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(2R, 3R)-2,3-butanediol dehydrogenase and coding gene and application thereof

A technology of butanediol and dehydrogenase, applied in the -2 field, can solve the problems of unsuitability for industrial production of butanediol, no industrial application value, low output and the like

Active Publication Date: 2012-12-05
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The only (2R, 3R)-2,3-butanediol dehydrogenase that has been reported now has only the bacterial strain isolated from Saccharomyces cerevisiae, but this bacterial strain is not suitable for industrial production of butanediol, and the yield is extremely low ( 1mM), this enzyme is only needed to maintain the physiological function of yeast itself, and has no practical industrial application value [J.Bio.Chem., 275(46), 35876-35885(2000)]

Method used

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  • (2R, 3R)-2,3-butanediol dehydrogenase and coding gene and application thereof
  • (2R, 3R)-2,3-butanediol dehydrogenase and coding gene and application thereof
  • (2R, 3R)-2,3-butanediol dehydrogenase and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1, (2R, 3R)-2, the construction of the acquisition of 3-butanediol dehydrogenase gene and its prokaryotic expression vector

[0052] The (2R,3R)-2,3-butanediol dehydrogenase gene from Paenibacillus polymyxa ATCC12321 was amplified from the Paenibacillus polymyxa ATCC12321 genome by PCR after whole genome sequencing and annotation analysis . Among them, the Paenibacillus polymyxa ATCC12321 strain was purchased from the American Biological Resource Collection (http: / / www.atcc.org / ), and the public can directly purchase it freely from the American Biological Resource Collection.

[0053] The cloning process of the gene and the construction process of its prokaryotic expression vector comprise the following steps:

[0054] 1. Cloning of (2R,3R)-2,3-butanediol dehydrogenase gene

[0055] After whole genome sequencing and annotation of genome information, a gene that may encode butanediol dehydrogenase was obtained, and the following primers were designed accordi...

Embodiment 2

[0058] Example 2, (2R, 3R)-2,3-butanediol dehydrogenase expression and purification in prokaryotic system

[0059] The prokaryotic expression vector pET22b-2R3R containing the (2R,3R)-2,3-butanediol dehydrogenase gene obtained in Example 1 was transformed into Escherichia coli E.coli BL21(DE3)pLysS, and the positive single clone was selected at 37°C Shake the bacteria until the OD600 is 0.6, add 1mM IPTG to induce, collect the bacteria by centrifugation after 5 hours, resuspend in ultrasonic buffer (containing 20mM phosphate buffer, 300mM NaCl, 10mM imidazole, pH 7.4) after repeated freezing and thawing , sonicated the cells, collected the supernatants and precipitates, and carried out SDS-PAGE electrophoresis analysis. The results showed that the prokaryotic expression vector carrying the target gene was expressed in large quantities in E. coli BL21(DE3)pLysS, and the expressed recombinant protein was single The molecular weight of the subunit is 38kDa, which is consistent wi...

Embodiment 3

[0061] Embodiment 3, enzymatic characteristic identification

[0062] Enzyme characterization was performed using the following reactions:

[0063]

[0064] This reaction is a reversible reaction, and the change of NADH is used as the basis for the determination of enzyme activity (the obtained recombinant protein can only use NAD+ / NADH as a coenzyme, and cannot use NADP+ / NADPH as a coenzyme).

[0065] Using NAD+ as a coenzyme, catalyze (2R,3R)-2,3-butanediol to (R)-acetoin (at 70°C, pH 11.0, with 100mM (2R,3R)-2,3- Butanediol is a substrate, 4mM NAD+ is a coenzyme), the result only generates (R)-acetoin; catalyzes meso-2,3-butanediol to generate (S)-acetoin (at 70 Under the conditions of ℃ and pH 11.0, using 100mM meso-2,3-butanediol as the substrate and 4mM NAD+ as the coenzyme), only (S)-acetoin was produced.

[0066] Using NADH as a coenzyme, catalyze (R)-acetoin to generate (2R,3R)-2,3-butanediol (at 70°C, pH 8.0, with 10mM (R)-acetoin as substrate , 0.2mM NADH is a...

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Abstract

The invention discloses (2R, 3R)-2,3-butanediol dehydrogenase from paenibacillus polymyxa ATCC12321 and a coding gene and application thereof. Zymology verifies that the dehydrogenase has new characteristics: (2R, 3R)-2,3-butanediol is catalyzed to generate (R)-acetoin and meso-2,3-butanediol is catalyzed to generate (S)-acetoin by taking NAD+ as coenzyme, and the (R)-acetoin is catalyzed to generate (2R, 3R)-2,3-butanediol, the (S)-acetoin is catalyzed to generate meso-2,3-butanediol, and butanedione is catalyzed to generate (R)-acetoin by taking NADH as coenzyme. Moreover, the dehydrogenasesubstrate has high specificity and wide industrial application prospect.

Description

technical field [0001] The present invention relates to enzymes and their coding genes and applications, in particular to a (2R, 3R)-2,3-butanediol dehydrogenase derived from Paenibacillus polymyxa ATCC12321 and its coding genes and applications . Background technique [0002] 2,3-butanediol (2,3-butanediol, BD) is widely used in chemical industry, food, aerospace fuel and other fields. Its dehydration product methyl ethyl ketone can be used as a solvent for resins, paints, etc.; the dehydration product 1,3-butadiene after esterification can be used for synthetic rubber, polyester and polyurethane; the higher calorific value (27,200kJ / kg) can be used as As a fuel additive; dehydrogenation with methyl ethyl ketone to form octane isomers can produce high-grade aviation oil; BD can also be used to prepare inks, perfumes, fumigants, moisturizers, softeners, plasticizers, explosives and chiral pharmaceutical carriers, etc. . The cost of BD chemical synthesis is high and the pr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/63C12N15/70C12N1/21C12P7/18C12P7/26C12R1/19
Inventor 于波孙际宾曾安平
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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