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Nitrile hydratase mutant and application thereof

A technology of nitrile hydratase and nitrile hydratase, which is applied in the field of bioengineering and can solve the problems of limited thermal stability and low catalytic efficiency

Active Publication Date: 2021-03-16
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current industrial production of bulk chemicals, such as acrylamide and nicotinamide, is not only limited by thermal stability, but also has low catalytic efficiency.

Method used

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  • Nitrile hydratase mutant and application thereof
  • Nitrile hydratase mutant and application thereof
  • Nitrile hydratase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Plasmid construction of nitrile hydratase mutants

[0033] Using the wild-type plasmid pET24a(+)-Cal.t WT (Zhang Sailan, Li Ting, Cheng Zhongyi et al. Study on heterologous expression and catalytic process of a new heat-resistant nitrile hydratase[J]. Food and Fermentation Industry, 2020, Volume 46 (14): pET24a (+)-Cal.t NHase) in 108-113 is a template, the mutation sites L48D and L48H are designed on the primers, and the plasmids with mutated base sequences are amplified by PCR. The primer sequences are shown in Table 1, and the amplification system is shown in Table 2.

[0034] The PCR amplification reaction conditions were pre-denaturation at 95°C for 3min, denaturation at 98°C for 15s, annealing at 55°C for 30s, extension at 72°C for 1min45s, and extension at 72°C for 5min, a total of 30 cycles. The PCR product was digested with DpnI digestive enzyme for 2-3h, transformed into E.coli DH5α, spread on LB medium plate containing 50mg / L kanamycin, and incuba...

Embodiment 2

[0042] Example 2: Expression and purification of wild enzyme WT and each mutant

[0043] Step 1: Transform the wild-type pET24a(+)-Cal.t WT of Cal.t NHase obtained in Example 1 and the reconstituted plasmids pET24a(+)-L48D and pET24a(+)-L48H into E.coli BL21(DE3 ), pick a single colony into 5mL LB medium, and culture at 37°C and 200rpm for 7-8h. The seed solution was transferred to 100mL 2×YT medium according to the inoculation amount of 1% (v / v), cultured at 37°C and 200rpm until the OD600 reached 0.6-0.8, and the final concentration was 0.4mM isopropylthiosemi Lactose (IPTG) and 0.1g / L CoCl 2 ·6H 2 O, change the culture temperature to 24°C, and induce expression for 12-16 hours.

[0044]Step 2: The wild-type WT and its two mutants were purified by affinity chromatography, and the purification column was a StrepTrap HP 1mL column from GE. The bacterial cells were collected by centrifugation at 10,000 rpm for 3 min, resuspended with 20 mL of binding buffer, and ultrasonica...

Embodiment 3

[0045] Example 3: Detection of wild type and mutant catalytic efficiency of Cal.t NHase

[0046] Dilute the pure enzyme concentration of WT and its mutants to 0.5 mg / mL with 10 mM KPB (pH 7.4) solution, take 10 μL into a 1.5 mL centrifuge tube, and place it on a metal bath at 25 °C. Add 490 μL of substrate (200 mM nicotinonitrile solution) to the centrifuge tube, vortex and mix thoroughly, react at 25° C. for 10 min, and then add 500 μL of pure acetonitrile solution to terminate. The reaction solution was diluted with pure acetonitrile solution to an appropriate multiple, and passed through a 0.22 μm filter membrane. Liquid phase detection method: the mobile phase composition is acetonitrile: water = 1:2 (v / v), the flow rate is 0.6mL / min, the detection wavelength is 215nm, the column temperature is 40°C, and the amount of nicotinamide produced in the reaction system is determined . The calculation results of specific enzyme activities of WT and mutants are as follows: figu...

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Abstract

The invention discloses a nitrile hydratase mutant and application thereof, and belongs to the technical field of bioengineering. According to the nitrile hydratase mutant, the enzyme activity of a single-point mutant L48D and the enzyme activity of a single-point mutant L48H constructed on by the 48th on a beta subunit in the amino acid motif of the nitrile hydratase are 750.26 + / -1.63 U / mg and 820.01 + / -0.98 U / mg respectively, and the enzyme activity of the single-point mutant L48D and the enzyme activity of the single-point mutant L48H are obviously improved in comparison with the specificenzyme activity 220.30 + / -2.28 U / mg of unmodified wild nitrile hydratase. the enzyme activity of the mutant L48D and the enzyme activity of the mutant L48H are respectively 3.4 times and 3.7 times ofthat of the wild nitrile hydratase mutant. The mutation of the two amino acid residues significantly improves the catalytic activity of nitrile hydratase, is beneficial to the production of industrialfine chemicals such as nicotinamide, improves the catalytic efficiency and reduces the production cost.

Description

technical field [0001] The invention relates to a nitrile hydratase mutant and its application, belonging to the technical field of bioengineering. Background technique [0002] Nitrile hydratase (NHase for short, EC 4.2.1.84) is a metalloenzyme that can catalyze the conversion of nitriles into high value-added amides through hydration reactions. It has a role in the industrial production of fine chemicals nicotinamide Huge application potential. The biological production technology of amides is a typical case of biological method replacing chemical method. It has the advantages of green environmental protection, mild reaction conditions and high safety factor, which is in line with the concept of sustainable development and green production. [0003] Amide products have great application value in the fields of industry, agriculture and medicine, especially the biocatalysis of nicotinamide and acrylamide is the most common. Among them, nicotinamide is an important chemical...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P17/12C12R1/19
CPCC12N9/88C12N15/70C12P17/12C12Y402/01084
Inventor 周哲敏江诗进程中一刘中美崔文璟周丽韩来闯郭军玲
Owner JIANGNAN UNIV
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