Nitrile hydratase lysine mutant HBA-K2H2R, coding gene and application

A nitrile hydratase lysine, encoding gene technology, applied in the application, genetic engineering, plant gene improvement and other directions, can solve the problem of artificially regulating the self-modification efficiency of nitrile hydratase

Active Publication Date: 2021-07-23
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-K2H2R, coding gene and application
  • Nitrile hydratase lysine mutant HBA-K2H2R, coding gene and application
  • Nitrile hydratase lysine mutant HBA-K2H2R, coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Site-directed mutagenesis of nitrile hydratase HBA

[0039] 1) Extract the recombinant plasmid pET-28-HBA containing the nitrile hydratase HBA gene from the recombinant Escherichia coli HBA 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.

[0040] 2) According to the nucleotide sequence of wild-type nitrile hydratase HBA (SEQ ID NO.4), design multi-site site-directed mutagenesis primers 5'-CAAACATGGTTTCGGCAAGATCATTCGTGAGAAACATGAGCCGCTGTTCC-3'(SEQ ID NO.5), 5'-GATCTTGCCGAAACCATGTTTGCCACCCAGGTCGTGG-3' (SEQ ID NO. 6), 5'-CAGCAGACCAAACGCGCGACGTTCCC AGTC-3' (SEQ ID NO. 7) and 5'-CGCGCGTTTGGTCTGCTGATTGGCACCGCGGGTC-3' (SEQ ID NO. 8).

[0041] 3) According to the instructions of the Fast MultiSite 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 ...

Embodiment 2

[0043] Embodiment 2: Enzyme preparation of nitrile hydratase mutant HBA-K2H2R and wild-type HBA

[0044] 1) Recombinant strains containing mutant HBA-K2H2R and wild-type HBA were inoculated in LB (50 μg / mL Kan) medium at an inoculum size of 0.1%, respectively, and cultured at 37° C. and 180 rpm for 16 hours with shaking.

[0045] 2) Inoculate the 1% inoculum of the activated bacterial liquid overnight into fresh LB (containing 50 μg / mL Kan) culture liquid, culture at 37°C with shaking at 180 rpm for 3 h (OD 600nm reach 0.6-1.0).

[0046] 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.

[0047] 4) Centrifuge at 8000rpm for 5min, collect the cells and suspend the cells with PBS buffer.

[0048] 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 crushing time ...

Embodiment 3

[0051] Embodiment 3: The property determination of the purified enzyme of nitrile hydratase mutant HBA-K2H2R and wild-type HBA

[0052] 1) Activity analysis of mutant HBA-K2H2R and wild-type HBA purified enzymes

[0053] The substrate 3-cyanopyridine was dissolved in 50mM boric acid-borax buffer solution (pH 8.0) to make the final concentration 10mM. After the reaction solution was preheated at 40°C for 5min, the final concentration of pure enzyme solution was added to make it final. The concentration was 7.2 μg / mL, and reacted at 40° C. for 10 min, and then 20 μL of 5M concentrated HCl was added to terminate 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.

[0054] The detection conditions of HPLC are as follows: Agilent InfinityLab Poroshell 120 EC-C18 column (4.6×150mm, 4μm) chromatographic column is used, the column temperature is 36°C, the mobile phase is 10%...

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Abstract

The invention discloses a nitrile hydratase mutant, and particularly relates to a nitrile hydratase lysine mutant HBA-K2H2R, a coding gene of the nitrile hydratase lysine mutant HBA-K2H2R, a plasmid and a recombinant bacterium containing the coding gene of the nitrile hydratase lysine mutant HBA-K2H2R, and an application of the nitrile hydratase lysine mutant HBA-K2H2R in catalyzing an organic nitrile compound to prepare an amide compound. The amino acid sequence of the nitrile hydratase lysine mutant HBA-K2H2R is as shown in SEQ ID NO. 1, the most suitable pH value of the nitrile hydratase lysine mutant HBA-K2H2R is 9.0, and the most suitable temperature 35 DEG C. Compared with a wild enzyme HBA, the nitrile hydratase lysine mutant HBA-K2H2R has the improved alkaline activity, and 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-K2H2R and its encoding gene, a plasmid and recombinant bacteria containing the mutant encoding gene, and the nitrile hydratase lysine Application of acid mutant HBA-K2H2R 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 u...

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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