Effects Of Structure Features On The Activity Of Aspergillus Kawachil α-L-Rhamnosidase

α-L-rhamnosidase is widely used in the food and biomedical industries because it can hydrolyze some of the natural glycosides such as naringin,rutin and myricitrin.As a significant green and economic catalyst,catalytic efficiency ofα-L-rhamnosidase is the critical factor determining the feasibility of using enzymes for industrial applications.So far,there is no report about the effects of structure features on the the catalytic efficiency ofα-L-rhamnosidase.In this study,a putativeα-L-rhamnosidase from Aspergillus kawachii was successfully identified,heterologous expressed and characterized.On the basis of this,the catalytic efficiency ofα-L-rhamnosidase was improved by molecular engineering.Then,the micro structural changes and properties of mutants were analyzed by molecular dynamics simulation（MD）.The main findings are as follows:（1）Recombinant expression and characterization ofα-L-rhamnosidase from A.kawachii:theα-L-rhamnosidase gene（AKrha）was cloned in p PIC9K vector and expressed in Pichia pastoris GS115.Then,the recombinant enzyme was purified by ultrafiltration and gel chromatography and the electrophoretic enzyme with a molecular weight of about 110 kDa was obtained.The results showed the optimum pH and temperature of recombinant enzyme were 4.0 and 50oC,the retained stable over the pH range of 3.0-8.0,and the half-lives at 60,65 and 70oC were 138,20 and 3 min,respectively.Sn²⁺,Cu²⁺,Mg²⁺,Fe³⁺,Al³⁺and SDS had significant inhibitory effects on the recombinant enzyme.The optimal substrate for the recombinant enzyme was naringin,which was not active against common artificial substrate p NPR.The K_m,k_cat,V_max and k_cat/K_m values of recombinant enzyme on narigin were 0.25 mM,0.62 s^-1,0.06μM min^-1 and 0.25×10^-2 s^-1μM^-1,respectively.（2）3-D structure modeling and molecular docking analysis ofα-L-rhamnosidase from A. kawachii:The 3-D structure of theα-L-rhamnosidase was established by using multi-template modeling method with Modeller software,and the model was further optimized by MD.The final complete and qualitative structural model conformation was evaluated by the Ramachandran Plot and Verify3D.Using the method of server domain search and references,the catalytic domain ofα-L-rhamnosidase was determined as a typical（α/α）₆-barrel domain of GH78 family.The results of molecular docking showed that naringin was located in the active cavity at the bottom of the（α/α）₆-barrel structure,and its interaction with the amino acid was similar to that of the crystal structure complex.（3）Improved the catalytic performance of recombinantα-L-rhamnosidase by site-directed mutagenesis:based on the sequence alignment and three-dimensional structure analysis,seven mutants were selected and constructed.The catalytic performance of three mutants was significantly enhanced.The specific activity of D555N,E617G and D555N-E617G increased by46.3%,174.0%and 585.5%respectively,compared with the wild type（WT）.The optimum pH and optimum temperature for all mutants were consistent with the WT,both at 4.0 and 50oC.The V_max of the D555N and E617G was slightly higher than that of the WT,and the k_cat/K_m were increased by about 0.5 and 3.5 times,respectively.The V_max,k_cat and k_cat/K_m of the D555N-E617G was increased by 5.3,9.9 and 9.0 times compared with WT.（4）The effects of mutations on the affinity of the substrate were analyzed by molecular dynamics simulations.The binding energies of WT,D555N,E617G and D555N-E617G with naringin were calculated by molecular dynamics simulations.The results showed that D555N and E617G significantly changed the movement of the loop around the substrate,while the effect of D555N-E617G on the loop region was narrower,but caused the fluctuation of individual amino acids.By observing the interaction,it was found that the decreasing in the amino acid interaction with the substrate of D555N mutations,causing the energy increased of electrostatic interaction and van der Waals,resulting decreseing in its affinity with the substrate.The increasing in the amino acid binding to naringin of E617G mutant,causing the energy significantly decreased of van der Waals and to enhance the ability to bind to naringin.The decreasing in the amino acid of the D555N-E617G mutant interacted with the substrate,causing the increased of electrostatic interaction energy and polar solvation energy,resulting in decreased affinity with naringin.The results of this affinity calculation are consistent with the K_m values of the mutants.