Breeding Of Bacillus Subtilis With High-activity Of Amylase, Protease And Screening Of Positive Clones Of Enzyme Genes

To screen Bacillus subtilis strains with high activity of amylase and protease, for the purpose of producing high-efficient microbial ecological agent to purify water. Two strains with high activity of amylase and protease were selected from ten strains of Bacillus subtilis collected from different sources by detecting the activities of amylase and protease in fermented liquor. They were named as H001 and H008 and characterized according to morphology, colony morphology, and physiology. The two strains H001 and H008 were primarily identified to be Bacillus sp., and they were suitable for producing high-efficient Microbial ecological agents to purify water.Plackett-Burman design and Box-Behnken design was employed to optimize a Solid multiplicated medium for Bacillus subtilis strain H001. Four important components were screened form eight related factors. The prime factors were optimized by response surface methodology so as to work out their optimum level. Resulted in 1.5-fold increased level of bacteria growth mass a maximum compared to initial level after 24 h of fermentation.The water purification functions of the strain Bacillus subtilis H001 by using 2 kinds of simulation pollution water samples was discussed. The results indicated that the strain could degrade the organic matter rapidly; the COD value and ammonia concentration of the water sample had dropped more efficient compare with other four standard Bacillus subtilis strains, total residual and filterability residual degeneration rate at 144 h was 63.3 % and 20.4 % seperately.A genome library of the strain Bacillus subtilis H001 was established by"shotgun"method in view of its high amylase and protease activity, the high molecular weight genome DNA was partially digested by Sau3AI restriction enzyme. Pieces of 1.5~7Kb DNA were extracted, the fragments were rccombinated and ligased with vector pUC18 which were totally digested with BamHI enzyme, and high efficiency competent DH5αE.coli host cells system were employed to increase the transformation efficiency, nearly 17000 transformants were got. Results of rapid Screening of transformants plasmid indicated that there were foreign DNA fragments in about 98% transformants. Seven positive clones were screened from library by using an improved positive transformant screening method. Plasmids extracted form positive transformants were retransformed, Three positive transformants aim gene inside surely were got finally, two of them expressed amylase activity, the rest one expressed protease activity.