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Bioconversion of L-Cysteine by Immobilization Protoplast

Author: ZhangSu
Tutor: WangFengShan
School: Shandong University
Course: Pharmaceutical Engineering
Keywords: L-cysteine DL-ATC Enzymatic conversion Pseudomonas Protoplast Mutagenic Immobilized Calcium alginate High Performance Liquid Chromatography Precolumn derivatization Phenyl isothiocyanate
CLC: TQ922.9
Type: Master's thesis
Year: 2009
Downloads: 44
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Abstract

L-cysteine ??is an important sulfur-containing amino acid, is widely used in medicine, food, cosmetics, and feed industry. The present test screening to obtain a Pseudomonas SDN625 DL-ATC synthesis of L-cysteine ??can be transformed. Protoplast preparation and mutagenesis screening from SDN625 strains protoplasts immobilized enzymatic continuous conversion of L-cysteine ??product separation and purification, analytical methods, product identification and other aspects of the study, the results are as follows : activated sludge sewage treatment center from Shandong New Time Pharmaceutical Co., Ltd. A strain can be transformed strains of DL-ATC synthesis of L-cysteine ??named SDN625, cultured cells 48 h after the synthesis of L-half The enzymatic activity of cystine can reach 3443 U / mL. After 18 h of culture cells collected was added to a final concentration of 2mg/mL of lysozyme, protoplast formation rate reached 87.3% in the the 37 ℃ enzymatic 1 h, the formed protoplasts added at a concentration of 50μg/mL mutagen NTG 28 ° C gentle shaking for 30 min, after the regeneration and filter to filter out a synthesis of L-cysteine ??the enzyme activity can reach 4492 U / mL of the strains, 30.5% higher than the highest value of the original strain SDN625 activity, named SDN625-1. Immobilized in calcium alginate and polyacrylamide way the protoplasm of strains SDN625-1 body immobilized, found that calcium alginate immobilized significantly better than polyacrylamide. Immobilized using calcium alginate, 28 h of culture cells were harvested by adding a final concentration of 2 mg / mL lysozyme, and incubated at 37 ° C for 4 h, the immobilized particles, the cell density was adjusted to 1 × 10 8 / mL, gel particle diameter of 2 mm, fixing the best effect. Optimal enzymatic reaction conditions, the study found that the DL-ATC in the substrate solution in the reaction concentration of 0.8%, the reaction temperature is 40 ° C, a pH value of 8.0 can be obtained when the maximum substrate conversion rate. By comparison, the immobilized protoplasts and repeated use of immobilized cells have the same performance, and can be used repeatedly more than 3 times. After 20 days 4 ° C low temperature refrigeration, then add fresh enzyme production medium enzyme activity recovery training, protoplast to resume in about 24 h of culture more than 90% of the enzyme activity. The study found that the sucrose hypertonic is more appropriate immobilized protoplast storage solution. The results also showed that the immobilized protoplasts first 3 uses the conversion rate is about 15% higher than the immobilized intact cells. Column fluidized bed reactor is designed continuous technical route transformed to produce L-cysteine. Protoplast immobilized particles, under the best conditions, loaded column after the preparation of substrate solution to the continuous conversion reaction carried out under optimum conditions, the conversion collected after 16 h to a solution containing a product concentration of 5.04 mg / mL solution of 4900 mL, molar rate of 75.7%. Dowex 50W × 2H ~ type cation exchange resin, the product was purified, to give a product of the L-cysteine ??crystals (19.6 g, purification, yield 79.4%). After crystallization of the product by polarimetry specific reaction of the mercapto group, DTNB, R_f value, and a high performance liquid chromatography after detection is characterized as L-cysteine. L-cysteine-high-performance liquid chromatographic assay methodology Discussion. Derivatization reagent phenyl isothiocyanate (PITC) pre-column derivatization of L-cysteine, derivatives product was purified by high performance liquid chromatography (HPLC) column separation with diode array (PDA) detector detection of L- The cysteine ??achieve qualitative and quantitative determination. Meanwhile, the enzymatic reaction liquid using the PITC DL-ATC enzymatic conversion of synthesis of L-cysteine ??derivative, and completed the accurate quantification of the enzymatic reaction solution of L-cysteine.

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