The Bioinformatics Analysis, Heterologous Expression And Research Of Enzyme Characteristics About Sorangium Cellulosum Cellulase Genes

Sorangium cellulosum is an efficient cellulose-degrading bacteria group.Its cellulase activity relies highly on the contact between cells and substrate.Many researchers are interested in the organization style of the cellulase in S.cellulosum.On the other side,due to the research done earlier by our lab,we found S.cellulosum So0157-2 strain can grow in alkaline environment(pH＞10) and degrade cellulose.So we assumed, there should be some alkaline cellulases within the strain,this discovery supplied good material for our research about alkaline cellulase.We used bioinformatics softwares such as Word document,Clustal and DNAman to found an ingenious bioinformatics method named "amino acid sequence probe hybridization".The method can screen for target cellulase genes quickly in S.cellulosum strain So0157-2 genome sequence(in 5202 contigs).As a result,we have got 26 cellulase gene sequences.We analyzed the genome sequence of S.cellulosum strain So0157-2 by BLAST and sorted the cellulose-degrading related enzymes out in genome sequence annotations of cellulosum So ce56 strain.After that we had a detailed assay of the species,quantities and distributions of cellulose-degrading related enzymes in S.cellulosum strains.Further more,we had a detailed research of the differences between cellulase genes in S. cellulosum and the ones in the microbe species which degrade cellulose by other strategies.On the basis of sequence analysis,we chose 3 genes code for cellulase in So0157-2 genome sequence and constucted 6 expression vectors carrying the genes.Through the optimization of expression conditions,we realized an endo-glucanase active expression in E.coli.We focused on an expressed endo-glucanase,finished the measurement of its optimal reaction temperature(50℃),optimal reaction pH(7.3-7.6),relative enzyme activity(1.662IU/mg) and the assay of enzyme thermal stability.These work accumulated experience for Sorangium cellulase heterologous expression,solved the difficulties of getting a single protein component in the original strain and provided high-quality materials for antibody preparation as well as analysis of protein biochemical characteristics.To study the composition and organization forms of Sorangium cellulases,we constucted a genome expression library of S.cellulosum So9733-1 strain in ZAP expression system.The average length of insert sequences was 3.98kb.The genome library contained 40000 clones and its coverage P=0.999982.The results lay the foundation for activity screening and sequence screening of So9733-1 cellulase genes. After the activity screen carried out in So9733-1 genome expression library,no positive clones were found.The result showed that further sequence screening and PCR amplification method might be the effective way to get its cellulase genes.Moreover,we isolated 17 myxobacteria strains in different environment soil samples. The discovery extended understanding of the living environment of myxobacteria species and provided more material for the research of the differences during different myxobacteria cellulase genes.In addition to the theoretical meanings above,there are certain potential application values in the study.Cellulase gene cloning,heterologous expression and analysis of enzyme activity as well as its biochemical characteristics lay the foundation for application.Especially,S.cellulosum So0157-2 strains alkaline cellulase may be possible for application in the paper and textile fields.