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A method for producing d-(-)-acetoin by enzymatic reaction in vitro

An acetoin and in vitro enzyme technology, which is applied in biochemical equipment and methods, botanical equipment and methods, and the introduction of foreign genetic material using vectors, which can solve the problems of low transformation rate and yield.

Active Publication Date: 2018-07-13
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the yield, conversion and yield of acetoin are also relatively low

Method used

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  • A method for producing d-(-)-acetoin by enzymatic reaction in vitro
  • A method for producing d-(-)-acetoin by enzymatic reaction in vitro
  • A method for producing d-(-)-acetoin by enzymatic reaction in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Overexpression of α-acetolactate synthase (alsS) using commercialized protein expression vector pET28a

[0027] Using the Bacillus subtilis B. subtilis 168 genome as a template, primers p-alsS1 and p-alsS2 were used to amplify the gene alsS fragment (about 1.7kp). The alsS fragment and the pET28a plasmid were double digested with Thermo Fast digest XhoI / BamHI, and after ligation and transformation, the expression vector pET28a-alsS of the alsS 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 α-acetolactate synthase (alsS).

Embodiment 2

[0028] Example 2 Overexpression of α-acetolactate decarboxylase (alsD) using commercialized protein expression vector pET28a

[0029] Using the genome of Bacillus subtilis B. subtilis 168 as a template, primers p-alsD1 and p-alsD2 were used to amplify the gene alsD fragment (768bp). Then the alsD fragment and the pET28a plasmid were digested with Thermo Fast digest XhoI / EcoRI, and after ligation and transformation, the expression vector pET28a-alsD of the alsD 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 α-acetolactate decarboxylase (alsD).

[0030] Table 1 The sequences of primers used for strain construction

[0031]

Embodiment 3

[0032] Example 3 Purification and Concentration of α-Acetolactate Synthase and α-Acetolactate Decarboxylase

[0033] 1. The specific steps for the purification and concentration of α-acetolactate synthase are:

[0034] 1) Escherichia coli BL21-1 was inoculated into 400mL LB medium, cultured on a shaker at 37°C and 220rpm until the OD600 was 0.6, and the inducer IPTG (isopropylthiogalactopyranoside) was added to a final concentration of 0.5mM, 16 Cultivate at ℃ for 12 hours, collect the bacterial cells by centrifuging at 4200 rpm for 20 minutes at 4℃, and suspend with 20 mL buffer A.

[0035]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.

[0036] 3) Using the crude enzyme solution obtained in step ...

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Abstract

The invention discloses a method of producing D-(-)-acetoin by in-vitro enzyme reaction, comprising the steps of (1) linking the carrier pET28a and alpha-acetolactate synthase encoding gene alsS to obtain pET28a-alsS, introducing the pET28a-alsS into Escherichia coli, fermenting, purifying, and concentrating to obtain alpha-acetolactate synthase concentrate; linking the carrier pET28a and alpha-acetolactate decarboxylase encoding gene alsD to obtain pET29a-alsD, introducing the pET29a-alsD to Escherichia coli, fermenting, purifying, and concentrating to obtain alpha-acetolactate decarboxylaseconcentrate; (2) mixing well the two concentrates, sodium pyruvate, Mg2+ and thiamine pyrophosphate, and allowing reaction to obtain the D-(-)-acetoin. The sodium pyruvate low in cost is used as a substrate to provide in-vitro efficient production of high-added-value chiral D-(-)-acetoin, and the finished D-(-)-acetoin has high purity.

Description

technical field [0001] The invention belongs to the field of bioengineering technology and application, in particular to a method for producing D-(-)-acetoin by in vitro enzyme reaction. Background technique [0002] Acetoin, also known as 3-hydroxy-2-butanone, is a colorless or light yellow liquid. The monomer is colorless or light yellow liquid with a milky aroma. It is a commonly used food-grade spice. Add Add it to food to enhance the milk flavor in food. At the same time, acetoin is one of the platform compounds prioritized by the U.S. Department of Energy, especially chiral acetoin (D-(-)-acetoin and L-(+)-acetoin) are widely used in functional materials , chiral drugs and chemical intermediate synthesis and other fields. [0003] At present, the main method of industrial production of acetoin is chemical synthesis, including oxidation of 2,3-butanediol, chlorination hydrolysis of butanone and partial hydrogenation of diacetyl. These production processes are relativ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N1/21C12P7/26C12N15/54C12N9/88C12N9/10
CPCC12N9/1022C12N9/88C12P7/26C12Y202/01006C12Y401/01005
Inventor 陈涛崔真真赵玉姣毛雨丰王智文
Owner TIANJIN UNIV
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