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Mutant of nitrile hydratase derived from caldalkalibacillus thermarum

A technology of nitrile hydratase and nitrile hydratase is applied in the field of enzyme engineering and can solve the problems of low concentration of final product, long growth cycle of Rhodococcus, low production efficiency and the like

Active Publication Date: 2020-03-31
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, in industrial production, Rhodococcus rhodochrous J1 is mainly used to catalyze the formation of nicotinamide, and the method of substrate feeding is adopted. However, the growth cycle of Rhodococcus is long, requiring 100 hours, and the production efficiency is not high. Amide production is up to 162g / L, while acrylamide production is up to 300g / L
At present, there are also reports on the production of nicotinamide by recombinant bacteria, but the concentration of the final product is low, only 240g / L

Method used

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  • Mutant of nitrile hydratase derived from caldalkalibacillus thermarum
  • Mutant of nitrile hydratase derived from caldalkalibacillus thermarum
  • Mutant of nitrile hydratase derived from caldalkalibacillus thermarum

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Kinetic simulation of nitrile hydratase (PtNHase) derived from Pseudonocardia thermophila and Cal.t NHase derived from Caldalkalibacillus thermarum found that the RMSF value of some amino acids was higher than that of It is speculated that these amino acids may affect its thermal stability. Therefore, the following mutants were constructed: Cal.t NHase-A20V, Cal.t NHase-H150S (the histidine at the 150th position of the β subunit shown in SEQ ID NO.2 is mutated into serine in the amino acid sequence) , Cal.t NHase-T104A (the threonine at the 104th position of the β subunit shown in SEQ ID NO.2 is mutated to alanine), Cal.t NHase-S152K (the amino acid sequence is as shown in SEQ ID NO.2 The serine at the 152nd position of the β subunit shown in .2 is mutated into lysine), Cal.t NHase-K185A (the lysine at the 185th position of the β subunit shown in the amino acid sequence as SEQ ID NO.2 is mutated to alanine).

[0036] (1) Construction of mutants:

[0037] Synthesize t...

Embodiment 2

[0049] Add 10 μL of 0.5 mg / mL mutant enzyme purified in Example 1 to the 500 μL buffer reaction system, and treat it in a metal bath at 70°C for 0 min, 10 min, 20 min, and 30 min, respectively, and measure the residual enzyme activity. is 100%.

[0050] like figure 1 As shown, it was found that the mutant enzyme Cal.t NHase-H150S was treated at 70°C for 10 minutes, and the enzyme activity of the mutant enzyme Cal. Properties of the tNHase-H150S mutant enzyme.

Embodiment 3

[0052] Prepare different concentrations of product nicotinamide solution: 0M, 2M, OD 600 =8 The wild enzyme and the mutant bacterial solution were respectively treated in solutions with different substrate concentrations at 30°C for 30 minutes, then the cells were resuspended and washed twice with KPB, and 10 μL was taken to measure the residual enzyme activity. The enzyme activity treated with 0M was defined as 100%.

[0053] like figure 2 As shown, the enzyme activity without product treatment is defined as 100%. It was found that after the mutant was treated with 2M product nicotinamide for 20 minutes, the remaining enzyme activity of the mutant enzyme Cal.t Nhase-A20V was increased from 40% of the wild enzyme to 69%. %, and the rest of the mutant enzymes Cal.t NHase-H150S, Cal.t NHase-T104A, Cal.t NHase-S152K, Cal.t NHase-K185A, compared with wild enzymes, showed different degrees of decline. The tolerance of the mutant enzyme Cal.t Nhase-A20V product was significantly ...

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Abstract

The invention discloses a mutant of nitrile hydratase derived from caldalkalibacillus thermarum, and belongs to the technical field of enzyme engineering. The half-life period of the nitrile hydratasemutant Cal.t Nhase-A20V at 70 DEG C is about 10 min, the thermal stability of the mutant Cal.t Nhase-A20V has no too large change compared with that of a wild enzyme, and the specific enzyme activityof the mutant Cal.t Nhase-A20V is 128% of that of the wild enzyme. At the same time, the mutant also has better substrate tolerance and product tolerance, the final yield of nicotinamide produced bywhole-cell catalysis reaches 598 g / L; therefore, the nitrile hydratase mutant Cal.t Nhase-A20V provided by the invention has very good enzymatic properties, and is beneficial to industrial productionin future.

Description

technical field [0001] The invention relates to a mutant of nitrile hydratase derived from Thermoalkali bacillus thermoalkali, belonging to the technical field of enzyme engineering. Background technique [0002] Nitrile hydratase (NHase) can be used to catalyze 3-cyanopyridine into nicotinamide with higher medicinal value. Nicotinamide is a vitamin that has been widely used in feed, food, pharmaceutical and other industries. The market demand for nicotinamide is very large, but the production level of nicotinamide in my country is not high and the scale is small. Therefore, there is great potential to use NHase for the production of nicotinamide. However, the reaction is an exothermic process, so high temperature in the production process will affect the performance of the enzyme activity, mainly because high temperature affects the structure of the enzyme, resulting in a decrease in enzyme activity, which in turn leads to a large amount of energy consumption and increases...

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 周哲敏刘中美李婷陈德智张赛兰周丽崔文璟程中一郭军玲
Owner JIANGNAN UNIV
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