D-pantolactone hydrolase, producing bacteria and genetically engineered bacteria thereof, and applications of D-pantolactone hydrolase and producing bacteria and genetically engineered bacteria

A technology of pantolactone and genetically engineered bacteria, applied in the field of D-pantolactone hydrolase and its producing bacteria, can solve the problem of few microorganisms producing lactone hydrolase and achieve high-efficiency expression

Active Publication Date: 2021-01-05
ZUNYI MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although the enzymatic separation of D,L-pantoate lactone to prepare D-pantoate has advantages, there are few lactone hydrolase-producing microorganisms that can be used in industrial production.

Method used

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  • D-pantolactone hydrolase, producing bacteria and genetically engineered bacteria thereof, and applications of D-pantolactone hydrolase and producing bacteria and genetically engineered bacteria
  • D-pantolactone hydrolase, producing bacteria and genetically engineered bacteria thereof, and applications of D-pantolactone hydrolase and producing bacteria and genetically engineered bacteria
  • D-pantolactone hydrolase, producing bacteria and genetically engineered bacteria thereof, and applications of D-pantolactone hydrolase and producing bacteria and genetically engineered bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Screening of D-pantolactone hydrolase-producing microorganisms (ie: D-pantolactone hydrolase-producing bacteria or Thielavia sp.zmu20201 microorganisms)

[0028] Preparation of PDA solid medium: take 200g of potatoes, wash and peel them, cut into pieces, add 1000mL of water and boil for 30 minutes, collect the filtrate through gauze filtration, replenish water to 1L, then add 20g of glucose and 15g of agar, pH neutral, after After high-pressure cultivation, a fixed medium was prepared.

[0029] Preparation of fermentation liquid medium: peptone 10g / L, yeast powder 5g / L, glycerol 10g / L, corn steep liquor 5g / L, neutral pH, cooled after high-pressure cultivation for later use.

[0030] Take 10-20g of the collected soil sample, add 100mL of sterile water, shake for 3 minutes, and then let it stand for 10 minutes; take the supernatant bacterial solution to dilute and apply, and dilute the supernatant to evenly apply to PDA medium; solid plate substrate Place in a...

Embodiment 2

[0036] Example 2: Fermentation of D-pantolactone hydrolase producing bacteria Thielavia sp.zmu20201 to produce enzyme

[0037] Strain activation culture: Pick an appropriate amount of Thielaviasp. zmu20201 microorganisms from the glycerol-preserved bacterial liquid or refrigerated seed slant and streak it on the potato dextrose agar (PDA) medium, and place the streaked eggplant bottle slant medium at a constant temperature of 28-35°C for cultivation Box, cultivated for 2-3 days.

[0038] Seed liquid culture: The liquid seed medium preparation scheme is as follows: peptone 10-20g / L, yeast extract 10-20g / L, sucrose 10-20g / L, corn steep liquor 1-5g / L, pH 6.5-7.5, extinguished at 115°C After inoculation for 30 minutes, cool down for later use; inoculate the activated seeds into a 250mL shaker, fill with 50-100mL liquid seed medium, and cultivate in a constant temperature shaker at 28-35°C for 48-60 hours.

[0039] Fermentation enzyme production culture: fermentation enzyme produc...

Embodiment 3

[0041] Example 3: D-pantoate lactone hydrolase-producing bacteria Thielavia sp.zmu20201 fermentation bacteria biotransformation to prepare D-pantoate

[0042] Using the bacteria collected from Thielavia sp.zmu20201 microbial fermentation as a biocatalyst, the hydrolysis experiment of D, L-pantolactone was carried out. The biocatalytic system is as follows: Dissolve 2g of D,L-pantolactone in 16mL of water, make up to 20mL with water, stir well and quickly add it into a 100mL three-necked bottle with a magnetic stirring speed of 200rpm; then add fermentation The collected wet mycelium 1g, starts the hydrolysis reaction, and the reaction temperature is controlled at about 30°C. The pH of the reaction was detected online with a pH meter, and the reaction pH was controlled at 7.0 ± 0.2 by adding 5% ammonia water. The reaction time was about 14 hours. When the reaction pH was basically stable, the reaction was terminated. The reaction process is shown in Table 2.

[0043] Table 2 T...

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Abstract

The invention discloses a D-pantolactone hydrolase producing bacteria in the technical field of bioengineering, and relates to a Thielavia sp. Zmu20201 serving as a microorganism, and the preservationnumber of the strain is CCTCC: M2020062. Meanwhile, the invention further discloses the D-pantolactone hydrolase from the Thielavia sp. Zmu20201 strain and an engineering bacterium strain of the D-pantolactone hydrolase, wherein the amino acid sequence number of the D-pantolactone hydrolase is as shown in SEQ ID NO.2. In addition, the invention also discloses an application of the Thielavia sp. Zmu20201 fermentation thallus or the lactone hydrolase gene engineering strain fermentation thallus as a biocatalyst in resolution of D, L-pantolactone to prepare optically pure D-pantoic acid.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a D-pantolactone hydrolase and its producing bacteria, genetically engineered bacteria and their application. Background technique [0002] D-pantothenic acid is a key chiral intermediate for the manufacture of calcium D-pantothenate, D-panthenol and D-pantethine. Calcium D-pantothenate is widely used in feed, medicine and food industries, especially as an important feed additive, and has become a vitamin second only to niacin in demand. With the development of food and feed industry, the current global market for Calcium D-pantothenate is in short supply. [0003] The main technical problem in the preparation of D-pantoic acid is the chiral resolution of D,L-pantoic acid lactone. At present, the chiral resolution methods of D, L-pantolactone include chemical resolution, physical resolution and biological enzymatic resolution. The chemical resolution technol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12N9/18C12P41/00C12P7/42C12R1/645
CPCC12N9/18C12P41/005C12P7/42Y02A50/30
Inventor 万南微陈永正张秋华
Owner ZUNYI MEDICAL UNIVERSITY
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