阅读说明：本技术 一种利用甲醇还原nad类似物的方法 (Method for reducing NAD analogue by using methanol ) 是由 赵宗保 王俊婷 于 2019-04-16 设计创作，主要内容包括：本发明公开了一种利用甲醇还原NAD类似物的方法及其应用。该方法中还原剂为甲醇,催化剂为可利用甲醇的甲醇脱氢酶,甲醇脱氢酶氧化甲醇的同时,将NAD类似物转化为其还原态。该方法可用于生产还原态NAD类似物或氘代的还原态类似物,也可为消耗还原态NAD类似物的酶促反应提供还原态辅酶,还原态的NAD类似物可作为辅酶应用于苹果酸酶ME-L310R/Q401C、D-乳酸脱氢酶DLDH-V152R、酿酒酵母醇脱氢酶等酶催化的还原反应,有利于NAD类似物的广泛应用。该NAD类似物还原方法可在温和条件下,再生还原态NAD类似物用于苹果酸或乳酸的制备；也可作为一种氧化还原力调控微生物体内苹果酸或乳酸的代谢强度。(The invention discloses a method for reducing NAD analogue by methanol and application thereof. In the method, the reducing agent is methanol, the catalyst is methanol dehydrogenase capable of utilizing the methanol, and the NAD analogue is converted into a reduction state while the methanol dehydrogenase oxidizes the methanol. The method can be used for producing reduced NAD analogues or deuterated reduced analogues, and can also provide reduced coenzyme for enzymatic reactions consuming the reduced NAD analogues, and the reduced NAD analogues can be used as the coenzyme for enzymatic reduction reactions catalyzed by malic enzyme ME-L310R/Q401C, D-lactate dehydrogenase DLDH-V152R, saccharomyces cerevisiae alcohol dehydrogenase and the like, and are beneficial to wide application of the NAD analogues. The NAD analogue reduction method can regenerate a reduced NAD analogue to be used for preparing malic acid or lactic acid under mild conditions; can also be used as a redox force to regulate the metabolic strength of malic acid or lactic acid in the microorganism.)

1. A method of reducing an NAD analog, comprising: taking a substrate of one or two of methanol and deuterated methanol combined in any proportion as a reducing agent, taking an enzyme of the reducing agent as a catalyst, mixing with the NAD analogue, reacting to obtain a reduced NAD analogue, and oxidizing the methanol into formaldehyde; the catalyst is a mutant of wild methanol dehydrogenase Bacillusstearothermophilus, DSM 2334(ADH 2334) which is modified by genetic engineering; the enzyme capable of utilizing methanol is one or more of BsMDH-V237, BsMDH-V237/N240, BsMDH-V237/N240/K241, BsMDH-Y171/V237/N240/K241, BsMDH-Y171/V237, BsMDH-Y171/I196, BsMDH-Y171/I196/V237/N240/K241, BsMDH-Y171, BsMDH-Y171/N240, BsMDH-Y171/I196/V237/N240/K241, BsMDH-V237/N240/K241, BsMDH-Y171/I196/V237/N240.

2. The method of claim 1, wherein: the reduced NAD analogue is Nicotinamide Cytosine Dinucleotide (NCD), Nicotinamide Thymine Dinucleotide (NTD) or Nicotinamide Uracil Dinucleotide (NUD), and the chemical structure of the reduced NAD analogue is as follows:

3. the method of claim 1, further characterized by: the enzyme capable of utilizing methanol is an active protein which takes methanol as a reducing agent and catalyzes and reduces NAD analogues into corresponding reduction states.

4. A method of reducing an NAD analog according to claim 1, further characterized by: in a buffer solution with the pH value of 4-9, the reaction temperature is 15-40 ℃, the initial final concentration of enzyme is 1 mu g/mL-600 mu g/mL, the initial concentration of NAD analogue is 0.001mM-20mM, and the initial concentration of methanol is 0.4mM-1000mM, wherein the buffer system comprises one or more than two of phosphate buffer solution, Tris-HCl buffer solution, HEPES buffer solution, MES buffer solution and PIPES buffer solution.

5. A method of reducing an NAD analog according to claim 1, further characterized by: the obtained reduced NAD analogue can be used as coenzyme by other enzymes for reduction reaction; such other enzymes include, but are not limited to, one or more of the following: malic enzyme ME (NCBI No. NP-415996.1) mutant ME-L310R/Q401C, lactate dehydrogenase DLDH (GeneBank No. CAA47255.1, PDB ID:2DLD) mutant DLDH-V152R, V152R/I177R/A212G or V152R/I177S/A212D; saccharomyces cerevisiae alcohol dehydrogenase catalyzing the reduction of acetaldehyde to ethanol, hydroxybutanone dehydrogenase catalyzing the reduction of diacetyl to hydroxybutanone, malate dehydrogenase (PDB id 1EMD) mutant MDH-L6R catalyzing the oxidation of malate to oxaloacetate.

6. A method of reducing an NAD analogue as claimed in claim 1 or 6 further characterized by: the enzyme that can utilize methanol is expressed in the cells of the microorganism, while the NAD analog and methanol are transported into the cells, and the NAD analog reduction reaction is carried out in the cells.

7. The method of claim 1 or 6, wherein: the reaction is carried out intracellularly, and the microorganisms expressing one or more of malic enzyme, lactate dehydrogenase, and the like and used for intracellular reduction of NAD analogs include, but are not limited to, prokaryotic microorganisms and eukaryotic microorganisms.

8. The method of claim 7, wherein: the prokaryotic microorganism includes but is not limited to one or two of Escherichia coli or lactococcus lactis.

9. The method of claim 7, wherein: the eukaryotic microorganism includes but is not limited to one or more than two of saccharomyces cerevisiae, trichoderma reesei or rhodotorula toruloides.

Technical Field

The invention belongs to the technical field of biology, and relates to an enzyme catalytic reduction method of coenzyme Nicotinamide Adenine Dinucleotide (NAD) analogue and application thereof, in particular to an enzyme catalytic reduction method for converting the NAD analogue into a reduction state thereof under the catalysis of enzyme by using methanol as a reducing agent, and the NAD analogue can be used as coenzyme by other enzymes to be applied to reduction reaction.

Background

Nicotinamide Adenine Dinucleotide (NAD) and its reduced NADH are important coenzymes in life processes, and participate in a series of redox metabolism and other important biochemical processes in life bodies. These coenzymes can be used for producing chiral chemicals and for preparing isotopic labels. Since many oxidoreductases use NADH or NADPH as a coenzyme, any manipulation to change the NAD concentration and its redox state can have a global effect on the cell, making it difficult to control a particular oxidoreductase in the biological system at the coenzyme level. Since NADH can be consumed by various pathways in the metabolic network, the efficiency of the target pathway's utilization of reducing power is affected. When NAD analogues are used for transferring reducing force, the analogues can only be recognized by mutant oxidoreductases, so that the target oxidoreduction process is regulated at the coenzyme level, and the analogue has important significance for biological catalysis and synthetic biology research (Ji DB, et al J Am Chem Soc,2011,133, 20857-20862; Wang L, et al ACCcatal, 2017,7, 1977-1983).

Several NAD analogues with good biocompatibility have been reported. Such as Nicotinamide Cytosine Dinucleotide (NCD), Nicotinamide Thymine Dinucleotide (NTD), Nicotinamide Uracil Dinucleotide (NUD) (Ji DB, et al, JAm Chem Soc,2011,133, 20857-20862; Ji DB, et al, Sci China Chem,2013,56, 296-300). Also, some enzymes recognizing NAD analogues have been reported, such as NADH oxidase (NOX, Genbank S45681) from Enterococcus faecalis, D-lactate dehydrogenase (DLDH, Genbank CAA47255) V152R mutant, malic enzyme (ME, Genbank P26616) L310R/Q401C mutant, and malic dehydrogenase (MDH, Genbank CAA68326) L6R mutant.

Using NAD analogs and enzymes that recognize them, more cost-effective biocatalytic systems can be constructed (catalytic dictionary, Edinbin et al, 2012,33, 530-. By selecting proper NAD analogues and recognizing enzymes thereof, the crude enzyme solution can be used for reaction to achieve the effect of pure enzyme catalysis, and the control of a complex biocatalytic conversion system at the coenzyme level is realized. Currently, regulation of intracellular metabolic reactions using NAD analogs has been achieved, and specific biocatalytic regulation has been achieved by intracellular transport of NCD, which is available for the reduction of pyruvate to lactate by DLDH-V152R (Wang L, et al ACS Catal,2017,7, 1977-.

Like the use of other redox coenzymes, NAD analogs also require regeneration cycles. The coenzyme regeneration methods mainly include an enzymatic method, an electrochemical method, a chemical method and a photochemical method. The enzyme method has the advantages of high selectivity, compatibility with synthetase, high conversion number and the like. Methanol dehydrogenase can convert methanol into formaldehyde, and formaldehyde can be converted into formate by catalysis of formaldehyde dehydrogenase endogenous to cells in microbial cells or enter a riboketone monophosphate pathway, a tetrahydromethopterin pathway and the like to generate metabolites and reducing power necessary for cell life activities, thereby realizing effective utilization of a carbon resource (Muller JE, equivalent. Metab Eng,2015,28, 190-.

Although the regeneration cycle of the NAD analogue has important significance in the fields of biological catalysis, synthetic biology and the like, few documents exist for efficiently reducing the NAD analogue by modifying the structure of enzyme at present, and no document reports how to modify methanol dehydrogenase to efficiently reduce the NAD analogue. The reported NAD analog reduction method (Zhao Zongbao et al, a reduction method of NAD analog, Chinese patent 201010524767.6) utilizes phosphite dehydrogenase to reduce NAD analog to NADH with phosphorous acid as a substrate, and at the same time, generates phosphoric acid as a byproduct. Compared with a phosphate compound which is a product generated by catalyzing a phosphorous acid compound by phosphorous acid dehydrogenase, a methanol dehydrogenase substrate has rich methanol sources and low price, and is a carbon compound with a larger prospect, and methanol can be reduced by the methanol dehydrogenase by utilizing microbial cells to finally generate a high value-added compound (Whitaker WB, et al. Therefore, the regeneration of NAD analogue by methanol dehydrogenase reduction is a new reduction method combining one-carbon resource utilization and NAD analogue regeneration, and carbon conversion and energy transfer are realized by one-carbon compound methanol while NAD analogue reduction is realized.

Based on the background and the advantages, the directed evolution method is utilized to obtain the mutant with the further improved reduction efficiency of the NAD analogue on the basis of the existing method for reducing the NAD analogue by using methanol and derivatives thereof.

Disclosure of Invention

The invention relates to an enzyme catalytic reduction method of coenzyme NAD analogue, in particular to a method for converting NAD analogue into a corresponding reduction state by taking one or two of methanol and deuterated methanol combined substrate in any proportion as a reducing agent and taking enzyme of the reducing agent as a catalyst. These reduced states of NAD analogs can be used as coenzymes for other oxidoreductases for reduction reactions. Therefore, the method can be applied to the fields of biological catalysis and biological conversion and has important value.

The invention relates to a method for reducing NAD analogue by methanol, which is characterized in that: taking a substrate of one or two of methanol and deuterated methanol combined in any proportion as a reducing agent, taking an enzyme capable of utilizing methanol as a catalyst, and reacting for 2-120min in a buffer system with pH4-9 at the reaction temperature of 15-40 ℃, at the final concentration of 1-600 [ mu ] g/mL of the enzyme, at the final concentration of 0.001-20 mM of the NAD analogue and at the final concentration of 1-1000 mM of the methanol compound to obtain the reduced NAD analogue. The buffer system comprises but is not limited to one or more than two of phosphate buffer, Tris-HCl buffer, HEPES buffer, MES buffer, PIPES buffer and acetic acid-sodium acetate buffer system.

NAD analogs include NCD, Nicotinamide Thymine Dinucleotide (NTD), and Nicotinamide Uracil Dinucleotide (NUD), which have the following chemical structure:

the NAD analogue related to the invention is prepared by reference method (Ji DB, et al. Sci China Chem,2013,56, 296-300).

The methanol dehydrogenase used in the invention is an active protein which takes methanol as a reducing agent and catalyzes and reduces NAD analogue into a corresponding reduction state. These enzymes are mutants of methanol dehydrogenase BsMDH (NCBI protein database No. P42327.1, containing complete amino acid sequences from 1 to 339) derived from Bacillus stearothermophilus (the mutation sites are represented by amino acid numbers and amino acid names before and after mutation, for example, V237T indicates that the 237 th amino acid is mutated from V to T, and the rest sites are similar), and include mutants BsMDH-V237T/N240E, BsMDH-V237T/N240E/K241A, BsMDH-Y171/171R/V237T/N240E/K241A, BsMDH-Y171R/I196V/V237T/N240E/K241A. V237T represents the mutation of the 237 th amino acid from V to T, N240E represents the mutation of the 240 th amino acid from N to E, and the like. Expression and purification of these enzymes was performed according to literature methods for expression of other oxidoreductases in E.coli (Ji DB, et al. J. Am ChemSoc,2011,133,20857).

The NAD analogs of the present invention contain nicotinamide mononucleotide units, as do NAD, the reduced form of which is 1, 4-dihydronicotinamide mononucleotide. Therefore, the NAD analogue reduced product has stronger absorption in the ultraviolet spectral region near 340nm and molar extinction coefficient₃₄₀About 6220M^-1·cm^-1(Ji DB, et al. creation of bioorchol redox systems pending on a nicotinamide flucytoside. J Am ChemSoc.2011,133, 20857-20862). The present invention utilizes this property to analyze the NAD analog reduction process. The conditions for quantifying the NAD analogue and its reduced product by liquid chromatography are as follows: the liquid chromatograph was Agilent 1100, the analytical column was Zorbax150mM X3.0 mM (3.5 μm), the mobile phase was 5mM tetrabutylammonium sulfate, and the flow rate was 0.5 mL/min. Each sample was tested for 20 min. The detection wavelengths are 260nm (the cofactor and the reduced coenzyme thereof have stronger light absorption at 260 nm) and 340nm (the reduced coenzyme has stronger light absorption at 340 nm).

The prepared reduced product of the NAD analogue can be used as coenzyme by other enzymes and applied to reduction reaction. Thus, the present invention can be viewed as a technique for regenerating the reduced state of a cyclic NAD analog. By the technique of the present invention, the reducing power of methanol is transferred and stored in the NAD analog reduced state to facilitate selective reduction of other substrates.

The buffer system used comprises one or more than two of phosphate buffer, Tris-HCl buffer, HEPES buffer, MES buffer and PIPES buffer, wherein the final concentration of the methanol dehydrogenase is 1 mu g/mL-600 mu g/mL (preferably 100 mu g/mL-500 mu g/mL, more preferably 100 mu g/mL-300 mu g/mL), the final concentration of the NAD analogue is 0.001mM-20mM (preferably 0.01mM-20mM, more preferably 0.1mM-10mM), and the final concentration of the methanol is 1mM-1000mM (preferably 10mM-1000mM, more preferably 100mM-900 mM).

Reducing NAD analogs using the methanol dehydrogenase to include, but not limited to, malic enzyme ME (NCBI No. NP-415996.1) mutant ME-L310R/Q401C which catalyzes the reduction of pyruvate; when the lactate dehydrogenase DLDH (GeneBank No. CAA47255.1, PDB ID:2DLD) mutant DLDH-V152R, V152R/I177R/A212G or V152R/I177S/A212D or saccharomyces cerevisiae alcohol dehydrogenase catalyzing reduction of pyruvate provides reduced coenzyme, a buffer system with pH4-9 is adopted, and the reaction temperature is 15-40 ℃.

The methanol-utilizing enzyme is expressed in the cells of the microorganism, and the NAD analogue can be transferred into the cells through an NAD transporter AtNDT2(Access NO. NC-003070) or NTT4(Haferkamp I, et al. Nature,2004,432, 622-; methanol permeates into the cells and the NAD analog reduction reaction proceeds inside the cells.

The microbial cells expressing methanol dehydrogenase and used for intracellular reduction of NAD analogue include but are not limited to prokaryotic microorganisms such as Escherichia coli, lactococcus lactis and the like or eukaryotic microorganisms such as Saccharomyces cerevisiae, Rhodotorula rubra or Trichoderma reesei and the like.

The invention has the advantages and beneficial effects that: methanol as a reducing agent is low in price and abundant in reserves, and is a carbon resource with a great prospect. The reaction of enzyme catalysis methanol can be used in a biological reaction system, and methanol is converted into chemicals with high added values by means of microbial fermentation and the like, so that the selective transfer of reducing power and the effective utilization of carbon are realized. In addition, by using deuterated methanol, the reduced state of the deuterated NAD analogue can be obtained and used for preparing high-purity deuterium-substituted biocatalytic products.

Description of the drawings:

FIG. 1 is a crystal structure of BsMDH-Y171R/I196E/V237T/N240E/K241A complex;

FIG. 2 is a crystal structure of BsMDH-Y171R/V237T/N240E/K241A complex.

Detailed Description

The following examples will assist one of ordinary skill in the art in further understanding the invention, but are not intended to limit the invention in any way.

Comparative example 1: reaction of methanol with NAD analogs in the absence of enzymes

NAD analogs (NCD, NTD and NUD) were prepared according to the literature methods (Ji DB, et al. Sci China Chem,2013,56, 296-300). The NAD analogue was made up to a 20mM concentration in water for use.

1mM NAD analogue substrate and 8mM methanol were dissolved in 1mL Tris-HCl buffer solution with a concentration of 50mM and pH 7.5, mixed, reacted at 30 ℃ for 2 hours, and 20. mu.L thereof was analyzed.

The NAD analogue substrate and its reduced product were detected by HPLC. The liquid chromatograph was Agilent 1100, the analytical column was Zorbax150 mM. times.3.0 mM (3.5 μm), the mobile phase was 5mM tetrabutylammonium sulfate, and the flow rate was 0.5 mL/min. Each sample was tested for 20 min. The detection wavelength is 260nm (strong absorption of cofactor and its reduced state at 260 nm) and 340nm (strong light absorption of reduced coenzyme at 340 nm).

Analysis revealed that all reaction samples had no characteristic peak at 340nm, and only a characteristic peak at 260nm was detected, which was identical to the retention time of the NAD analogue. Indicating that methanol cannot directly reduce the NAD analog without the enzyme.

Comparative example 2: reaction of methanol with NAD analogs under enzyme-inactivating conditions

Methanol dehydrogenase BsMDH (NCBI protein database No. P42327.1, containing complete amino acid sequences from 1 to 339) from Bacillus stearothermophilus was heated in a water bath at 98 ℃ for 60min for use. The reference describes a method for measuring NADH (Guo Q, et al biochemistry,2016,55,2760-2771), in which NAD is used as a substrate, and the detection shows that the sample loses the activity of catalytically reducing NAD into NADH.

The NAD analogs NCD, NTD and NUD were reacted one by one as follows: 1mM NAD analogue, 8mM methanol and 80. mu.g of inactivated methanol dehydrogenase BsMDH were dissolved in 1mL of Tris-HCl buffer solution of 50mM concentration, pH 7.5, mixed, reacted at 30 ℃ for 2 hours, and 20. mu.L thereof was analyzed.

All samples of the reaction were found to have no characteristic peak at 340nm and only a characteristic peak at 260nm was detected which was identical to the retention time of the NAD analogue, as analyzed by the method of comparative example 1. Indicating that the heat-inactivated enzyme is unable to catalyze the reduction of the NAD analog by methanol.