Fluid Simulation And Study On Immobilized Enzyme Packed-bed Reactor Based On TUDCA Transformation

Currently tauroursodeoxycholic acid(TUDCA) mainly extracted from bear bile, but the scarcity and particularity of bear bile makes TUDCA production can not content with the needs of clinical medicine. In this report, based on using the method of double enzyme coupling immobilized enzyme to produce TUDCA raised by our team, focused on efficient immobilized enzyme reactor development, FLUENT software was applied to simulate the flow field inside of the packed bed reactor, in addition, discussed the effects of flow rate and height-diameter ratio on reactor distribution of flow field, further tested and analyzed the simulation results through experiments, obtained a CFD model that can replace experiment to some extent. This research will provide theoretical and experimental bases for reactor design.This original of the project from the national science and technology major special project and the number of the project is 2014ZX09301306-007, the main researches and results contents are as follows:(1)The expression and purification of 7?-HSDH and 7?-HSDH were performed by using pGEX-6p-1 and a GST Gene Fusion System, besides, chitosan immobilized the 7?-HSDH and 7?-HSDH as a carrier. 1) One-step affinity purification by GST obtained the 7?-HSDH and 7?-HSDH free enzyme, the results of electrophoretic analysis are unique and clear, the enzyme concentrations were 1.776mg/mL and 2.059mg/m L, specific enzyme activities were 24.16U/mg and 49.36U/mg. 2) Using glutaraldehyde-activated chitosan microspheres to immobilized 7?-HSDH and 7?-HSDH, the results showed that both of the enzymes immobilized rates were greater than 98%.(2)Using double co-immobilized enzyme to transform TUDCA, obtained the optimum pH value, temperature and substrate concentration, ascertained optimum parameters of enzymatic reaction for further study. The results indicated that: 1)The value of PH has ranged from 7 to 11,the efficiency sorting of TUDCA transformed by immobilized enzyme is pH9> Ph11> Ph10> p H8> pH7, so the optimum pH value was 9. 2)The temperature has ranged from 5? to 60?, immobilized enzyme catalytic efficiency increased slowly at the beginning, then decreased rapidly, the efficiency sorting of TUDCA transformed by immobilized enzyme is 25?> 15?> 5?>37?> 45?> 60?, so the optimum temperature is 25?. 3) The substrate concentrations has ranged from 5mM to 25 mM, immobilized enzyme catalytic ability enhanced and then decreased, the efficiency sorting of TUDCA transformed by immobilized enzyme is 10mM> 15mM> 20 m M> 25Mm> 5Mm, so the optimum substrate concentration is 10 mM.(3)Simulated packed-bed reactor flow field by FLUENT 6.3, and studied the influences of flow rate and height-diameter ratio of reactor. We found that: 1) When the reactors height-diameter ratio were constant, the uniformity of the flow field with the flow rate increased, which would make contact of the substrate with the enzyme more fully, more conducive to enzymatic reaction. 2) Under certain flow rate, changed the height-diameter ratio, with the higher ratio, the liquid flowed through the same relative cross-sectional, however, the longer liquid residence time in the reactor, resulted in longer contacted time of enzyme and substrate, the more conducive to the enzymatic reaction.(4)Flow rate and height-diameter ratio of reactor have effects on the biotransformation of TUDCA verified by immobilized enzyme packed-bed reactor study. 1)When the height-diameter ratio of reactor was 30:16, the flow rates were 1mL/min, 2mL/min, 4m L/min respectively, comparing the production efficiencies of volume, the results showed that: 4mL/min> 2m L/min> 1m L/min, consistent with the simulation results.2)At the flow rate of 2mL/min, height-diameter ratio of 30:16, 60:16, 100:16, comparing the average single cycle yield of TUDCA, the results showed that: 100:16> 60:16> 30:16, consistent with the simulation results. Through the experiments, this assignment shows that the CFD model can be used to establish the numerical simulation of a packed-bed reactor, a solid-liquid two-phase flow process, be able to replace the experiment to a certain extent.