Isolation And Identification Of The Strain Bacillus Cereus OPF0031and Cloning Of The Pullulanase Gene

The ocean sediment is another field for the research on pullulanase and the interrelatedstrains. A certain amount of strains had been screened and one of them has been selected forthe research.The adjustment of culture, temperature and pH offers the opportunity for bacterialstrain isolation and growth.The strains with higher pullulanase activity was determined byobserving the clear halo and the DNS method.There were40strains had been selectedinclonding9strains with higher pullulanase activity.They were numbered OPF0031、P14、Y15、A36、A39、P51、P13、Y17、A33、Y11、Y12、P12and A35.16S rRNA gene and physiological characteristics of the bacterium were used toidentify the strain OPF0031.Since the analysis of the16S rDNA gene sequence and thedetailed physiological characteristics of OPF0031showed highest similarity with these ofBacillus cereus, the strain was identified to be genus Bacillus cereus. The sequence wasdeposited in the GenBank database with accession no. JQ824137.A singlefactor experiment design and Response surface methodology based on theCenter Composite Design (CCD) were applied to enhance pullulanase production. Glucosegaves the maximum pullulanase activity. Soluble starch, dextrin, potato starch and thepowder of glutinous were also the good source of carbon for production of pullulanase.Medium with beef extract or peptone showed higher levels of pullulanase than that withother nitrogen sources. To investigate the effects of minerals, different minerals weresupplemented in the basal medium. K+、Al3+、 NH4+and Mg2+had the biger effect onpullulanase production.The optimized medium containing (g/L): starch15, peptone10,(NH4)2SO49, MgSO40.9, Fe2(SO4)30.018and KH2PO40.5.The strain were incubated at30℃with rotate speed at180rmp. The Pullulanase activity was1.3871U and had nearly14-fold increase.Homology search was performed using BLAST search algorithm and publicdatabases.And conserved sequences were observed on the3′-terminal and5′-terminal,which the primers were designed with. The genomic DNA of Bacillus cereus OPF0031wasused as template in PCR profile. The sequence was deposited in the GenBank database withaccession no. JQ707952.