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Proline racemase as well as preparation and application thereof

A racemase and proline technology, applied in the field of genetic engineering, can solve problems such as difficult access, small catalytic sites, and increased difficulty in inhibitor screening and design

Pending Publication Date: 2022-08-05
YUNNAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main reasons may be two aspects: (1) the enzyme is only found in some bacteria and parasites, which is not easy to obtain, and there are few research sources, so it has not been widely studied; (2) the catalytic site of the enzyme is too small, which is not conducive to The design of any drug increases the difficulty of inhibitor screening and design

Method used

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  • Proline racemase as well as preparation and application thereof
  • Proline racemase as well as preparation and application thereof
  • Proline racemase as well as preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0037] Experimental Example 1 Acquisition of proline racemase gene NParc2

[0038] (1) Screening of gibbon fecal microbial metagenomic proline racemase genes

[0039] From the constructed gibbon fecal microbial library, according to the analysis results of gene prediction, functional annotation and secreted protein prediction, the gene with the annotation result of proline racemase was screened, and the proline racemase gene NCPrac2 was obtained. The gene sequence is as shown in SEQ ID NO.1.

[0040] (2) Cloning of proline racemase gene NCPrac2

[0041] PCR amplification was performed using NCPrac2-F / NCPrac2-R as primer pair and Western black crested gibbon fecal microbial metagenomic DNA as template.

[0042] PCR reaction system (20.0 μL): metagenomic DNA 0.5 μL, PrimeSTAR Max 10 μL, NCPrac2-F 0.5 μL, NCPrac2-R 0.5 μL, ddH 2 O make up 20.0 μL.

[0043] PCR reaction parameters were: 98°C, 10s; 55°C, 15s; 72°C, 90s; 30 cycles; 72°C, 10 min; 4°C, 10 min.

[0044] PCR results ...

experiment example 2

[0050] Experimental Example 2 Preparation of proline racemase NCPRAC2

[0051] The proline racemase gene NCPrac2 prepared in Example 1 was connected with the plasmid pEASY-E2 to obtain a recombinant expression vector pEASY-E2-NCPrac2, and then transformed into E. coli BL21(DE3) to obtain a recombinant E. coli strain BL21(DE3) / NCPrac2. Take the E. coli strain BL21(DE3) / NCPrac2 containing the recombinant expression vector pEASY-E2-NCPrac2, inoculate it with 0.1% inoculum in LB (containing 100 μg / mL Amp) medium, and cultivate overnight at 37°C and 180rpm. Then this activated bacterial solution was inoculated into fresh LB (containing 100 μg / mL Amp) medium with 1% inoculum, and cultured at 37°C and 180 rpm for about 5-6 h (OD). 600 After reaching 0.8-1.0), IPTG with a final concentration of 0.7 mmol / L was added for induction, and cultured at 16 °C and 180 rpm for about 16 h. The cells were collected by centrifugation at 5000 rpm for 10 min. After suspending the cells with an app...

experiment example 3

[0053] Experimental Example 3 Determination of Properties of Proline Racemase NCPRAC2

[0054] The enzymatic activity of the recombinant proline racemase NCPRAC2 was determined by a two-step method of racemization and oxidation.

[0055] One unit of enzyme activity (U) was defined as the amount of enzyme required to catalyze the production of 1 μmol of D-Pro within 1 min.

[0056] (1) Determination of optimal pH and pH stability of proline racemase NCPRAC2

[0057] Optimum pH determination of the enzyme: The proline racemase NCPRAC2 purified in Example 2 was subjected to enzymatic reaction at 37°C in a buffer of pH 3-pH 13.

[0058] Determination of pH stability of the enzyme: The purified enzyme solution was placed in a buffer solution of pH=3-13, treated at 37°C for 1 h, and then subjected to enzymatic reaction, and the untreated enzyme solution was used as a control.

[0059] The buffer solution was: 20 mmol / L Brittan-Robinson buffer solution (pH=3-13). Using L-proline a...

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Abstract

The invention discloses preparation and application of proline racemase. The amino acid sequence of the proline racemase is shown as SEQ ID NO.2, and the optimum pH of the proline racemase is 9.0; after the enzyme is treated by a buffer solution with the pH value of 6.0-12.0 for 1 hour, the residual enzyme activity is more than 55%; the optimum temperature is 40 DEG C; the enzyme activity still keeps 100% or above after the enzyme is tolerant for 1 h under the conditions of 30 DEG C, 37 DEG C and 40 DEG C, and the half-life period is reached after the enzyme is tolerant for 50 min under the condition of Under the conditions that the pH is 9.0 and the temperature is 40 DEG C, Km and Vmax of the enzyme are 0.06 mmol / L and 2.1610 mu mol / L / min respectively, the prepared proline racemase has good thermal stability and a wide pH action range, the enzyme has been determined as a drug screening target for treating the exacerbasid disease, and development of an inhibitor of the proline racemase has huge research value.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to the preparation and application of a proline racemase. Background technique [0002] Proline racemase (PRAC, EC5.1.1.4) is a dimeric enzyme that catalyzes the interconversion of L-proline and D-proline. Catalysis", which is performed jointly by two cysteine ​​residues at the N- and C-termini, does not require cofactors or other known coenzymes, and has been described so far in only three species. It was first isolated from Clostridium sticklandii by Cardinale and Abeles in 1968. The study found that PRAC can regulate the energy metabolism of Clostridium bacteria through the Stickland pathway. It is the main energy source of Clostridium and belongs to bacteria. PRAC. In 2000, Reina-San-Martin B et al. isolated and cloned the first eukaryotic PRAC from the human pathogenic parasite Trypanosoma cruzi. The study found that the secreted form of the enzyme is an effective...

Claims

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Application Information

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IPC IPC(8): C12N9/90C12N15/61C12N15/70C12N1/21C12Q1/533C12R1/19
CPCC12N9/90C12Y501/01004C12N15/70C12Q1/533G01N2333/99Y02A50/30
Inventor 许波杨金茹黄遵锡张呈波唐湘华
Owner YUNNAN NORMAL UNIV
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