Improved Soluble Expression And Catalytic Activity Of A Thermostable Esterase Using A High-throughput Screening System Based On A Split-GFP Assembly

Thermostable esterase is one of the most promising green biocatalysts.Thermostable esterase offers a series of advantages,such as high thermophilicity,pH stability and organic solvent tolerance.Therefore,it has wide application in ole-chemical industry,paper industry and pharmaceutical industry.In this study,the thermostable esterase gene,named Aaeo1 was screened out from the genome of Aquiex aeolicus VF5.The expression level of Aaeo1 was still low and formed a great amount of inclusion body and low catalytic activity.The technique of directed evolution mimicking natural selection has been shown to be effective in modifying the thermostable esterase functions.The mutants with improved soluble expression and catalytic activity were obtained by two-step high-throughput screening system.Then,the enzymatic properties of the mutants were determined.?1?Optimum of error-prone PCR?epPCR?.The optimal system of epPCR was as following:Mg²⁺7 mmol·L^-1,Mn²⁺0.2 mmol·L^-1.The error rate has an average of 3.1 base pairs/kb,which meets the requirement of a mutant library;?2?Determination of effective,sensitive,fast and accurate screening approach.Using split-GFP as reporter to construct the mutant library by directed evolution.Compared to the full-length Enhanced GFP?EGFP?as reporter,the high-throughput screening of esterases based on the split-GFP assembly system offered a series of advantages,such as decreased fluorescence background and false positives.The error rate of positive colonies was greatly improved from previous 88%to 95%.The mutants with improved soluble expression were screened from the library which capacity was no less than 10⁴ through FACS.96-well plate fermentation was utilized for further mutant certification;?3?The obtain of mutants with improved soluble expression and catalytic activity.Through two-step high-throughput screening method,two of the best mutant?named EP1 and EP2?were obtained.According to the sequencing,EP1 and EP2 contained the amino acid mutations,I8V/I51V/F127I/E170D and V17G/H22R/K92N/I158K,respectively.The mutant enzymes formed fewer inclusion bodies and the soluble fractions were improved by 2-fold than that of parent Aaeo1.The specific activity of the purified mutants were 3.14 and 3.2-fold higher than that of parent Aaeo1,respectively.Through site-directed mutation,the optimum mutants I51V,E170D,I51V/E170D were obtained.The enzyme activity of the mutants were 2.35,2.46 and4.5-fold higher than that of parent Aaeo1,respectively.Through saturated mutation,the optimum mutants I51L/E170D was obtained.The enzyme activity was 4.8-fold higher than that of parent Aaeo1.The specific activity of the purified mutants I51V,E170D,I51L/E170D were6.86,10.01 and 14.86-fold higher than that of parent Aaeo1,respectively;?4?The enzymatic properties of the mutants were determined.The optimum temperature of Aaeo1 and the mutants were 80 ^oC and 70 ^oC.It was found that the mutants were more stable at high temperatures compared to the parent Aaeo1.The optimum pH value of the Aaeo1 was around 8.0 and no distinct changes were found in the mutants.Both the parent and the mutants displayed the similar profiles in pH stability and were very stable at slightly alkaline pH.The affinity to pNPC₄ of EP1 and EP2 were increased according to the lower K_m values.With a decrease in K_m and an increase in k_cat,the k_cat/K_m values of the EP1 and EP2 were about 2.42,2.35-fold higher than that of Aaeo1.The K_m values of the mutants I51V,E170D,I51L/E170D were increased while the k_cat/K_m values of the mutants were about 5.65,4.79 and 10.7-fold higher than that of Aaeo1;?5?According to the simulated protein structure of Aaeo1,the distance between E170D and the active site Asp163 was about 8.9?,and the enzyme activity might be affected within this short distance by the mutation.The effect of I51V at the surface may alter the enzyme structure through a long-distance affect.