Improved The Catalytic Activity Of Aspergillus Niger ?-L-Rhamnosidase Based On Site-Directedmutation

In the previous study,the gene of Aspergillus niger?-L-rhamnosidase r-Rha1 was cloned and expressed.Three-dimensional model of r-Rha1 was also simulated by Modeller.In this study,rational design was used for selected amino acid sites to contruct mutants,the enzymatic properties such as relative enzyme activity,kinetic parameter,optimum temperature,thermal stability,optimum pH and pH stability were characterized.Furthermore,circular dichroism,fluorescence spectroscopy and molecular dynamics analysis were applied to illustrate the mechanism involved with the improvement of catalytic activity.First,two mutant sites,including five mutants,were obtained based on the multiple sequence alignments of r-Rha1 and six characterized?-L-rhamnosidases from different sources.After the screening,the relative activity of R404S and N578D had increased by 30%and 130%,respectively.Kinetics experiments showed that the Michaelis constant?K_m?of mutants were lower than the wild type.The catalytic efficiency of R404S and N578D have increased by 6%and 16%.The results of MM/PBSA showed that the binding energies of mutants with pNPR were reduced.The higher affinity of substrate and enzymes were illustrated by the results of experimental data and MM/PBSA.By circular dichroism and fluorescence spectroscopy,we found that the structure of mutants was similar to wild type.The changed flexible of mutants and several loops around catalytic pocket were analyzed by molecular dynamics may be the reason for the increase in catalytic activity.Then,six mutant sites,including fourteen mutants,were designed by analyzing molecular docking of r-Rha1 with pNPR.The relative activity of A355N,S356Y and D525N had increased by 45%,77%and 185%,respectively.Kinetics experiments showed that the Michaelis constant?K_m?of mutants were lower than the wild type.The catalytic efficiency of A355N,S356Y and D525N have increased by 4%,9%and 53%.The calculated of MM/PBSA demonstrated the binding energies of mutants were decreased than wild type,the tendencies were similar with the results of K_ms.The thermalstability of D525N was slightly increased,which had increased by 0.8h,2 min and 1 min at 60oC,65oC and 70oC than half-lives of wild type.By circular dichroism and fluorescence spectroscopy,we found that the structure of mutants was similar to wild type.The molecular dynamics indicated that flexible of mutants,loops at the bottom of??/??₆ barrel and?-helix of??/??₆ barrel were reinforced.The involved amino acid regions may be related to the improvement of catalytic efficiency.Finally,according to the results of previous two strategies,ten double-points mutants were constructed and six mutants with improved catalysis efficiency were selected.The relative activity of A355N-R404S,A355N-D525N,A355N-N578D,S356Y-N578D,R404S-D525N and D525N-N578D had increased by 45%,57%,81%,20%,47%,34%,respectively.The K_m and binding energy of mutants were lower than wild type.MM/PBSA showed that the binding energies of mutants with pNPR were reduced.The higher affinity of substrate and enzymes were illustrated by the results of experimental data and MM/PBSA.The results of circular dichroism and fluorescence spectroscopy showed that the structure of mutants were similar to wild type.The molecular dynamics indicated flexible of mutants and loops at the bottom of??/??₆-barrel were reinforced.The flexible of protein may be improved by the changed loops,which will increase the catalytic activity.