Method for preparing D-pantoic acid

A pantothenic acid and dinucleotide technology, applied in fermentation and other directions, can solve the problems of great limitation and low cost, achieve high yield, simplify the extraction process, and facilitate large-scale industrial production

Active Publication Date: 2021-01-08
天津工微生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, NADPH reductase needs to use expensive NADPH as a coenzyme to drive the reaction, which is very limited in industry, so it is necessary to further study the synthesis method of pantoate with lower cost

Method used

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  • Method for preparing D-pantoic acid
  • Method for preparing D-pantoic acid
  • Method for preparing D-pantoic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1, NADH reductase preparation D-pantoic acid activity screening assay

[0025] Dissolve ketopantoic acid, NADH and NADH reductase protein solution (NADH reductase purchased from Tianjin Gong Microbial Technology Co., Ltd. for amino acid reduction reaction, product number is GWOR001-GWOR096, a total of 96 different NADH reductases) In 20 mM (pH 7.5) phosphate buffer, the quantification is 1 mL. The concentration of ketopantoic acid in the reaction system was 100 mM, the concentration of NADH in the reaction system was 1 mM, and the concentration of NADH reductase protein solution was 1 mg / mL.

[0026] After online monitoring with a spectrophotometer (instrument model: UV-1800PC) for 5 min, the enzyme activity was calculated according to the extinction coefficient (6.22 cm-1mmol-1) of NADH at 340 nm. It was found that among the 96 different NADH reductases tested, GWOR005 enzyme had the highest activity in catalyzing the reaction of NAD and ketopantoate to genera...

Embodiment 2

[0027] Embodiment 2, use NADH reductase to prepare D-pantoic acid

[0028] Based on the results of Example 1, the following experiments were further carried out.

[0029] Reaction system (100 ml): composed of reaction solution, ketopantoic acid, NADH reductase and NADH.

[0030] Among them, the reaction solution: 0.5 M Tris-HCl (pH 8.0), 0.6 M glucose, 0.5 mg / mL glucose dehydrogenase; the concentration of ketopantoic acid in the reaction system is 500 mM; NADH reductase: use such as The GW005 commercial enzyme used in Example 1 (purchased from Tianjin Gong Microbiology Technology Co., Ltd.) had a concentration of 1 mg / mL in the reaction system; the concentration of NADH in the reaction system was 1 mM.

[0031] Reaction conditions: 30°C, 200 rpm for 12 hours.

[0032] Product detection: After the reaction was completed, the reaction system was diluted 10 times, and the product concentration was detected by HPLC.

[0033] HPLC chromatographic conditions: Instrument model: Sh...

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Abstract

The invention discloses a method for producing D-pantoic acid by using NAD reductase. According to the method, the cheap NAD coenzyme is used for driving the reaction for the first time, so that the production of the D-pantoic acid is efficiently realized. According to the method, the D-pantoic acid can be prepared through one-step reaction of all the raw materials and enzymes in one reactor, intermediate steps and reaction are not involved, the technological process is greatly simplified, the catalytic reaction is basically not inhibited by the product D-pantoic acid, the high yield can be obtained through high-concentration conversion, and therefore, according to the method, the process is simplified, the cost is greatly reduced by adopting the low-price NAD coenzyme, and large-scale industrial production is facilitated.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a preparation method of chiral D-pantoic acid. Background technique [0002] D-pantothenic acid is an important chemical raw material, mainly used in the synthesis of B vitamins calcium pantothenate, cosmetic raw material panthenol, pharmaceutical raw material high pantothenic acid, and coenzyme A and other chemicals. Its structural formula is as follows: [0003] [0004] The current synthesis of pantoate is mainly achieved by chemical and enzymatic hydrolysis kinetic resolution. In industrial production, the chemical method has cumbersome steps and pollutes the environment; while the kinetic resolution of enzymatic hydrolysis requires the use of DL-pantolactone as a raw material, and then uses a chiral-selective pantolactone hydrolase to convert DL- -D-pantoate lactone in pantoate lactone is hydrolyzed to D-pantoate acid, then D-pantoate acid is recovered and re-formed D-pantoa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/42
CPCC12P7/42
Inventor 不公告发明人
Owner 天津工微生物科技有限公司
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