Optimization Of Mannanase Production By Bacillus Amyloliquefaciens T27 And Investigation Of The Enzyme Properties

P-mannanase hydrolyzes mannan specially, liberating mannan-oligosaccharides and mannose. It widely spreads among plants, animal and microorganism, which shows extensive purpose in the food industry, paper industry, fabric industry and feed additive industry. As industrial process often demand the enzyme to be of high stability, thermostablity, and even alkaline or acid stability, researchers have focused on the extreme environments, such as high-pressure, high temperature, and extreme pH environments, to find extremophiles which may produce enzyme with special properties. Screening of marine microorganisms with P-mannanase producing ability from the special nature is a new trend to mine novel mannanase.In this research, one marine strain that shows remarkablely mannan-degrading abilty is screened from sea mud.16srDNA sequence of this strain is determined to be of 100% identity to species Bacillus amyloliquefaciens, and thus the strin is designated Bacillus amyloliquefaciens T27. Optimization of enzyme production using different parameters found that using konjac flour as carbon source, yeast extract as nitrogen source, at 37℃,50ml medium volume,250rpm,24 hours,Bacillus amyloliquefaciens T27 poduces the best enzyme activity, with activity of 98U/ml in the crude fermentation broth. To further study the enzyme properties of this strain, we amplified the mannanase genes from Bacillus amyloliquefaciens T27 by PCR with primers, which are designed according to the published homologous sequences from NCBI database. Sequencing analysis showed that this mannanase gene has 1083bp, encoding 360 amino acids, and the gene is designated Man27. Database blast show that this gene shares high indentity with mannanases gene origninated from bacillus, which is attributed to glycosyl family 26.The mannanases coding gene Man27 was cloned into expression vector pGEX-6p-l and transformed into E. coli (BL21) DE3 strain, and the activity of recombinant enzyme is determined. It shows the optimal activity at pH 7,40℃and above 60% activity at at 80℃. Pretreating the enzyme at pH4.0-10.0 for one hour, the recombinant mannanase shows nearly full activity, indicating that the recombinant enzyme is relatively pH stable. Such properties of the mannanase should lay a basis for further use of the enzyme in a suitable industrial areas.