Study Of Site-directed Mutagenesis And Enzymatic Properties Of Escherichia Coli L-glutamate Decarboxylase

L-glutamic acid decarboxylase (L-glutamate decarboxylase, GAD) is a key enzyme involved in thebiological production of gama-aminobutyric(GABA). Widely utilized GAD from Escherichia coli hasstrict substrate specificity to L-glutamic. However, the low working pH of wild-type GAD seriouslylimited its industrial application; wild-type GAD optimum pH is too low, the optimum pH of enzymeactivity is3.8to4.5, when pH above6.0, GAD molecules easy happening depolymerization to inactivation.Due to the industrial production of GABA used for L-glutamate as substrates, L-glutamate solution is closeto pH neutral, in order to achieve optimal pH of GAD, with large amount of hydrochloric acid or sulfuricacid, this caused the additional raw material cost and the subsequent cost of wastewater treatment, on theother hand, the acid reaction conditions to the production equipments of GABA has much more corrosiveand accelerated aging equipments, reduce the service life of the production equipments, it has alsoincreased the extra production cost. Thus, to avoid of use of corrosive acids in the biological production ofGABA, protein engineering on GAD to raise its optimal working pH is indispensable and becomes urgentdemands of industrial bioproduction of GABA.To achieve the above purpuse. As reported previously by Eugenia Pennacchietti, changes of twoamino acid residuals on Escherichia coli glutamate decarboxylase can significantly improve pH limitationof WT enzyme, and the specific activity of the mutant enzyme at pH of5.9is four times of that fromwild-type enzyme, however, detailed biochemical analysis of the mutant enzyme was not reported or fullyinvestigated in that study. Therefore in this study, we cloned E. coli gadB and performed site-directedmutagenesis followed by overexpression of the recombinant enzymes, for the purpose of thoroughcharacterization of enzymatic properties, particularly its pH stability and thermostability in reactionmixture, which could help us better understanding in its catalytic mechanism and development of betterindustial GAD.Conclusion:1. E. coli gene gadB, encoding glutamate decarboxylase, was cloned and performed site mutation,both wild type and mutant of gadB was inserted into expression plasmids for production of recombinantenzymes.2. Both enzymes, GadB and GadB△HT, were obtained after overexpression in E coli, and, their pHor temperature optimum, themo-stability or pH stability were paralleled compared and systematicallyinvestigated, it was confirmed that mutant enzyme GadB△HT did exhibit better pH adaptation, workingwell in pH rang of3.4-6.2, and can still have50%of full catalytic activity at disfavored pH.3. Converted products in solution were detected by HPLC, it was found that in30min,0.1g of wet E.coli cells expressing wild-type GAD can yield10.34g/L of GABA, and the specific activity in host cellswas33460U/g of wet cells; with0.1g of wet E. coli cells expressing GadB△HT, the accumulated GABAwas11.28g/L and specific activity of host cells was measured as36500U/g. Therefore, at pH of4.2and 30℃, there was no significant difference between GAD and its mutant.4. Scaled up experiments were performed using10g of wet cells, that can convert182.5g/L of sodiumglutamate to123.63g/L of GABA in22h, the conversion efficiency reached88.61%; by using10g ofmutant GAD expressing E. coli cells, up to117.50g/L of γ-aminobutyric acid can be obtained from176.61g/L of sodium glutamate, and the conversion efficiency was as high as95.06%.5. It demonstrated that bioengineered is better in industrial application of GABA production, whichdisplayed higher performance in our studies and could reduce producing costs as well as discharging ofindustrial waste.