Cloning, Expressinn And Mutation Of The Heat-stable α-Amylase Gene

The physiological and genetic characteristics of Bacillus licheniformis 020401 were studied, and the enzymic character of the heat-stable α-amylase produced by this strain was analyzed. The heat-stable α-amylase gene was amplified from B. licheniformis with PCR and cloned in Escherichia coli. The gene was mutated by recombinant PCR. The mainly researching contents were showed as below:1. The optimum pH and temperature of the heat-stable a-amylase produced by B. licheniformis 020401 was 6.5 and 85℃, separately, thought it could remain superior activity at 95℃.2. The chromosome DNA of B. lichenformis 020401 was extracted., The gene of 1.9kb was obtained by PCR with appropriate primers. Thegene was then connected with pUC19 plasmid and the recombinant plasmid was transformed into E. coli JM109. A positive clone JM109/pUAM was screened by the blue-white choose and halo method.3. It was proved that the JM109/pUAM could secret heat-stable α-amylase into the medium through measuring the activity of the a-amylase in the cell-free supernatant of the culture. The relative molecular weight of the a-amylase was about 60000 by SDS-PAGE.4. The sequence of the gene was menstruated and compared with the known sequence of the heat-stable a-amylase gene. The homology was above 99%.5. The aim gene was connected with pGEM-3Zf(+) plasmid and cloned into E. coli JM109, and as a result, a positive clone JM109/pGAM was obtained. It had the same protein producing ability as JM109/pUAM and the activity of the a-amylase was higher than that of the JM109/pUAM.6. The codes of the a-amylase gene were mutated at the amino acid 134 and 320, respectively. The two kinds of gene were separately connected with pGEM-3Zf(+) plasmid and transformed into E. coli JM109. Two positive clones, JM109/pGATl and JM109/pGAT2, were obtained.