Studis On The Homologous Expression And Truncated Expression Of Mannanase Gene From Erwinia Carotovor

Bio-extraction of fiber from crops is a method using bacteria which producing series of enzymes to decompose hemicellulose of the crops.Two Erwinia carotovora CXJZ95-198 and CXJZU-120 have been isolated in this laboratory, which are reported as the most efficient deguming bacteria. In this study, the property of mannanase from CXJZ95-198,and the mannanase expression level on CXJZU-120 were investigated to promote their production, the results are as follows.manA, the mannanase gene of CXJZ95-198 has been analysed by bioinformatics method in homology alignment, signal peptide prediction, transmembrane region prediction, phylogenic tree foundation, second structure and crystal structure foundation. The results show that manA has low similarity in the alignment with other source of mannanase genes, and its translation product is extracellular. Signal peptides of 27AA from the N-terminal were found at the manA's translation product. Four truncated mannanase genes were designed according to the homologous model, in which 0,81,111,141bp were deleted from the N-terminal respectively. N-truncated gene expression proved the 27aa aside the N terminal were signal peptide, and loss of the 37aa from the N terminal show little effect on mannanase activity. However, the enzyme will be inactivatied while the number of amino acid deltion was more than 47.The manA was cloned into plasmid pEASY El express and transferred into E.coli BL21 (DE3).The recombinant plasmids were extracted from the positive transformant, and transferred into Erwinia carotovor variant CXJZU-120. Measured by circle decomposing method, the mannanase expression level of the homologous recombinant was 2/3 higher than the original host CXJZU-120, and the promotion of mannanase activity is 2.1 times.