Screening, Characterization And Application Of α-Glucosidases From Environmental Metagenomic DNA

Glycosidase is an attractive enzyme due to its hydrolyzing and transglycosylation capability. To further explore and develop its important function in industrial field, this study is aiming to screen novel α-glucosidase from metagenomic DNA. In this study, a pair of low degeneracy primers based on the amino acid sequences of α-Glucosidase from Xanthomonas campestris was designed.23α-Glucosidase homologous genes were identified from the environmental microorganism metagenomic DNA by gene specific primered PCR.Two homologous gene fragments Glu33and Glu50share75%and76%sequence identity to a-glucosidase Xanthomonas campestris (xcg) were retrieved from metagenomic DNA samples. The chimerical genes, termed as xcg-33and xcg-50, were created by substitution of corresponding region of xcg gene from X, campestris pv.campestris strain B100. The length of xcg-33and xcg-50were1617and1629bp, respectively, encoding539and543amino acids, respectively. Both the xcg-33and xcg-50together with xcg genes were successfully over-expressed in E. coli under control of the T7promoter, resulting in expression of the active. soluble protein that constituted35%of the total cell protein content. Characterization of enzymatic properties shows that the specific activity of XcG-33and XcG-50increased6-8folds than XcG towards p-nitrophenyl-α-D-glucoside (pNPG). Besides the pH optimum of both XcG-33and XcG-50showed a shift to neutral about1.5compared that of wild type XcG. To study the thermostability of the α-glucosidases they were kept in30℃, at the beginning60min, above50%activities were lost for XcG-33and XcG-50. The study of organic reagent tolerance showed that they were remarkable stable in normal hexane, but can hardly tolerate glutaraldehyde, they lost90%activities at the concentration of10%. The study of metal ions tolerance showed most metal ions can largely restrain the activities of α-glucosidase, particularly Cu2+, Fe3+and Ni2+. The hydrolyzalion of disaccharides showed that only maltose and lactose can be hydrolyzed by these α-glucosidases. Furthermore, transglycosylation reaction using hydroxyphenyl and maltoside as acceptor and donor molecules showed that all of the enzyme can synthesize α-arbulin efficiently, while XcG-33can produce both α-arbutin and α-arbutin-α-glycosides,which was significantly distinct from the other enzymes.Besides,eugenol,menthol,puerarin and elemene can also be utilized as glycosyl receptor for transglycosylation reaction.In summary,we identified two novel α-glucosidases from metagcnomic DNA by PCR based metagenomic screening,XcG-33showed specific activities for producing new compounds in transglucosylalion reaction.We conclude that PCR-based screening of metagenome is an extremely effective method for seeking enzymes with functional diversity.At the same time,the retrieved homologous gene fragments provide a valuable material for the following work such as function of research on glycosidases family or cvolution by by DNA shuffling.