Method for generating L(+)-acetoin by in-vitro enzyme reaction

A technology of acetoin and in vitro enzyme, which is applied in the field of in vitro enzyme reaction to produce L-acetoin, can solve the problems of many by-products and affect downstream separation, and achieve effective regeneration

Active Publication Date: 2017-07-21
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are many by-products in this re

Method used

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  • Method for generating L(+)-acetoin by in-vitro enzyme reaction
  • Method for generating L(+)-acetoin by in-vitro enzyme reaction
  • Method for generating L(+)-acetoin by in-vitro enzyme reaction

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Example 1 Overexpression of 2,3-butanediol dehydrogenase (bdhA) using commercial protein expression vector pET28a

[0028] Using the Bacillus subtilis B. subtilis 168 genome as a template, primers p-bdhA1 and p-bdhA2 were used to amplify the gene bdhA fragment (about 1.0 kp). The bdhA fragment and the pET28A plasmid were digested with Thermo Fast digest NheI / BamHI, and after ligation and transformation, the expression vector pET28a-bdhA of the bdhA gene was obtained (see figure 1 ), and the sequence detection was correct. The plasmid with the correct sequencing result was transformed into commercially competent E. coli BL21(DE3) by the traditional calcium chloride method to obtain BL21-1 overexpressing 2,3-butanediol dehydrogenase (bdhA).

Embodiment 2

[0029] Example 2 Overexpression of NADH oxidase (yodC) using commercialized protein expression vector pET28a

[0030] Using the Bacillus subtilis B. subtilis 168 genome as a template, primers p-yodC1 and p-yodC2 were used to amplify the gene yodC fragment (609bp). Then the yodC fragment and the pET28a plasmid were digested with Thermo Fast digest NdeI / XhoI, and after ligation and transformation, the expression vector pET28a-yodC of the yodC gene was obtained (see figure 2 ), and the sequence detection was correct. The plasmid with the correct sequencing result was transformed into commercially competent E. coli BL21(DE3) by the traditional calcium chloride method to obtain BL21-2 overexpressing NADH oxidase (yodC).

[0031] Table 1 Primer sequences used for strain construction

[0032]

Embodiment 3

[0033] Example 3 Purification and concentration of 2,3-butanediol dehydrogenase and NADH oxidase

[0034] 1. The specific steps for the purification and concentration of 2,3-butanediol dehydrogenase are:

[0035] 1) Inoculate Escherichia coli BL21-1 into 400mL LB medium, culture on a shaker at 37°C, 220rpm until the OD600 is 0.6, add the inducer IPTG to a final concentration of 0.5mM, culture at 16°C for 12h, centrifuge at 4°C, 4200rpm for 20min Collect the cells and suspend them with 20mL buffer A.

[0036] 2) Collect the suspension of BL21-1 obtained in step 1), break the cells under the action of a high-pressure cell breaker, at 4°C, 1200bar, oil pressure 18Kg / cm 3 Treated 3 times under the same conditions, centrifuged at 4°C and 8000rpm for 30min after crushing, and collected the supernatant to obtain the crude enzyme solution.

[0037] 3) Using the crude enzyme solution obtained in step 2) to purify the protein by using a gravity nickel column purification method. At 4...

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Abstract

The invention discloses a method for generating L(+)-acetoin by in-vitro enzyme reaction. The method comprises the following steps: (1) connecting a carrier pET28a with 2,3-butanediol dehydrogenase coding gene bdhA to obtain pET28a-bdhA, and importing escherichia coli, fermenting, purifying, and concentrating to obtain 2,3-butanediol dehydrogenase concentrated solution; connecting a carrier pET28a with NADH oxidase coding gene yodC to obtain pET28a-yodC, and importing the escherichia coli, fermenting, purifying, and concentrating to obtain NADH oxidase concentrated solution; and (2) uniformly mixing the 2,3-butanediol dehydrogenase concentrated solution, the NADH oxidase concentrated solution, meso-2,3-butanediol, NAD+ and FAD+, reacting to obtain the L(+)-acetoin. The method is capable of using the cheap meso-2,3-butanediol as a substrate, and realizing the in-vitro generation of the L(+)-acetoin with high added value. The yield and purity are high.

Description

technical field [0001] The invention belongs to the field of bioengineering technology and application, in particular to a method for producing L(+)-acetoin by in vitro enzyme reaction. Background technique [0002] Acetoin, the chemical name is 3-hydroxy-2-butanone, also known as methyl acetylmethanol, is a colorless or light yellow liquid, the monomer is colorless or light yellow liquid, milky aroma, dimer It is white crystalline powder, capable of spontaneous combustion, easily soluble in water, soluble in ethanol, propylene glycol, slightly soluble in ether, almost insoluble in vegetable oil. Acetoin is a commonly used food-grade spice, which is added to food to enhance the milk flavor in food. As one of the platform compounds prioritized by the U.S. Department of Energy, acetoin is widely used in functional materials, pharmaceutical production, and chemical synthesis. [0003] At present, the main method of industrial production of acetoin is chemical synthesis, inclu...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C12N9/02C12N9/04C12P7/26C12R1/19
CPCC12N9/0006C12N9/0036C12N15/70C12P7/26C12Y101/01004C12Y101/01076C12Y106/03001
Inventor 王智文崔真真毛雨丰马红武赵玉姣陈涛
Owner TIANJIN UNIV
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