Industrial biotechnology has become the third wave of modern biotechnology development.Enzyme preparations have been applied to various industrial fileds,the efficiency and stability of enzyme catalysis are still the main bottleneck and restriction for the industrial application of enzymes,therefore,stable and efficient enzyme preparations have always been a research hotspot in the field of biotechnology.Fortunately,the emergence of bioinformatics has promoted the upgrading of enzyme molecular design strategies from directional evolution to the design of modified enzyme molecules through semi-rational design and rational design strategies.α-L-rhamnosidase is a glycoside hydrolase and an important biocatalyst in the food and pharmaceutical industries.In this paper,we used evolutionary and structural biological analysis to explore the function of the GH78 family α-L-rhamnosidase active site architecture.The mechanism of action of substrate binding was elucidated through molecular dynamics simulation of α-L-rhamnosidase-naringin and MMPBSA calculation,and the pH stability of GH78 familyα-L-rhamnosidase was improved by rational design strategy.The main results are as follows:First,the active-site sequence profile of GH 78 family were built by sequence and structural alignments.The function of 6 key residues were identified from the active architecture.Asp249 and Glu520 were catalytic residues,Asp249 was a proton donor,Glu520 was a nucleophile,and GH78 family α-L-rhamnosidase was a single-substitution reverse-catalytic type;Asp244 and Asp254 can regulate the pKa value of proton donor;Trp263 and Trp359 were involved in substrate recognition and binding,and the ruthenium structure formed a "sandwich" structure with the L-rhamnose in the substrate recognition process.Then,through the alanine and virtual saturation mutations,9 pH stability-related mutations were designed.Two experimentally improved pH stability sites were obtained,the enzyme activity of R307Y was also significantly improved compared with the wild type.The activity of E603F was slightly reduced compared with that of wild-type.The structural changes of mutants were analyzed by circular dichroism and fluorescence spectroscopy.The molecular dynamics simulation was used to elucidate the intrinsic reason of mutation-induced protein conformation and related amino acid interaction changes.Finally,through molecular dynamics simulation and MMPBSA calculation,it is clarified that var der Waals force was main driving force,electrostatic interaction and non-polar solvents contribute less to the combination.Trp236、Trp253、Ala340、Trp359、Ile462、Phe461、Tyr516、Val522、Trp528 and so on were key residues for hydrophobic effect,Ser286、Phe465、Pro521 formed 3 steady hydrogen bonds with naringin in the analysis of hydrogen bonds after the equilibrium of molecular dynamics simulation.This study analyzed the A.niger a-L-rhamnosidase driving forces combined with naringin,and important residues formed hydrogen bond and hydrophobic effect in the process of the combination.In the meantime,the results provided key amino acid sites for remoulding α-L-rhamnosidase in protein engineering. |