Isolation and Identification of Isoamylase Producing Strains and Cloning and Expression of Isoamylase Gene

Objective:In order to further improve the utilization rate of starch and gain high yield isoamylase from microbial strains. Methods:A stain with high isoamylase activity, named as strain LZ-5, was isolated from the soil that rich in starch. The strain was identified by morphologic observation, physiology and biochemistry experiments, 16S rDNA and gyrB gene sequence analysis. With the prime designed on the basis of the published converse nucleotide sequence of isoamylase gene from Bacillus lentus, the isoamylase gene was obtained from strain LZ-5 and amplified by PCR, cloned into vector pMD-18T and construct recombinant plasmid, then transformed into E.coli DH5a and identified by restriction enzyme digestion with positive recombinant plasmid. At last, the expressed product was detected. Results:The strain LZ-5 was identified as Bacillus subtilis and the isoamylase activity was 10.9 U/mL. Sequencing results showed that this isoamylase gene had a homology of 96% with the isoamylase gene sequence of Bacillus lentus, indicate that the isoamylase gene was successfully expressed in prokaryotic expression vector E.coli DH5a. After induced by IPTG, the isoamylase activity was 28.4 U/mL that the engineering bacteria crushed by cell disruptor, was 2.6 times as much as that produced by original strain. Conclusion:Bacteria isoamylase gene recombinant expression is one of the main strategy to solve low isoamylase activity.