Levo lactone hydrolase producing fungus, and its method for preparing chiral hydroxy acid

A hydrolase and hydroxy acid technology, applied in the field of highly selective L-lactone hydrolase producing bacteria, can solve the problems of low substrate concentration, affecting the application effect, and insufficient optical purity of the product, and achieve stable enzyme production and stereoselectivity Good results

Inactive Publication Date: 2007-03-28
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] In summary, in the existing chiral hydroxy acid synthesis technology, both the chemical resolution method and the existing biological method have made some progress, but there are still relatively low

Method used

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  • Levo lactone hydrolase producing fungus, and its method for preparing chiral hydroxy acid
  • Levo lactone hydrolase producing fungus, and its method for preparing chiral hydroxy acid
  • Levo lactone hydrolase producing fungus, and its method for preparing chiral hydroxy acid

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The fermentation culture of embodiment 1 Fusarium proliferatum ECU2002

[0041] 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 2 days. Fermentation medium (g / L): glycerol 30; peptone 10; yeast extract 10; NH 4 NO 3 3; Inorganic salt (g / L) (NaCl1; MgSO 4 ·7H 2 O1; FeSO 4 ·7H 2 O0.02; ZnSO 4 ·7H 2 O0.03; CuSO 4 ·5H 2 00.005); pH7.5. Sterilized at 121 DEG C for 15 minutes, cooled and inoculated after sterilization, with an inoculum size of 2%, fermented at 30 DEG C, under the condition of a rotating speed of 160r / min, and cultivated for 2 days when the dry weight of the thalline reached 18g / L, the enzyme production can reach more than 90U / L, and the specific activity is 5U / g.

Embodiment 2

[0042] The immobilization of embodiment 2 cell debris

[0043] 1) Take 50g of Fusarium proliferatum ECU2002 cells, add 5g of quartz sand to grind for 1h to break the cell wall, centrifuge at 12,000rpm for 15min, the supernatant is cell-free extract, and the precipitate is cell debris.

[0044] 2) Take 5 g of cell fragments, add different carriers, measure the viability after immobilizing the cell fragments, and measure their residual viability after storing at 4° C. for 1 week (results are shown in Table 1).

[0045] 3) Use glutaraldehyde-immobilized cell fragments as the enzyme source, (±)-pantolactone as the substrate, the reaction volume is 20mL, the substrate concentration is 2M, 10g of immobilized cell fragments are added, and the reaction temperature is 30°C. 3M ammonia water was added dropwise to maintain the pH of the reaction solution at 7.0. After a reaction time of 24 hours, the conversion rate was 34.4%, and the optical purity of the product (-)-pantolactone was 94...

Embodiment 3～13

[0048] Example 3-13 The hydrolysis activity of Fusarium proliferatum ECU2002 immobilized cells to a series of lactone compounds

[0049] A series of lactones in the following list 2 are substrates, and the substrate concentration is 100 mM , the reaction volume was 10 mL, and 0.2 g of immobilized cells (cells obtained by cross-linking with 15 mM glutaraldehyde at 30° C. for 3 h) were added, and the viability was measured after reacting at 30° C. for 0.1 to 0.5 h 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 n-butylphenyl peptide) is not obvious, and the activity of the enzyme is only 25%. Immobilized cells showed high activity to α-substituted lactone, and when the substrate was α-hydroxy-γ-butyrolactone, the activity was the highest, which was 54 times th...

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Abstract

The invention relates to laevorotation lactone specificity hydrolytic enzyme producing strain and the method used it to make chirality oxyacid. The enzyme producing is Fusarium proliferatum Nirenberg ECU2002 with storage number CGMCC 1494. The chirality oxyacid preparing method includes the following steps: using the fungous mycelium, rough enzyme extract or their immobilization derivative as biocatalyst; processing antipode selectivity hydrolysis resolution for a series of racemic chirality lactone to gain many optically active (+)-oxyacid and (+)-lactone which can be hydrolyzed into (-)-oxyacid; (+)-alpha-hydroxyl-beta, beta-dimethyl-gamma-butyric acid which are D-(+)-pantoic acid; simple acidizing to gain chirality intermediate D-(-)-pantoic acid lactone widely used in preparing feed and daily chemical engineering industry.

Description

technical field [0001] The invention relates to a high-selectivity L-lactone hydrolase-producing bacterium and a technical method for preparing chiral hydroxyacids or corresponding lactones using the strain. technical background [0002] Hydroxy acids, such as lactic acid, malic acid, tartaric acid, citric acid and gluconic acid, etc., are a very important class of organic acids, which have unique physiological functions in living organisms and are very useful in the food / feed industry functional additives. This situation is very similar to amino acids, and the two can also be transformed into each other under certain conditions. Some unnatural hydroxy acids, especially optically active chiral hydroxy acids, can also be used as "structural building blocks" for the chemical synthesis of various natural products and drug molecules. [0003] In addition to pure α-hydroxy acids, β-hydroxy acids, γ-hydroxy acids, and ω-hydroxy acids with hydroxyl at the end can all be dehydrate...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12P41/00C12P7/42C12N11/00
CPCC12R1/77C12N9/18C12P7/42C12P7/40C12N1/145C12R2001/77
Inventor 许建和张仙潘江徐毅
Owner EAST CHINA UNIV OF SCI & TECH
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