Agrobacterium strain and method for preparing left-lateral lactone compounds thereby

A technology of Agrobacterium and D-lactone, which is applied in the field of high-selectivity D-lactone hydrolase-producing bacteria, can solve the problems of environmental protection and economy, and achieve the effect of stable enzyme production and good stereoselectivity

Inactive Publication Date: 2008-08-27
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The whole process consumes a lot of acid and alkali and organic solvents...

Method used

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  • Agrobacterium strain and method for preparing left-lateral lactone compounds thereby
  • Agrobacterium strain and method for preparing left-lateral lactone compounds thereby

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Screening of Agrobacterium CGMCC 2371

[0037] Collected 400 soil samples under different environmental conditions, respectively using γ-butyrolactone, DL-pantotolactone, DL-sodium pantoate, or L-pantotolactone as the only carbon source for enrichment Cultivate and screen L(+)-lactonohydrolase-producing bacteria.

[0038] (1) Put the soil sample in a test tube, add 2mL enrichment medium A (g / L) (substrate 1.0, NaNO 3 4.0, KH 2 PO 4 4.0, MgSO 4 0.1, KCl 0.5, ZnSO 4 ·7H 2 O 0.1, CuSO 4 ·5H 2 O 0.05), at 30°C, 200r / min enrichment culture for 1 day, keep the test tubes with obvious signs of microbial growth, take 0.2mL enrichment culture solution from each tube, add 1.8mL sterilized enrichment medium B (g / L) (substrate 5.0, NaNO 3 4.0, KH 2 PO 4 4.0, MgSO 4 0.1, KCl 0.5, ZnSO 4 ·7H 2 O 0.1, CuSO 4 ·5H 2 O 0.05), culture at 30°C, 200r / min for 1-2 days, take 0.1mL enriched culture solution from each tube and spread it on plate medium C (...

Embodiment 2

[0041] The fermentation culture of embodiment 2 Agrobacterium CGMCC 2371

[0042] Slant and plate medium (g / L): glycerol 30, yeast extract 7.5, peptone 7.5, agar 20. Sterilize at 121°C for 15 minutes, cool down after sterilization, plate, inoculate, and incubate at 30°C for 1 day. Fermentation medium (g / L): 20 dextrin; 10 glycerol; 10 peptone; 10 yeast extract; Fermentation is carried out under the condition of 160r / min, and the dry weight of the bacteria reaches 5.2g / L after 36 hours of culture, the enzyme production can reach more than 366U / L, and the specific activity is 70U / g.

Embodiment 3

[0043] The immobilization of embodiment 3 Agrobacterium CGMCC 2371 cells

[0044] 1) Take 5 g of wet cells of Agrobacterium CGMCC 2371, add 15 mM glutaraldehyde, cross-link at 30° C. for 3 h, and then centrifuge to obtain immobilized cells. After the cells are immobilized, the activity recovery rate of the enzyme is generally above 90%.

[0045] 2) A series of lactones in the following list 1 are substrates, and the substrate concentration is 100 mM , with a reaction volume of 10 mL, 1 g of immobilized cells were added, and the catalytic activity was measured after reacting for 0.1 to 0.5 h at 30°C under magnetic stirring. Table 2 lists the relative viability of immobilized cells when different lactones are hydrolyzed. Taking the activity of γ-butyrolactone as 100%, the hydrolysis effect of immobilized cells on lactones with relatively complex structures (such as vitamin C) is not obvious. When the substrate is β-butyrolactone, the viability of immobilized cells is the...

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Abstract

The invention discloses a strain of agrobacterium radiobacter of specificity hydrolytic dextral lactone and the application of the agrobacterium radiobacter in preparing dextral lactone compound such as D-(-)-pantoic acid lactone and the like. The enzyme-producing bacterium is the agrobacterium radiobacter and the preservation number is CGMCC2371. Thalli of the bacterium and an immobilized derivative of the bacterium are taken as biocatalyst, dexpazufloxacin which are not needed in pantoic acid lactone racemic mixture is hydrolyzed into (-)-hydroxy acid directly, useful D-(-)-pantoic acid lactone is preserved, and the latter is widely used in producing feedstuff and D-calcium pantothenate and D-panthenol which are useful in the daily chemical industry.

Description

technical field [0001] The invention relates to a highly selective D-lactone hydrolase producing bacterium, a cultivation method and the application of the strain to prepare L-lactone compounds or corresponding chiral hydroxy acids. technical background [0002] Hydroxy acids are natural products similar to amino acids and play a very important role in the physiological activities of organisms. Hydroxy acids and their derivatives are also intermediates in the synthesis of many drugs. Hydroxy acids can form lactones through simple acidification. At present, there are many reports on the preparation methods of optically pure hydroxy acids or lactones for commercial use. The predecessors used chemical resolution to separate (R)-α-hydroxy -β,β-Dimethyl-γ-butyrolactone, ie D-(-)-pantolactone. D-pantothenolactone is an important synthetic intermediate for the preparation of D-calcium pantothenate and D-panthenol. Calcium D-pantothenate (also known as vitamin B5) is one of the i...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N9/16C12P7/42C12P7/62C12P41/00C12R1/01
Inventor 许建和张仙潘江陈兵
Owner EAST CHINA UNIV OF SCI & TECH
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