Identification Of Gene For Pullulanase From Bacillus Thuringiensis

Starch is so far the most extensive used industrial material, and Amylase is the enzyme which can hydrolyze this kind of material. There are many kinds of Amylase such asα-Amylase,β-Amylase,Glucoamylase and De-branching enzymes and ect. And De-branching enzymes become more important because of the existence of amylopectin. However, few de-branching enzymes have been used for many reasons. Pullulanase (EC 3.2.1.41), a de-branching enzyme capable of hydrolyzingα-1,6-linkages of polymers and give amylose as the product. Pullulanase is one of de-branching enzymes. 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.The overall goal of this research is to try to express gene BtP from Bacillus thuringiensis in Escherichia coli and Bacillus subtilis. It indicated that there were two genes which probably code the pullulanase, amyX and pulA. The two genes were amplified from genomic DNA of Bacillus thuringiensis. And gene BtP had been cloned into Escherichia coli and Bacillus subtilis through the expressing vector pET-28a-amyX and pHSG-pulA individually, and the pullulanase activity had been detected in the two kinds of recombinants. Enzymatic properties analysis of the recombinant pullulanase of Bacillus subtilis (pHSG-pulA) revealed that an enzymatic activity optimum at pH 6.5, and its optimal temperature for enzymatic activity was about 50℃. Research on the stability of recombinant plasmids indicated that pET-28a-amyX was stable in the Escherichia coli, while plasmid lost during the transfer of culture of recombinant Bacillus subtilis (pHSG-pulA), and almost the whole plasmids had lost after the five times of transfer of culture without use of selection pressure.The fermentation conditions of recombinants were optimized. Carbon source,nitrogen source,incipient pH of the culture medium,volume of medium,inoculum size and inducing time analysis gave a optimum culture medium: soluble starch 2%, yeast extract 1%, peptone 1%, NaCl 1%, K2HPO4 0.017%, MgSO4·7H2O 0.012% , MnSO4·7H2O 0.012%, incipient pH of the culture medium 8.0, volume of medium 20 mL/250 mL, 10% of inoculum size, and added IPTG after 4 hours of incubation . And activity of the recombinant pullulanase was up to 3.4 U/mL.