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L-threonine aldolase from enterobacter cloacae and application thereof

A technology of threonine aldolase and Enterobacter cloacae, applied in the field of chemical engineering, can solve problems such as high cost, serious environmental pollution, and complicated process

Inactive Publication Date: 2015-06-03
数谱科技浙江有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the chemical synthesis process, the synthesis of this type of β-hydroxy-α-amino acid requires a multi-step process to separate isomers, which is complex, high cost, and serious environmental pollution

Method used

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  • L-threonine aldolase from enterobacter cloacae and application thereof
  • L-threonine aldolase from enterobacter cloacae and application thereof
  • L-threonine aldolase from enterobacter cloacae and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Isolation of the nucleotide sequence of L-threonine aldolase from Enterobacter cloacae

[0043] The total DNA was extracted from Enterobacter cloacae cells cultured for 36 hours according to the method provided in the "Molecular Cloning Experiment Guide", and 7 μg was used as a template for polymerase chain reaction. According to the published L-threonine aldolase sequence design, PCR amplification was carried out on a T-Gradient PCR instrument (Biometra Company) using the above-mentioned extracted DNA as a template. The primers, components and amplification conditions used in the reaction were as follows:

[0044] Primer 1: 5'-ATGATTGATTTACGCAGTGATACCG-3'

[0045] Primer 2: 5'-TTAACGCTGTAAAAACGCCTGCCAG-3'

[0046]

[0047] Amplification conditions: Denaturation at 94°C for 3min, followed by 30 cycles of 94°C for 1min, 58°C for 1min, 72°C for 1min, and finally 72°C for 10min. The results of agarose gel electrophoresis showed that a fragment with a size of...

Embodiment 2

[0048] Embodiment 2: Construction of Escherichia coli recombinant expression vector

[0049] According to the coding region sequence shown in SEQ ID NO:1, a pair of gene-specific amplification primers is designed to separate its potential open reading frame sequence:

[0050] Primer 3: 5'-GGCTCTAGA ATGATTGATTTACGCAGTGATACCG-3';

[0051] Primer 4: 5'-GCCGGATCC TTAACGCTGTAAAAACGCCTGCCAG-3';

[0052] The 5' ends of these two primers contain XbaI and BamHI restriction sites respectively. The amplification conditions and reaction components used are the same as above, and the sequencing result of the amplified product shows that it is consistent with the sequence shown in SEQ ID NO:1. Then, 50 μl of the PCR product and 1 μl of pET11a (Invitrogen) were subjected to double enzyme digestion, and the large fragment was recovered and ligated with T4 ligase overnight in a 4-degree refrigerator. The ligation product was transformed into Escherichia coli DH5α, and positive clones were s...

Embodiment 3

[0053] Example 3: Preparation of L-threonine aldolase crude enzyme

[0054] Inoculate a single colony of the identified positive transformant in SOC medium containing Amp (100 μg / ml), culture it with shaking at 37°C overnight, and the bacterial concentration reaches OD 600 When =1, inoculate 1% in LB medium containing Amp (100μg / ml), and continue to culture at 37°C until OD 600 At 1.2-1.5, the cells were harvested by centrifugation. The cells were resuspended in 0.1M phosphate buffer solution, ultrasonically disrupted, and then centrifuged at 20,000 rpm for 1 hour. The supernatant was harvested as the crude enzyme of L-threonine aldolase, aliquoted, and stored at -20°C.

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Abstract

The invention relates to a nucleotide sequence for coding L-threonine aldolase separated from the enterobacter cloacae. The nucleotide sequence is a nucleotide sequence shown in SEQ ID NO:1, or a fragment, an analogue and a derivative of the nucleotide sequence. The nucleotide sequence for coding L-threonine aldolase is connected with an exogenous regulation sequence; and a carrier for functional expression, a cellular organism containing the carrier and a descendant of the organism are contained. A method for preparing the L-beta-hydroxy alpha-amino acid or D-beta-hydroxy alpha-amino acid from the nucleotide sequence, or polypeptide sequence, or cellular organism containing the carrier and descendant of the organism is provided.

Description

technical field [0001] The invention belongs to the field of chemical engineering and enzyme engineering, and relates to an L-threonine aldolase from Enterobacter cloacae tzyx2, specifically L-threonine aldolase from Enterobacter cloacae tzyx2 Nucleotide sequences and their applications. Background technique [0002] Aldolases are a class of reversible lyases that were first recognized by humans in 1934. Acid shrinkases can catalyze the reversible aldol condensation of donors (nucleophiles, usually ketones) and acceptor aldehydes (electrophiles). Most acid condensing enzymes are very specific for their donors, but have a large degree of freedom for acceptor aldehydes. According to the different specificity of aldolase to donor, aldolase can be divided into five categories: dihydroxyacetone phosphate (DHAP)-dependent aldolase, pyruvate and phosphoenolpyruvate-dependent aldolase, Acetaldehyde-dependent aldolase, glycine-dependent aldolase and dihydroxyacetone-dependent aldo...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N9/88C12N15/70C12N1/21C12P13/04
Inventor 张昕欣于红艳王玉新吴翰桂
Owner 数谱科技浙江有限公司
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