Engineering bacterium, treatment method of ornithine-containing solution and kit

A treatment method, ornithine technology, is applied in the biological field to achieve the effects of high product added value, high catalytic activity, and reduced separation costs

Pending Publication Date: 2021-03-19
ZHEJIANG ZHONGSHAN CHEM IND GRP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a treatment method and kit for e

Method used

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  • Engineering bacterium, treatment method of ornithine-containing solution and kit
  • Engineering bacterium, treatment method of ornithine-containing solution and kit
  • Engineering bacterium, treatment method of ornithine-containing solution and kit

Examples

Experimental program
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Effect test

Embodiment 1

[0075] Embodiment 1 Construction of Ornithine Racemase Engineering Bacteria

[0076] Construction of genetically engineered bacteria expressing ornithine racemase: according to the amino acid sequence (NCBI accession number: CBH21408.1) of the reported ornithine racemase orr derived from Clostridium sticklandii (ATCC12662), after codon optimization, the obtained Such as the nucleotide sequence of SEQ ID: NO.1.

[0077] The whole gene was synthesized, and restriction sites NcoI and XhoI were designed at both ends, and subcloned into the vector pET28a to obtain the recombinant plasmid pET28a-orr.

[0078] The constructed recombinant plasmid pET28a-orr was transformed into the Escherichia coli expression host BL21(DE3) by the calcium chloride method to obtain the ornithine racemase expression strain E.coli BL21(DE3) / pET28a-orr.

[0079] The transformation step of the recombinant plasmid can be,

[0080] S1. Add kanamycin (50 μg / ml) to the LB solid medium, the day before the tra...

Embodiment 2

[0087] The construction of embodiment 2 ornithine decarboxylase engineering bacteria

[0088] Construction of genetically engineered bacteria expressing ornithine decarboxylase: select primers, replicate ornithine decarboxylase on Escherichia coli MG1655 by PCR, and obtain the nucleotide sequence containing SEQ ID: NO.2.

[0089] The nucleotide sequence containing SEQ ID: NO.2 was connected to the vector pET28a to obtain the recombinant plasmid pET28a-speC.

[0090] The constructed recombinant plasmid pET28a-speC was transformed into Escherichia coli expression host BL21(DE3) by calcium chloride method to obtain ornithine racemase expression strain E.coli BL21(DE3) / pET28a-speC.

[0091] The transformation step of the recombinant plasmid can be,

[0092] S1. Add kanamycin (50 μg / ml) to the LB solid medium, the day before the transformation reaction, invert the plate and save;

[0093] S2, prepare two experimental groups respectively,

[0094] The first group, plasmid DNA gro...

Embodiment 3

[0099] Embodiment 3 cultivates ornithine decarboxylase engineering bacteria

[0100] Cultivate genetically engineered bacteria expressing ornithine decarboxylase: pick a single colony of genetically engineered bacteria expressing ornithine decarboxylase, inoculate it in LB medium, and culture it overnight at 37°C; then transfer the bacteria to LB medium, The inoculum size is 1% of the volume of LB medium, cultured to OD at 37°C 600 When the concentration is 0.4, add IPTG to a final concentration of 0.8mmol / L, culture overnight at 25°C, collect the bacteria by centrifugation and crush to obtain a crude enzyme solution of ornithine decarboxylase.

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Abstract

The invention provides an engineering bacterium capable of expressing ornithine decarboxylase, a treatment method of an ornithine-containing solution and a kit. The engineering bacterium comprises a nucleotide sequence for encoding ornithine decarboxylase. The treatment method of the ornithine-containing solution comprises the following steps: obtaining the ornithine-containing solution which comprises D-ornithine and L-ornithine; culturing engineering bacteria capable of expressing ornithine decarboxylase to obtain an ornithine decarboxylase crude enzyme solution; and conducting enzyme treatment: adding the ornithine decarboxylase crude enzyme solution and pyridoxal phosphate into the ornithine-containing solution, and reacting to generate a final product which contains D-ornithine and/orbutanediamine. The kit contains the ornithine decarboxylase crude enzyme solution. According to the treatment method of the ornithine-containing solution, DL-ornithine is converted into butanediamineand D-ornithine, the butanediamine and the D-ornithine are easy to separate, the product purity and the optical purity are improved, the product yield is increased, and the separation cost is reduced.

Description

Technical field: [0001] The invention relates to the field of biotechnology, in particular to an engineering bacterium, a treatment method for ornithine-containing solution and a kit. Background technique: [0002] Ornithine is widely used in the fields of medicine, food and pesticides. The development goal of optically pure ornithine derivatives is to be used as drugs or pharmaceutical intermediates. L-Ornithine is an important amino acid, which is widely used in medicine, food and feed additives. D-ornithine is an unnatural amino acid, but it has unique biological characteristics and plays an important role in peptide synthesis. Substituting some L-amino acids in the polypeptide with D-ornithine can make the polypeptide produce special biological properties, such as: D-ornithine can replace the second amino acid of enkephalin to form Tyr-D-Orn- The analogue of Gly-PheNH2 has dozens of times the physiological effect of enkephalin. When some amino acid antibiotics are mi...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/60C12N15/61C12N15/70C12N9/80C12N9/90C12P13/10C12P13/00C12R1/19
CPCC12N9/88C12N9/90C12N15/70C12P13/10C12P13/001C12Y401/01017C12Y501/01012C12N2800/22
Inventor 王昕张云云吴文娟
Owner ZHEJIANG ZHONGSHAN CHEM IND GRP
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