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Method for producing D-lactic acid by enzyme resolution of D,L-lactic Acid

A technology of lactic acid and enzymatic method, applied in the field of D-lactic acid preparation, achieves the effects of simple separation, easy access and high substrate concentration

Inactive Publication Date: 2010-12-15
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] After searching, there is no report on the method of splitting racemic lactic acid to prepare D-lactic acid by using microbial NAD-independent lactate dehydrogenase (iLDH)

Method used

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  • Method for producing D-lactic acid by enzyme resolution of D,L-lactic Acid

Examples

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Effect test

Embodiment 1

[0039] Example 1: The method of using P.stutzeri SDM crude enzyme liquid to resolve racemic lactic acid to produce D-lactic acid

[0040] (1) Preparation of crude enzyme solution containing NAD-independent lactate dehydrogenase: select Pseudomonas stutzeri (Pseudomonasstutzeri) SDM CCTCC No.M206010 bacterial strain (the applicant of the bacterial strain has been preserved in China Center for Type Culture Collection) were inoculated on solid slant basic medium containing 1.5% agarose and added with 0.5% DL-sodium lactate, and cultured at 30°C for 20 hours. Put the above-mentioned cultured strains into 50 ml of liquid basic medium containing 0.5% DL-sodium lactate with an inoculation loop and inoculate 1 to 2 loops under aseptic conditions, and culture it on a shaker at 180 rpm for 10 hours at 30°C. , to produce seeds;

[0041] With a 5% (volume ratio) inoculum size, the inoculum was placed in 150 ml of liquid basic medium containing 1% DL-sodium lactate, and cultured on a shak...

Embodiment 2

[0048] Example 2: The method of using P. stutzeri SDM crude enzyme solution to resolve racemic lactic acid to produce D-lactic acid

[0049] (1) Preparation of crude enzyme solution containing NAD-independent lactate dehydrogenase: select Pseudomonas stutzeri (Pseudomonasstutzeri) SDM CCTCC No.M206010 strain to inoculate in 1.5% agarose containing 0.5% DL- Sodium lactate solid slant minimal medium, cultured at 30°C for 20 hours. Put the above-mentioned cultured strains into 50 ml of liquid basic medium containing 0.5% DL-sodium lactate with an inoculation loop and inoculate 1 to 2 loops under aseptic conditions, and culture it on a shaker at 180 rpm for 10 hours at 30°C. , to produce seeds;

[0050] With a 5% (volume ratio) inoculum size, the inoculum was placed in 150 ml of liquid basic medium containing 1% DL-sodium lactate, and cultured on a shaker at 30°C until the enzyme activity of NAD-independent lactate dehydrogenase reached 220 units / liter, stop the fermentation cul...

Embodiment 3

[0057] Example 3: The method of using P.stutzeri SDM crude enzyme liquid to resolve racemic lactic acid to produce D-lactic acid

[0058] (1) Preparation of crude enzyme solution containing NAD-independent lactate dehydrogenase: select Pseudomonas stutzeri (Pseudomonasstutzeri) SDM CCTCC No.M206010 strain to inoculate in 1.5% agarose containing 0.5% DL- Sodium lactate solid slant minimal medium, cultured at 30°C for 20 hours. Put the above-mentioned cultured strains into 50 ml of liquid basic medium containing 0.5% DL-sodium lactate with an inoculation loop and inoculate 1 to 2 loops under aseptic conditions, and culture it on a shaker at 180 rpm for 10 hours at 30°C. , to produce seeds;

[0059] With a 5% (volume ratio) inoculum size, the inoculum was placed in 150 ml of liquid basic medium containing 1% DL-sodium lactate, and cultured on a shaker at 30°C until the enzyme activity of NAD-independent lactate dehydrogenase reached When the concentration is 200 units / liter, th...

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Abstract

The invention discloses a method for producing D-lactic acid by splitting racemized lactic acid by adopting an enzyme method, comprising steps of: (1) preparation of an intact cell suspension or a crude enzyme liquid containing NAD independent lactic acid dehydrogenase, (2) inactivating the NAD independent D-lactic acid dehydrogenase by a thermal denaturation method, (3) splitting the racemized lactic acid, (4) preprocessing of transformation liquid, (5) separation of D-lactic acid and pyruvic acid, (6) refining the D-lactic acid and the pyruvic acid, etc. The method has advantages of simple culture medium, short growth cycle, low cost, low expenses of the follow-up separation and extraction, high substrate concentration resistance and high enantiomeric excess value of the product D-lactic acid, and lays the foundation for the development and application of low-priced racemized lactic acid and the high-efficient production of D-lactic acid.

Description

technical field [0001] The present invention relates to a method for preparing D-lactic acid, in particular to a method for producing D-lactic acid by splitting racemic lactic acid using NAD (nicotinamide adenine dinucleotide)-independent L-lactate dehydrogenase produced by microorganisms method. Background technique [0002] Lactic acid is widely used in chemical, pharmaceutical and other industries and scientific research. Lactic acid is divided into L-lactic acid, D-lactic acid and racemic lactic acid (DL-lactic acid). Synthesis of biodegradable polymer polylactic acid by polymerizing lactic acid monomers is a hotspot in the research of ecological plastics "Appl. Microbiol. Biotechnol. 2007, 74, 524-534. Fermentative production of lactic acid from biomass: an overview on process developments and future perspectives.”. Since the blending of L-polylactic acid and D-polylactic acid can greatly improve the heat resistance of polylactic acid, the research on D-lactic acid p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/56C12R1/38C12P41/00C12P7/40
Inventor 马翠卿高超邱建华许平
Owner SHANDONG UNIV
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