The Molecular Modification Of Glutamate Decarbox-ylase Derived From Lactobacillus Brevis Lb85and Its Bio-synthesis Study Of γ-aminobutyric Acid

Glutamate decarboxylase (GAD) transforms L-glutamate into γ-aminobutyric acid(GABA) with concomitant consumption of a proton. GAD derived from lactic acid bacteria(LAB) exhibits optimum activity at pH4.0-5.0and loses its activity greatly at near-neutral pH.This property has a negative effect on the use of GAD for the production of GABA. In thisstudy, to broaden the active range of a GAD, i.e. GadB1from Lactobacillus brevis Lb85toward a near-neutral pH, irrational design using directed evolution and rational design usingsite-specific mutagenesis were performed. In addition, GABA biosynthesis in recombinantCorynebacterium glutamicum strains expressing the mutant gadB1Mgene were researched.The main contents and results are as follows.1. gadB1derived from L. brevis Lb85was cloned and over-expressed in Escherichia coliBL21(DE3). After purification, the enzymatic properties of GadB1were analyzed. ThroughSDS-PAGE and gel filtration chromatography analysis, GadB1is a monomer with amolecular weight of approximately54kDa. The optimum activity of GadB1was shown at pH4.6and40-50℃. GadB1sharply losed activity at pH values higher than4.6. It was morestable at pH4.5-5.5and40-60℃. The presence of the coenzyme PLP within the concentrationof0-100μmol·L-1increased its activity. The metal ion Ba2+, Ca2+, K+, and Na+had a positiveeffect on the enzymatic activity. Its kmand Vmaxvalues were22.90mmol·L-1and38.46U·mg-1,respectively.2. Based on the monomer homology model of GadB1, several improved mutants wereobtained through site-specific mutagenesis, with M1(GadB1E312S) the best one. Its specificactivity was53.90%higher than that of wild-type GadB1and its catalytic efficiency increased2.30-folds at pH6.0.3. For directed evolution of GadB1, a sensitive high-throughput screening strategy basedon a pH indicator was established. One improved mutant, i.e. M2(GadB1T17I/D294G/Q346H) wasselected from800variants after one round of error prone-PCR. It exhibited3.94-foldincreased activity at pH6.0and11.32-and25.26-folds increased catalytic efficiency at pH4.6and6.0, respectively. In M2mutant, the T17I and D294G mutation contributed to thebroadening of its range toward near-neutral pH, whereas the Q346H mutation contributed tothe increased substrate affinity at acidic pH.4. E312S mutation was then performed in mutant M2to obtain the combined mutant M3(GadB1T17I/D294G/E312S/Q346H). The mutant M3showed even higher catalytic efficiency,13.12-and43.16-folds than of wild-type GadB1at pH4.6and6.0, respectively. It indicated thatmutant site E312S derived from directed mutagenesis and mutant sites derived from error-prone PCR had the synergistically improved effect. At pH6.0, the GABA biosynthesized byGadB1M3was6.32-folds that of wild-type GadB1.5. Recombinant C. glutamicum strains ATCC13032/pEC-gadB1, ATCC13032/pEC-gadB1M1, ATCC13032/pEC-gadB1M2and ATCC13032/pEC-gadB1M3were constructed.After fermentation in shake flask, GABA production in gadB1M1-, gadB1M2-and gadB1M3- expressing ATCC13032strains from their endogenous L-glutamate increased by9.60%,20.30%and63.90%, respectively compared to that in the wild-type gadB1-expressing strain.After fermentation in a3L fermentor for72h, GABA production reached to10.37g·L-1and16.70g·L-1in gadB1-and gadB1M3-expressing ATCC13032strains, respectively, with themutant61%higher than that of wild-type. These result indicated that the amino acidsubstitutions in these mutants have beneficial effect on expanding the active pH range andenhancing GABA biosynthesis.