Isolatio, Optimalization Of Fermentation Conditions And Construction Of Genomic DNA Library Of Alkaline Lipase-producing Bacterial Strains

Two alkaline lipase-producing bacterial strains(WA and WP) were isolated from soil by means of solid plate method. Strain WA was identified as Bacillus amyloliquefaciens and firstly reported to produce alkaline lipase enzyme, and WP as Photobacterium logei.Factors affecting the lipase fermentation of B.Amyloliquefaciens were investigated. The composition of the fermentation medium was optimized by orthogonal test and the optimal lipase-producing conditions were found to be as follows: 1% olive oil, 1% soluble starch as carbon source, 2% soy bean meal and 2% corn meal as nitrogen source, initial pH7.0 and the optimal cultivation temperature 30C for 72h with 15 mL medium in 100 mL flask at 180rpm. Under these conditions, the alkaline lipase activity of the supernatant can reach a maximum yield of 12 U/mL.The alkaline lipase activity is optimal at pH8.5 and 37℃, and stimulated by Ca2+ and K+ but inhibited by Cu2+ and Fe3+. The lipase produced by this strain has good thermo-stability at a certain extent and was stable ranging pH7.0 to pH10.0.DNA from B. amyloliquefaciens was partially digested with Sau3 Al. The DNA fragments ranging from 4~10kb were recovered from agarose gel and inserted into BamHI site of plasmid vector pUC18. The recombinant DNA was transformed intoEscherichia coli DH5a and about 5000~6000 clones were obtained. The agarose gel electrophoresis showed that 80% of clones contain inserted fragment with 4-10 kb in size. This genomic library is useful for gene cloning, including lipase gene.