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Method for co-production of 5-keto-D-gluconic acid and ethyl (S)-4-chloro-3-hydroxybutyrate

A technology of ethyl hydroxybutyrate and gluconic acid, which is applied in the field of biocatalysis, can solve the problems of difficult to achieve complete conversion of gluconic acid, low oxygen concentration of the substrate, low binding efficiency, etc., and achieve green reaction, high selectivity, and process simple effect

Pending Publication Date: 2019-01-29
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But in this process, there is competition between Ga5DH and NAD(P)H oxidase for the oxidation of NAD(P)H, in which the binding efficiency of NAD(P)H oxidase and NAD(P)H is low, and the substrate oxygen is in The concentration in the water is low and difficult to increase, resulting in a large amount of Ga5DH catalyzing the main reaction in reverse during the reaction process, reducing 5KGA to gluconic acid. When the reaction reaches a certain level, the reaction tends to balance, and the remaining gluconic acid cannot be further processed. Converted to 5KGA, it is difficult to achieve complete conversion of gluconic acid at this time

Method used

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  • Method for co-production of 5-keto-D-gluconic acid and ethyl (S)-4-chloro-3-hydroxybutyrate
  • Method for co-production of 5-keto-D-gluconic acid and ethyl (S)-4-chloro-3-hydroxybutyrate
  • Method for co-production of 5-keto-D-gluconic acid and ethyl (S)-4-chloro-3-hydroxybutyrate

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Experimental program
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Effect test

Embodiment 1

[0027] Example 1: Derived from Gluconobacteroxydans The gluconate dehydrogenase gene ( gdh ) clone

[0028] Gluconobacteroxydans Purchased from China General Microorganism Culture Collection and Management Center (CGMCC 1.637). Gluconobacter oxydans culture adopts the medium formula (g / L): glucose 100, yeast extract 10, calcium carbonate 20, agar 15, completely dissolved in deionized water, adjusted to pH 6.8, constant volume, high pressure steam sterilization .

[0029] Gluconobacter oxydans was inoculated into LB liquid medium, cultured at 37°C to logarithmic growth phase, and the genome of Streptococcus thermophilus was extracted using DNAKit (TIANGEN, China). The primers used to construct the expression vector are added with restriction enzyme sites, and the primer sequences are as follows:

[0030]Upstream primers (gluconate dehydrogenase-sense with EcoRI) are:

[0031] 5'-CCGGAATTCATGAGTACCTCTCTTTTTG-3'

[0032] Downstream primers (gluconate dehydrogenase-anti ...

Embodiment 2

[0035] Example 2: Recombinant expression vector pET-28a- gdh build

[0036] Use EcoRI and HindIII to digest (purchased from Novagen Merck China) and the amplified target gene containing two restriction sites, respectively, and recover the double-digested target fragment and expression vector, respectively. The expression vector pET-28a and the target gene (the accession number in Genbank is NC_019396.1) were ligated overnight with T4-DNA ligase (purchased from TaKaRa company) to obtain the recombinant vector pET-28a- gdh ; Add 10 μL of the ligation product to E. coli BL21 competent cells, place on ice for 30 min, and heat shock at 42°C for 90 s. Place on ice for 2min. Add pre-warmed 0.45 mL of LB medium. 220rpm, 37°C, 1h. Add 200 μL of bacterial solution to a plate containing 30 μg / mL kanamycin, and culture at 37°C for 12-16 hours overnight to obtain recombinant bacteria. E.coli BL21 (with pET-28a- gdh ).

Embodiment 3

[0037] Example 3: Carbonyl reductase gene derived from ScheffersomycesstipitisCBS6054 ( sou2 ) source and acquisition

[0038] This example provides a polynucleotide molecule encoding carbonyl reductase activity, the nucleotide molecule encoding Genbank accession number XP-001387287, and artificially synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

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Abstract

The invention discloses a method for co-production of 5-keto-D-gluconic acid and ethyl (S)-4-chloro-3-hydroxybutyrate. Gluconic acid and 4-chloro-3-ethyl acetoacetate are adopted as raw materials, through a double-enzyme coupling system composed of gluconic acid dehydrogenases and carbonyl reductases, the gluconic acid is oxidized to generate the 5-keto-D-gluconic acid, and at the moment, 4-chloro-3-ethyl acetoacetate is reduced to generate the ethyl (S)-4-chloro-3-hydroxybutyrate. By establishing the double-enzyme coupling system, high-yield, low-cost and fast co-production of the 5-keto-D-gluconic acid and the ethyl (S)-4-chloro-3-hydroxybutyrate is achieved; and the system is further suitable for other alcohol dehydrogenase and the carbonyl reductases and is an economical, convenient and effective biocatalysis system with wide applicability.

Description

technical field [0001] The invention relates to the technical field of biocatalysis, in particular to a method for co-producing 5-keto-D-gluconic acid and 4-chloro-3-hydroxybutyric acid ethyl ester. Background technique [0002] 5-keto-D-gluconic acid (5-keto-D-gluconic acid, 5KGA) is a keto acid compound formed by further dehydrogenation of gluconic acid at the 5-position. After catalytic oxidation, it can synthesize bio-based platform molecules2,5 -furandicarboxylic acid, as well as food additives, pharmaceutical resolving agents L(+)-tartaric acid and glycolic acid. [0003] As early as 1922, German scientist Kiliani et al. proposed the chemical method of nitric acid for oxidizing glucose to prepare 5KGA, with a yield of only 10-12%, and this process needs to consume a lot of acid and alkali, and the product separation and purification process is complicated and time-consuming. In contrast, biological methods can selectively dehydrogenate the 5-hydroxyl group of gluconic...

Claims

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Application Information

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IPC IPC(8): C12P7/58C12P7/62
CPCC12P7/58C12P7/62
Inventor 曹飞陈姣武红丽沙凤陆凡
Owner NANJING UNIV OF TECH
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