Gene Cloning and Expression of Rhizopus or yzae α-Amylase

Based on Rhizopus oryzae complete genome sequence published by Broad Institute,two 1389-bp gene sequences coding a-amylase were cloned from two R.oryzae strains.Sequences alignment indicated that there was no intron existed in the two amylase genes,and the two genes have a similarity of 95.54% and the similarity between the gene products was 97.84%.The gene products belongs to the amylase family 13 and have four classical highly conserved regions that observed in most a-amylases.Recombinant plasmids for expression of the cloned a-amylase genes were constructed based on the plasmid vector pET-28a(+).a-Amylases displayed higher expression levels in E coli BL21(DE3) codon plus strain,the a-amylase activity were achieved at 1.3 U/mL and 0.5 U/mL,respectively.One of the a-amylases was purified through Ni affinity column and subsequently used for hydrolyzation of(potato) starch,and there was approximately 10% glucose,74% maltose and negligible maltotriose or other oligosaccharides remained in the end-products.