Explored The Functions Of Domain B Region In A ?-amylase AmyP From Marine Microorganism

Alpha-amylase is widely distributed in animals,plants and microorganisms,which is one of the indispensable enzymes in industrial production today.In the previous experiments,we laboratory found a novel amylase AmyP(belonging to the new glycoside hydrolase subfamily GH13 37)was isolated from the marine macro-gene library and the sequence similarity to the known a-amylase was less than 20%.It was found that the domain size of the Domain B region of AmyP(26 amino acid residues)was only about 20%of the Domain B of the other subfamily a-amylase reported in the present study compared with the other subfamilyamylases of GH13,And the sequence is too short to form a typical Domain B structure as well as the usual a-amylases,which the function of the exercise with the current report that the function may also not the same.Therefore,the study of this specific Domain B region can enrich people's understanding of the function of the domain B of a-amylase,and also provide new information for understanding the catalytic mechanism of a-amylase.To explore the functional of a-amylase AmyP in Domain B region.We first designed the whole region deletion mutations and 26 site single point mutation experients.It was found that AmyP amylase lost activity after deletion mutation,indicating that although domain B region is very short,it is an indispensable part of AmyP.The enzymatic properties of 26 site single point mutations were summarized,It was found that the enzymatic activities of the mutants on the different substrates were decreased as a whole.The kinetics constants(Km,Kcat)of the mutants were further determined.It was found that the enzyme activity of the mutant was mainly due to the decrease of the enzyme reaction rate(kcat value decreased significantly),but on the affinity of the enzyme is not obvious(Km values did not change significantly).In order to further explore the factors that redvce the rate of enzyme reaction.First,it is found that the amino acid in domain B region may reduce the catalytic reaction rate of the enzyme by affecting the overall structure of the enzyme.Therefore,?wild-type AmyP,Domain B deletion mutants,multi-point mutant mutants(9 sites with large decrease in enzymatic activity)were detected under excitation light of wild-type AmyP,single point mutation The fluorescence spectra of the mutant(9 sites with large enzymatic activity decrease)were found to be less obvious than those of the Domain B region,and the environmental changes of the aromatic residues were less obvious.The overall structural changes will be less obvious.Thus,the amino acid in the Domain B region slightly affects the overall structure of the enzyme.Secondly,through the literature analysis also found Domain B region of the amino acid may also affect the catalytic microenvironment(dissociation)to reduce the enzyme catalytic reaction rate.Thus,an experiment to detect the optimal pH of the mutant was designed(the change in enzyme ionization was shown by the change in the optimum pH of the enzyme),and it was found that the optimum pH of the mutant was different from that of the wild type AmyP Sex shift,which indicates that mutations in enzyme activity are associated with changes in enzyme ionization.In order to further verify that the amino acid changes in the Domain B region did affect the ionization of the enzyme,the ionization constant pKa of the mutant and the wild type AmyP was detected and it was found that the acid dissociation constant of the mutant compared with the wild type AmyP,The alkali dissociation constant did not change significantly,which indicated that the amino acid change in Domain B did affect the ionization of a-amylase AmyP,which in turn affected the decrease of the catalytic reaction rate.