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Production of L-phenylalanine by Immobilized Cells

Author: LengYong
Tutor: SunZhiHao
School: Jiangnan University
Course: Fermentation Engineering
Keywords: L-phenylalanine Immobilized cells Packed-bed reactor Extraction
CLC: TQ922
Type: Master's thesis
Year: 2006
Downloads: 351
Quote: 3
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Abstract


Aspartate aminotransferase activity of the laboratory preserved recombinant E. coli E. coli SW0209-52 cell immobilization methods, conditions, immobilized cells packed-bed reactor continuous conversion of L-phenylalanine conversion solution extraction research and conducted a preliminary estimate of the cost of raw materials in the whole process. Identified by binding carrageenan embedded with 0.05% glutaraldehyde immobilization method, a carrageenan concentration of 2.5% and 7.5% of the amount of bacterial cells, immobilized cells obtained in the activity recovery was 73%. Investigated the influence of various factors in the the immobilized cell transformation process, identified the most suitable conversion conditions pH 8.0, temperature 37 ° C, to determine the optimum pyridoxal phosphate (PLP) concentration of 100 micromol / L. The cells were immobilized after operation significantly improve the stability and storage stability than free cells. Compared PLP and the vitamin B 6 as aspartate aminotransferase effect of coenzyme vitamin B 6 in the role of ATP can be transformed into pyridoxal phosphate, vitamin B 6 can replace to some extent the stability of the immobilized cells in the coenzyme as a coenzyme PLP stability phase immobilized cells PLP, but as to the vitamin B 6 relatively poor. By examining the impact of height to diameter ratio of continuous reaction, height to diameter ratio of 13.3 packed column of immobilized cells packed-bed reactor L-phenylalanine continuous transformation. Were investigated the influence of the substrate concentration and the duration of continuous transformation, considering the rate of substrate conversion and volume yield, identified by the flow of substrate (phenylketonuria) concentration of 30 g / L and a residence time of 10 h as ScS filled bed reactor stability conditions. Under this condition, the packed bed reactor with continuous operation for 40 days, benzo-keto acid conversion rate of more than 85%, the average volume in a yield of L-phenylalanine 1.85g/L/h, shows the immobilized cells packed-bed reactor better operational stability. 19 kinds of resin for the selective adsorption of L-phenylalanine and L-aspartic acid and L-phenyl-alanine, identified by strong acidic styrene cation exchange resin JK006 are holes to separate transforming the mixture of L-phenylalanine, to examine the different adsorption, elution, and decolorizing the influence of extraction conditions on the L-phenylalanine to determine the optimum operating conditions: the conversion solution to pH 2.0, L-phenylalanine concentration of 5 g / L, adsorption temperature of 20 ° C, 1 mol / L of ammonia eluted at a flow rate of 1 mL / min, D4006 resin bleaching. Conversion solution after bleaching was concentrated, crystallized and washed with ethanol, and recrystallized to give a purity of 96.7% of L-phenylalanine crystals, the entire extraction process yield was 82.8%. Preliminary estimate of the cost of raw materials required by the use of immobilized cells of the whole process of continuous transformation of the preparation of L-phenylalanine, and 5'-phosphate pyridoxal and vitamin B 6 As aminotransferase coenzyme The continuous conversion and partial conversion process cost comparison. Due the vitamin B 6 Price low use vitamin B 6 of batch conversion process, although immobilized cells cost rise, but the total conversion cost is still a certain degree of decline. In order to further reduce production costs, to achieve industrialization immobilized the cells preparation of L-phenylalanine, pyridoxal phosphate coenzyme immobilized through process optimization, supplementary ATP ways to improve the vitamin B 6 instead of pyridoxal phosphate operational stability of immobilized cells for further research.

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