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Method for enzymatic synthesis of nicotinamide uracil dinucleotide

A technology of uracil dinucleotide and uridine triphosphate, which is applied in the field of enzymatic synthesis of nicotinamide uracil dinucleotide, which can solve the problems of difficult separation and purification, difficulty of NUD entering cells, and cumbersome steps

Active Publication Date: 2017-06-23
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
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Problems solved by technology

[0009] Aiming at the disadvantages of cumbersome chemical synthesis of NUD, difficult separation and purification, NUD is difficult to enter cells, etc., and no enzyme that can efficiently catalyze the synthesis of NUD has been found so far. The purpose of the present invention is to provide a method for enzymatic synthesis of NUD. The mutant of nucleotide adenosyltransferase is used as a catalyst to catalyze the synthesis of NUD with nicotinamide mononucleotide and uridine triphosphate as substrates, and transfer NUD by expressing nicotinamide mononucleotide adenosine in the cell Enzyme mutants can directly synthesize NUD in cells, which can be used to selectively regulate metabolic processes and increase the yield of target metabolites

Method used

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  • Method for enzymatic synthesis of nicotinamide uracil dinucleotide
  • Method for enzymatic synthesis of nicotinamide uracil dinucleotide
  • Method for enzymatic synthesis of nicotinamide uracil dinucleotide

Examples

Experimental program
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Effect test

Embodiment 1

[0071] Example 1 NadD 3G10 Catalytic synthesis of NUD

[0072] 1. Construction of mutant library

[0073] The mutation library was constructed by RF cloning (F. van den Ent, et al. Journal of Biochemical and Biophysical Methods. 2006, 67, 67). In the first step, the template is a wild-type NadD expression vector (see Comparative Example 1), and the primers replace the base of the amino acid site to be mutated with a degenerate base NNK, and the PCR product is recovered; in the second step, the template is the same as the first step, The primers were the PCR products recovered in the first step, and the obtained products were digested with DpnI restriction enzyme, and transferred into BL21(DE3) competent cells, and the obtained transformants were the mutant library expressing NadD mutants, each mutant The expression bacteria were named BL21(DE3)(pET-NadD-xxx), and the expressed protein product was named NadD xxx . Among them, xxx is a combination of numbers and letters, and...

Embodiment 2

[0080] Example 2 NadD 3G10 Pure Enzyme Preparation of NUD

[0081] 10mL pure enzyme NadD 3G10 The reaction system for synthesizing NUD contains Tris 50mM, UTP 2mM, NMN 4mM, MgCl 2 10mM, MnCl 25mM, pure enzyme NadD 3G10 10mg, pH 8.0, reacted at 37°C, 200rpm for 1h, and then centrifuged at 5000g, 4°C for 15min to remove protein with an ultrafiltration centrifuge tube, reference method (D.Ji, et al.Journal of the American Chemical Society.2011 , 133, 20857), isolated and purified to obtain 5.8 mg of NUD with a yield of 45%.

[0082] Product NMR data: 1 H NMR (D2O, 400MHz): δ9.36(s, 1H), 9.20(d, J=6.2Hz, 1H), 8.88(d, J=8.1Hz, 1H), 8.22(pseudo t, J=6.6Hz , 1H), 7.79(d, J=8.2Hz, 1H), 6.10(d, J=5.4Hz, 1H), 5.82-5.80(m, 2H), 4.52(brs, 1H), 4.48-4.46(m, 1H), 4.39-4.37(m, 1H), 4.34-4.32(m, 1H), 4.25-4.21(m, 2H), 4.18-4.15(m, 3H), 4.07-4.04(m, 1H).13CNMR( D2O, 100MHz): δ166.1, 165.6, 151.7, 146.1, 142.6, 141.7, 139.9, 133.9, 128.7, 102.5, 99.9, 88.5, 87.0, 82.9, 77.6, 73.7, 70.7,...

Embodiment 3

[0083] Example 3 NadD 3G10 Preparation of NUD from crude enzyme solution

[0084] NadD 3G10 The expression engineered bacteria BL21(DE3)(pET-NadD-3G10) was streak cultured, and a single colony was picked and inoculated in 5mL LB (containing kanamycin 50ng / μL) liquid medium, activated overnight at 37°C and 200rpm, All of them were inoculated in 50 mL of fresh LB (containing 50 ng / μL of kanamycin and 1 mM of IPTG), and cultured at 30° C. and 200 rpm for 24 h. The cells were collected by centrifugation, added with 5 mL of cell lysate (50 mM HPEPS pH 7.5, 1% Triton X-100, 1 mg / mL lysozyme), and lysed at 37° C. for 1 h at 200 rpm. Centrifuge (12000g) for 10min, collect the supernatant to get NadD 3G10 crude enzyme solution.

[0085] 300μL crude enzyme solution reaction system contains: Tris 50mM, UTP 100μM, NMN 4mM, MgCl 2 10mM, MnCl 2 5mM, NadD 3G10 50 μL of crude enzyme solution, pH 8.0, reacted at 37°C, 200 rpm for 2 hours. Add 150 μL of ice-cold 1.2M HClO 4 After mixin...

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Abstract

The invention relates to a method for enzymatic synthesis of nicotinamide uracil dinucleotide and an application of the method. The nicotinamide uracil dinucleotide is prepared by catalyzing a coupling reaction between nicotinamide mononucleotide and uracil nucleoside triphosphate with a nicotinamide mononucleotide adenylyltransferase mutant serving as a catalyst. A coding gene of the nicotinamide mononucleotide adenylyltransferase mutant is expressed in microorganism cells; the nicotinamide uracil dinucleotide is synthesized by engineering bacteria on the basis of an endogenous metabolite; and the intracellular nicotinamide uracil dinucleotide, as a coenzyme, can selectively mediate an oxidation-reduction reaction, so that the yield of a target metabolite is improved. According to a typical embodiment, a succinic acid yield is improved by 35%.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a method for enzymatically synthesizing nicotinamide uracil dinucleotide (NUD) and its application, and expressing a mutant of nicotinamide mononucleotide adenylyltransferase in microbial cells The coding gene of the engineered bacteria uses endogenous metabolites to synthesize NUD, and intracellular NUD can act as a coenzyme to selectively mediate redox reactions and increase the yield of target metabolites. Background technique [0002] Nicotinamide adenine dinucleotide (NAD) and its reduced state (NADH) all play a very important role in cell reproduction, growth, differentiation, apoptosis and other life activities (W.Ying, et al.AntioxidantsRedox Signaling .2008, 10, 179). In addition, NAD is also the coenzyme of many important redox enzymes, which plays the role of transferring hydrogen and electrons. Its structural formula is as follows: [0003] [0004] Since N...

Claims

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Application Information

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IPC IPC(8): C12P19/36
CPCC12P19/36
Inventor 赵宗保王雪颖刘玉雪
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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