Study On The Thermostability And Alkalistability Of ?-glucuronidase Based On Structural Design

?-glucuronidase can hydrolyze and break the glucuronidase bonds of glycyrrhizin to produce Glycyrrhetic acid 3-O-mono-?-D-glucuronide or glycyrrhetinic acid derivatives with higher added value.Compared with glycyrrhizin,the product has more pharmacological activities and is a good sweetener,so it has great production significance.However,in industrial environment,the production of GAMG or GA by biological method requires ?-glucuronidase with good thermostability and alkalistability.At present,the efficiency improvement of Glycyrrhetic acid 3-O-mono-?-D-glucuronide or glycyrrhetinic acid from the conversion of substrate glycyrrhizin is still limited,either by improving its alkalistability or its thermostability.Therefore,this topic combined enzymatic,thermostability and alkalistability,according to two different sources of ?-glucuronidase(TpGUS79A from Talaromyces pinophilus and PGUS from Aspergillus oryzae).By analyzing the sequence and structure information of enzyme molecules and modifying amino acids directionally,the mutants with higher heat resistance and alkali resistance were obtained.The main work is as follows.1.First of all,using online prediction software hotspots wizard 3.0 to analyzes the sequence information of TpGUS79A-P-6Ps(6 disulfide bond designed into the structur to improve its thermostability).Combined with PyMOL on enzyme simulation analysis of the structure,eight amino acids for improve the stability of TpGUS79A-P-6Ps,E118,E155,E182,D230,D273,D382,E409 and D502 were selected as candidates(6 of them were located on the surface long loop and 2 were located near the active center).They were mutated into arginine,so as to improve the alkalistability.2.Secondly,the activity of the mutated TpGUS79A-P-6Ps at different temperatures and pH was tested.The results showed that the constructed mutants showed no activity,and the activity of mutants WT and starting enzyme TpGUS79A-P-6Ps was also low activity.Theoretically,the reason may be the change of secondary structure caused by the mutation,which squeezes the channel and prevents the substrate from entering the active center of the enzyme normally.In addition,enzyme production was increased by reactivation to supplement sugar fermentation.3.We used VMD software to build the alkalistability and thermostability mutant model after the combination.Dynamics simulation was play on the thermostability mutants(amputated C end 14 amino acids of PGUSE),alkalistability mutants(mutation nine PGUSE surface acidic amino acids arginine)and dual strategy combination mutant(thermostability and alkalistability strategy combination)to find the effects of mutations on the overall stability of the enzyme.It can be seen from the simulated parameters of RNSD and RMSF that the stability of the enzyme has been improved after the combination.4.Based on the predicted results of molecular dynamics simulation,we constructed 6 mutants PGUSE-D591-604-E79 R ? PGUSE-D591-604-E124 R ?PGUSE-D591-604-E135R?PGUSE-D591-604-E150R?PGUSE-D591-604-D200 R and PGUSE-D591-604-5Rs that may improve the thermo and alkali stability.Experimental verification showed that the mutant still showed the maximum activity at 40 ?,while the optimal pH was moved from 4.5 to 6.5,which increased by 2 units.In terms of thermo and alkali stability,the dual-strategy combination mutant PGUSE-D591-604-5Rs showed the best performance.After heat treatment at 65 ? for 120 min,it could still maintain an activity 30% higher than that of the wild type,while after treatment at pH 7.5 for 5 h,it still had an activity residue 12% higher than that of the wild type.