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Butanedione reductase application

A diacetyl and reductase technology, which is used in the introduction of foreign genetic material using a carrier, recombinant DNA technology, fermentation, etc., can solve the problems of various applications of enzymatic properties, such as the lack of further research, and achieve substrate specificity. Good, high enzymatic activity, broad prospects for industrial applications

Inactive Publication Date: 2017-01-18
GUANGXI ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the relevant research on this enzyme, although its application in the biosynthesis of (S, S)-2,3-butanediol has been reported, it is limited to the application of butanedione as a substrate [Bioresource Technol. , 2012, 115: 111-116; Scientific Reports, 2013, 3: 2643], but its enzymatic properties and other applications have not been further studied

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Preparation of (S)-acetoin, (R)-acetoin, (S, S)-2,3-butanediol, meso-2,3-butanediol and ethyl lactate.

[0062] Using pETDuet-1 as the starting vector, the fragment of the gene encoding diacetyl reductase bdh obtained by PCR was double digested, and then connected to pETDuet-1 that had been treated by the same double digestion to construct pETDuet-bdh. On this basis, the same method was used to construct pETDuet-bdh-fdh. Introduce pETDuet-bdh-fdh into Escherichia coli E.coli BL21(DE3) to obtain recombinant Escherichia coli E.coli BL21(DE3)(pETDuet-bdh-fdh). Pick positive single clones and shake to OD at 37°C 600 = 0.6-0.8, adding 0.2mM IPTG, inducing at 25°C for 10h, collecting the cells by centrifugation, resuspending in binding buffer (containing 20mM phosphate, 300mM NaCl, 10mM imidazole, pH7.4), and ultrasonically disrupting the cells.

[0063] Carry out protein purification by His-tag label, specific method is: obtain supernatant through Ni-NTA gel col...

Embodiment 2

[0068] Example 2 Application of recombinant Escherichia coli containing diacetyl reductase BDH encoding gene bdh to catalyze diacetyl synthesis (S)-acetoin

[0069]1) Using pETDuet-1 as the starting vector, construct the recombinant E. coli expression vector pETDuet-bdh-fdh containing the gene bdh encoding diacetyl reductase and the gene fdh encoding formate dehydrogenase, and introduce it into E. coli BL21(DE3) , to obtain recombinant Escherichia coli E.coli BL21(DE3) / pETDuet-bdh-fdh;

[0070] 2) After a single colony of E.coli BL21(DE3) / pETDuet-bdh-fdh was transferred to LB liquid medium for activation culture, protein expression was induced at 25°C and 0.2mM IPTG to obtain Cells with enzyme BDH activity and formate dehydrogenase FDH activity as biocatalysts;

[0071] 3) Using the biocatalyst obtained in step 2), using diacetyl as a substrate, sodium formate as a hydrogen donor, and reacting at pH 6.0-9.0 at 33°C.

[0072] Gas chromatography was used to determine the conce...

Embodiment 3

[0073] Example 3 Application of recombinant Escherichia coli containing diacetyl reductase BDH encoding gene bdh to catalyze (S)-acetoin synthesis of (S, S)-2,3-butanediol

[0074] 1) Using pETDuet-1 as the starting vector, construct the recombinant E. coli expression vector pETDuet-bdh-fdh containing the gene bdh encoding diacetyl reductase and the gene fdh encoding formate dehydrogenase, and introduce it into E. coli BL21(DE3) , to obtain recombinant Escherichia coli E.coli BL21(DE3) / pETDuet-bdh-fdh;

[0075] 2) After a single colony of E.coli BL21(DE3) / pETDuet-bdh-fdh was transferred to LB liquid medium for activation culture, protein expression was induced at 25°C and 0.2mM IPTG to obtain Cells with enzyme BDH activity and formate dehydrogenase FDH activity as biocatalysts;

[0076] 3) Using the biocatalyst obtained in step 2), using (S)-acetoin as a substrate, sodium formate as a hydrogen donor, and reacting at pH 6.0-9.0 at 33°C.

[0077] The concentration and optical ...

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Abstract

The invention provides an application of butanedione reductase BDH of enterobacter cloacae subsp. dissolvens SDM. Through enzymology analysis, the BDH has the advantages of high enzyme activity, good substrate specificity, and single optical selectivity, and has wide industrial application prospect. According to the specific property of the BDH, The invention provides a plurality of applications of BDH or cell organism containing the BDH coding gene bdh to carry out catalyzed synthesis to obtain the chiral pure (S)-acetoin, (R)-acetoin, (S,S)-2,3-butylene glycol, meso-2,3-butylene glycol and ethyl lactate, and the butanedione reductase has important meaning for researching synthesis of chiral pure (S)-acetoin / (R)-acetoin and (S,S)-2,3-butylene glycol / meso-2,3-butylene glycol.

Description

technical field [0001] The invention relates to the application of enzymes, in particular to the application of a diacetyl reductase BDH derived from Enterobacter cloacae subsp.dissolvens SDM. Background technique [0002] Acetoin has obvious cheese aroma and fat aroma. It is mainly used in the production of spices such as cream, dairy products, coffee and yogurt. It is a food spice additive that meets the national standard GB2760-86. In addition, acetoin is an important chemical synthesis intermediate. In 2004, the US Department of Energy listed acetoin as one of the 30 priority platform compounds for development and utilization, which can be widely used in pharmaceutical, chemical and other fields. There are two optical isomers of acetoin (S)-acetoin and (R)-acetoin. In addition to the above applications, chiral pure acetoin also has special uses, such as the synthesis of chiral drugs and liquid crystal composites [Lett.Appl.Microbiol., 2013, 57: 274-281; ​​Bioresource Te...

Claims

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Application Information

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IPC IPC(8): C12P7/62C12P7/26C12N15/70
Inventor 李检秀谢能中黄日波黄艳燕刘祺霞王青艳严少敏陆迪陈先锐
Owner GUANGXI ACAD OF SCI
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