Cloning, Expression And Characterization Of The Sai Gene Encoding α-Amylase Inhibitor

Alpha-amylase inhibitor is a kind of alpha-Glucosidase inhibitor that can inhibit the alpha-amylase of mammals, insects and a few microorganisms. It has become one of the new research hotspots for their value in medicine and agriculture. Alpha-Amylase inhibitor has potential value as a medicine against diabetes, hyperglycemia, hyperlipaemia and adiposity. The inhibitors can inhibit the activity of alpha- amylases of insect pest, its gene can be used as insect-resistant genes in crops breeding, and biology pesticide.In this study, we separated actinomyces from soil, cloned gene of alpha-amylase inhibitor and prokaryotic expressed, purificationed a recombination protein. We established a method to prepare alpha-amylase inhibitor recombination protein, and study on its activity. The main results are as followed:1. Some actinomyces are obtained from soil which have activity in inhibit of alpha-amylase, most of these are Streptomyces. The fermentation broth of actinomyces inhibiting the alpha-amylase, indicates that some stuff was exist in the broth. And salting-out protein in the broth indicates that the protein is one of element that can inhibit alpha-amylase. Cloning and expression gene of alpha-amylase inhibition have been conducted.2. The gene encoding alpha-amylase inhibitor was cloned from the genome of actinomyces,named sai. Prokaryotic expressed the recombination protein, named SAI, and purified by affinity chromatograph. It was found that the coding sequence of the gene consisted of the 360 bp nucleotides which encoded 119 amino acids. The molecular weight of the protein was 12568.1 Da, and the isoelectric point was 6.54. Bioinformatics software predicted that the main secondary structure of SAI wasα-helix and random coil. A signal peptide was exist in N-terminatio of this protein.The SAI could combine and transport protein, and also have immune response.3. The SAI is specificity active against alpha-amylases of mammals, insects, but not of plants and fungus. The SAI are of high thermal stability and pH stability, and the proportion of inhibitor and alpha-amylase was 1:1. The SAI is high safety for Marc 145 cell. The animal experiment has showed that the hyperglycemia is restrained with the SAI. The SAI is also active against alpha-amylases of Helicoverpa armigera, and inhibit the growth of larvae, but inactive towards those of wheat seeds.