Comparison Of Characterizations Between D-mannose Isomerase And D-lyxose Isomerase And Rational Engineering Of D-mannose Isomerase

As a kind of functional sugar,D-mannose has many beneficial functions,and is widely used in the fields of food,medicine,and cosmetics.At present,there are three methods for preparing D-mannose,namely,plant extraction,chemical synthesis,and biological enzyme method.Compared with the former two methods,biological enzyme method has the advantages of environmental friendliness and easy operation,and has become a research trend in the preparation of D-mannose.D-mannose isomerase（D-MIase）and D-lyxose isomerase（D-LIase）can catalyze D-fructose to D-mannose.In this study the D-MIase from Escherichia coli DH5αand the D-LIase from Bacillus licheniformis ATCC 9945a were analyzed and compared with their structural characteristics and then were heterologously expressed and characterized.Further,D-MIase was rationally engineered,and mutants with significantly improved thermostability were obtained.And its enzymatic analysis and application were carried out.The main research content and results are as follows:（1）The characterizations of D-MIase and D-LIase were analyzed and compared.The results showed that the optimal reaction temperature and p H for D-MIase was 30℃and6.0-7.0,respectively.The optimal reaction temperature and p H for D-LIase was 50℃and7.5,respectively,and the optimal metal ion was Mn²⁺.The two isomerases showed significant differences in temperature and acid-base tolerance.D-LIase was more heat-resistant and sensitive to acid environment while D-MIase was contrary.Under the optimal conditions,D-mannose was synthesized.The results showed that the space-time efficiency and conversion rate of D-MIase reached 167 g·L^-1·h^-1and 30.9%,respectively,which was 28 times of the catalytic process of D-LIase.D-MIase showed more efficient production capacity and had the potential for further development.（2）D-MIase were rational engineered by virtual amino acid mutation in Discovery Studio and Fire Prot.According to the results of enzyme activity modification,HIS-176,HIS-383 and TRP-51 were key active sites,and the change of amino acids at this location would bring about a significant reduction of enzyme activity.The Fire Prot strategy predicted 17 hotspots for thermostability modification.After re-screening,5 favorable mutation sites were identified and then iteratively combined to obtain the optimal mutant M4（A241P/A116V/G253A/Q379P）.Compared with wild enzyme,the half-life of M4 at50℃had been increased to 628.4 minutes,which was 33 times that of wild enzyme.And the relative enzyme activity had also been increased by 64%,overcoming the problem of trade off in enzyme thermostability engineering.（3）The characterization of M4 showed that,the optimal reaction temperature and p H was 45℃and 7.5,respectively.Compared with wild enzyme,the optimal reaction temperature increased by 15℃.Through the molecular dynamics,replace of the ALA-241and GLN-379 sites reduced the skeleton entropy of protein unfolding due to the proline effect,resulting in an increase in protein rigidity.And change of ALA-116 and GLY-253enhanced the hydrophobicity,which improved stability.The results of application of WT and M4 in the production of D-mannose at 50℃indicated that the conversion rate and spatiotemporal efficiency of M4 were 29.9%and 161.6 g·L^-1·h^-1,respectively.Compared with WT,the conversion rate of M4 increased by 22.0%,and the spatiotemporal efficiency was 3.8 times that of WT.The catalytic efficiency of M4 has been significantly improved,indicating its potential for industrial application.