Biocatalytic Preparation Of L-tert-leucine

Tert-leucine is a kind of non-proteinogenic chiral amino acid, it has a tertiarybutyl c-hain that has occupied a large space, so that it can control the molecular conformati-on completely, and it can increase the hy-drophobic of polypeptide and stability of en-zyme degradation. Therefore, in clinical, it can be used for the synthesis of anti-cance r,anti-AIDS drugs. Chemical synthesis of L-tert-leucine has some drawbacks, such as p-ollution, low yield, multiple steps and so on. In recent years, biosynthesis of L-tert-le-ucine has been extensively studied.Three methyl and ammonium formate are used as substrate in this study, bacterial c-ells are used as a catalyst to synthesis L-tert-leucine. According to the experimental study, through the experimental study, to determine the product transformation and an-alysis method, fermentation medium and the conversion process.Firstly, The detection method has been studied by this thesis, after a discussion on the thin layer chromatography, OPA derivative method and HPLC direct method, ulti-mately direct detection of HPLC is used as characterization method. The detection co-nditions of HPLC are as followed:Kromasil700-5C18colum、0.25%(NH4)H2PO4a-nd methanol are used as mobile phase, the volume ratio is100:5> flow rate is0.8ml/min, detection wavelength is205nm.Secondly, the two kinds of gene engineering bacterium E.coli Ri&E.coli R2are used as experimental strain. After the experiment, optimized the fermentation medium of the E.coli R1&E.coli R2f:beef extract1%, peptone1%, yeast extract3%, glucose0.3%, two of0.3%potassium hydrogen phosphate, ammonium nitrate0.5%, NaCl0.4%, ma-gnesium sulfate0.01%. And the best cultivation and enzyme production conditions we-re obtained:5%inoculation quantity, pH7.2, fermentation temperature32℃, the incub-ation time is21hours.Thirdly, by optimizing the conversion process to obtain the optimum conversion rea-ction conditions:reaction temperature30℃, bacteria quantity15%, two kinds of enzy-me producing bacteria ratio of wet weight (E.Coli R1:E.Coli R2)2:1(g/g), reaction i-n pH7.2phosphate buffer,0.1mol/L, reaction time24h, substrate ratio,0.15M:0.2M, L- tert-leucine yield reached68%.Finally, polyvinyl alcohol (PVA) as entrapping agents, adding sodium alginate (CA) aims at helps the shape function, the mixed solution of boric acid as crosslinking a-gent, immobilized bacteria. A preliminary study on the immobilization conditions has b-een studied, the concentration of three methyl pyruvate is0.15mol/L, the concentrati-on of ammonium formate is0.2mol/L, the concentration of NADH is lmmol/L, the a-mount of bacteria was15%(g/ml), two potassium hydrogen phosphate buffer solution0.1mol/L, reaction temperature30℃, Shaker oscillating reaction time72h.