Gene Expression, Characteristics And Molecular Modification Of Three Pullulanases From Bacillus Cereus Gxbc-3

Exploreing excellent new pullulanase genes, and enriching pullulanase theory were of great significance to realize the localization of pullulanase. A strain producing pullulanase was separated from compost mixture of different soil samples, by useing of plate screening for the purpose of industrial pullulanase development. After morphological characteristics and physiological&biochemical characteristics observations, and combined with the phylogenetic analysis of16SrDNA, it was identified as Bacillus cereus GXBC-3.The sequences encoding putative pullulanase Ⅰand Ⅱ from Bacillus cereus in GenBank database were analyzed. pulA, pulB and pulC, three genes coding for pullulanase from Bacillus cereus GXBC-3, were cloned and expressed in Escherichia coli XL-10Gold. These recombinant pullulanases, PulA、PulB and PulC were purified by nickel affinity chromatography. Their protein expression and molecular weigh were4.23mg/mL,84.56kDa;6.43mg/mL,94.71kDa and3.33mg/mL,95.37kDa, respectively. Five missing mutants were gained by using inverse PCR to modificate the recombint pullulanases PulA, PulB and PulC. Two of these mutants, QSPulA and QSPulB didn’t express; while the other threes, QSPulC-1, QSPulC-2and QSPulC-3, had a small amount of expression. Enzyme properties of the three mutants indicated that:(1) The N-terminal domain Pfm PUD could transforme type Ⅰpullulanase into type Ⅱ, cause of QSPulC-1, QSPulC-2, and QSPulC-3didn’t only had pullulanase activities, but also had low amylase activities.(2) The N-terminal domain Pfm PUD could seriously reduce the protein expression, because the expression of QSPulC-1, QSPulC-2, and QSPulC-3were less than1mg/mL, which was less than one third of PulC s expression.(3) The N-terminal domain Pfm PUD could significantly drop down the activity of pullulanase, while the specific activity, Km and Vmax of QSPulC-1, QSPulC-2, and QSPulC-3were greatly reduced.