of the submitted xylanase gene sequence by NCBI reached more than 95%.It showed that the cloned xylanase belongs to the glycosylhydrolase family 11(GH11)which is a hydrophilic,alkaline,extracellular secretory protein with a signal peptide by bioinformatics analysis.The xylanase gene was linked to the expression vector pET-30 a and transformed into E.coli BL21 to induce the expression of 28 KDa protein,the fusion protein was detected by SDS-PAGE protein electrophoresis.By using the DNS method to determine the activity of xylanase,the results showed that the activities of xylanase in BL21(pET-30a-xylanase)strain increases progressively with time.The maximum enzyme activity of xylanase reached 387.26 U at 24 h,and the change of enzyme activity was significant differences.This study also cloned and named EG,with a size of 1500 bp.It revealed that EG belongs to glycosylhydrolase family 5(GH5)and also contains carbohydrate binding domain 3(CBM3),which is also a hydrophilic alkaline extracellular secretory protein with a signal peptide by bioinformatics analysis.