| Pullulanase (pullulanase-6-glucanohydrolase, EC 3.2.1.41) is usually considered a debranching enzyme that specifically cleavesα-1,6 glycosidic linkages in pullulan, starch, amylopectin, and related oligosaccharides. The enzyme is widely used in starch-processing industry, because it can promote the utility efficiency and productivity of starch on a largescale.The most important industrial application of pullulanase is used in combination with glucoamylase orβ-amylase, respectively, in the saccharification process, and it's full-blown application is used for the production of dextrose, high-maltose syrups and beer from starch hydrolyases. Pullulanase can improve the utilization rate of starch material, decrease the consumption for its hydrolyzing character and this make it very important and promising in the industry. Analysis of the complete genome sequence of Bacillus licheniformis, which has been published in 2004, revealed a putative pullulanase gene which was 2133 bp and annotated as amyX. For many years, Bacillus licheniformis as a host for heterologous gene expression was used for expressing variety heterologous protein including pullulanse. However, there is no report on the study of pullulanase and it's gene of Bacillus licheniformis in domestic and overseas.In this research paper, the coding region of amyX gene was amplified from genome DNA of B.licheniformis by PCR and inserted into the downstream of T7 promoter of the expression vector pET28a. The positive transformants were characterized by pullulanase activity determination, SDS-PAGE, and PCR amplification against E.coli total DNA. It demonstrated that gene of pullulanse got proper expression in E.coli. The transformants named BL21 (pET-28a-BlP) secreted recombinant pullulanase, which had the activity of 5.6 U/mL.The experiment of fermentation and PCR method showed that BL21 (pET-28a-BlP) had a good genetic stability.The molecular weight of the pullulanase was about 80 kD. The optimum reaction temperature and pH rang for the enzyme activity was found to be from 35 to 45 degrees C and from pH 5.5 to 6.5, respectively. Furthermore, the pullulanase exhibited a stable activity under abroad pH from 5 to 7 at 40 degreesC, a stable activity between 35 and 55 degrees C at pH 6.0, and good thermostability, which had a residual activtity after 64 hours as measured at 40 degrees C and pH 6.0 of 40 percent. Metal ions were needless in the enzymatic reaction, however, the pullulanase activity was inhibited more or less by the addition of some ions such as Ag+,Cu2+,Zn2+,Co2+,Mn2+,Fe2+,Cd2+ and Pb2+, was improved by the addition of Mg2+ and Ca2+, and was slightly affected by the addition of K+,Na+,Li+,Ni2+ and Ba2+. In addition, may be because that the pullulanse molecular had less disulfide-bond, the addition of SDS (5×10-3mol/L) inhibited the enzyme activity absolutely.The fermentation conditions of recombinant were optimized. Anlysis the effect of nitrogensource,inductiontime,temperature,pH,broth amount and inoculation amount on the expression of pullulanase. The results indicated that the optimum culture medium: yeast extract 1%, peptone 1%, NaCl 1%, K2HPO4 0.017%, MgSO4·7H2O 0.012%, MnSO4·7H2O 0.012%; the optimum condtions: induction after cultivation for 3 hours, cultivated temperature and pH respectively was 20 degrees C and pH 6.0, volume of medium 20mL/250mL, and 10% of inoculum size. Under the optimum conditions show that the activity of the recombinant pullulanase was up to about 6.5 U/mL.According to above, the yield of the pullulanase from there combinant E.coli was 5.6U/mL; ezyme properties of the crude enzyme were studied; by optimization of fermentation conditions show that the average pullulanase activity was(6.5±0.1)U/mL. So the recombinant pullulanase had wide foreground in industial production and utilization.
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