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Biocatalyst for producing d-lactic acid

A lactic acid and microorganism technology, applied in the field of microorganisms producing D-lactic acid, can solve problems such as inactivation of aspA, difficulty in decomposing aspartic acid or glutamine, research on the amount of fumaric acid production, and the like. The effect of suppressing by-products and reducing purification costs

Active Publication Date: 2006-11-01
MITSUI CHEM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0028] In addition, the effect of aspA inactivation has only been disclosed in the past in the case of Yersinia pestis (Dreyfus, L.A., et.al., J.Bacteriol., Vol.136(2), pp757-764(1978))
However, the gist of this thesis is that the inactivation of aspA makes it difficult to decompose aspartic acid or glutamine in cells, and did not study the amount of fumaric acid produced

Method used

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  • Biocatalyst for producing d-lactic acid
  • Biocatalyst for producing d-lactic acid
  • Biocatalyst for producing d-lactic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0129] [Example 1] Utilize MT-10364 bacterial strain to produce lactic acid

[0130] The composition of the medium used for the culture is shown in Table 1 below.

[0131] Table 1

[0132] glucose

10％

Cereal extract (manufactured by Nippon Food Chemical Industry Co., Ltd.)

ammonium sulfate

Disodium hydrogen phosphate · 12 hydrate

Potassium dihydrogen phosphate

Sodium chloride

Magnesium sulfate 7 hydrate

Adecanol LG126

5％

0.5％

0.3％

0.15％

0.15％

0.1％

0.1％

[0133] This culture medium contains 0.34% of reducing sugar derived from grain extract after acid hydrolysis, 0.31% of D-lactic acid, 0.31% of L-lactic acid, 0.33% of free amino acid and trace amounts of various organic acids.

[0134] As pre-cultivation, 25ml LB Broth, Miller culture solution (Difco244620) was installed in the Erlenmeyer flask to carry out the planting of Escherichia coli MT-10934, stirred and cultivated...

Embodiment 2

[0140] [Example 2] Construction of ldhA expression vector and lactic acid producing bacteria

[0141] In order to obtain the promoter of serine hydroxymethyl transferase (glyA), the genomic DNA of Escherichia coli was used as a template, and sequence number 1 and sequence number 2 were used as probes to amplify by PCR method, and use restriction The resulting fragment was cut with endonuclease EcoRI, thereby obtaining a fragment encoding the glyA promoter of about 850 bp. Moreover, in order to obtain the structural gene of ldhA, the genomic DNA of Escherichia coli was used as a template, and sequence number 3 and sequence number 4 were used as probes to amplify by PCR method and cut with restriction enzymes EcoRI and HindIII The obtained fragment, thereby obtaining a ldhA structural gene fragment of about 1.0 kbp. The above two fragments were mixed with the fragment obtained by cutting plasmid pUC18 with restriction enzymes EcoRI and HindIII, ligated with DNA ligase, and then...

Embodiment 3

[0144] [Example 3] Lactic acid production by lactic acid-producing bacteria MT-10934 / pGlyldhA strain

[0145] As pre-cultivation, carry out the planting of the lactic acid producing bacteria MT-10934 / pGlyldhA bacterial strain obtained in Example 2 in an Erlenmeyer flask equipped with 25ml LB Broth, Miller culture solution (Difco244620), and utilize the same method as Example 1 to cultivate . After the cultivation, the content and optical purity of lactic acid were measured by HPLC according to conventional methods. The results are shown in Table 3.

[0146] D-lactic acid accumulation

[0147] In the above results, the reason why the amount of total lactic acid exceeds the amount of glucose added at the beginning of the culture is that the carbon source in the grain extract was utilized. However, even if all the reducing sugars, organic acids, and amino acids in the grain extract are used, a conversion rate of 90% or more can be achieved.

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Abstract

It is intended to provide a process for highly producing D-lactic acid. It is also intended to provide a process for highly selectively producing D-lactic acid having a high optical purity with the formation of little organic acids as by-products. D-Lactic acid is produced by culturing a microorganism having inactivated or lowered pyruvate formate-lyase activity and elevated Escherichia coli-origin NADH-dependent D-lactic acid dehydrogenase (ldhA) activity, a microorganism having inactivated or lowered FAD-dependent D-lactic acid dehydrogenase activity, or a microorganism as described above having a TCA cycle, inactivated or lowered malic acid dehydrogenase activity and inactivated or lowered aspartic acid ammonia-lyase activity. The ldhA activity is elevated by ligating a gene encoding ldhA to a promoter of a gene controlling the expression of a protein, which participates in a glycolysis system, a nucleic acid biosynthesis system or an amino acid biosynthesis system, on genome.

Description

technical field [0001] The present invention relates to a microorganism capable of producing D-lactic acid with high selectivity and high efficiency, and a method for producing D-lactic acid using the microorganism. Specifically, it relates to a method for efficiently producing high-purity lactic acid, and particularly relates to an efficient method for producing D-lactic acid with a small accumulation of pyruvic acid. [0002] The present invention also relates to a method for producing D-lactic acid, characterized in that the method uses a microorganism with inactivated or reduced activity of FAD-dependent D-lactate dehydrogenase. [0003] In addition, the present invention also relates to a D-lactic acid-producing microorganism that does not produce succinic acid or fumaric acid as an impurity when producing D-lactic acid, and a method for producing D-lactic acid using the same. Background technique [0004] The biodegradable polymer polylactic acid triggers CO 2 While ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/56C12N1/21C12N15/09
Inventor 和田光史及川利洋望月大资德田淳子川岛美由贵安乐城正阿部玲子三宅仁基高桥均泽井秀树耳塚孝森重敬东庸介
Owner MITSUI CHEM INC
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