Isolation and Identification of the Amylase-producing Bacteria in Silkworm Intestine and Cloning and Expression of Their Amylase Genes

Amylase-producing bacterias in larval guts of the Bombyx mori L.at 5 th year phase were isolated and identified to application for probiotics.Cloning,sequences analysis and prokaryotic expression in Escherichia coli of this amylase gene was conducted.NA starch medium was used to isolate amylase-producing bacterias.Strains identification which was on 16S rDNA sequences analysis and morphology was carried out.Enzymatic activity of them was measured by DNS method.Primers of amylase genes were designed to clone the genes.The full-length open reading frame of the amylase gene from strain DZ-a was fused into the prokaryotic expression vector pET28a and was introduced into E.Coli Transetta (DE3)cells.As a result,two amylase-producing strains were isolated.The result of 16S rDNA analysis in Genbank sug- gested that the strains both have 99%homologous proportion to Bacillus cereus.The enzymatic activity of amylase was strain DZ-a 20.5 U/mL and strain DZ-h 24.2 U/mL respectively.Sequences analysis of the amylase genes showed that the cloned sequences of the two strains were 3 414 bp and 3 378 bp respectively.They both contained complete open reading frames encoding 586 amino acid residues.The SDS-PAGE showed that the molecular weight of the expressed protein was about 66 kD.The two strains DZ-a and DZ-h were identified as Bacillus eereus.The cloned sequences both were complete Bacillus eereusα-amylase genes.And one amylase gene of them was successfully expressed.These results will provide foundation for further purification and identication of the target protein and for the function study of this amylase.