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Enzyme-producing microorganism used for hydrolyzing D,L-pantoyl lactone and application and sifting method of enzyme-producing microorganism

A technology of pantolactone and screening method, which is applied in the application and screening field of hydrolysis D, L-pantolactone reaction, can solve the problems of complicated steps, low efficiency and low accuracy, and achieves the screening method Simple, high column throughput results

Active Publication Date: 2018-05-08
ZHEJIANG NHU CO LTD +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This traditional screening method, which first judges the catalytic activity and then detects the stereoselectivity, has the disadvantages of cumbersome steps, heavy workload, and low efficiency.
In addition, due to many factors affecting the determination of optical rotation value, errors often occur in the optical rotation value measured during the screening process, and the stereoselectivity of microbial hydrolyzed pantolactone cannot be accurately evaluated. It is necessary to further use chromatographic methods to determine the optical purity.
Therefore, the traditional screening method is cumbersome and has low accuracy.

Method used

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  • Enzyme-producing microorganism used for hydrolyzing D,L-pantoyl lactone and application and sifting method of enzyme-producing microorganism
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  • Enzyme-producing microorganism used for hydrolyzing D,L-pantoyl lactone and application and sifting method of enzyme-producing microorganism

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Screening of enzyme-producing microorganisms for hydrolysis of D, L-pantolactone (1)

[0058] Strains: 100 strains of Fusarium sp.

[0059] Implementation steps: Unseal the Fusarium mold stored in the ampoule according to the standard procedure, draw 0.3 to 0.5 milliliters of the aforementioned liquid culture medium of the present invention used for Fusarium mold cultivation with a sterile straw, drop it into the ampoule, gently Shake to dissolve the freeze-dried bacteria into a suspension, and spread it on a solid plate. At the same time, suck a small amount of bacterial suspension and transplant it into a medium test tube, and cultivate the activated strain at 25°C. Then the activated and rejuvenated strains were inoculated into 24-well plates for culture, and the mycelium obtained from the culture was used as an enzyme catalyst to react with D,L-pantolactone solution.

[0060] Take the mycelium of the same quality as D,L-pantolactone and add it to 5mL...

Embodiment 2

[0066] Example 2: Screening of enzyme-producing microorganisms for hydrolysis of D, L-pantolactone (2)

[0067] Strains: 125 Fusarium strains collected by the inventor and isolated from soil near the Xinchang River in Xinchang County, Zhejiang Province

[0068] According to the steps in Example 1, 125 Fusarium strains were subjected to high-throughput screening for hydrolyzing D, L-pantolactone, and 5 Fusarium strains with high hydrolytic activity and stereoselectivity were obtained. The results are shown in the following table 2.

[0069] Table 2 Enzyme-producing ability and stereoselectivity of five strains of Fusarium sp.

[0070] Strain number

[0071] It can be seen from the above results that the strains RPH22, RPH94, and RPH115 showed excellent stereoselectivity of the product D-pantolactone, and the ee value of the product D-pantolactone was significantly higher than that of other strains, all of which were 90 %above.

Embodiment 3

[0072] Embodiment 3: the re-screening of the Fusarium fungus producing D-pantolactone hydrolase

[0073] The strains RPH22, RPH94, RPH115, AS3.4712, AS3.2626 and AS3.1818 with high ee value of the product D-pantolactone obtained from the preliminary screening of the above 225 Fusarium strains were re-screened, Investigate its hydrolytic activity and substrate tolerance.

[0074] The above-mentioned 6 bacterial strains were inoculated into 200mL of the aforementioned liquid culture medium of the present invention, and cultured with shaking at 28°C and 200r / min for 48h, and the fermentation liquid was removed by Buchner funnel suction filtration, and the mycelium was collected. Take the mycelia of the same quality as D, L-pantoolactone and add them to the solutions with D, L-pantoolactone concentrations of 1%, 20%, 50% and 70% respectively for reaction, and use ammonia water The pH value of the reaction was controlled to be 6.8-7.0, and the reaction was stopped after 10 hours...

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Abstract

The invention provides an enzyme-producing microorganism for hydrolyzing D,L-pantoyl lactone and application and a sifting method of the enzyme-producing microorganism. The method specifically comprises the steps of using an enzyme-producing microorganism strain to hydrolyze D,L-pantoyl lactone, then acidizing D,L-pantoyl lactone to form a converting solution, and conducting high-performance-liquid chromatography chirality online detection on the formed converting solution to quickly sift out strains which have the catalytic activity and stereoscopic selectivity on hydrolyzing D,L-pantoyl lactone. Through the directed sifting method, it is found that a bacterial strain Fusarium verticillioides CGMCC NO.14552 has an obvious and unique advantage in the aspect of hydrolyzing D,L-pantoyl lactone, the hydrolyzing conversion rate of 1-70% D,L-pantoyl lactone within 5-10 h can reach 35% or above, and the ee value of a target product D-pantoyl lactone is larger than 98%.

Description

technical field [0001] The invention belongs to the field of biocatalysis research, and specifically relates to an enzyme-producing microorganism for hydrolyzing D, L-pantolactone, and its application and screening method in the hydrolysis of D, L-pantolactone. Background technique [0002] D-pantothenolactone is a precursor substance for the synthesis of coenzyme A in organisms, and it is also an important chiral intermediate used in the industry to prepare biochemical products such as pantothenic acid and panthenol. The latter is used in the fields of medicine, food, feed and health products Has a wide range of applications. [0003] Utilizing enzyme-producing microorganisms (wild bacteria or engineering bacteria) to catalyze the preparation of D-pantolactone is the current mainstream production method, which has many advantages such as environmental protection, economy, and high optical purity of the product. Based on comprehensive literature and industrial production pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P41/00C12P17/04C12N1/14C12R1/77
CPCC12N1/14C12P17/04C12P41/001C12N1/145C12R2001/77
Inventor 耿英龙吴胜胡柏剡吴晓东彭俊华王钰李伟峰刘晓庆
Owner ZHEJIANG NHU CO LTD
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