Screening Of Cellulase-related Genes From Metagenomic Library

With the progress of human civilization,global greenhouse effect continues to worsen,environmental pollution is becoming more and more serious,and energy consumption and utilization are becoming more and more prominent.How to effectively reduce the use of petroleum and fossil fuels in order to reduce environmental pollution has become the research direction of scientific researchers in various countries.Cellulose,as the most abundant renewable resource on earth,decomposes macromolecular cellulose into oligosaccharides by cellulase,so as to make efficient use of cellulose resources widely existing in nature.At the same time,the degradation of cellulose by cellulase can also be used to produce industrial ethanol fuel for human beings,which can not only solve part of the environmental pollution problem,but also provide help for us to alleviate the energy crisis.At this stage to the common use of the genetic screening for macro genomics,macro genomics refers to extract samples from a specific environment,and then get the microbial total DNA samples,sample build metagenomic library methods,studies have shown that in nature for human direct study of less than 1%of the total microbial cultivation,therefore cannot cultivate microliter occupied most of,however,they are often not cultured microbes can bring us more unknown new enzyme gene,has wide biological research value.The problem of energy shortage and environmental pollution can be solved more effectively by using the method of macrogenomics to screen cellulase genes.This experiment adopts the method of feature selection in laboratory building of yunnan soil library for cellulose enzyme gene screening,culture medium with hydroxymethyl cellulose sodium as filter medium,the purpose of bacteria can be decomposed sodium carboxymethyl fibers,after Congo red stain has apparent change,so as to produce cellulose enzyme microbial screening separation.After screening about 980,000 clones of yunnan library,one cellulase gene was successfully screened and named ZFYN184.The amino acid sequences encoded by ZFYN184 were found to belong to the 44th family of glycosidase(GH44)by Blastp analysis and evolutionary tree analysis.By linking the pet-30a(+)expression vector with the target gene fragment,the recombinant plasmid pet-30a(+)-184 was successfully constructed in the laboratory,and e.coli BL21(DE3)was selected as the protease expression vector for a series of protein expression and purification.After purification,the enzymatic characteristics of the obtained protein were analyzed,and the optimal pH of the protein numbered ZFYN184 was 4.The protein was acidic and stable under acidic conditions.Optimum temperature of 40? 30,40? under the condition of treatment after 60 min,enzyme activity remained more than 80%,has the very high thermal stability.In addition,this enzyme has good tolerance to metal ions,and to organic reagents such as urea,EDTA,ethanol and DMSO.The above research will provide technical reference for further research on cellulase.