Recombinant nitrile hydratase and application thereof in preparation of nicotinamide by coupling ion exchange resin

An ion-exchange resin, nitrile hydratase technology, applied in the application, recombinant DNA technology, lyase and other directions, can solve the problems of limiting the application of nitrile hydratase catalyst, difficult product separation and purification process, slowing down of enzyme catalytic reaction speed, etc. The effect of improved substrate and product tolerance, mild conditions, and non-polluting energy consumption

Pending Publication Date: 2022-04-05
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the characteristics of the nitrile hydratase gene cluster and the characteristics of partial nitrilase, and also due to the protein characteristics of the enzyme, any factors that can cause protein denaturation may cause enzyme inactivation, such as pH, heavy metal salts, heat, ultraviolet rays, severe Shock, etc.
Even under optimal catalytic conditions, the enzyme-catalyzed reaction rate will gradually decrease as the reaction time increases
In addition, the free enzyme produces impurities after catalyzing the hydration hydrolysis reaction, which causes difficulties in the product separation and purification process, resulting in increased production costs
These factors all greatly limit the application of nitrile hydratase catalyst in modern industry

Method used

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  • Recombinant nitrile hydratase and application thereof in preparation of nicotinamide by coupling ion exchange resin
  • Recombinant nitrile hydratase and application thereof in preparation of nicotinamide by coupling ion exchange resin
  • Recombinant nitrile hydratase and application thereof in preparation of nicotinamide by coupling ion exchange resin

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026]Embodiment 1, nitrile hydratase gene cloning and construction of engineering bacteria

[0027] 1. Engineering bacteria pET28a-NHaseYpB 1

[0028] (1) Wild-type nitrile hydratase

[0029] According to the nitrile hydratase (nitrolehydratase H-Nhase, GenBank: D67027.1) from Rhodococcus rose (R.rhodochrous J1) in NCBI, it contains β subunit (NHase-B), α subunit (NHase-A) and The three parts of the activator gene (NHase-G) were synthesized by Beijing Qingke Biotechnology Co., Ltd. after codon optimization according to the preference of Escherichia coli. The nucleotide sequence of the β subunit (NHase-B) is shown in SEQ ID 1-690 in NO.2, the alpha subunit (NHase-A) nucleotide sequence is shown in 691-1302 in SEQ ID NO.2, and the activator gene (NHase-G) nucleotide sequence is shown in Shown in positions 1303-1617 in SEQ ID NO.2.

[0030] (2) Fragment

[0031] β subunit fragment: take step (1) to synthesize the nitrile hydratase gene β subunit (NHase-B) fragment as a temp...

Embodiment 2

[0083] Embodiment 2, the induced expression of recombinant nitrile hydratase genetically engineered bacteria

[0084] The correct recombinant Escherichia coli engineering bacteria E.coil BL21(DE3)-pET28a-NHaseYpB sequenced in Example 1 1 , E.coil BL21(DE3)-pET28a-NHaseYpB 2 , E.coil BL21(DE3)-pET28a-NHaseYpB 3 , E.coil BL21(DE3)-pET28a-NHaseYpB 4 They were respectively inoculated in 10 mL liquid LB medium containing Kan (final concentration 50 μg / mL), and cultured at 37° C. for 10 h. The cultured seed solution was transferred to a new 100mL liquid LB medium containing Kan (final concentration 50μg / mL) at a volume concentration of 1% inoculum, cultivated under the same conditions until the OD600 of the bacterial solution was 0.6, and added a final concentration of 0.5 mM IPTG, then add 400 μL of 100 mM CoCl 2 Aqueous solution (final concentration 0.4mM), induced at 28°C for 12h; centrifuged at 9000rpm for 10min to collect bacteria. The cells were washed twice with PBS buff...

Embodiment 3

[0090] Embodiment 3, the impact of resin on enzyme-catalyzed reaction

[0091] Resin pretreatment: (1) Resins D201, D301, JKA915, BA765, D101, D311 (all purchased from Jiangsu Jinkai Resin Chemical Co., Ltd.) in Table 8 were mixed with 10% NaCl aqueous solution at a mass concentration about twice the volume of the resin in Soak in a beaker, stir the resin layer at a low speed of 20rpm for 1 hour, place the liquid level at a place 200-300mm above the resin layer and soak for 20 hours, then rinse with water until the effluent is colorless. (2) Then carry out backwashing to remove tiny impurities mixed in the resin, soak the resin layer with 4% HCl aqueous solution approximately equal to twice the volume of the resin for 1 hour, and put the liquid surface to 200-300mm above the resin layer to soak After 2-4 hours, rinse with clean water until the pH value of the effluent is 5-6. (3) After soaking the resin layer with a 2% NaOH aqueous solution with a mass concentration of about ...

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Abstract

The invention provides recombinant nitrile hydratase and application thereof in preparation of nicotinamide by coupling ion exchange resin, which comprises the following steps: by taking a concentrated solution of supernate obtained by ultrasonically crushing wet thalli obtained by fermentation culture of recombinant nitrile hydratase gene engineering bacteria as a catalyst and 3-cyanopyridine as a substrate, adding the ion exchange resin, and reacting at the temperature of between 20 and 30 DEG C to obtain the nicotinamide. The preparation method comprises the following steps: forming a reaction system by taking a phosphoric acid buffer solution with the pH value of 6-9 as a reaction medium, carrying out hydration reaction under the conditions of 20-30 DEG C and 100-1000rpm, and after the reaction is completed, separating and purifying the reaction liquid to obtain the product nicotinamide. The recombinant nitrile hydratase coupled ion exchange resin has the advantages of mild conditions, high efficiency, high chemical selectivity, regioselectivity and the like, the biological catalysis process has the characteristics of no toxicity, no pollution, low energy consumption and the like, the method is an environment-friendly synthesis method, 95% or more of nicotinic acid impurities are adsorbed, and the method is suitable for industrial production. The nicotinamide product with the purity of 99.99% or above is obtained.

Description

technical field [0001] The invention belongs to the field of biochemical industry, and relates to a recombinant nitrile hydratase and the application thereof to prepare high-purity nicotinamide by catalyzing hydration of 3-cyanopyridine coupling ion exchange resin. Background technique [0002] Nicotinamide (nicotinamide, trade name nicotinamide) is a water-soluble, stable, small-molecule vitamin that can easily penetrate the stratum corneum. As a pharmaceutical ingredient, it has high safety and is also a basic vitamin supplement in clinical dermatology treatment. Studies in recent years have proved that nicotinamide, as a functional ingredient of cosmetics, has good effects in inhibiting melanin, anti-inflammation, anti-aging, and treating acne. Generally speaking, the added amount of niacinamide must reach more than 3% to exert the whitening effect. High content of niacinamide may cause skin irritation to a certain extent. The main reason may be caused by a small amount ...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N1/21C12N15/70C12P13/02C07D213/82C12R1/19
Inventor 郑建永王祎丁宋问孙杰于欣君
Owner ZHEJIANG UNIV OF TECH
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