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Nitrile hydratase lysine mutant HBA-K2H1, coding gene and application

A nitrile hydratase lysine, encoding gene technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve problems such as artificial regulation of the self-modification efficiency of nitrile hydratase

Active Publication Date: 2021-06-11
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no example of cobalt-type nitrile hydratase actively completing post-translational modification without the assistance of molecular chaperones, nor is there any specific example of artificially adjusting the post-translational self-modification efficiency of nitrile hydratase. method

Method used

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  • Nitrile hydratase lysine mutant HBA-K2H1, coding gene and application
  • Nitrile hydratase lysine mutant HBA-K2H1, coding gene and application
  • Nitrile hydratase lysine mutant HBA-K2H1, coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Site-directed mutagenesis of nitrile hydratase HBA

[0038] 1) Extract the recombinant plasmid pET-28-HBA containing the nitrile hydratase HBA gene from recombinant Escherichia coli according to the instructions of the plasmid mini-extraction kit from Axygen Company. The amino acid sequence of wild-type nitrile hydratase HBA is shown in SEQ ID NO.3.

[0039] 2) Design site-directed mutagenesis primers 5'-CAAACATGGTTTCGGCAAGATCATTCGTGAGAAAAACGAGCCGCTGTTCC-3'(SEQ ID NO.5) and 5'-GATCTTGCCGAAACCATGTTTGCCACCCAGG TCGTGG-3'(SEQ ID NO.5) according to the nucleotide sequence (SEQ ID NO.4) of wild-type nitrile hydratase HBA ID NO.6).

[0040] 3) According to the instructions of the Fast Mutagenesis System Kit of Beijing Quanshijin Biotechnology Co., Ltd., the recombinant plasmid pET-28-HBA obtained in step 1 was used as a template, and the primers synthesized in step 2 were used for single-site site-directed mutagenesis PCR.

[0041] 4) Transform the site-directed m...

Embodiment 2

[0042] Embodiment 2: Enzyme preparation of nitrile hydratase mutant HBA-K2H1 and wild-type HBA

[0043] 1) Recombinant strains containing mutant HBA-K2H1 and wild-type HBA were respectively inoculated in LB (50 μg / mL Kan) culture medium at an inoculation amount of 0.1%, and cultured with shaking at 180 rpm for 16 hours at 37°C.

[0044] 2) Inoculate the 1% inoculum of the activated bacterial solution overnight into fresh LB (containing 50 μg / mL Kan) culture solution, culture at 37°C with shaking at 180 rpm for 3 h (OD 600nm reach 0.6-1.0).

[0045] 3) Add IPTG (isopropylthiogalactopyranoside) at a final concentration of 0.5 mM for induction, and culture at 20° C. with shaking at 150 rpm for about 20 h.

[0046] 4) Centrifuge at 8000rpm for 5min, collect the cells and suspend the cells with PBS buffer.

[0047] 5) Ultrasonic disrupt the bacteria in a low-temperature ice-water bath. Ultrasonic crushing conditions are: power 300W, 5s on, 10s off, temperature 4°C, effective cru...

Embodiment 3

[0050] Embodiment 3: the property determination of the purified enzyme of nitrile hydratase mutant HBA-K2H1 and wild-type HBA

[0051] 1) Activity analysis of mutant HBA-K2H1 and wild-type HBA purified enzymes

[0052] Dissolve the substrate 3-cyanopyridine in 50mM boric acid-borax buffer solution (pH 8.0) to a final concentration of 10mM. After preheating the reaction solution at 40°C for 5min, add the enzyme solution to a final concentration of 3.2μg / mL, react at 40°C for 10min, then add 5M concentrated HCl

[0053] 20 μL to stop the reaction. After removing impurities by filtration with a 0.22 μm filter membrane, the amount of the product 3-pyridinecarboxamide formed was detected by HPLC. The detection condition of HPLC is to use Agilent InfinityLab Poroshell 120EC-C18 column (4.6×150mm, 4μm) chromatographic column, the column temperature is 36°C, the mobile phase is 10% (v / v) acetonitrile, and the flow rate is 0.5mL / min. The wavelength is 215nm; 1 enzyme activity unit (U...

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Abstract

The invention discloses a nitrile hydratase mutant, and in particular relates to a nitrile hydratase lysine mutant HBA-K2H1 and a coding gene thereof, a plasmid and a recombinant bacterium containing the coding gene of the mutant, and application of the nitrile hydratase lysine mutant HBA-K2H1 in catalyzing an organic nitrile compound to prepare an amide compound. The amino acid sequence of the mutant HBA-K2H1 is as shown in SEQ ID NO.1, the optimum pH value of the mutant HBA-K2H1 is 8.5, and the optimum temperature of the mutant HBA-K2H1 is 35 DEG C. Compared with a wild enzyme HBA, the alkaline activity of the mutant nitrile hydratase HBA-K2H1 is improved, and the mutant nitrile hydratase HBA-K2H1 can be applied to the biotechnical fields of production of acrylamide, nicotinamide and other amide compounds with high added values through biological catalysis, chemical fiber surface modification, alkaline sewage treatment and the like.

Description

[0001] (1) Technical field [0002] The present invention relates to a nitrile hydratase mutant, in particular to a nitrile hydratase lysine mutant HBA-K2H1 and its coding gene, a plasmid and recombinant bacteria containing the mutant coding gene, and the nitrile hydratase lysine Application of acid mutant HBA-K2H1 in catalyzing the preparation of amide compounds from organic nitrile compounds. [0003] (2) Background technology [0004] Nitrile hydratase can convert the cyano groups of various organic nitrile compounds into amide groups through the action of water and hydrolysis. Compared with chemical hydrolysis methods, biotransformation methods have high efficiency, mild conditions, less environmental pollution, and can be introduced without additional protection / modification. Chemical, regio and enantioselective advantages are achieved in the case of groups. In order to catalyze the production of amide compounds such as acrylamide, nicotinamide and pyrazinamide by using n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P17/12C12R1/19
CPCC12N9/88C12N15/70C12P17/12C12Y402/01084
Inventor 柳志强沈骥冬蔡雪金利群徐建妙郑裕国
Owner ZHEJIANG UNIV OF TECH
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