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Functions and application of short chain dehydrogenase TsrU

A short-chain dehydrogenase, a technology for use, applied in the fields of biotechnology and engineering, capable of solving problems such as complex structures

Active Publication Date: 2013-07-17
SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Thiostrepton is a member of the family of thiopeptide antibiotics. Although Thiostrepton was first discovered in 1954, due to its complex structure, its molecule was not determined until 1989 with the help of X-crystal diffraction technology. structure

Method used

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  • Functions and application of short chain dehydrogenase TsrU
  • Functions and application of short chain dehydrogenase TsrU
  • Functions and application of short chain dehydrogenase TsrU

Examples

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preparation example Construction

[0093] In a preferred example of the present invention, the preparation method of TsrU comprises steps:

[0094] (1) Obtain a 0.85kb DNA fragment encoding the short-chain dehydrogenase TsrU from the Streptomyces laurentii genome by PCR, then connect it to a commercially available plasmid vector, and introduce the successfully constructed plasmid into an E. coli host to obtain recombinant strain. Induce the expression of host TsrU;

[0095] (2) Centrifuge the cultured strain, collect the bacteria, resuspend the bacteria in the bacteriostasis buffer (50mM Tris-Cl, 200mM NaCl, pH=8.0), and sonicate the bacterium. After breaking the bacteria, the suspended liquid was centrifuged, and the supernatant was combined with Ni-NTA overnight, washed with the bacteria breaking buffer containing different concentrations of imidazole, and the protein TsrU was obtained in the washing liquid of the imidazole solution. TsrU can be stored at -80°C for a long time.

[0096] Catalytic substrate...

Embodiment 1

[0137] Construction of Escherichia coli Recombinant Strain SL1121

[0138] The sequence of primers for cloning the gene encoding the TsrU protein is as follows:

[0139] TsrU-F (SEQ ID NO: 3) 5'-CGA AGC TTC AGG TGG TGC CGT CAG ACG-3';

[0140] TsrU-R (SEQ ID NO: 4) 5'-CAT ATG ACC GCC CCC GCG CTC CCG CTC-3'.

[0141] Using the total DNA of Streptomyces laurentii ACTCC31255, a thiostrepton-producing bacterium, as a template, the PCR reaction system was composed of dNTP, DMSO, enzyme-free water, high-fidelity Primestar DNA polymerase and its buffer, and the TsrU The 0.85kb fragment expressed by the gene was amplified by PCR. After the amplification reaction, the reaction product was separated by gel electrophoresis, recovered and purified by cutting the gel, after purification, restriction endonuclease HindIII and NdeI were added to digest and recover the fragment, and it was connected into pET28a treated with the same enzyme to construct a recombinant vector . The vector was...

Embodiment 2

[0143] Heterologous expression of short-chain dehydrogenase TsrU

[0144] Inoculate Escherichia coli SL1121 in 3ml LB medium (Kanamycin 50μg / ml) and incubate at 37°C for 8 hours, then inoculate in 800ml LB medium (Kanamycin 50μg / ml), and incubate at 37°C for 2.5 hours to A 600nm 0.5-0.7. Then transfer to 25° C. to continue culturing for 0.5 hours, add IPTG to a final concentration of 0.1 mM, and culture at 25° C. for more than 6 hours (or overnight). The bacterial liquid was centrifuged (5000 rpm, 20 minutes), and the bacterial cells were collected. The bacteria were disrupted by ultrasonic waves, the broken bacteria were centrifuged (15000 rpm, 30 minutes), and the supernatant solution was mixed with 3 mL of Ni-NTA (QIAGEN) overnight. Finally, wash with imidazole solutions with gradient concentrations of 100, 150, 200, 250, and 300 mM respectively, collect eluate from 150 to 200 mM, concentrate and desalt, and obtain purified TsrU protein.

[0145] figure 2 The SDS-PAGE ...

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Abstract

The invention discloses functions and application of a short chain dehydrogenase TsrU. Specifically, the short chain dehydrogenase TsrU can catalyze the carbonyl chiral single reduction reaction of 4-acetyl-2-quinolinic acid or its analogues, can be used for in-vitro efficient production of 4-(1-hydroxymethyl)-2-quinolinic acid or its structural analogues, and in a side chain reaction of thiostrepton biosynthesis, the enzyme has a high tolerance to a substrate, so that the short chain dehydrogenase TsrU can be widely applied to chiral reduction reactions of other similar substrates.

Description

technical field [0001] The invention belongs to the field of biotechnology and engineering, and in particular relates to the function and application of a short-chain dehydrogenase TsrU. Background technique [0002] Thiopeptide antibiotics are a class of cyclic peptide compounds with highly modified amino acid residues rich in sulfur. Thiopeptide antibiotics have a three- or four-substituted pyridine core, multiple nitrogen heterocycles such as thiazole, oxazole or thiazoline, and dehydrated amino acids, and some have highly modified aromatic ring side chains. Most of the thiopeptide antibiotics can inhibit the growth of Gram-positive bacteria, and have a strong killing effect on multidrug-resistant opportunistic pathogens. The mechanism of action of thiopeptide antibiotics is achieved by binding to ribosomes to inhibit protein synthesis. According to the different binding sites, the mechanism of action can be divided into two categories: (1) binding to the 23S rRNA-L 11 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/12C12N9/02
Inventor 刘文段炼王守锋廖日晶
Owner SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI
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