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Construction of Co-expression SHMT and TPase Recombinant Vector and Dual-enzymatic Synthesis of L-tryptophan

Author: LiZuo
Tutor: LiuJun
School: Wuhan Polytechnic University
Course: Microbial and Biochemical Pharmacy
Keywords: Serine hydroxymethyl transferase Tryptophanase Co-expression Lactose - induced The dual enzymatic synthesis of L- tryptophan
CLC: TQ922
Type: Master's thesis
Year: 2010
Downloads: 52
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Abstract


Constructed in this study a single expression of serine hydroxymethyl transferase (SHMT) genetically engineered bacteria, a single expression of the tryptophan enzymes (TPase) genetically engineered bacteria and co-expression of SHMT and TPase genetically engineered bacteria. Lactose-induced expression of three recombinant engineered bacteria enzyme production and optimize the induction of enzyme production. Genetically engineered bacteria using recombinant enzyme production of two bacteria twice and single bacteria once the enzyme production, the use of the dual enzymatic and single bacteria Streptococcus two enzymes are two ways to complete the enzymatic synthesis of L-tryptophan, and compare the two The enzymatic synthesis of L-tryptophan pathway characteristics. 1 of Escherichia coli K-12 genome as a template, PCR amplification of the gene encoding SHMT and TPase coding. The PCR product purification and recycling, Nco Ⅰ and BamH I double digested recovery fragments, respectively, with the same by Nco Ⅰ and BamH I double digestion of the plasmid pET-28a carrier phase connection, constructed recombinant plasmid pET-SHMT and pET-TPase . The recombinant plasmids were transformed into E. coli BL21 (DE3) competent cells to construct a single expression the the SHMT gene engineering strain BL21 (DE3) / pET-SHMT and single expression the TPase gene engineering strain BL21 (DE3) / pET-TPase. The constructed plasmid pET-TPase Bgl Ⅱ and Hind III restriction enzyme digestion, and recovery of DNA fragments containing the T7 promoter and the TPase gene. Constructed with BamH Ⅰ and Hind Ⅲ double digestion of plasmid pET-SHMT and purified cleavage products connected with the DNA fragment containing the T7 promoter and gene TPase Construction of Expression of the recombinant plasmid pET-ST. The recombinant plasmids were transformed into E. coli BL21 (DE3) competent cells, build co-expression of SHMT and TPase genetically engineered bacteria E. coli BL21 (DE3) / pET-ST. Fermentation of a single expression SHMT single expression of TPase, co-expression of SHMT and TPase three genes, engineering bacteria in lactose-induced expression of lactose-induced concentration, the amount of induced initial growth, induction temperature induction time of the production activity. Single expression of the SHMT gene engineering bacteria BL21 (DE3) / pET-SHMT optimal conditions: optimal lactose concentration 8g / L; the best initial growth OD600 value of about 1.0; optimal induction temperature of 37 ° C, The optimal induction time of 6h. Optimal induction conditions develop engineered bacteria production SHMT activity reached 220.7 (U / mL), increased 6.4-fold compared to wild mushroom. Single expression of of TPase genetically engineered bacteria BL21 (DE3) / pET-TPase optimized conditions: optimal lactose concentration of 12g / L; the best initial growth OD600 value of about 1.0; optimal induction temperature of 37 ° C, The optimal induction time of 6h. Culture engineered bacteria to the optimal induction conditions TPase activity of its production reached 113.7 (U / mL), increased 8.4-fold compared to wild mushroom. Co-expression of SHMT and TPase gene engineering strain BL21 (DE3) / pET-ST, the optimal conditions: optimal lactose concentration of 12g / L; optimal amount of initial growth until OD600 is about 1.0; optimal induction temperature of 30 ℃, the best induction time of 6h. Culture engineered bacteria to the optimal induction conditions, its production SHMT activity reached 210.2 (U / mL) increased 6.1-fold compared to wild mushroom; TPase activity of its production reached 93.5 (U / mL), compared to the wild mushroom improve 6.9 times. Three pairs of bacteria double enzyme produced first single expression SHMT genetically engineered bacteria SHMT as the enzyme source, the use of glycine and formaldehyde synthesized from L-serine, glycine added to 30g, formaldehyde to fed-way join and take advantage of its control the pH value of the reaction solution is between 6.8 ~ 7.2. L-serine reaction liquid was collected after the completion of the reaction were added to 9g indole synthesis of L-tryptophan, in a single expression of the TPase gene engineering bacteria produced TPase catalytic reaction. This approach glycine conversion rate was 83.3%, the indole conversion was 92.5%, HPLC determination of L-tryptophan accumulated amount of 41.5g / L. 4 single strain two enzymes produced by co-expression of the genetically engineered bacteria SHMT and TPase as the enzyme source, L-serine and L-tryptophan two-step synthesis reaction at the same time in the same reaction tank. Design three different groups of the addition amount of raw materials, wherein the raw material added in an amount of 25 g of glycine and 7g indole, the the feedstock conversion rate and the product was higher cumulative volume, the glycine and the indole conversion rate was 82.7% and 82.9%, respectively, high performance liquid Phase Determination of the amount of accumulation of L-tryptophan was 28.9 g / l.

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