Reserch On Improvement Of Catalytic Efficiency Of Glutamate Decarboxylase By Error-prone PCR

γ-aminobutyric acid(GABA)is a non-protein amino acid widely found in plants,animals and microorganisms,which has many physiological functions,such as lowering blood pressure,resisting anxiety,regulating hormone secretion and so on.Glutamate decarboxylase(GAD)transformes glutamic acid to GABA by decarboxylation.Therefore,it is of great significance to screen GAD mutants with high specific enzyme activity and establish the corresponding high-density fermentation process.First of all,a high-throughput screening system for glutamic acid decarboxylase mutants was established.According to the pH variation rule of the whole-cell biocatalysis that transformed Glu to GABA,the indicator of chlorophenol-bromcresol green sodium acid was selected,the indicator ratio was 1:1,the screening concentration was 0.6%,and the concentration of Glu was 10 g/L,The high throughput screening system was established through the three steps:the screening of resistant plate,the culture of single colony with 96-well plate combined with the comparison and analysis of OD575/OD600 scanned by the microplate reader,the screening of the mutant strain in shaking flask.Secondly,engineering bacteria with improved transformation efficiency of Glu were obtained.GAD mutants library was constructed by error-prone PCR technique,three engineering strains with improved transformation efficiency and heat resistance were obtained.The train BW(AC)/PYB1S-LbgaBV209V/T237S/C379C/V406V was incubated at 55 ℃for 3 hours.After 4 hours of whole-cell catalytic reaction,the GABA content reached 270.36 ±4.48 g/L,and the transformation efficiency of GABA was 67.62 g/L/h,which was 21.2%higher than that of the original strain(55.73 g/L/h);the engineering strain BW(AC)/PYB1S-LbgadBA38S/s162S was incubated at 45℃ for 3 hours.Under the same conditions,the transformation efficiency of GABA reached 88.15 g/L/h(264.33 ±6.78 g/L)after 3 hours,which was 25.9%higher than that of the original strain(64.51 g/L/h).After heat treatment at 35℃,the transformation efficiency of GABA of the engineering strain BW(AC)/PYB1S-LbgadBD227D/G311G/V425 reached 90.64 g/L/h(271.92±5.74 g/L)with 13.7%improvement.Finally,the high density fermentation process was established by inorganic salt culture.pH was controlled at 6.8 after induction beginning that the value of OD600 was 50,induction temperature was 30℃ and induction time was 18 hours,the value of OD600 reached 146 after 32 hours culture,the strain BW(AC)/PYB1S-LbgadBA38s/s162S completely transformed Glu to GABA for only 3 hours,the GABA content was 299.16 g/L(99.72 g/L/h),which was 26.5%higher than that of the original strain(77.26 g/L/h)in the system,the GABA content of the strian BW(AC)/PYB1S-LbgadBD227D/G311G/V425 reached 299.73 g/L(99.91 g/L/h),which was 15.9%higher than that the original strain(95.87 g/L/h).