Computer-aided Simulation,Design And Analysis Of The Domain Functions Of ?-L-rhamnosidase

?-L-rhamnosidase,a member of glucoside hydrolase family,can hydrolyze glycosides with terminal rhamnosyl groupscatalyze such as naringin and rutin.It plays an important role in the application of food and medicine industrial.As a significant green and economic catalyst,catalytic efficiency of?-L-rhamnosidase is the critical factor determining the feasibility for industrial applications.In this study,the 3D structure of the?-L-rhamnosidase was established and the recombinant?-L-rhamnosidases combined with the domains from different resources were then heterologous expressed and characterized.The functions of these domains were explored by analyzing the enzyme activity,optimum pH,optimum temperature,pH stability and the kinetic constants.This study provides a theoretical basis for the extension of the scope and depth of application of this enzyme in food processing.The main experimental results were as follows:?1?The amino acid sequence of?-L-rhamnosidase?AT-rha?from Aspergillus tubingensis had a similarity of 92%to the?-L-rhamnosidase?AK-rha?from Aspergillus kawachii.The Modeller 9.15 was used to simulated the three-dimensional structure of?-L-rhamnosidase with multi-template modeling method and the new simulation of?-L-rhamnosidase was optimized by energy minimization.The?-L-rhamnosidases from A.tubingensis and A.kawachii were consisted of two?-sheet domains and an??/??₆-barrel domain.The results of molecular docking showed that naringin was located in the active cavity at the bottom of the??/??₆-barrel domain.?2?By using computer-aided analysis of the interaction of amino acid residues at the junction of domains,the replacement sites for domain exchange were determined at Gly400 and Asn787.Then the barrel domain of A.kawachii?-L-rhamnosidase was replaced with the corresponding domain of A.tubingensis?-L-rhamnosidase and the recombinant AK-rha_?TT was constructed and heterologous expressed in strain Pichia pastoris GS115.The specific activity of AK-rha_?TT increased to 38 folds compared with the wild type?-L-rhamnosidase from A.kawachii.The K_m and k_catat of the recombinant AK-rha_?were 0.33 m M and 3.06×10⁴ s^-1,respectively.?3?On the basis of the replacement of barrel domains,the remaining two domains of?-L-rhamnosidase from A.kawachii were replaced with from A.tubingensis?-L-rhamnosidase.The recombinants AK-rha_?T?T1?AK-rha_?T?T2T?T2 were constructed.The specific activity of the recombinant AK-rha_?T?T1?AK-rha_?T?T2T?T2 changed into 129.3 and 132.4 times,compared with the wild type A.kawachii?-L-rhamnosidase,and changed into 0.73,0.75 times,compared with the wild type A.tubingensis?-L-rhamnosidase.The K_m and k_catat of the recombinant AK-rha_?T?T1?AK-rha_?T?T2T?T2 was 0.37 mM,1.5 mM and 3.67×10⁴ s^-1,9.17×10⁴ s^-1.?4?The optimum temperature and pH of recombinant?-L-rhamnosidase was 60?and 4.0.Enzyme retained stable over the pH range of 4.0-6.0,and the half-lives at 60?were 4.5 h,6 h,4.5 h,and the half-lives at 65?were 50 min,60 min,55 min.By analyzing the hydrogen bonding forces of the amino acid residues at the differential sites of the A.kawachii?-L-rhamnosidase and A.tubingensis?-L-rhamnosidase in sequences alignment.The number of hydrogen bonds in two?-sheet domains of A.tubingensis?-L-rhamnosidase is more than that of A.kawachii?-L-rhamnosidase.This quantitative relationship is reversed in??/??₆-barrel domain.The difference in the number of hydrogen bonds on domains may be the main reason why the catalytic activity of A.tubingensis?-L-rhamnosidase is higher than that of A.kawachii?-L-rhamnosidase.