Gene Cloning, Expression, Characteristics And Molecular Modification Of Two Alkaline Proteases From Bacillus Licheniformis GXT-1

Alkaline protease plays an important role in the industrial application, which accounts for about30%of the globle enzyme market. It is practically important for alkaline enzyme’developing and application by exploring and further studying new alkaline enzymes.This research successfully screened a wild-type strain with an ability to produce alkaline protease from nature. The strain was identified as the genus Bacillus licheniformis, named Bacillus licheniformis GXT-1. Two alkaline protease genes bll,bl2were successfully cloned from the this strain, then the operation of construction and expression were held in the host E.coli. These enzymes BL1, BL2were purified by nickel affinity chromatography and the molecular weigh was33kD,27KD, respectively.By studying the enzymatic properties reveals that both enzymes BL1, BL2belong protease S8family. When using casein as the substrate, the BL1optimal condition was pH10.0and70℃,the relative activity was more than80%when the pH value was between7.0and11.0, the specific activity was14643U/mg, Km values was3.80mg/mL, the maximum velocity Vmax was19803U/mg. The BL2optimal condition was pH10.0and70℃, the relative activity was more than80%when the pH value was between8.0and11.0, the specific activity was21855.90U/mg, Km values was13.59mg/mL, the maximum velocity Vmax was48278U/mg.Seven mutants M118A, M134S, M221S, M94S, M248S, M255S, M284S were gained by using inverse PCR to modificate the recombint alkaline protease BL1and BL2. It can not detect any enzyme activity from mutuants M94S and M134S. The other four mutants had some changes in their enzyme properties: the Km value of mutants M248S, M255S, M118A, M221S had decreased, but only the mutant M248S increased its specific activity, the rest mutants M118A, M221S,, M255S M284S decreased their specific activity.