The Site-directed Mutagenesis Of AgaH Gene From Lactococcus Spp.and Its Enzymatic Activity Variation

The a-galactosidase is a kind of exoglycosidase that can catalyzes the hydrolysis of a-galactoside banded in oligosaccharides. It is extracted from yeast fermentation broth firstly. A-galactosidase is widely distributed in nature, such as in animal, plant and microbes. Both carbohydrate utilization ratio and growth performance of animals could be improved when a-galactosidase was added to beans feed. It has great potential application in feed industry, sugar production, and medical industry, etc, so it is considered to be one of the most promising enzyme. Especially in recent years, the research and applications of a-galactosidase in the field of conversion of blood type, organ transplant, and Fabry Disease have shown an exhilarating prospect.We predicted the active site of the enzyme by bioinformatics analysis. The site-directed mutagenesis technique is utilized to change some amino acid residues in the a-galactosidase to illustrate its activity site. In this article a-galactosidase gene is used as start molecule that was cloned previously from Lactococcus spp.. We predict the enzyme active sites by homology modeling and the catalytic center of this enzyme are designed.Homology modeling estimated that the174amino acid residue may be a key residue for its catalysis or substrate reorganization. We change the GAC code of Asparagine (D174)into GGC that code for Glycine (G174) and then to CAC that code for Histidine (H174). The site-directed mutations of the gene are obtained by overlapping PCR. Then the mutant gene was cloned into expression vector pET28a thus the vectors pET28a-aga(D174-G) and pET28a-aga(D174-H) are constructed. The two vectors are transformed into E. coli BL21(DE3). The mutated enzymes are induced expression in BL21(DE3) by IPTG induction. The expressed protein band was observed through SDS-PAGE. The enzyme activity was detected by spectorphotometry with p-NPH as substance. The mutated enzyme activities showed no obvious difference with their control by T-test. And the substrate character has not changed either. It is concluded that the D174residue amino acid on a-galactosidase was not an activity residue for its catalytical activity nor substrate recognization. The active site of a-galactosidase needs to be further researched.