Cloning And Overexpression Of Gene Encoding The Pullulanase

Pullulanase (pullulan-6-glucanohydrolase, EC 3.2.1.41) is usually considered a debranching enzyme that specifically cleaves α-1, 6 glycosidic linkages in pullulan,starch,amylopectin,and related oligosaccharides. The enzyme is widely used in the starch-processing industry, because it can promote the utility efficiency and productivity of starch on a large scale. The most important industrial application of pullulanase is used in combination with glucoamylase or β-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 hydrolysates.In this research paper, the gene encoding pullulanase from Bacillus naganoensis(ATCC53909) had been cloned and expressed in Pichia pastoris host SMD1168. Moreover, the fermentation conditions of the engineering yeast strain and the properties of the recombinant pullulanase had also been studied in our research. This study would offer the theoretical basis to the protein engineering of the pullulanase and its overexpression and future application.This thesis consisted of four parts:1. Cloning the Pullulanase gene from Bacillus naganoensis(ATCC53909);2. Expression the Pullulanase gene in Pichia pastoris;3. Optimization of the fermentation conditions of the recombinant Pichia pastoris;4. Studying on properties of the crude recombinant pullulanase.The detailed results and conclusions were as follows:1. The specific primers were designed according to the reported nucleotide sequence of the Pullulanase. The gene fragment encoding the pullulanase was clonedsuccessfully from Bacillus naganoensis{ATCC53909) total DNA with the specific primers. Sequence analysis showed that the cloned gene was identical with the reported PuUulanase.2. The expression vector pPIC9K—PuUulanase was constructed by ligating the cloned gene with 3'-terminal of a-factor signal sequence on the pPIC9K in EcoR. I site. The recombinant expression vector was introduced into Pichia pastoris SMD1168 by electroporation. The positive transformants were characterized by puUulanase activity determination, SDS-PAGE, and PCR amplification against yeast total DNA. It demonstrated that the gene of the puUulanase got proper expression and modification correctly in SMD1168. The transformant named P8 secreted recombinant puUulanase, which had the highest enzyme activity of 350.8U/mL, about 129.9 times higher than that of the original strain. The experiment of fermentation and PCR method showed that P8 had a good genetic stability.3. Improved expression level of recombinant pullulanase by Pichia pastoris was achieved by optimization both the growth phase and induction phase culture conditions. The results indicated that the optimum fermentation condition of the growth phase is as follows: 2.5% glycerol, 2.5% (NH4) 2SO4, 0.00004% Biotin, 10%lmol/L phosphate buffer, pH5.0, 240rpm,30°C,48h,and 10% inoculation amount. The orthogonal test results of induction phase showed that the optimum desired fermentation conditions were as follows: 3.0% methanol, 0.25% glycerol, 2.0%(NH4) 2SO4, 0.00004% Biotin, 10%lmol/L phosphate buffer, pH7.0, 240rpm, 30°C,48h,l:l inoculation ratio, and the same concentration of methanol and glycerol were supplemented every 24 hours. Five times repeated fermentation experiments of strain P8 under the optimum conditions showed that the average pullulanase activity was (507.34±2.76) U/mL.4. The molecular weight of the pullulanase was about 119.9kDa. The optimum reaction temperature and pH range for the enzyme activity were found to be from 40 to 60 degrees C and from pH 4.5 to 5.0, respectively. Furthermore, the puUulanase exhibited a stable activity under a broad pH range from 3.5 to 6.0 at 60 degrees C, a stable activity between 40 degrees C and 65 degrees C at pH4.5, and goodthermostability, which had a residual activity after 64 hours as measured at 60 degrees C and pH4.5 of 45 percent. Metal ions were needless in the enzymatic reaction, but the pullulanase activity was inhibited more or less by the addition of some ions such asAg\ Cu2\ Zn2+> Co2\ Mn2\ Fe2\ Cd2\ Pb2+and Hg2+, was improved by the addition of Mg2+and Ni2+ , and was slightly affected by the addition of K+> Na+> Li+> Ca2+and Ba2+. In addition, may be because that the pullulanase molecular had less disulfide-bond, the addition of SDS (5X10 Wl/L) inhibited the enzyme activity absolutely.According to above, the yield of the pullulanase from the recombinant yeast was more productive than that of the original strain. Application properties of the crude enzyme were studied. It showed that the expression products of recombinant yeast were more stable in the pH range of 3.5 to 6.0 and exhibited higher thermostability than the original strain. So the recombinant pullulanase had a wide foreground in industrial production and utilization. The extraordinary high substrate specificity together with its thermostability made this enzyme a good candidate for biotechnological applications in the starch-processing industry.