The Function Research About The Key Amino Acid Sites Of Pullulanase Combined

Pullulanases belong to a family of 13 glycosyl hydrolase(so called a-amylase family).Because it can hydrolysis specificity alpha-1,6-glycosidic bond of the amylopectin branch points and has important value for industrial application.For Anoxybacillus sp.LM18-11 sources of heat resistant pullulanase(PulA)crystal structure analysis found that the structure of a new domain(CBM68).The domain truncation experiment found that the structure have influence the enzyme properties.In order to further study the domain structure,studied the structure function of domain sites.Based on the analysis of the crystalline structure of pullulananse(PulA)-maltotetraose complex,nine key amino acid sites(Y14,D16,K62,V91,G92,R96,N370,G371 and Y372)in a new carbohydrate-binding module CBM68 which closely related to substrate binding were found.Nine mutants of Y14A,D16A,K62A,V91A,G92A,R96A,N370A,G371A and Y372A were obtained respectively by site-directed mutation.Among them,Y14A and R96A showed lower affinity for substrate and lower catalytic efficiency than those of the wild-type pullulanase(PulA).The Km values of Y14A and R96A were 4.2 times and 2.5 times higher than that of PulA,respectively.The kcat/Km values of Y14A and R96A only remained 29%and 37%of the original kcat/Km value(PulA),respectively.Moreover,the optimum temperatures of both Y14A and R96A were 10℃ lower than that of the wild-type pullulanase(PulA).Especially the half-life of R96A reduced to 8h from 48h at 60℃.The mutants properties changed larger because of van der Waals force,hydrogen bond or flexible changes based on the analysis of the protein structure.The Km values of Y14A and R96A remained 30%and 38%of the original Km value(PulA),respectively.The kcat/Km values of V91A increased by 16.7%,while the kcat/Km values of G92A only remained 18.4%of the PulA.But the enzyme activity of G92A remained 78.5%at 65℃.That increased by 43%than the wild-type PulA.The experimental of isothermal titration calorimetry were using the interactions of mutants V91A,G92A with substrate.The result shows that the two mutants affinity change was dominated by van der Waals force.All the results in this work have proved the significance of the key amnio acid sites Y14,V91,G92 and R96 on the structure CBM68.The method of directed evolution was used according to the nature of the amino acid.The four sites designed for the properties of different amino acids respectively.Succeeded in building 16 mutants.Determination of kinetic and thermodynamic parameters,the result shows that enzyme catalysis is negative influence changed the conservative aromatic amino acids.The value of Km and kcat/Km are opposite about the V91 and G92 mutation for the same amino acid.It is not same about the interaction mechanism of the two amino acid with substrates.Compared to the other properties of amino acids(Ser,Glu),the same nature of amino acids(Lys,His)increased the Km and kcat/Km about the site R96 mutations.Visible,the interaction is very complex between the amino acids.Its mechanism is not clear now.The research could enhance the understanding of the functional mechanism of CBM68.