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Double-enzyme system for efficiently synthesizing S-adenosylhomocysteine and application method thereof

A kind of adenosine homotype, cysteine ​​technology, applied in the fields of pharmaceutical engineering, genetic engineering, bioengineering, molecular biotechnology, enzyme engineering, can solve the problems of complex process, fast NAD consumption, low enzyme activity, etc., to improve yield Effect

Inactive Publication Date: 2014-08-06
南京仙奕基因科技有限公司
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AI Technical Summary

Problems solved by technology

[0007] In order to solve the problems of low enzyme activity, poor stability, fast NAD consumption and complicated process in the existing SAH preparation method, the present invention develops a high-activity high-temperature enzyme for the synthesis of SAH, and utilizes the extremely heat-resistant lactic acid dehydrogenation Enzyme for NAD regeneration and establishment of a dual-enzyme system for efficient production of SAH

Method used

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  • Double-enzyme system for efficiently synthesizing S-adenosylhomocysteine and application method thereof
  • Double-enzyme system for efficiently synthesizing S-adenosylhomocysteine and application method thereof
  • Double-enzyme system for efficiently synthesizing S-adenosylhomocysteine and application method thereof

Examples

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Effect test

example 1

[0035] Example 1. Cloning, expression and purification of SAHase and LDH derived from Thermotoga maritima

[0036] (1) Cloning of extremely heat-resistant SAHase and LDH genes

[0037] The extraction of Thermotoga maritima genome, PCR amplification, DNA isolation, purification, digestion, ligation, transformation of Escherichia coli and other genetic operations were carried out according to the standard methods in the third edition of the "Molecular Cloning Handbook".

[0038] (2) Overexpression of extremely heat-resistant SAHase and LDH genes in Escherichia coli

[0039] Recombinant plasmid pHsh- sahase and pHsh- ldh convert E. coli In BL21, culture at 30°C, pick a single colony and inoculate into LB medium containing 0.1 mg / ml ampicillin, culture at 30°C with shaking until OD 600 When the temperature reaches 0.6-1.0, transfer the test tube to a 42°C water bath shaker to induce gene expression, continue to culture for 2-6 h, break the cells with ultrasonic waves, a...

example 2

[0043] Example 2. Enzyme activity assay

[0044] (1) Determination of S-adenosyl homocysteine ​​hydrolase activity

[0045] Using adenosine and homocysteine ​​as substrates, the amount of SAH produced in the reaction system was determined by HPLC. 1 mL of reaction solution contained 1 mM adenosine, 2 mM homocysteine, 1 mM NAD, and 50 mM Tris-HCl (pH 8.0). Add an appropriate amount of enzyme solution, react at 85 °C for 20 min, and quickly transfer to an ice bath to terminate the reaction. The production of SAH was detected by HPLC at 260 nm. Definition of SAHase enzyme activity unit: The amount of enzyme required to generate 1 μmol of SAH per minute is defined as an activity unit. SAH was used as a standard curve.

[0046] (2) Determination of lactate dehydrogenase enzyme activity

[0047] LDH activity was determined by spectrophotometry, with sodium pyruvate and NADH as substrates, and the decrease of NADH in the reaction system was measured. The reaction system is 20...

example 3

[0048] Example 3. Enzymatic preparation of SAH

[0049] (1) Optimum conditions for the production of SAH in a single-enzyme reaction system

[0050] The reaction system was 10 ml, containing 20 mM adenosine, 40 mM homocysteine, 1 mM NAD, 0.5 mg purified SAHase, and 50 mM Tris-HCl. The reaction conditions were 85 °C, pH 8.0, 12 h.

[0051] (2) Optimum conditions for the production of S-adenosylhomocysteine ​​by a dual-enzyme reaction system

[0052] The reaction system is 10 ml, containing 20 mM adenosine, 40 mM homocysteine, 40 mM sodium pyruvate, 1 mM NAD, 0.5 mg purified SAHase enzyme solution, 0-2 mg purified LDH, 50 mM Tris-HCl. The reaction conditions were 85 °C, pH 8.0, 12 h.

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Abstract

The invention relates to the field of bioengineering, particularly relates to the technical fields such as a molecular biotechnology, genetic engineering, enzyme engineering and pharmaceutical engineering, the high-efficiency expression and enzymology qualitative analysis of extremely heat resistant recombinant enzymes and a method for efficiently producing S-adenosylhomocysteine (SAH) by using the enzymes under a high-temperature reaction condition. The SAH belongs to a type of effective sedative and antiviral factor. The method is characterized by comprising the following steps: (1) finding out the optimal reaction conditions and highest catalytic activity of a thermotoga maritime S-adenosylhomocysteine hydrolytic enzyme, so as to obtain an enzyme seed with excellent activity and stability for enzymic method production of SAH; (2) coupling SAH synthetic reaction to coenzyme regeneration reaction, so as to establish a double-enzyme system and technology for efficiently producing SAH; (3) with recombinant thermotoga maritime lactic dehydrogenase as a coenzyme, regenerating the reaction catalyst, so as to realize high yield of a main product and low consumption; and (4) establishing unique technological conditions for efficient production of SAH according to the nature of the thermotoga maritime S-adenosylhomocysteine hydrolytic enzyme.

Description

Technical field [0001] The present invention involves the field of biological engineering; specific technical fields such as molecular biotechnology, gene engineering, enzyme engineering, pharmaceutical engineering, etc.; more specific expression and enzyme qualitative of extremely thermal support enzymes, as well as the application of these enzymes at high temperature efficient at high temperature efficiencyMethods to produce S-adenosyl-type cysteine. Background technique [0002] S-adenosyl-type cysteine (SAH) is an amino acid derivative, which is related to disease resistance in organisms. It is an important indicator of early diagnosis of related pathological disorders.The exogenous SAH has a sedative effect. It is effective sleeping pills and anticonid drugs, and it is also a antiviral factor.With the extensive research and application of SAH in the medical field, its efficient production and preparation technology has also attracted much attention. [0003] SAH can be synth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/14C12P13/02C12N15/70C12N9/04
CPCC12N9/0006C12N9/14C12P19/40C12Y303/01001Y02P20/584
Inventor 邵蔚蓝钱国军王洪成
Owner 南京仙奕基因科技有限公司
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