Genes of D-lactic dehydrogenase from serratia marcescens and research of cloning and expressing recombinant strains and recombinant enzymes

A technology of lactate dehydrogenase and lactic acid, applied in the field of biotechnology and medicine, can solve the problem of lack of various D-lactate dehydrogenase

Inactive Publication Date: 2011-01-12
EAST CHINA UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0007] However, little is known about D-lactate production at present, and there is a lack of diverse and efficient D-lactate dehydrogenases. Therefore, there is an urgent need in this field to develop new D-lactate dehydrogenases for use in D-lactate Fermentation production of

Method used

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  • Genes of D-lactic dehydrogenase from serratia marcescens and research of cloning and expressing recombinant strains and recombinant enzymes
  • Genes of D-lactic dehydrogenase from serratia marcescens and research of cloning and expressing recombinant strains and recombinant enzymes
  • Genes of D-lactic dehydrogenase from serratia marcescens and research of cloning and expressing recombinant strains and recombinant enzymes

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Experimental program
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Effect test

Embodiment 1

[0119] The extraction of embodiment 1 Serratia marcescens genomic DNA

[0120] Genomic DNA of Serratia marcescens H3010 (purchased from ATCC) in the logarithmic growth phase was extracted with the Genomic DNA Purification Kit (Biodev, Beijing) according to the instructions provided by the manufacturer, and purified with 0.8% agar The obtained bacterial genomes were detected by sugar gel electrophoresis.

Embodiment 2D

[0121] Cloning of embodiment 2D-lactate dehydrogenase gene, construction and induced expression of expression plasmid

[0122] 1. Cloning of D-lactate dehydrogenase gene

[0123] Synthesize the following primers LDHf and LDHr, the primer sequence is:

[0124] LDHf: 5'-CCCATATGAAATTGGCGATATC-3' (SEQ ID NO: 3);

[0125] LDHr: 5'-TCAGGCGTTCAGCTGGTTC-3' (SEQ ID NO: 4)

[0126] Using the Serratia marcescens genomic DNA obtained in Example 1 as a template, the Serratia gene was amplified.

[0127] The PCR amplification system is: 2 μl of genomic DNA, 2 μl of primers LDHf and LDHr, 1.6 μl of dNTP, 2 μl of 10×Tag buffer, 0.4 μl of TAKARA Tag polymerase, ddH 2 O 10 μl.

[0128] The PCR reaction program was: pre-denaturation at 97°C for 5 min, denaturation at 95°C for 30 s, annealing at 56.5°C for 1.5 min, 30 cycles, and extension at 72°C for 10 min.

[0129] Perform agarose gel electrophoresis on PCR amplification products, some results are as follows image 3 shown.

[0130] Th...

Embodiment 3D

[0134] The construction of embodiment 3D-lactate dehydrogenase expression system

[0135] Cultivate the recombinant strain JM83 / pMD-ldh, extract the plasmid by alkaline method, cut it with restriction endonuclease BamHI and NdeI, and then combine the 1.0kb exogenous fragment recovered by the gel recovery kit with restriction endonuclease The pET-28a(+) vector recovered by BamHI and NdeI double enzyme digestion gel was ligated under the action of T4DNA Ligase, and the plasmid pET-ldh could be obtained. The construction procedure was as follows: Figure 4 shown.

[0136] Perform XbaI digestion on the constructed pET-ldh (there are two XbaI restriction sites on the recombinant plasmid) to verify that two fragments of 1.1 kb and 5.2 kb in size should be obtained, and when the vector pET-28a (+) is used as a control Only the 5.4kb linearized fragment should appear, as in Figure 5 shown. The pET-ldh expression plasmid was constructed correctly.

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Abstract

The invention relates to genes of D-lactic dehydrogenase from serratia marcescens and the research of cloning and expressing recombinant strains and recombinant enzymes, in particular provides new D-lactic dehydrogenase ('D-LDH protein' for short), polynucleotide for encoding the D-LDH protein and a method for generating the D-LDH protein through recombination technology. The invention also discloses application of the polynucleotide for encoding the D-LDH protein. The D-LDH protein has the function of catalyzing pyruvic acid to form D-lactic acid, and can be used for producing the D-lactic acid of which the price is 8 to 10 times higher than that of L-lactic acid efficiently, so the D-LDH protein has huge application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology and medicine, in particular, the invention relates to a new D-lactate dehydrogenase (D-LDH) polypeptide and its coding gene from Serratia marcescens. The present invention also relates to a recombinant vector containing the gene, a host cell transformed with the vector (recombinant bacterial strain), and its use in catalyzing pyruvate to generate D-lactic acid. Background technique [0002] Lactic acid widely exists in human body, animals, plants and microorganisms. Lactic acid can be divided into D-lactic acid, L-lactic acid, D, L-lactic acid according to its different optical activity. [0003] D-lactic acid and its derivatives are widely used in the fields of food, medicine, feed and chemical industry, especially in the field of medicine, D-lactic acid polymers can be used as materials for drug sustained-release dosage forms. This is because the polymer of D-lactic acid cannot be digested and abs...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/63C12N1/21C12N1/19C12N5/10C12P7/56C12R1/19C12R1/43
Inventor 沈亚领邱昱张燎原魏东芝张国钧周文瑜朱家文
Owner EAST CHINA UNIV OF SCI & TECH
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