Nitrile hydratase mutant, genetically engineered bacteria containing mutant and application of mutant

A technology of nitrile hydratase and mutants, applied in the field of enzyme engineering, can solve the problems of low thermal stability and achieve good enzymatic properties, good product tolerance, and improved thermal stability

Active Publication Date: 2019-04-09
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most nitrile hydratases are not thermally stable. Therefore, selecting a nitrile hydratase with improved

Method used

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  • Nitrile hydratase mutant, genetically engineered bacteria containing mutant and application of mutant
  • Nitrile hydratase mutant, genetically engineered bacteria containing mutant and application of mutant
  • Nitrile hydratase mutant, genetically engineered bacteria containing mutant and application of mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Construction of recombinant E. coli

[0055] (1) Construction of mutant αL6T / A19V / F126Y-βM46K / G47N / E108R / S212Y:

[0056] The nitrile hydratase mutant αL6T / A19V / F126Y-βM46K / E108R / S212Y gene was synthesized by chemical synthesis, and the gene was cloned at the NdeI and Bpu10I restriction sites of the pET24a plasmid, completed by General Biological Systems (Anhui) Co., Ltd. , Obtained pET24a-αL6T / A19V / F126Y-βM46K / E108R / S212Y recombinant plasmid. Using pET24a-αL6T / A19V / F126Y-βM46K / E108R / S212Y as a template, PCR was performed under the conditions shown in Table 1. The sequence information of the upstream primers used is shown in SEQ ID NO.9, and the sequence information of the downstream primers used is shown in SEQ ID NO. Shown in .10. The PCR product was transformed into E. coli JM109 to obtain the recombinant plasmid pET24a-αL6T / A19V / F126Y-βM46K / G47N / E108R / S212Y carrying the gene encoding the mutant. The recombinant plasmid pET24a-αL6T / A19V / F126Y-βM46K / G47N / E108R / ...

Embodiment 2

[0066] Example 2 Expression of Nitrile Hydratase

[0067] The BL21 / pET24a-αL6T / A19V / F126Y-βM46K / G47N / E108R / S212Y recombinant E. coli was inoculated into 5 mL of LB medium with a kanamycin concentration of 100 μg / mL, and cultured overnight at 37° C. with 200 r / min shaking. The above-mentioned overnight culture was inoculated into LB medium containing 100μg / mL kanamycin at the inoculum amount of 1%, and cultured with shaking at 37°C and 200r / min to the OD of the bacterial solution 600 To 0.6-0.8, add IPTG to a final concentration of 0.4mmol / L, induce culture at 20°C for 16-20h, collect the bacteria and ultrasonically break, analyze and identify the expression level of nitrile hydratase recombinant protein by Tris-tricine SDS-PAGE method, the results are as follows figure 1 Shown. After sonication and centrifugation at 12000 rpm for 60 minutes, the protein was purified with affinity chromatography column Strep Trap FF. The specific enzyme activity of the recombinant nitrile hydratase...

Embodiment 3

[0068] Example 3 Determination of thermal stability

[0069] In 500μL buffer reaction system (20mmol / L Na 2 HPO 4 , 280mmol / L NaCl and 6mmol / L KCl) was added 0.5mg / ml of the purified mutant enzyme in Example 1 10μL, stored in a metal bath at 50°C, and sampled every 20 minutes to determine the residual enzyme activity.

[0070] Such as figure 2 As shown, it was found that after the mutant was treated at 50°C for 80 minutes, the remaining enzyme activity of the mutant enzyme increased from 37% of the control (residual enzyme activity 333U / mg) to 53% (remaining enzyme activity 424U / mg); at 50°C After 100 minutes of treatment, the relative enzyme activity of the mutant enzyme increased from 24% of the control (remaining enzyme activity 216 U / mg) to 45% (remaining enzyme activity 360 U / mg). The thermal stability of the mutant is significantly improved.

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Abstract

The invention discloses a nitrile hydratase mutant, genetically engineered bacteria containing the mutant and application of the mutant, and belongs to the technical field of enzyme engineering. According to the invention, a 47th glycine of the nitrile hydratase mutant alphaL6T/A19V/F126Y-betaM46K/E108R/S212Y (disclosed in the invention patent CN102216455A) mutates into asparagine; the obtained new mutant enzyme has better temperature tolerance and product tolerance, which is conducive to industrial production in the future; a recombinant strain containing the nitrile hydratase mutant is fermented at a high density, and nicotinonitrile is used as a substrate to perform whole-cell catalytic reaction to prepare nicotinamide. Compared with a chemical production method, the method is safe andclean in production process and free of environmental pollution; and compared with an enzymatic method, the method has low price of the substrate and high catalysis efficiency, obtains a final productnicotinamide at a yield of over 95% and a concentration up to 680 g/L, and simplifies separation and purification steps of the product.

Description

Technical field [0001] The invention relates to a nitrile hydratase mutant, genetic engineering bacteria containing the mutant and applications thereof, and belongs to the technical field of enzyme engineering. Background technique [0002] Nitrile hydratase (NHase) can be used to catalyze 3-cyanopyridine to nicotinamide with higher medicinal value. Niacinamide is a vitamin and has been widely used in feed, food, pharmaceutical and other industries. Nicotinamide is in great demand in the market, and it is estimated that it needs more than 2,000 tons per year. However, the current production level of niacinamide in my country is not high and the scale is small, requiring a large amount of imports, about 1,000 tons. Therefore, the use of NHase in the production of nicotinamide has great potential. However, the reaction is an exothermic process, so the high temperature in the production process will affect the performance of the enzyme activity. The main reason is that the high tem...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N1/21C12N15/70C12P17/12C12P13/02C12R1/19
CPCC12N9/88C12N15/70C12P13/02C12P17/12C12Y402/01084
Inventor 周哲敏刘中美周丽崔文璟郭军玲蓝瑶
Owner JIANGNAN UNIV
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