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Symbiobacterium thermophilum meso-diaminopimelate dehydrogenase mutants

A technology of diaminopimelate dehydrogenase and mutants, which is applied in the field of protein engineering and can solve problems such as research reports without the preference modification of DAPDH family enzymes and coenzymes.

Inactive Publication Date: 2016-08-03
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In terms of coenzyme preference, the coenzyme preference of the known DAPDH enzymes and mutants is NADP(H), and so far there is no research report on the modification of DAPDH family enzyme coenzyme preference

Method used

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  • Symbiobacterium thermophilum meso-diaminopimelate dehydrogenase mutants
  • Symbiobacterium thermophilum meso-diaminopimelate dehydrogenase mutants
  • Symbiobacterium thermophilum meso-diaminopimelate dehydrogenase mutants

Examples

Experimental program
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Effect test

Embodiment 1

[0018] Example 1: Gene mutation

[0019] Used St Dapdh The Genbank number of the gene is AP006840.1. First, the gene is fully synthesized and connected to the pET32 vector to obtain a plasmid: pET32- St Dapdh , and soluble express the wild-type gene in Escherichia coli BL21 (DE3), and the N-terminus of the expressed protein has 6*histag. According to the site to be mutated, refer to the instructions of the QuickChangeMutagenesisKit kit, synthesize the PCR mutation primers used in Table 1, extract the plasmid DNA from the wild-type strain, and use it as a template to amplify the PCR product. Dpn1 cutting, nucleic acid recovery after cutting and transformation into TOP10 competent, single colonies were picked and sent for sequencing, and for the samples with correct sequencing, the BL21 strain was transformed.

[0020] Table 1: Mutant PCR Primers

[0021]

[0022] by p ET32- St Dapdh The plasmid was used as a template, and primers 1 and 2 were used to introduce th...

Embodiment 2

[0023] Example 2: Expression and preliminary purification of mutant enzymes

[0024] The mutant strains obtained in Example 1 were cultured in 2LLB liquid medium, and cultured at 37°C to OD 600 After about 0.8, add isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.5 mM to induce expression, the induction temperature is 25°C, and the induction time is 20 hours. After induction of expression, the cells were collected by centrifugation at 5000 rpm for 5 minutes, resuspended and washed with buffer A (20 mM Tris-Cl pH 8.0, 50 mM sodium chloride), and centrifuged again. All subsequent purification experiments were performed at 4°C, and all buffers were pre-cooled to 4°C. Resuspend the washed cells with 100mL of buffer A, homogenate under high pressure, and centrifuge at 14,000rpm for 30 minutes to remove the broken precipitate. B (20mM Tris-Cl pH8.0, 50mM imidazole, 500mM sodium chloride) removes impurity proteins, and buffer C (20mM Tris-Cl pH8.0, 250mM imi...

Embodiment 3

[0025] Example 3: Viability assay of mutants

[0026] The activity of the mutants was determined using a SPECTRAMAXM2e (MD, USA) microplate reader, and the activity measurement system used was as follows: the final concentrations of each component were: 20mM substrate pyruvate, 200mM substrate ammonium chloride, 1mM coenzyme NADH (or NADPH ), an appropriate amount of StDapdh mutant pure enzyme, the assay activity buffer was 100 mM sodium carbonate / sodium bicarbonate buffer solution pH 9.0, and the final volume was 200 μL. The substrate and protein samples were first added to a 96-well plate and equilibrated at 30°C for 10 minutes, then the coenzyme NADH was added to initiate the reaction, and the enzyme activity was determined by measuring the decrease in absorbance at 340nm (the molar extinction coefficient of NADH at 340nm is 6.22 mM -1 ?cm -1 ), the enzyme activity unit is defined as the amount of enzyme required to consume 1 μmol of coenzyme NADH per minute when cataly...

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Abstract

The invention discloses five symbiobacterium thermophilum meso-diaminopimelate dehydrogenase mutants after coenzyme specific turning. The source of used template enzyme is symbiobacterium thermophilum NADP(H) dependent meso-diaminopimelate dehydrogenase (StDapdh), after enzyme mutation, the amino acid residue with the homology comparison equivalent to the template enzyme site 35 is replaced by glutamic acid which is R35E, the amino acid residue with the homology comparison equivalent to the female parent enzyme site 35 and site 36 are respectively replaced by glutamic acid and valine which are R35E / R36V, the amino acid residue with the homology comparison equivalent to the female parent enzyme site 35 and site 36 are respectively replaced by aspartic acid and valine which are R35d / R36V, the amino acid residue with the homology comparison equivalent to the female parent enzyme site 35 and site 36 are respectively replaced by aspartic acid and glutamine which are R35d / R36Q, the amino acid residue with the homology comparison equivalent to the StDapdh site 35, site 36 and site 76 are respectively replaced by glutamic acid, valine and valine which are R35E / R36V / Y76V, according to the mutant enzyme, the coenzyme specificity is mutated from NADP(H) to NAD(H).

Description

technical field [0001] The invention belongs to the field of protein engineering and relates to a biocatalyst meso - Coenzyme preference transformation of diaminopimelate dehydrogenase, and obtained meso - A mutant in which the coenzyme preference of diaminopimelate dehydrogenase changes from NADP(H) to NAD(H). Background technique [0002] D-amino acid is a kind of unnatural amino acid, and it is also an important chiral intermediate, which is widely used in food, cosmetics and pharmaceutical industries (Wang Ying, Li Yunzheng, Liaoning Chemical Industry 2003, 32 , 58-60.). Most of the known D-amino acid dehydrogenases are membrane proteins (TanigawaM., ShinoharaT., etal , Amino Acids 2010, 38 ,247-255; Xu S.J., JuJ.S., MaY.H., WeiShengWuXueBao 2007, 47 , 634-638; Jones H., Venables W.A., Biochimie 1983, 65 ,177-183; JonesH.,VablesW.A., FEBS Lett. 1983, 151 ,189-192; Wild J., Obrepalska B., Mol. Gen. Gent. 1982, 186 , 405-410; Wild J., Klopotowski T., ...

Claims

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Application Information

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IPC IPC(8): C12N9/06C12R1/01
Inventor 刘卫东陈曦赵雷明李键煚冯进辉吴洽庆朱敦明马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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