Expression And Characterization Of Multifunctional Amylase OPMA-N

Isomaltooligosaccharide, also called branchingoligosaccharide, is one type of oligosaccharide consisting of 2-5 glucosyl residues and at least oneα-1, 6 glycosidic bond. Isomaltooligosaccharide mainly includes isomaltose, panose, isomaltotriose and isomaltotetraose. Compared with common oligosaccharide, isomaltooligosaccharide has its own unique character and physiological effects.This thesis is based on the research achievements of the amylase-producing mesophilic strain ZW2531-1,newly isolated from Chinese soil, was screened according to its ability to digest starch by clear zone method. The amylase secreted by ZW2531-1 degraded starch to produce several oligosccharides including maltose, maltotriose, and isomaltotriose as the major end-products, and perhaps other oligosaccharides such as isomaltotetraose. According to its morphological characteristics observed by an electron microscopy and its phylogenetic tree built with its 16S rDNA gene, strain ZW2531-1 belonged to genus Bacillus, and could be proposed as Bacillus sp. ZW2531-1. Based on the catalytic characters of the amylase from ZW2531-1 and the conserved sequences of the general amylases, our primers were designed,a DNA fragment named OPMA-N was obtained by PCR using the genomic DNA of Bacillus sp. ZW2531-1 as templet, then, was cloned into pET28a. Amino acid sequence alignment and three-dimensional structure simulation showed that OPMA-N gene contained 1767bp and its open reading frame (ORF) encoded 588 amino acids (GenBank accession number ABY71030). The OPMA-N had four highly conserved regions and a (β/α)8 barrel, its catalytic residues were thought to be Asp327-Glu356-Asp423. OPMA-N was homologous to maltogenic amylases. OPMA-N should be a novel member ofα-amylase family or GH13 family, or of multifunctional amylases subfamily.In order to investigate OPMA-N catalytic character and rules, and the function of its N domain, the expression vectors pET28a-OPMA-N, pET28a-?OPMA-N (lacking N domain) and pET28a-OPMA-N*(V328I) were constructed, then OPMA-N, ?OPMA-N and OPMA-N* were expressed in E.coli Bl21(DE3). Through Ni2+-NTA column or by the refolding and renaturation of inclusion bodies, the active OPMA-N, ?OPMA-N and OPMA-N* were purified to over 95% homogeneity with a renaturation yield of about 38%.The enzymatic characterization of OPMA-N, ?OPMA-N and OPMA-N* were analyzed and the results showed that their optimum temperature was 60℃and their optimum pH was 7.5, their activity was independent on Ca2+ . The kinetic analysis showed that OPMA-N only degraded starch, rather than pullulan orβ-CD, to produce several oligosccharides including maltose, maltotriose, and isomaltotriose, and perhaps other oligosaccharides such as isomaltotetraose, but did not produced glucose. The N domain played a very important role in binding starch. V328 residues of OPMA-N had a greater impact on the type of the glycosidic bond of its substrate OPMA-N act on. These results provided a theoretical basis for the industrial application of OPMA-N.Our country is very rich in starch resources which is widely distributed, large output, low price and no seasonal restrictions on its production. Thus, isomaltooligosaccharide produced from starch will become the main functional oligosaccharide in our country. Obviously, the absence of glucose in OPMA-N's products not only advanced the value of OPMA-N's products, but also simplified the purification process of OPMA-N's products from starch. Therefore, OPMA-N was expected as a more excellent candidate enzyme for the industrial production of isomaltooligosaccharides from starch than the traditional ones.