Biocatalysis Of ?-ketoglutaric Acid To Glutamic Acid

Biocatalysis of a-ketoglutaric acid(2-KG)to glutamate has improtant application prospects in organic synthesis and detection reagents.At present,there is little research on biocatalyzing 2-KG to Glu.The key of realizing this reaction is to find glutamate dehydrogenase(GDH)with excellent properties and produce efficiently the enzyme catalyst.Firstly,GDH was screened,and six kinds of GDH genes were chemically synthesized on pET-28a(+)plasmid and were imported into BL21(DE3).The corresponding GDH-producing engineering bacteria were successfully constructed.AxyGDH which can express solubly and have high specific enzyme activity was screened by enzyme activity of crude extract,heat stability of crude extract and so on.Subsequently,the enzymatic properties of AxyGDH were characterized.The optimum temperature was 60? and the optimum pH was 8.0.Under this condition,the specific enzyme activity reached 903.1±24.6 U/mg,and the enzyme half-life was 167 h.With the concentration increase of substrate 2-KG and NH4+ in the reaction,the catalytic activity of the enzyme increase first and then decrease,the enzyme activity reached the highest when the concentration of 2-KG was 10 mmol/L and the concentration of NH4+was 250 mmol/L.Metal ions choosed in experiment inhibit the catalytic activity of the enzyme.Then,the reaction conditions of AxyGDH?glucose dehydrogenase(GLDH)catalyse together were studied.The enzymatic properties of AxyGDH provide a design basis for catalyzing the reaction of 2-KG to Glu.The optimum reaction temperature was 40?,pH was 8.0,enzyme activity ratio was 1:2(AxyGDH:GLDH),the add ratio of amino donor NH4+ to the substrate 2-KG was 1:1(mol/mol),and the add ratio of glucose to substrate 2-KG was 1.25:1(mol/mol).Under the optimum reaction conditions,when the concentration of the substrate 2-KG is 10 mmol/L and adding 0.04 mmol/L NAD+,the substrate is completely converted after 28 min.Finally,the enzyme fermentation conditions of the engineering strain E.coli(DE3)/pET-28a(+)-AxyGDH were optimized.At first,the enzyme production conditions of the engineering bacteria were optimized,and the initial OD600 of the induction was 1.2,the IPTG concentration of the inducer was 0.7 mmol/L,and the induction time was 24 h.Then,the optimal medium was got by single factor carbon and nitrogen source optimization and response surface medium composition optimization:glycerin 11.3 g/L,Angel Yeast powder 16.3 g/L,MgSO4·7H2O 0.62 g/L,Na2HPO4·12H2O 17.1 g/L,KH2P04 3 g/L,NH4Cl 1.5 g/L,NaCl 0.5 g/L.The engineering bacteria were fermented in a 10 L fermenter to produce AxyGDH,and the enzyme activity of the fermentation broth reached 9066±24.6 U/mL when controlling the specific growth rate is 0.2 h-1,which was about 63.5 times of the initial enzyme activity of 142.7±3.2 U/mL.At this time,the dry weight of cells was 44.8g/L.This greatly reduces the production cost of glutamate dehydrogenase which provides reference for the industrial production of glutamate dehydrogenase.