Studies On Construction Of Escherichia Coli Glutamate Decarboxylase Thermostable Strain And Its Extracellular Dispaly

?-aminobutyric acid,a non-protein amino acid widely exits in nature,is an important inhibitory neurotransmitter involved in a variety of physiological responses in the central nervous system.It is also a raw material for synthesizing the precursor of nylon 4,and widely used in food,medicine,feed and chemical fields.Glutamate decarboxylase(GAD)can specifically catalyze the production of ?-aminobutyric acid from L-glutamic acid,which is the only key enzyme for the preparation of y-aminobutyric acid by biocatalysis.However,the enzyme has two main defects:First,the optimum reaction pH is low(3.6?5.4),and the enzyme easily inactivated at pH above 6.0;Second,the enzyme has the poor thermal stability,and is easy to lose its activity.These drawbacks greatly limit the industrial applications of GAD.In this study,recombinant glutamate decarboxylase mutants from Escherichia coli strains with high pH stability and thermal stability were obtained by site-directed saturation mutation and high through-put screening.The properties of wild-type GAD and its mutants were compared.In addition,recombinant GAD surface display expression Escherichia coli on the help of the ice nucleoprotein(INP)was constructed.The whole cell apparent activities and their GABA converting abilities of intracellular GAD expression and cell surface display GAD expression recombinant bacteria were studied.The details are as follows:Firstly,the recombinant strain with wild type glutamate decarboxylase B(GadB)from E.coli,recombinant strain with GadB the C-terminal deletion mutant and recombinant strain with N-terminal site-directed mutant were constructed.Recombinant GadB and its mutants were successfully expressed in Escherichia coli.Secondly,the enzyme characters of wild-type GadB,C-terminal deletion mutant GadB(GadB?HT)and thermostable mutant GadB(M3,M4,M5)were compared.The optimal reaction temperature of wild type GadB,M3,M4,M5 is 30,32,37,35?,respectively.Except for GadB?HT which has the optimum reaction pH of 4.8,the optimum reaction pH of wild-type GadB,M3,M4 and M5 were all 3.8.The mutants(M3,M4 and M5)showed higher thermostability than wild-type GadB at 40?,50? and 60? with M4 the best.The T1550,Tm value and half-life of M4 were 54.5?,51.6? and 160.05 min,respectively,which was 14.3?,10.8? and 5.62 times higher than that of wild type GadB(40.2?,40.8 ?,24.24 min),respectively.Homology modeling analysis showed that the increased hydrogen bonds both inside a subunit chain and between subunit chains of GadB hexamers may be main cause of increased thermal stability of M3,M4 and M5.Finally,the recombinant bacteria with wild-type GadB or its mutants surface display expression were successfully constructed.The apparent activity of recombinant bacteria with GAD surface display expression was significantly higher than that of GadB intracellularly expression ones.In a 100 mL GABA preparation experiment with 10 g/L recombinant cells addition,the GABA yield and L-Glu molar conversion rate of wild type GadB intracellular expression bacteria were 254.8 g/L and 82.36%,respectively,at 6 h,while those of wild type GadB cell surface display expression ones were 270.8 g/L and 87.4%,respectively,which were 16 g/L and 5%increase,respectively.At 12 h,the yield of GABA produced by surface display expressed GadB was 27 g/L more than the intracellular expression one.At 24 h,the molar conversion rate was nearly 98%,and the GABA yield was 303.98 g/L.Bacteria with cell surface display M4 expression showed higher conversion speed,which can completely convert 3 M L-Glu within 12 h.