Construction Of Mutant Of Erwinia Carotovora And Cloning Of β-mannanase Gene From Bacillus Subtilis And Its Expression

Fiber is the second most necessary material for human being's survival. with the shortages of oil, forest and land resource and the improvement of the quality of human's life, it is urgent to develop non-cotton,and non-wood fiber resources.Because herbaceous fiber extracted biologically is the developmental trend which can reduce the heavy pollution, upgrade the product quality and lower the production costs, This study has been carried out widely at home and abroad.Construction of bacterial mutant is one of the most important way to discover new genes, analyze functions of genes and find out certain biological mechanism. In this research, 5523 mutants were obtained by Mini-Tn10 mediated insertional mutagenesis in CXJZU120, the mutant of Erwinia carotovora which is the most powerful strain reported for the bio-extration of herbaceous materials. Going with halo-producing assay we used the mutants to degrade non-cellulose and screened three non-cellulose degrading reduced or free mutants. It laid the foundation for studying the mechanism of non-cellulose degrading, searching the genes which are related to biological non-cellulose degrading and constructing a new generation of efficient strain.A strain of Bacillus subtilisBE-91 which can hydrolyze the glucomannan of Amorphophallus was used as material to isolateβ-mannanase Gene, manA, by PCR, A DNA fragment was amplified and cloned from BE-91 genomic DNA. Sequence analysis and SDS-PAGE of the extracellular protein showed that this DNA fragment encoded a putative protein containing the conserved domain ofβ-mannanase, it belonged to the family of mannanase, and an expected protein about 35kD was found. The recombinant vector which came from the ligation of the fragment and expression vector PET28a was transformed into powerful strain of herbaceous bio-extraction, Erwinia carotovoraCXJZU-120 by electroporation. DNS showed that the mannanase activity was 183-259% higher than the original strain, and a number of parameters had markedly been improved compared with the original strain.