Structure And Function Of Multifunctional Amylase OPMA-G

Amylases (EC 3.2.1) are the type of enzymes that hydrolyze starch. Some of them have not only transglycosylation and hydrolysis activities and are named multifunctional amylases. The multifunctional amylases OPMA-G which are cloned and expressed in our laboratory possess the four high conserved regions, and it has dual activities of glycoside hydrolase and glycosyltransferase. The amylase degraded starch to produce maltose, maltotriose, isomaltotriose, isomaltotetraose. Isomaltotriose and isomaltotetraose belong to isomaltooligosaccharide(IMO). High-IMO syrups have characteristics as bifidus factor. Compared with common oligosaccharide, they have the function to improve intestinal microbial flora and prevent dental caries. Therefore, the study on the structure and function of OPMA-G can provide theoretical guidance to similar enzymes for molecular modification and function optimization.In order to investigate the role of its C-terminal domain of OPMA-G, the gene encoding C-terminal domain-truncatedΔOPMA-G was cloned from full-length OPMA-G gene, then the expression vectors pET28a-AOPMA-G was constructed and AOPMA-G was expressed as inclusion body in E.coli BL21(DE3). The expression yield ofΔOPMA-G was 42%. The collected inclusion bodies were denatured with 8 mol/L urea and refolded with dialysis in gradient urea. The purity of refolded AOPMA-G was more than 85% by SDS-PAGE. Compared with the enzymatic properties of OPMA-G, the specific activities of AOPMA-G was decreased from 8.22 U/mg to 6.53 U/mg, the optimum temperature drifted from 50℃to 40℃, the optimum pH was still 7.5. Like OPMA-G, AOPMA-G had also a strong digesting ability towards soluble starch, rather than pullulan or p-CD, it also degraded starch to produce maltose, maltotriose, isomaltotriose and isomaltotetraose with no difference in the proportion of these products. In addition,ΔOPMA-G was also a calcium-independent enzyme, and Zn2+, Ni2+ and K+ could also activate it, whereas Fe3+, Fe2+, Cu2+ and EDTA could inhibit it. Native-PAGE showed thatΔOPMA-G could exist in the form of monomer, dimer, trimers and more polymers, which indicated that C-terminal domain had no effect on oligomerization of OPMA-G. However, the starch adsorption rate of OPMA-G and AOPMA-G were 8.5% and 5.8%, respectively, which indicated that C-terminal domain of OPMA-G associated with the adsorption of the substrate, the missing of C-terminal domain caused the reduced of absorption rate, consequentially decrease the activity of the enzyme.Furthermore, the residue V200 in OPMA-G was selected for site-directed mutagenesis in order to investigate its effect on OPMA-G function based on the sequence alignment and homology modeling. Two expression vectors pET28a-OPMA-GM3 (V200G) and pET28a-OPMA-GM4 (V200K) were constructed using overlapping PCR method, and OPMA-GM3 and OPMA-GM4 were expressed in E.coli BL21(DE3) to the yield of 37% and 38%, respectively. Soluble OPMA-GM3 and OPMA-GM4 were purified by Ni2+-NTA chromatography to over 95% purities by SDS-PAGE.OPMA-GM3 and OPMA-GM4 also had a strong digesting ability towards soluble starch, the specific activity of OPMA-GM3 and OPMA-GM4 were 9.51U/mg and 8.63U/mg compared with 8.22U/mg of OPMA-G. Similar to OPMA-G, OPMA-GM4 degraded starch to produce maltose, maltotriose, isomaltotriose and isomaltotetraose, however,OPMA-GM3 did to produce some glucose besides the products above. Moreover, more maltose or more isomaltotriose was detected in OPMA-GM3's or OPMA-GM4's product system. The optimum temperature of two mutants was 50℃, and they might maintain more than 70% activities after incubated at 30℃-70℃for 1h. The optimal pH and pH stability of OPMA-GM4 was unchanged, but that of OPMA-GM3 showed an acid pH shift of about 1.0 unit. In addition, the ions shown above had the same effects on the activities of OPMA-GM3 and OPMA-GM4. These results showed that steric hindrance and charge at residue 200 may have the effects on the catalytic activity and enzymatic properties of OPMA-G, the larger steric hindrance or and positive charge are benefit for the formation of branches of products.Until now, the structure-function relationship of multifunctional amylases have not been fully understand, and the possible catalytic mechanisms and transglycosylation activity of these enzymes have not been fully elucidated. In recent decades, many researchers study the structure-function relationship of multifunctional amylases through directed-site mutagenesis. With the development of homology modeling, the rational design of the directed-site mutagenesis was also been developed. This thesis analyzes the function of C-terminal and the residue V200 of OPMA-G. All of the above results of this thesis provided a theoretical basis for the function optimization and the use of OPMA-G in industrial applications.