Bioconversion System And Process Regulation For Synthesis Of 4-Hydroxyisoleucine By Recombinant Dioxygenase

4-hydroxyisoleucine?4-HIL?is a non-protein amino acid with functions such as promoting the secretion of insulin,regulating dyslipidemia,and promoting fat metabolism,and has potential value in the treatment of diabetes.In the previous study,a Bacillus strain containing the ido gene has been screened from the soil,and the gene has been transferred into E.coli to construct a recombinant strain for one-step catalysis of the synthesis of L-isoleucine?L-ILe?to 4-HIL.In this study,the reaction process was regulated based on the characteristics and conditions,under which the recombinant IDO catalyzed the hydroxylation of isoleucine.The conversion system was expanded to investigate the conversion efficiency at high substrate concentration and further increase the yield of 4-HIL.The main results are listed as follows:?1?L-Ile was added exogenously,and the recombinant E.coli BL21/pET28a-ido was used as the catalytic enzyme.Based on the biotransformation conditions under which the IDO catalyzed the hydroxylation of isoleucine,single factor optimization was carried out at shake flask level.It was found that the best conversion result was observed after induction with the static whole cell as catalyst for 16 h.The whole-cell showed the best catalytic efficiency under the conditions of a 1:1 of molar ratio of?-ketoglutaric acid??-KG?to substrate,2 g·L^-1of Fe²⁺,5 mmol·L^-1 of Vc,and 300 mmol·L^-1 of L-Ile was catalyzed once to produce 190mmol·L^-1 of 4-HIL.?2?The catalytic reaction is based on the cell as the enzyme source,therefore,the concentration of the bacteria after the end of the fermentation can reflect the amount of the IDO to some extent.In order to obtain more cells to save cost savings,the fermentation conditions of the recombinant E.coli BL 21/pET28a-ido were optimized.The cell activity and the accumulation of product 4-HIL were the best after 10 h of fermentation under the conditions of initial pH 7.0,4%of the inoculum size,10 g·L^-1 of glycerol as the carbon source.The optimal conditions of lactose induction included 5 g·L^-1 of lactose,induction temperature of 45°C.In addition,the chance of lactose addition was after incubation for 12 h,and the induction time was 20 h,the OD₆₀₀ was up to 12 after fermentation.The recombinant E.coli was cultured in 3 L fermenter,and the OD₆₀₀ of the cells reached 44 after fermentation for 24h,resulting in a 90%of catalytic efficiency of the cells.The IPTG was replaced by lactose to overcome the defect that IPTG was expensive and toxic to cells.?3?According to the condition optimization of shake flask,the whole-cell catalytic system was amplified,and the concentration of the recombinant bacteria and the stirring speed was optimized by single factor in a 500 mL reactor.The cells were cryopreserved to catalyze 300 mmol·L^-1 of the substrate to produce 250 mmol·L^-1 of 4-HIL,which increased the yield of 4-HIL by 20%compared to the shake flask level.By increasing the substrate concentration and adding the cosubstrate?-KG in the late stage,630 mmol·L^-1 of 4-HIL was produced from 800 mmol·L^-1 of substrate,and the yield was 78%.The recombinant cells were reacted in batches at the maximum solubility of the substrate.After 3 batches of reaction,there was 220 mmol·L^-1 of 4-HIL produced from 300 mmol·L^-1 of substrate as before,which overcame the problem of low product yield under high substrate concentration and laid foundation for the large-scale production of 4-HIL by whole cells.