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Method of enzymatic co-production

An enzymatic and enzymatic reaction technology, applied in the direction of peptides, fermentation, etc., can solve the problems of expensive substrates, difficult to use in large quantities, etc., and achieve the effect of saving reaction time, saving raw material costs, and simplifying reaction procedures

Active Publication Date: 2019-01-29
BEIJING TIANKAI YIDA BIOLOGICAL SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the substrates used by these enzymes are expensive, such as phosphoenolpyruvate used by pyruvate kinase; and the by-products generated have certain biological toxicity and pollution, such as the products of acetate kinase and ammonia kinase catalyzed reactions They are acetic acid and ammonia respectively, so it is difficult to use them in large quantities in industrial production

Method used

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Experimental program
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Effect test

Embodiment 1

[0044] The preparation of embodiment 1 crude enzyme

[0045] The ATP regenerating enzyme and AK enzyme in the method of the present invention can be obtained commercially, or are artificially modified enzymes having the same catalytic function.

[0046] The enzyme preparation process is as follows:

[0047] Primers were designed according to the gene sequences of PPK enzyme, ADK enzyme, PAP enzyme and AK enzyme, and gene fragments were respectively amplified by PCR and connected to expression vectors (commercially available). .coli BL21(DE3) strain (commercially available).

[0048] Insert the transformed E.coli BL21(DE3) monoclonal into LB medium, culture to the logarithmic phase, add 1mM isopropyl-β-D-thiogalactopyranoside (IPTG) for induction, induce The cells were collected after 5 hours, and high-expression strains were screened by sodium dodecylsulfonate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0049] Insert the screened high-expression strains into the seed m...

Embodiment 2

[0053] Embodiment 2 co-production of S-adenosylmethionine and glutathione

[0054] The operation steps of co-producing S-adenosylmethionine and glutathione are as follows:

[0055] Add substrates to a 100L reaction system: 2.5kg L-glutamic acid, 2.5kg L-cysteine, 1.5kg glycine, 1.5kg L-methionine, 2.5kg adenosine, and 4.2kg hexameta Sodium phosphate, 0.27kg ammonium chloride, 0.37kg potassium chloride, 1.0kg sodium p-toluenesulfonate, 1.0kg magnesium chloride hexahydrate, 0.1kg manganese chloride monohydrate and 0.5kg disodium hydrogen phosphate, stir well and adjust the pH value to 7.0, temperature to 35°C. Add bifunctional glutathione synthase (GshF) 800U / L, PPK enzyme 500U / L, ADK enzyme 500U / L, PAP enzyme 500U / L and AK enzyme 1000U / L in the reaction system to start the reaction, the added enzyme See Example 1 for preparation.

[0056] After reacting for 2 hours, 650 U / L of methionine adenosyltransferase (MAT) was added to continue the reaction, controlling the pH value t...

Embodiment 3

[0058] Example 3 co-production of creatine phosphate and glutamine

[0059] The operation steps for co-production of phosphocreatine and glutamine are as follows:

[0060] Add substrates to a 100L reaction system: 2.0kg creatine, 2.5kg glutamic acid, 0.2kg adenosine, 2.0kg tetrapolyphosphate, 0.4kg potassium chloride, 0.5kg magnesium chloride hexahydrate and 0.6kg Tris, stir Evenly, adjust the pH value to 8.8 and the temperature to 32°C. Add creatine kinase 1000U / L, glutamine synthetase 800U / L, PPK enzyme 500U / L, ADK enzyme 500U / L and AK enzyme 1000U / L to the reaction system to start the reaction. For the preparation of the added enzymes, see Example 1 . During the reaction, the pH value was controlled to be 8.8, and the temperature was 32°C.

[0061] After reacting for 6 hours, the amount of creatine phosphate produced by high performance liquid chromatography (HPLC) was 30 g / L. The HPLC detection conditions are: Kromasil C18 chromatographic column (purchased from AKZO NO...

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Abstract

The invention discloses a method of enzymatic co-production. The method includes the following steps: adding two or more of reaction substrates, salt ions and adenosine in proportion in and enzymaticreaction system; and adding an ATP regeneration enzyme, an AK enzyme and two or more of production enzymes to the system for reaction. The method of enzymatic co-production has the following advantages: 1) two or more of the reaction substrates and corresponding production enzymes are added into the reaction system requiring ATP to generate a plurality of products with economic value, which simplifies a reaction process and has higher industrial value; 2) the co-production saves the ATP regeneration enzyme and AK enzyme and reaction time and reduces water, electricity and labor costs; and 3) the adenosine is used to replace conventional ATP or AMP, which saves a plurality of raw material costs for industrial production, and the adenosine has the advantages of low price and wide range of sources.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an enzymatic coproduction method. Background technique [0002] Adenosine triphosphate (ATP) consists of one adenosine (A) and three phosphate groups (Pi), with a molecular weight of 507 and molecular formula C 10 h 16 N 5 o 13 P 3 . The ATP molecule has a structure of A-Pi~Pi~Pi, and the two phosphate bonds away from adenosine are represented by ~, which are high-energy phosphate bonds, which are broken during the reaction to release a large amount of energy: when a high-energy phosphate bond is broken, the ATP molecule is converted into Adenosine diphosphate (ADP) releases energy of 30.5kJ / mol; when the second high-energy phosphate bond is broken, the ADP molecule is converted into adenosine monophosphate (AMP), which can release energy of 27.6kJ / mol. ATP is the converter and storage of energy in living organisms, known as "energy currency". [0003] ATP participates in many...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/40C12P13/00C12P13/14C12P13/04C12P17/10C12P19/32C12P19/02C12P21/02
CPCC12P13/001C12P13/04C12P13/14C12P17/10C12P19/02C12P19/32C12P19/40C07K5/0215
Inventor 刘珊珊刘辉周稳文秦永发
Owner BEIJING TIANKAI YIDA BIOLOGICAL SCI & TECH
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