D-lactic acid dehydrogenase of Sporolactobacillus inulinus, coding gene and application thereof

A lactate dehydrogenase and encoding technology, applied in the field of D-lactate dehydrogenase, can solve problems such as inability to obtain a single configuration compound, impure product, complex technology, etc., and achieve the effect of broad industrial application prospects

Inactive Publication Date: 2012-11-07
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods all have disadvantages such as complex technology, polluting the environment, impure products, low optical purity, and the inability to obtain single-configuration compounds to varying degrees. Therefore, in recent years, they have turned to the development and research of biosynthetic methods.

Method used

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  • D-lactic acid dehydrogenase of Sporolactobacillus inulinus, coding gene and application thereof
  • D-lactic acid dehydrogenase of Sporolactobacillus inulinus, coding gene and application thereof
  • D-lactic acid dehydrogenase of Sporolactobacillus inulinus, coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1, the acquisition of D-lactate dehydrogenase gene and its protein

[0053] 1. Cloning of D-lactate dehydrogenase gene

[0054] After sequencing the entire genome of Lactobacillus inulinus (Sporolactobacillus inulinus) CASD CGMCC No.2185 and annotating the genome information, a gene that may encode lactate dehydrogenase was obtained, and the following primers P1 and P2 were designed according to the gene sequence.

[0055] P1: 5'-GG TCATGA AAATCATTATGTTCAG-3' (the underline is the recognition sequence of the restriction site BspH I, the 5th-24th is the 1st-20th of the sequence 2)

[0056] P2: 5'-GC AAGCTT GTTTTCTACAGCTACTTTGTT-3' (underlined is the recognition sequence of the restriction site Hind III, and the following sequence is the reverse complementary sequence of the 985-1005th position of sequence 2)

[0057] Using the total gene of Sporolactobacillus inulinus (Sporolactobacillus inulinus) CASD CGMCC No.2185 as a template, and using P1 and P2 as p...

Embodiment 2

[0069] The enzymatic characteristic identification of embodiment 2, embodiment 1 gained object protein

[0070] Identify the enzymatic properties of the resulting protein of interest in Example 1 with the following reactions:

[0071] (Phenyl)pyruvate+NAD(P)H+H + →D-(phenyl)lactic acid+NAD(P) +

[0072] With 20mM pyruvate or 20mM phenylpyruvate as substrate, 0.2mM NADH or 0.2mM NADPH as coenzyme, and 15μg D-lactate dehydrogenase were added to 100mM phosphate buffer (pH5.5) and mixed (the concentrations of the above substances are the final concentrations in 100mM phosphate buffer), and react at 30°C for 1 hour. After the reaction is finished, the contents of D-lactic acid and L-lactic acid, or D-phenyllactic acid and L-phenyllactic acid in the reaction liquid are detected.

[0073] The results show that: the target protein obtained in Example 1 can use NADH as a coenzyme, and can also use NADPH as a coenzyme, specifically:

[0074] Using NADH as a coenzyme to catalyze pyr...

Embodiment 3

[0087] Embodiment 3, utilize D-lactate dehydrogenase to produce D-phenyllactate

[0088] Add 15 μg of the target protein (D-lactate dehydrogenase) obtained in Example 1, 5 mM phenylpyruvate (substrate), and 2.1 mM NADH (coenzyme) to 100 mM phosphate buffer (pH5.5 ) (the concentrations of the above substances are the final concentrations in 100mM phosphate buffer), and react at 30°C for 1 hour. After the reaction, the content of D-phenyllactic acid and L-phenyllactic acid in the reaction solution was detected. Calculate substrate conversion and optical purity of D-phenyllactate. The experiment was repeated three times, and the results were averaged.

[0089] The results are shown in Table 2. D-phenyllactic acid at a concentration of 2.1 mM was detected in the reaction solution, but the presence of L-phenyllactic acid was not detected.

[0090] Table 2 Results of 3 repeated experiments using D-lactate dehydrogenase cells to produce D-phenyllactate (1)

[0091]

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Abstract

The invention discloses a D-lactic acid dehydrogenase derived from Sporolactobacillus inulinus, a coding gene and application thereof. The D-lactate acid dehydrogenase provided by the invention is (a) or (b) as below: (a) a protein composed of an amino acid sequence shown in a sequence 1 in the sequence table; and (b) a sequence 1 derived protein, with D-lactate acid dehydrogenase activity, obtained by substitution and / or deletion and / or addition of one or several amino acid residues on the amino acid sequence shown as the sequence 1. The D-lactate acid dehydrogenase provided by the invention can use NADH or NADPH as a coenzyme to catalyze pyruvic acid for generation of D-lactic acid, and can also use NADH as a coenzyme to catalyze phenyl pyruvic acid for generation of D-phenyl lactic acid; and a generated product has optical purity reaching 100% (without L-product generation). Therefore, the invention has wide industrial application prospects.

Description

technical field [0001] The invention relates to a D-lactate dehydrogenase derived from Sporolactobacillus inulinus, its encoding gene and application. Background technique [0002] 3-Phenyllactic acid, namely 2-hydroxy-3-phenylpropionic acid, is an important precursor for chemical synthesis and is widely used in the fields of medicine, chemical industry and biosynthesis. In recent years, it has been widely concerned by the food industry as a new type of antibacterial agent. The second carbon atom of 3-phenyllactic acid is a chiral carbon, which has two configurations, namely D-phenyllactic acid and L-phenyllactic acid. The effects of these two configurations on organisms and their application in medicine , Chemical applications are different. D-phenyllactic acid, also known as D-3-phenyllactic acid, D-2-hydroxy-3-phenylpropionic acid or (R)-2-hydroxy-3-phenylpropionic acid (CAS No. 7326-19-4 ). D-phenyl lactic acid can be used to synthesize hypoglycemic drugs, anthelmint...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C12P7/56C12P7/42
Inventor 马延和于波王丽敏朱凌峰
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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