Method for preparing D-cystine and L-tryptophane by using DL-cysteine split by microbial enzyme method

A cysteine, microbial enzyme technology, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve problems such as enantiomer consumption, achieve good temperature tolerance, high catalytic efficiency, The effect of good industrial value

Inactive Publication Date: 2010-08-25
天津启仁医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But often under the action of the enzyme, one of the enantiomers is consumed, which is the disadvantage of this method

Method used

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  • Method for preparing D-cystine and L-tryptophane by using DL-cysteine split by microbial enzyme method
  • Method for preparing D-cystine and L-tryptophane by using DL-cysteine split by microbial enzyme method
  • Method for preparing D-cystine and L-tryptophane by using DL-cysteine split by microbial enzyme method

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Experimental program
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Effect test

Embodiment 1

[0034] Construction of Genetic Engineering Bacillus subtilis and High Expression of Tryptophanase

[0035] According to the tryptophanase gene sequence of Escherichia coli K12 strain, design the upstream primer P1 of the tryptophanase gene: 5'-CCG AAGCTT ATG GAA AAC TTT AAA CAT CTC C-3' and the downstream primer P2: 5'-CCC GGA TCC TFA AAC TFCTTT CAG TTT TGC GG-3', using the genomic DNA of Escherichia coli JM109 strain as a template, the tryptophanase gene was amplified by PCR, and the recombinant expression plasmid pWB980-tnaA was constructed respectively.

[0036] The recombinant plasmid was transformed into Bacillus subtilis WB600, and a tryptophanase genetically engineered strain was constructed.

[0037] Inoculate the positive recombinant strain into 4 mL of LB medium containing 20 μg / mL of kanamycin for overnight culture, then inoculate 50 mL of fresh LB medium containing 20 μg / mL of kanamycin at 37°C Shake culture for 24 hours, remove the bacteria by centrifugation, tak...

Embodiment 2

[0039] Determination of D / L-cysteine ​​by pre-column derivatization HPLC

[0040] The pre-column derivatization method is as follows: with 100μL 10mmol / L Na 2 CO 3 solution (pH9) to dissolve cysteine ​​to a final concentration of 10-40mmol / L, then add 1% FDAA 200μL acetone solution, incubate at 40°C for 1h, add 2N HCl 20μL to terminate the reaction, and inject 20μL for HPLC analysis. Chromatographic conditions: C 18 Chromatographic column (Phenomenex Luna 5μ, 100A, 250×4.6mm), using phase A water (containing 0.1% TFA) and phase B acetonitrile (containing 0.1% TFA) as mobile phases, gradient elution: 0.0min, 55% phase A ; 11.0min, 47% phase A; room temperature, detection wavelength 340nm, flow rate 1mL / min. Inject 5 times continuously, calculate the peak area and take the average value, draw the standard curve with the D-cysteine ​​concentration (X, mmol / L) as the abscissa and the peak area (Y) as the ordinate. The D-cysteine ​​produced by the enzymatic reaction was dilute...

Embodiment 3

[0042] Determination of L-Tryptophan Content by HPLC

[0043] Precisely configure L-tryptophan standard solution with a concentration of 50-200 mg / L, take 20 μL of samples for injection, and use HPLC method to determine the content of L-tryptophan. Chromatographic conditions: C18 chromatographic column (Phenomenex Luna 5μ, 100A, 250×4.6mm), mobile phase: methanol: 0.001mol / L potassium dihydrogen phosphate (30:70), room temperature, detection wavelength 225nm, flow rate 1mL / min. Inject 5 times continuously, calculate the peak area and take the average value, draw the standard curve with the L-tryptophan concentration (X, mg / L) as the abscissa and the peak area (Y) as the ordinate.

[0044] The L-tryptophan produced by the enzymatic reaction and the refined L-tryptophan can be accurately quantified after being detected by high performance liquid chromatography and compared with the standard curve. Figure 2.

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Abstract

The invention discloses a method for preparing D-cystine and L-tryptophane by using DL-cysteine split by a microbial enzyme method. The method comprises the following steps of: splitting the DL-cysteine to produce D-cysteine and L-tryptophane through an enzymatic reaction by using fermentation liquor of recombined Bacillus subtillis which efficiently express tryptophanase as an enzyme source and using the DL-cysteine and indole as a substrate; and oxidizing the enzymatic reaction liquor to ensure that the D-cysteine is oxidized into D-cystine, then regulating pH to be 5 and performing isoelectric point crystallization to separate the D-cystine out, and collecting the precipitation to obtain the D-cystine. A method for purifying the L-tryptophane comprises the following steps of: removing the residual indole from the supernatant which is subjected to the isoelectric point crystallization by adopting an S-8 type macroporous resin, then absorbing the L-tryptophane in the supernatant by adopting an NKA-II type macroporous resin, eluting by using 50 percent ethanol, reducing pressure, concentrating and drying to obtain the L-tryptophane. The invention provides a novel process route of green production of the D-cystine and the L-tryptophane.

Description

【Technical field】: [0001] The invention belongs to the technical field of amino acid production by biotechnology, and relates to a method for producing D-cystine and L-tryptophan by splitting DL-cysteine ​​with microbial enzymes. 【Background technique】: [0002] Cystine is an important pharmaceutical intermediate, and is also commonly used in the preparation of compound amino acid oral preparations or infusions, dyes, cosmetics, dairy product additives, oil antioxidants, etc.; it can also be used in heavy metal antidotes, hepatitis and other drugs. Cystine can promote the redox ability of body cells, strengthen white blood cells to prevent pathogenic bacteria infection, and can be used to treat leukopenia, coronary heart disease, prevent fatty liver, liver cirrhosis, promote hair growth, prevent skin aging, and can also be used for typhoid fever, dysentery, and influenza , asthma, neuralgia, eczema and various poisoning and other diseases. [0003] D-cystine is a dimer of D...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/22C12P13/12C12R1/125
Inventor 张奇侯洁白芳段静静高智慧黎霞张玺
Owner 天津启仁医药科技有限公司
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