Nicotinamide riboside kinase mutant with enhanced thermal stability and activity, encoding gene of nicotinamide riboside kinase mutant, and application

A technology of nicotinamide riboside and ribokinase, which is applied in the field of biological enzyme engineering, can solve the problems of low thermal stability of NRK, difficulty in realizing industrial production, and long reaction time, so as to prolong storage time, reduce production cost, and reduce reaction time Effect

Active Publication Date: 2021-03-26
中山俊凯生物技术开发有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Aiming at the deficiencies in the prior art, the technical problem to be solved by the present invention is to provide a mutant of nicotinamide ribokinase with enhanced thermostability an

Method used

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  • Nicotinamide riboside kinase mutant with enhanced thermal stability and activity, encoding gene of nicotinamide riboside kinase mutant, and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Construction and cultivation of embodiment 1 mutant

[0034] Selection of mutation sites: Using the nicotinamide ribokinase NRK derived from Homo sapiens as the starting template, the possible effective mutation sites are predicted to be S31C, K49E, E59D, N62D, E64D, A69T, S71Q, S76N, D109N, T110D, S139P, Q159E, D176E, L179N, I186Q. The gene containing 15 mutation sites was then fully synthesized.

Embodiment 2

[0035] The construction of embodiment two prokaryotic expression system

[0036] The mutant NRK (named NRK15) gene fragment was synthesized by Changzhou Jiyu Biotechnology Co., Ltd., and recombined into the PUC57 vector. After double digestion with restriction endonucleases NdeI and HindIII (purchased from New England Biolabs, NEB) at 37°C for 4 hours, they were separated by 1% agarose gel electrophoresis and recovered by gel cutting (the gel recovery kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.). Subsequently, it was ligated with the expression vector pET28a(+) (Novagen Company) that had also undergone double enzyme digestion, under the action of T4 DNA ligase (purchased from Takara Company) overnight at 16°C. The connection solution was used to transform DH5a competent cells, and colony PCR screening and sequencing verification were performed to obtain the positive recombinant plasmid NRK15-pET28a(+).

[0037] The positive recombinant plasmid N...

Embodiment 3

[0039] Shake flask fermentation of embodiment three enzymes prepares enzyme freeze-dried powder

[0040] The expression strain NRK15-pET28a(+) / BL21(DE3) constructed above was added with a final concentration of 30 μg / mL kanamycin sulfate in 5 mL LB liquid medium [10 g / L tryptone (OXIOD), 5 g / L Yeast powder (OXIOD), 10g / L sodium chloride (Sinopharm Reagent)], after overnight shaking culture at 37°C and 200rpm, inoculate in a 1% (V / V) ratio of 1% (V / V) containing Kanamyces sulfate with a final concentration of 30μg / mL In 400mL LB liquid culture medium, shake culture at 37°C and 200rpm. Waiting for OD 600 Between 0.8-1.0, the inducer IPTG (isopropyl-β-D-thiogalactopyranoside) was added at a final concentration of 0.1 mM, and induced overnight at 25°C. The bacteria were collected by centrifugation at 4°C and 8000rpm, then suspended in 50mM pH7.0 sodium phosphate buffer, ultrasonically disrupted (200W, 3s / 5s, 30min), centrifuged at 4°C and 12000rpm for 20min, and the supernatant ...

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Abstract

The invention belongs to the technical field of biological enzyme engineering, and particularly relates to a nicotinamide riboside kinase mutant with enhanced thermal stability and activity, an encoding gene of the nicotinamide riboside kinase mutant, and application. Compared with wild nicotinamide riboside kinase of which the amino acid sequence is disclosed by SEQ ID NO:4, the amino acid sequence of the nicotinamide riboside kinase mutant is characterized in that one-point or multiple-point combined mutation is carried out on any one or a plurality of sites in 15 pieces of sites of the amino acid sequence disclosed by SEQ ID NO:4. The nicotinamide riboside kinase mutant provided by the invention can be used for synthesis and preparation of [beta]-nicotinamide mononucleotide. Compared with wild enzymes, the nicotinamide riboside kinase mutant constructed by the invention is characterized that the thermal stability and the unit enzyme activity of the mutant are greatly improved, so that the reaction temperature of the enzyme can be obviously improved, a reaction rate is quickened, reaction time is shortened, production cost is lowered, an NMN yield is improved, and the nicotinamide riboside kinase mutant has a wide prospect of large-scale industrial application.

Description

technical field [0001] The invention relates to the technical field of biological enzyme engineering, in particular to a mutant of nicotinamide ribokinase with enhanced thermostability and activity, its coding gene and its application. Background technique [0002] Nicotinamide mononucleotide (NMN) is a naturally occurring biologically active nucleotide. NMN has two irregular forms, the α isomer and the β isomer. Among them, the β isomer is the active form of NMN with a molecular weight of 334.221 g / mol. NMN is the nicotinamide adenine dinucleotide (Nicotinamide adenine dinucleotide, NAD + , also known as coenzyme I) is an important intermediate in the synthetic pathway. In recent years, relevant research reports in international authoritative academic journals such as Science, Nature, and Cell have shown that supplementing NMN can effectively increase and restore the level of coenzyme I in the body, greatly delay aging and prevent various neuron degenerative diseases such...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/54C12N15/70C12N1/21C12P19/30C12R1/19
CPCC12N9/1205C12N15/70C12P19/30C12Y207/01022
Inventor 戴维周嘉莹余允东刘慧徐广见
Owner 中山俊凯生物技术开发有限公司
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