Improvement Of The Activity And Thermostability Of L-glutamate Oxidase By Directed Evolution

L-glutamate oxidase(LGOX)is a type of flavin protease that catalyzes the deamination of L-glutamic acid to alpha-ketoglutarate,ammonia,and hydrogen peroxide.Glutamate oxidase with high enzymatic activity and thermal stability will have a great application space in industrial biological applications,for example for the production of plants,biosensors,biochemical clinical testing.In this study,a directed library was constructed by error-prone PCR using directed evolution technology,and a high-throughput screening method was established.Four mutants of glutamate oxidase were obtained by screening:F94L,S280T,1282M,H533R,compared with the wild type,its enzyme activities increased by 18%,52.5%,18%,30%.By randomly saturating these mutants,two mutants,1282L and H533L,were obtained,and their enzymatic activities were increased by 12%and 30%,respectively,which was 30%and 60%higher than that of wild type.Thereafter,the site-directed mutagenesis techniques combining mutation sites mutated to obtain multi-site mutants,mutant enzyme activity was highest S280TH533L,increased activity relative to the wild type 90.7%.As a result of the thermostability measurement of the mutant of glutamate oxidase,the remaining enzyme activities of the mutants S280T and S280TH533L at 50℃ for half an hour were 34.8%,34.6%,and 8.2%,respectively,while the wild type had no activity.It was also found by the measurement of Td value,mutants S280T,H533L,S280TH533L 2.1℃were higher than the wild-type,0.8℃,1.9℃.In addition,measurement of the kinetic constants of the mutant was found to significantly reduce the Km value,Kcat value increased while the described substrate mutant enhanced affinity and catalytic efficiency is increased.Analysis of the results of homologous imodcling revealed that the mutant mutation site is located near the ion channel or the active center,resulting in an increase in catalytic activity;and the change in hydrogen bonding may lead to an increase in stability.The results of whole cell transformation verification experiments showed that,in the same time,the mutant is capable of more catalyst substrate,its molar conversion rate of 20%.Therefore,in this paper,the enzymatic activity and thermal stability of glutamate oxidase were improved by directed evolution technology.