Preliminary Analysis Of The Key Protein Structures That Related To The High Maltose-forming Ability Of Rhizopus Oryzae A-amylase

In the process of industrial production of high maltose syrup,the concentration of maltose in the final product produced from starch by a-amylase is an important index to evaluate its application performance.Presently,the research of fungal a-amylase with high maltose forming ability was mainly focus on enzyme-substrate catalytic pattern or other biochemical properties,while the research on the relationship between fungal amylase structure and its high maltose producing ability has rarely been reported.There were few reports to investigate the enzyme protein structure that related to the high maltose forming ability at the molecular level,and the maltose forming mechanism of fungal?-amylase has not obtained a reasonable explanation.Therefore,in this study,the computer-assisted simulation technique was used to perform molecular docking for Rhizopus oryzae a-amylase with maltotriose.The amino acid residues that may relate to the high maltose-forming ability of the enzyme were selected and mutated.The enzymatic properties of the mutantants were determined and the key amino acids that may relate to the high maltose-forming ability were analyzed.After computer simulation and analysis for R.oryzae a-amylase with maltotriose docking,simulation of the mutants and the interactions of amino acids with maltotriose were analyzed,and the docking results were used as the theoretical basis for further experiments.Site-directed mutagenesis of R.oryzae ?-amylase was done with overlapping PCR technology,and the mutants were screened and prepared by scale-up fermentation and protein purification strategy.Analysis of enzymatic properties showed that YHB,in which Y80,H286 and R333 were substituted by Leu,has the optimum temperature at 60?,which was 10? higher that of the native enzyme.The optimum temperature for H286L was found at 55? and the optimum pH for H286L was 5.0,which was decreased by 0.5 with comparing with the native enzyme,while the optimum temperature and pH of Y80L and R333L were almost the same with the native enzyme,respectively.With comparing with the wild-type,mutant of YHR could generate higher concentration of maltose after incubation for 8 h and formed maltose was increased by 11.67%with soluble starch as the substrate,and the maltose concentration was increased by 18.87%with maltotriose as the substrate.The maltotriose and starch were hydrolyzed by Y80L,H286L and R333L,maltose in the final products mutant H286L was increased by 10.32%and 16.87%,respectively.The results showed that the mutation of H286(L)had a promoting effect on the substrate conversion ability.In addition,the saturated mutation was perforemed for H286 and related mutants were studied for maltose forming from maltotriose and starch.It was found that the mutant H286M had a positive effect on maltose forming.Based on the analysis of the docking results of ROAmy with maltotriose,it was found that there were four special histidine residues in the presence of the core catalytic region and these four histidines were mutated and four mutants were constructed,respectively.Analysis results showed that those histidine residues may present little function on the maltose forming ability for ROAmy except for H286.Ten mutants were obtained by mutation of amino acids that located in the conserved regions of ROAmy.Compared with the native enzyme,only the maltose concerntration formed by D284L and N285L were increased 12.56%and 10.84%,respectively,when maltotriose was used as the substrate,and the yield of maltose was increased by 8.38%and 7.45%,respectively,when soluble starch was used as the substrate.For other mutants,no significant differences were observed for maltose concerntration formed from maltotriose or starch.