WO2022217695A1 - 烟酸或其衍生物的单核苷酸及其生物产物的制备方法 - Google Patents

烟酸或其衍生物的单核苷酸及其生物产物的制备方法 Download PDF

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WO2022217695A1
WO2022217695A1 PCT/CN2021/094844 CN2021094844W WO2022217695A1 WO 2022217695 A1 WO2022217695 A1 WO 2022217695A1 CN 2021094844 W CN2021094844 W CN 2021094844W WO 2022217695 A1 WO2022217695 A1 WO 2022217695A1
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phosphate
enzyme
nicotinamide
glucose
atp
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PCT/CN2021/094844
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French (fr)
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潘永强
卢锦春
王骏
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百瑞全球有限公司
潘永强
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides

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  • the present invention relates to the field of biotechnology, specifically, the present invention belongs to the field of biochemistry, in particular to a method for preparing a mononucleotide of nicotinic acid or its derivatives and using the mononucleotide of nicotinic acid or its derivatives as an intermediate Methods of making various biological products.
  • Nucleosides and mononucleotides of niacin or its derivatives are important intermediates in the synthesis of various biological products required by the body.
  • a representative example is ⁇ -nicotinamide mononucleotide (NMN).
  • ⁇ -Nicotinamide mononucleotide is a nicotinamide adenosine dinucleotide intermediate that is required in all living things, and is converted into nicotinamide in various organ cells by nicotinamide mononucleotide adenosyltransferase in the body Adenosine dinucleotide.
  • nicotinamide adenosine dinucleotide which is a necessary substance and an indispensable coenzyme for maintaining life: the biochemical reaction of hydrogen ions in cells, the regulation of energy metabolism in the nucleus, and the mechanism of maintaining the biological clock.
  • Adenosine dinucleotides are all involved and their levels in the body have a decisive impact on the above physiological effects, so at the same time, nicotinamide adenosine dinucleotide shoulders the function of gene repair and telomere stabilization, enhancing the body's Immunity and the promotion of microfilament blood vessels in proliferating muscles.
  • the level of nicotinamide adenosine dinucleotide in the body is closely related to the health and aging of the body: the level of nicotinamide adenosine dinucleotide in the body increases with age. Decrease, then reduce metabolism and cause related diseases, and it is proposed that increasing the level of nicotinamide adenosine dinucleotide can help the body to metabolize and resist the effects of aging, and it is believed that the use of ⁇ -nicotinamide mononucleotide can achieve to the relevant purpose.
  • Beta-nicotinamide mononucleotide is a natural substance that can be absorbed in daily meals.
  • the content of avocado and broccoli is relatively high, but if you only consume the amount your body needs every day through your diet, you need to eat dozens of kilograms. Only the fruits and vegetables can meet the standard, I am afraid it is difficult to achieve. Therefore, it is more appropriate to take ⁇ -nicotinamide mononucleotide as a nicotinamide adenosine dinucleotide supplement.
  • Nucleosides and mononucleotides of nicotinic acid or derivatives thereof can be extracted from yeast or synthesized using chemical and traditional enzymatic methods.
  • the content of nucleosides and mononucleotides (such as ⁇ -nicotinamide mononucleotide) of niacin or its derivatives in yeast is very small, and it takes multiple steps to extract products with lower purity, so it is sold for sale.
  • the high price and low quality are not suitable for the general public, and the chemical method has the same disadvantages as the extraction method, so these two methods are gradually eliminated.
  • Enzymatic synthesis has the advantages of higher productivity and energy saving, and can produce products with higher purity, which will become nucleosides and mononucleotides (such as ⁇ -nicotinamide mononucleotide) for the production of niacin or its derivatives mainstream technology.
  • the traditional enzymatic synthesis of nucleosides and mononucleotides of nicotinic acid or its derivatives eg ⁇ -nicotinamide mononucleotide
  • ribose The demand for ribose grows rapidly every year, and in addition to the production of nucleosides and mononucleotides (such as ⁇ -nicotinamide mononucleotide) that can be used for niacin or its derivatives, it is more often used in dietary supplements, food and beverages. In the field of functional nutrition, ribose has been used to improve the flavor of emerging "artificial meat" in recent years, and is an indispensable ingredient in the production of this trendy food. The demand for ribose is far higher than its supply, creating a situation of rising prices and unstable supply in recent years.
  • nucleosides and mononucleotides such as ⁇ -nicotinamide mononucleotide
  • nucleosides and mononucleotides of niacin or its derivatives (such as ⁇ -
  • nicotinamide mononucleotide is able to improve the above-mentioned pressure, delaying and reversing aging is a win-win opportunity that meets the wishes of the general public and meets the needs of society.
  • nucleosides and mononucleotides such as ⁇ -nicotinamide mononucleotide
  • the demand for nucleosides and mononucleotides (such as ⁇ -nicotinamide mononucleotide) of niacin or its derivatives will increase year-on-year, and the supply and demand of ribose will be more severe, resulting in unstable prices and supply
  • Unfavorable production of nucleosides and mononucleotides of niacin or its derivatives causes great losses to both users and society, so it is necessary to develop nucleosides that use diversified and sustainable niacin or its derivatives. Enzymatic preparation of glycosides and mononucleotides.
  • nucleosides and mononucleotides of nicotinic acid or its derivatives are the precursors for the formation of various biological products (especially nicotinamide adenosine dinucleotide) in organisms. in-depth research.
  • An improved method for providing a single nucleotide of nicotinic acid or its derivative and its biological product can facilitate quantitative production of the substance and facilitate research in this field.
  • the present invention provides a novel nicotinic acid or its derivative mononucleotide and an industrial quantitative preparation method of its biological product, as well as the use of nicotinic acid or its derivative mononucleotide as an intermediate to prepare various biological products Methods of producing products, particularly nucleosides of nicotinic acid or derivatives thereof.
  • the present invention provides:
  • a method for preparing the mononucleotide of nicotinic acid or its derivatives comprising the steps of: using a reaction substrate comprising a hexose source and nicotinic acid or its derivatives, in the presence of a phosphate donor, biologically Enzyme-catalyzed reaction to generate mononucleotides of nicotinic acid or its derivatives.
  • the mononucleotide of nicotinic acid or its derivative is selected from at least one of ⁇ -nicotinamide mononucleotide and nicotinic acid mononucleotide.
  • the source of hexose is derived from a monosaccharide having six carbon atoms, a polysaccharide capable of producing hexose, or a mixture thereof.
  • the monosaccharide is selected from any one of D-glucose, D-mannose, D-galactose, D-fructose or a mixture thereof, preferably D-glucose, D-mannose and D-fructose, more D-glucose and D-fructose are preferred.
  • the hexose source is derived from polysaccharides linked with multiple hexose units through glycosidic bonds, preferably sucrose, maltose, inulin, raffinose, maltodextrin, starch or mixtures thereof, more preferably sucrose, maltose and cotton subsaccharides, more preferably sucrose and maltose.
  • the biological enzyme is a single biological enzyme or a biological enzyme group comprising multiple biological enzymes.
  • the reaction conditions include: the temperature is 25-40°C, preferably 30-39°C, more preferably 35-38°C; and/or the pH of the reaction system is 6.0-8.5, preferably pH 7.0-8.0, more It is preferably pH 7.5-7.8.
  • the reaction substrate further comprises auxiliary ions, and the auxiliary ions include metal ions, chloride ions, magnesium ions, calcium ions, potassium ions, sodium ions, zinc ions, fluoride ions, sulfide ions, carbonate ions, sulfites At least one of ions and phosphorus-containing ions is preferably at least one of sodium ions, magnesium ions, potassium ions, carbonate-based ions, sulfite-based ions, and phosphorus-containing ions.
  • the phosphate donor is selected from ATP or its salt, ADP or its salt, AMP or its salt, CTP or its salt, GTP or its salt, UTP or its salt, ITP or its salt and polyphosphoric acid or its salt At least one of ATP or its salt, ADP or its salt, AMP or its salt and at least one of polyphosphoric acid or its salt is preferred.
  • the phosphoric acid donor can be polyphosphoric acid or ATP or its salt
  • the hexose source is derived from D-glucose
  • the biological enzyme is a biological enzyme group
  • the biological enzyme group comprises polyphosphate-glucose phosphotransferase ( EC 2.7.1.63), Glucose-6-phosphate isomerase (EC 5.3.1.9), Hexulose 6-phosphate isomerase (EC 5.3.1.27), Hexulose 6-phosphate synthase (EC 4.1.2.43 ), ribose 5-phosphate isomerase (EC 5.3.1.6), phosphoribosyl diphosphate kinase (EC 2.7.6.1) and nicotinamide phosphoribosyltransferase (EC 2.4.2.12); among which polyphosphate- Glucose phosphotransferase uses polyphosphate and D-glucose as substrates to convert and synthesize glucose-6-phosphate and polyphosphate (n-1).
  • the glucose-6-phosphate isomerase in the biological enzyme group uses glucose-6-phosphate as the substrate.
  • -Phosphate is converted into fructose-6-phosphate as a substrate
  • ketohexose 6-phosphate isomerase in the biological enzyme group is converted into arabinose-3-hexulose-6 using fructose-6-phosphate as a substrate - Phosphate
  • hexose 6-phosphate synthase uses arabinose-3-hexulose-6-phosphate as the substrate to convert to ribulose-5-phosphate and formaldehyde
  • the ribose 5-phosphate isomerase in ribulose-5-phosphate is converted to ribose-5-phosphate as the substrate
  • the phosphoribosyl diphosphate kinase in the biological enzyme group is based on ribose-5-phosphate and phosphate donors.
  • the nicotinamide phosphoribosyltransferase in the biological enzyme group uses nicotinamide and 5-phosphorylribose-1-pyrophosphate as substrates to convert and synthesize to ⁇ - Nicotinamide mononucleotide and pyrophosphate, or converted to nicotinic acid mononucleotide and pyrophosphate using nicotinic acid as a substrate.
  • the hexose source is derived from D-glucose
  • the biological enzyme is a biological enzyme group comprising glucose isomerase (EC 5.3.1.5), hexokinase (EC 2.7.1.1), glucose-6 - Phosphate isomerase (EC 5.3.1.9), Hexulose 6-phosphate isomerase (EC 5.3.1.27), Hexulose 6-phosphate synthase (EC 4.1.2.43), Ribose 5-phosphate isomerase (EC 5.3.1.6), phosphoribosyl diphosphate kinase (EC 2.7.6.1) and nicotinamide phosphoribosyltransferase (EC 2.4.2.12).
  • the hexose source is derived from D-fructose
  • the biological enzyme is a biological enzyme group comprising hexokinase (EC 2.7.1.1), ketohexose 6-phosphate isomerase (EC 5.3.1.27 ), ketohexose 6-phosphate synthase (EC 4.1.2.43), ribose 5-phosphate isomerase (EC 5.3.1.6), phosphoribosylbisphosphate kinase (EC 2.7.6.1) and nicotinamide phosphoribosyltransferase ( EC 2.4.2.12).
  • the hexose source is derived from D-mannose
  • the biological enzyme is a biological enzyme group comprising mannose kinase (EC 2.7.1.7), mannose-6-phosphate isomerase (EC 5.3.3. 1.8), ketohexose 6-phosphate isomerase (6EC 5.3.1.27), ketohexose 6-phosphate synthase (EC 4.1.2.43), ribose 5-phosphate isomerase (EC 5.3.1.6), ribose phosphate Diphosphokinase (EC 2.7.6.1) and nicotinamide phosphoribosyltransferase.
  • the hexose source is derived from at least one of sucrose, disaccharide maltose, raffinose, maltodextrin and starch and wherein prior to the biological enzyme catalyzed reaction, the hexose source is treated with a compound selected from the group consisting of heat, acid, alkali At least one method in hydrolyzing sucrose into at least one monosaccharide selected from D-glucose, galactose, D-fructose and D-mannose, and then adding the monosaccharide to the reaction substrate; or
  • the hexose source is at least one of sucrose, disaccharide maltose, raffinose, maltodextrin and starch
  • directly using the biological enzyme group will include the hexose source and niacin or its derivatives Nucleosides and reaction substrates are converted into ⁇ -nicotinamide mononucleotides, wherein the biological enzyme group comprises amylases (EC 3.2.1.1-3), pullulanase (EC 3.2.1.41), maltase ( EC 3.2.1.20), isomaltase (EC 3.2.1.10), alpha-galactosidase (EC 3.2.1.22), sucrase (EC 3.2.1.26), sucrose alpha-glucosidase (EC 3.2.1.48) ), polyphosphate-glucose phosphotransferase (EC 2.7.1.63), glucose-6-phosphate isomerase (EC 5.3.1.9), hexokinase (EC 2.7.1.1
  • the sucrose comprises at least one of white granulated sugar, brown granulated sugar, brown granulated sugar, soft white sugar, monocrystalline rock sugar, polycrystalline rock sugar, brown sugar, brown sugar, borneol, sugar cube, icing sugar, and liquid syrup; and
  • the disaccharide maltose comprises at least one selected from maltose and isomaltose.
  • the biological enzyme comprises ATP regenerating enzyme
  • ATP regenerating enzyme comprises polyphosphate-AMP phosphotransferase (EC 2.7.4.B2), polyphosphate kinase (EC 2.7.4.1), adenosine kinase (EC 2.7.1.20) At least one of ); and wherein the method further comprises a step of ATP regeneration, the step comprising: polyphosphoric acid-AMP phosphotransferase converts polyphosphoric acid and AMP as substrates to generate ADP, and polyphosphorylation kinase converts polyphosphoric acid to polyphosphoric acid and AMP/ADP as a substrate to generate ADP/ATP, and adenosine kinase converts two molecules of ADP as a substrate to generate ATP and AMP.
  • polyphosphokinase (EC 2.7.4.1) includes two groups of enzymes in class II and class III, in which class II polyphosphokinase is used to convert polyphosphate and ADP as substrates to generate ATP, and class III is used.
  • Polyphosphokinase converts polyphosphoric acid and AMP/ADP as substrates to generate ADP/ATP; when using class II polyphosphokinase, it also includes the use of polyphospho-AMP phosphotransferase and/or when converting from AMP to ATP. or adenosine kinase, and when a class III polyphosphokinase is used, the method further includes using the enzyme alone to convert AMP to ADP and ATP.
  • the reaction substrate further comprises at least one of polyphosphate, adenosine and adenine
  • the phosphoric acid donor is ATP
  • the biological enzyme comprises ATP regeneration enzyme
  • the ATP regeneration enzyme comprises adenylate kinase (EC 2.7.4.3), at least one of adenine phosphoribosyltransferase (EC 2.4.2.7),
  • the method further includes an ATP regeneration step, which includes: firstly, adenylate kinase converts a molecule of adenosine and ATP as substrates to generate AMP and ADP, and then uses the ATP regeneration enzyme group to generate polyphosphate as the main
  • the substrate converts AMP and ADP to generate two molecules of ATP or one molecule of ADP and ATP; adenine phosphoribosyltransferase converts one molecule of adenine and 5-phosphorylribose-1-pyrophosphate as substrates to generate one molecule.
  • the reaction substrate further comprises at least one of nicotinamide and nicotinic acid
  • the mononucleotide is at least one of ⁇ -nicotinamide mononucleotide and nicotinic acid mononucleotide.
  • the method further comprises the step of adding an inert material for absorbing or decomposing formaldehyde in the reaction substrate, preferably the inert material is zeolite.
  • the biological enzyme group is recombinase, and the recombinase is synthesized in the bacterial species to which it belongs and expressed and extracted in Escherichia coli HB101;
  • the biological enzyme is in at least one form selected from cell disrupting liquid, supernatant enzyme liquid, pure enzyme and immobilized enzyme/cell prepared in any way.
  • a method for preparing a biological product comprising the steps of:
  • reaction substrate comprising a hexose source and nicotinic acid or a derivative thereof, in the presence of a phosphate donor, a mononucleotide of nicotinic acid or a derivative thereof is catalyzed by a biological enzyme reaction;
  • the biological product is selected from the group consisting of nicotinamide riboside or any of its salt derivatives, oxidized nicotinamide adenine dinucleotide or any of its salt derivatives, reduced nicotinamide adenine dinucleotide or any of its salts
  • nicotinamide riboside or any of its salt derivatives oxidized nicotinamide adenine dinucleotide or any of its salt derivatives
  • reduced nicotinamide adenine dinucleotide phosphate or any of its salt derivatives nicotinic acid riboside or any of its salts
  • the method of the present invention adopts a novel enzymatic method to prepare or produce the mononucleotide of nicotinic acid or its derivatives and use the mononucleotide as an intermediate to prepare biological products such as the nucleosides of nicotinic acid or its derivatives, overcoming The disadvantages of extraction methods and chemical synthesis methods are eliminated.
  • the present invention utilizes biological enzymes to prepare mononucleotides of nicotinic acid or its derivatives and its biological products, in addition to inheriting the advantages of the production method, it is more cost-effective than the traditional enzymatic process, and the preparation method is the same as that of biological products.
  • the nucleosides of nicotinic acid or its derivatives and the production of mononucleotides (eg ⁇ -nicotinamide mononucleotide/nicotinic acid mononucleotide) in nicotinic acid are echoed, and the products are more suitable for biological use than chemical production.
  • the present invention selects various hexose sources to replace ribose as one of the substrate raw materials to prepare ⁇ -nicotinamide mononucleotide and nicotinic acid mononucleotide, and use various enzyme combinations to carry out ⁇ -nicotinamide mononucleotide.
  • using different hexoses and hexose-producing polysaccharides only need to use the matching biological enzyme combination without the need to make large-scale production equipment changes, which is conducive to flexible industry production operations.
  • the source of hexose can be preferably D-glucose and D-fructose: these two hexoses are globally distributed and inexpensive, and are ideal choices to replace ribose as raw materials, and can be used in combination with various biological enzymes.
  • Polysaccharides, including sucrose, starch, maltose, etc., are also very common in daily life, so the diversification and sustainability of the preparation method of the present invention are unmatched by traditional enzymatic methods.
  • the preparation method of the present invention can also use an ATP regeneration enzyme or a combination thereof: the ATP regeneration enzyme or a combination thereof can recycle the ATP in the reaction system, further reducing the production cost, while both ADP and AMP in the enzymatic reaction are by-products , using the enzyme group to convert and synthesize the by-product to the substrate is more favorable for the production of ⁇ -nicotinamide mononucleotide/nicotinic acid mononucleotide.
  • the ATP regenerating enzyme or its combination makes the preparation method more diversified, and the use of adenosine and adenine to assist ATP can reduce the use of ATP, reduce the production cost, and reduce the dependence on the amount of ATP in traditional enzymatic production. .
  • preparation method of the present invention can also use inert material to remove the formaldehyde produced in the preparation method, and inert material can be zeolite or a material with an equivalent effect such as activated carbon; removing by-products can be harmful to nicotinic acid or its derivative.
  • inert material can be zeolite or a material with an equivalent effect such as activated carbon; removing by-products can be harmful to nicotinic acid or its derivative.
  • Figure 1 shows the results of SDS-PAGE gel electrophoresis of polyphosphoglucose phosphotransferase, wherein
  • Figure 2 shows a graph of the results of SDS-PAGE gel electrophoresis of glucose-6-phosphate isomerase, wherein
  • Figure 3 shows the result of SDS-PAGE gel electrophoresis of hexokinase
  • Prestained Protein Ladder (PageRuler TM Prestained Protein Ladder, 10 to 180 kDa, Thermo Fisher Scientific).
  • FIG. 4 shows a graph of the results of SDS-PAGE gel electrophoresis of 6-phosphate hexose isomerase, wherein
  • Column 1 is 6-phosphate hexose isomerase cell fragmentation liquid
  • Figure 5 shows a graph of the results of SDS-PAGE gel electrophoresis of ketohexose 6-phosphate synthase, wherein
  • Prestained Protein Ladder (PageRuler TM Prestained Protein Ladder, 10 to 180 kDa, Thermo Fisher Scientific).
  • Figure 6 shows a graph of the results of SDS-PAGE gel electrophoresis of ribose 5-phosphate isomerase, wherein
  • Prestained Protein Ladder (PageRuler TM Prestained Protein Ladder, 10 to 180kDa, Thermo Fisher Scientific);
  • Figure 7 shows a graph of the results of SDS-PAGE gel electrophoresis of phosphoribosyl diphosphate kinase, wherein
  • Prestained Protein Ladder PageRuler TM Prestained Protein Ladder, 10 to 180kDa, Thermo Fisher Scientific
  • Figure 8 shows a graph of the results of SDS-PAGE gel electrophoresis of nicotinamide phosphoribosyltransferase, wherein
  • Prestained Protein Ladder PageRuler TM Prestained Protein Ladder, 10 to 180kDa, Thermo Fisher Scientific
  • Figure 9 shows a graph of the results of SDS-PAGE gel electrophoresis of polyphosphate-AMP phosphotransferase, wherein
  • Prestained Protein Ladder PageRuler TM Prestained Protein Ladder, 10 to 180kDa, Thermo Fisher Scientific
  • Figure 10 shows a graph of the results of SDS-PAGE gel electrophoresis of adenylate kinase, wherein
  • Prestained Protein Ladder PageRuler TM Prestained Protein Ladder, 10 to 180kDa, Thermo Fisher Scientific
  • FIG 11 shows the results of SDS-PAGE gel electrophoresis of polyphosphokinase, wherein
  • Prestained Protein Ladder (PageRuler TM Prestained Protein Ladder, 10 to 180 kDa, Thermo Fisher Scientific);
  • Figure 12 shows a graph of the results of SDS-PAGE gel electrophoresis of mannokinase cells, wherein
  • Prestained Protein Ladder (PageRuler TM Prestained Protein Ladder, 10 to 180 kDa, Thermo Fisher Scientific).
  • Figure 13 shows a graph of the results of SDS-PAGE gel electrophoresis of mannose-6-phosphate isomerase, wherein
  • Prestained Protein Ladder (PageRuler TM Prestained Protein Ladder, 10 to 180kDa, Thermo Fisher Scientific);
  • Figure 14 shows a graph of the results of SDS-PAGE gel electrophoresis of polymaltase, wherein
  • Prestained Protein Ladder PageRuler TM Prestained Protein Ladder, 10 to 180kDa, Thermo Fisher Scientific
  • Figure 15 shows a graph of the results of SDS-PAGE gel electrophoresis of sucrase, wherein
  • Prestained Protein Ladder (PageRuler TM Prestained Protein Ladder, 10 to 180 kDa, Thermo Fisher Scientific);
  • Figure 16 shows a graph of the results of SDS-PAGE gel electrophoresis of 5'-nucleotidase, wherein
  • Prestained Protein Ladder (PageRuler TM Prestained Protein Ladder, 10 to 180 kDa, Thermo Fisher Scientific);
  • the present inventors have developed a method for preparing a mononucleotide of nicotinic acid or its derivatives, comprising the following steps: using a reaction substrate comprising a hexose source and nicotinic acid or its derivatives, in the presence of a phosphate donor Next, the mononucleotide of nicotinic acid or its derivatives is generated by the catalyzed reaction of biological enzymes.
  • hexose sources such as monosaccharides such as polysaccharides, D-glucose and D-fructose
  • monosaccharides such as polysaccharides, D-glucose and D-fructose
  • D-glucose and D-fructose as raw materials for the production process of mononucleotides of niacin or its derivatives, breaking the traditional enzymatic process using ribose or rarer and more expensive
  • the compound is the practice of raw materials.
  • the invention also opens up the convenience in technology: when two hexose sugars are used as raw materials, when using different hexose sugars, it is only necessary to use or replace the matched enzyme combination for production, and the production equipment therein does not need to be replaced and can be used interchangeably, Industrial feasibility is ensured.
  • the inventors believe that the long-term development of nicotinic acid or its derivative mononucleotide (eg ⁇ -nicotinamide mononucleotide) or various biological products prepared therefrom as an intermediate must be based on the preparation method that can provide diversity and sustainability, both of which are linked to the availability and price of raw materials.
  • the traditional enzymatic process can only rely on ribose as a single raw material, which is contrary to the above two major points.
  • the inventors also realize that the current supply and demand of ribose is far behind the technological development of mass production. If the mononucleotide of nicotinic acid or its derivatives (such as ⁇ -nicotinamide mononuclear nucleoside) is added nucleotides) or various biological products prepared with them as intermediates, the situation will be more severe in the foreseeable future, directly attacking the single nucleotides of nicotinic acid or its derivatives (such as ⁇ -nicotinamide mononucleoside acid) or the popularity of various biological products prepared with it as an intermediate in society.
  • nucleosides and mononucleotides such as ⁇ -nicotinamide mononucleotide
  • nucleosides and mononucleotides such as ⁇ -nicotinamide mononucleotide
  • the method developed by the present inventor reduces the cost while solving the current problem, and reduces the amount of adenosine triphosphate (ATP) that needs to be used in the traditional process, and then uses polyphosphoric acid to regenerate adenosine triphosphate in an auxiliary combination to further reduce the cost. more favorable conditions.
  • ATP adenosine triphosphate
  • the method of the present invention uses a reaction substrate comprising a hexose source and nicotinic acid or its derivatives, and in the presence of a phosphate donor, a single nucleotide of nicotinic acid or its derivatives is generated by a biological enzyme-catalyzed reaction .
  • Niacin or derivatives thereof may be niacin and niacinamide. Therefore, the mononucleotide of nicotinic acid or its derivatives and its biological products can be ⁇ -nicotinamide mononucleotide, nicotinic acid mononucleotide, oxidized nicotinamide adenine dinucleotide or any salt thereof.
  • Derivatives Derivatives of Reduced Nicotinamide Adenine Dinucleotide or any of its Salts, Oxidized Nicotinamide Adenine Dinucleotide Phosphate or Derivatives of any Salt, Reduced Nicotinamide Adenine Dinucleotides Phosphoric acid or any of its salt derivatives, nicotinic acid riboside or any of its salt derivatives, nicotinic acid mononucleotide, nicotinic acid adenine dinucleotide or any of its salt derivatives and nicotinic acid adenine dinucleotide Derivatives of glucosinolates or any salt thereof, preferably beta-nicotinamide mononucleotides.
  • the structures of niacin and niacinamide are known in the art and are readily available in the marketplace.
  • a significant advantage of the present invention is that sources of hexose sugars (such as polysaccharides, monosaccharides such as D-glucose and D-fructose) are raw materials for the production process of mononucleotides of niacin or its derivatives and its bioproducts , which replaces the use of ribose or rarer and more expensive compounds as raw materials in traditional enzymatic processes.
  • the source of hexose can be from a monosaccharide having six carbon atoms, a polysaccharide capable of producing hexose, or a mixture thereof.
  • the monosaccharide with six carbon atoms can be selected from any one of D-glucose, D-mannose, D-galactose, D-fructose or mixtures thereof, preferably D-glucose, D-mannose and D- Fructose, more preferably D-glucose and D-fructose.
  • the source of hexose can also be derived from polysaccharides in which various hexose units are linked by glycosidic bonds, as long as the polysaccharides can be degraded to hexose monosaccharides (such as D-glucose and/or D-fructose) catalyzed by the activity of biological enzymes .
  • the polysaccharide may be sucrose, maltose, inulin, raffinose, maltodextrin, starch or mixtures thereof, more preferably sucrose, maltose and raffinose, most preferably sucrose and maltose.
  • Sucrose may comprise at least one of white granulated sugar, brown granulated sugar, brown granulated sugar, soft white sugar, monocrystalline rock sugar, polycrystalline rock sugar, brown sugar, brown sugar, borneol, sugar cube, icing sugar, liquid syrup; and maltose may comprise selected from maltose and at least one of isomaltose.
  • the polysaccharide Before the biological enzyme catalyzes the reaction, the polysaccharide can be converted into at least one monosaccharide selected from the group consisting of D-glucose, galactose, D-fructose and D-mannose by hydrolyzing the polysaccharide by at least one method selected from the group consisting of heat, acid and alkali. , and then the monosaccharide is put into the reaction substrate.
  • Phosphate donor refers to any compound capable of donating phosphate in an enzymatic reaction.
  • Phosphate donors can be selected from ATP or its salts, ADP or its salts, AMP or its salts, CTP or its salts, GTP or its salts, UTP or its salts, ITP or its salts, and polymers with different phosphate chain lengths. At least one of phosphoric acid or its salts.
  • the reaction substrate may further comprise at least one of auxiliary ions and polyphosphoric acid or salts thereof.
  • the polyphosphoric acid or its salt is preferably a sodium salt of polyphosphoric acid.
  • the degree of polymerization of polyphosphoric acid may be 3-20,000; preferably, the degree of polymerization of polyphosphoric acid may be 3-7,000, more preferably 3-75.
  • the auxiliary ions may comprise metal ions, chloride ions, magnesium ions, calcium ions, potassium ions, sodium ions, zinc ions, fluoride ions, sulfide ions, carbonate ions, sulfite ions, and phosphorus-containing ions At least one of them is preferably at least one of sodium ions, magnesium ions, potassium ions, carbonate-based ions, sulfite-based ions, and phosphorus-containing ions.
  • the auxiliary ion can be in the state of its inorganic salt or organic salt, preferably at least one of magnesium chloride hexahydrate, sodium chloride, manganese chloride, magnesium sulfate and potassium carbonate, more preferably magnesium chloride hexahydrate, sodium chloride and carbonic acid at least one of potassium.
  • reaction substrate may also contain other additives, for example, pH adjusters, such as buffers/salts, preferably sodium phosphate buffer, potassium phosphate buffer and tris buffer, More preferred are sodium phosphate buffer and tris buffer.
  • pH adjusters such as buffers/salts, preferably sodium phosphate buffer, potassium phosphate buffer and tris buffer, More preferred are sodium phosphate buffer and tris buffer.
  • concentration of the pH adjusting agent can be 0.001M-1M, preferably 0.01M-0.5M, more preferably 0.05M-0.3M.
  • the biological enzyme is a single biological enzyme or a biological enzyme group comprising multiple biological enzymes.
  • the biological enzyme group is a recombinase, which is synthesized in the bacterial species to which it belongs and expressed and extracted in a vector.
  • the biological enzyme is in at least one form selected from cell disrupting liquid, supernatant enzyme liquid, pure enzyme and immobilized enzyme/cell.
  • the vector may include E. coli (eg, E. coli HB101), yeast.
  • E. coli eg, E. coli HB101
  • yeast cells containing the recombinase or fragments thereof can be used as the biological enzyme.
  • the cells can be E. coli cells, yeast cells.
  • the vector can include Escherichia coli, yeast, Bacillus and other methods commonly used in biological science to express the recombinase.
  • the biological enzyme or biological enzyme group can be used in cells, disrupted liquid, supernatant liquid or liquid state of purified enzyme, or can be made into immobilized cells or immobilized enzymes with its corresponding carrier in any way for enzymatic reaction.
  • a suitable or compatible biological enzyme or group of biological enzymes can be selected according to the composition of the substrate.
  • the biological enzyme group contains polyphosphate-glucose phosphotransferase (EC 2.7.1.63), glucose-6 - Phosphate isomerase (EC 5.3.1.9), Hexulose 6-phosphate isomerase (EC 5.3.1.27), Hexulose 6-phosphate synthase (EC 4.1.2.43), Ribose 5-phosphate isomerase (EC 5.3.1.6), at least one of phosphoribosyl diphosphate kinase (EC 2.7.6.1) and nicotinamide phosphoribosyltransferase (EC 2.4.2.12).
  • the specific reaction process includes: the polyphosphate-glucose phosphotransferase in the biological enzyme group uses polyphosphate and D-glucose as substrates to convert and synthesize glucose-6-phosphate and polyphosphate (n- 1), the glucose-6-phosphate isomerase in the biological enzyme group is converted to fructose-6-phosphate with glucose-6-phosphate as the substrate, and the ketohexose 6-phosphate isomerase in the biological enzyme group uses fructose-6-phosphate isomerase. -6-Phosphate is converted to arabinose-3-hexulose-6-phosphate as a substrate. Esters are converted to ribulose-5-phosphate and formaldehyde as substrates.
  • Ribulose-5-phosphate isomerase in the biological enzyme group is converted to ribose-5-phosphate as substrates.
  • Biological enzymes The phosphoribosyl diphosphate kinase in the group uses ribose-5-phosphate and phosphate donors as substrates to convert and synthesize to 5-phosphoribose-1-pyrophosphate and AMP, and the nicotinamide phosphoribosyltransferase in the biological enzyme group uses nicotinamide phosphoribosyltransferase as a substrate.
  • Amide and 5-phosphorylribose-1-pyrophosphate are converted to ⁇ -nicotinamide mononucleotide and pyrophosphate, or nicotinic acid is converted to nicotinic acid mononucleotide and pyrophosphate.
  • the biological enzyme group may include hexokinase (EC 2.7.1.1), hexose 6-phosphate isomerase (EC 5.3.1.27), hexose 6-phosphate synthase (EC 5.3.1.27) 4.1.2.43), at least one of ribose 5-phosphate isomerase (EC 5.3.1.6), phosphoribosyl diphosphate kinase (EC 2.7.6.1) and nicotinamide phosphoribosyltransferase (EC 2.4.2.12).
  • the group of biological enzymes may include glucose isomerase (EC 5.3.1.5), hexokinase (EC 2.7.1.1), ketohexose 6-phosphate isomerase (EC 5.3.1.27) , ketohexose 6-phosphate synthase (EC 4.1.2.43), ribose 5-phosphate isomerase (EC 5.3.1.6), phosphoribosylbisphosphate kinase (EC 2.7.6.1) and nicotinamide phosphoribosyltransferase (EC 2.4.2.12) at least one.
  • glucose isomerase EC 5.3.1.5
  • hexokinase EC 2.7.1.1
  • ketohexose 6-phosphate isomerase EC 5.3.1.27
  • ketohexose 6-phosphate synthase EC 4.1.2.43
  • ribose 5-phosphate isomerase EC 5.3.1.6
  • phosphoribosylbisphosphate kinase EC
  • the biological enzyme group may include mannokinase (EC 2.7.1.7), mannose-6-phosphate isomerase (EC 5.3.1.8), hexulose 6-phosphate isomerase (6EC 5.3.1.27), hexulose 6-phosphate synthase (EC 4.1.2.43), ribose 5-phosphate isomerase (EC 5.3.1.6), phosphoribosyl diphosphate kinase (EC 2.7.6.1) and nicotinamide at least one of phosphoribosyltransferases.
  • the nucleosides comprising the hexose source and niacin or its derivatives can be directly used in the biological enzyme group And the reaction substrate is converted into ⁇ -nicotinamide mononucleotide/mononucleotide.
  • the group of biological enzymes includes amylases (EC 3.2.1.1-3), pullulanase (EC 3.2.1.41), maltase (EC 3.2.1.20), isomaltase (EC 3.2.1.10) ), alpha-galactosidase (EC 3.2.1.22), sucrase (EC 3.2.1.26), sucrose alpha-glucosidase (EC 3.2.1.48), polyphosphate-glucose phosphotransferase (EC 2.7.1.63 ), glucose-6-phosphate isomerase (EC 5.3.1.9), hexokinase (EC 2.7.1.1), hexose 6-phosphate isomerase (EC 5.3.1.27), hexose 6-phosphate isomerase At least one of enzymes (EC 4.1.2.43), ribose 5-phosphate isomerase (EC 5.3.1.6), phosphoribosyl diphosphate kinase (EC 2.7.6.1) and nicotinamide phosphoribos
  • the biological enzyme may further comprise an ATP-regenerating enzyme, and the ATP-regenerating enzyme may comprise a polyphospho-AMP phosphotransferase (EC 2.7.4.B2), a polyphosphokinase (EC 2.7.4.1), adenosine At least one of the kinases (EC 2.7.1.20).
  • the method of the present invention may further comprise a step of ATP regeneration, the step comprising: polyphosphate-AMP phosphotransferase converts polyphosphate and AMP as substrates to generate ADP, and polyphosphokinase converts polyphosphate and AMP to ADP.
  • AMP/ADP is converted to ADP/ATP as a substrate
  • adenosine kinase is converted to ATP and AMP using two molecules of ADP as a substrate.
  • polyphosphokinases include two groups of enzymes in class II and class III, wherein polyphosphokinases of class II are used to convert polyphosphate and ADP as substrates to generate ATP, using III Class II polyphosphokinases can use polyphosphate and AMP/ADP as substrates to convert to ADP/ATP; when using class II polyphosphokinases, the conversion of AMP to ATP also includes the use of polyphosphate-AMP phosphate Transferases and/or adenosine kinases, and when a class III polyphosphokinase is used, the methods of the invention may also include the use of the enzymes alone to convert AMP to ADP and ATP.
  • the reaction substrate of the present invention may further comprise at least one of adenosine and adenine, and the ATP regenerating enzyme comprises adenylate kinase (EC 2.7.4.3), adenine phosphoribosyltransferase (EC 2.4.2.7) at least one,
  • the method of the present invention may further comprise a step of ATP regeneration, the step comprising: adenylate kinase first converts a molecule of adenosine and ATP as substrates to generate AMP and ADP, and then uses the ATP to regenerate
  • the enzyme group uses polyphosphate as the main substrate to convert AMP and ADP into two molecules of ATP;
  • adenine phosphoribosyltransferase uses one molecule of adenine and 5-phosphorylribose-1-pyrophosphate as substrates to convert to generate A molecule of AMP and phosphoric acid is then used to convert AMP to ATP using polyphosphate as the main substrate using the ATP-regenerating enzyme group.
  • the method of the present invention may also comprise any one of the following steps or a combination thereof:
  • the biological enzyme group includes polyphosphate-glucose phosphotransferase (EC 2.7.1.63), glucose-6-phosphate isomerase (glucose- 6-phosphate isomerase; EC 5.3.1.9), hexulose 6-phosphate isomerase (6-phospho-3-hexuloisomerase; EC 5.3.1.27), hexulose 6-phosphate synthase (3-hexulose-6- phosphate synthase; EC 4.1.2.43), ribose-5-phosphate isomerase (EC 5.3.1.6), ribose-phosphate diphosphokinase (EC 2.7.6.1), and nicotinamide phosphate At least one of ribosyltransferase (nicotinamide phosphoribosyltransferase; EC 2.4.2.12); wherein the polyphosphate-glucose phosphotransferase in the enzyme group
  • 5-phosphoryl ribose-1-pyrophosphate is converted into ⁇ -nicotinamide mononucleotide and pyrophosphate as substrate, or converted into nicotinic acid mononucleotide and pyrophosphate by using nicotinic acid as substrate;
  • the biological enzyme group includes glucose isomerase (glucose isomerase; EC 5.3.1.5), hexokinase (hexokinase; EC 2.7.1.1), hexulose 6 - Phosphate isomerase (6-phospho-3-hexuloisomerase; EC 5.3.1.27), 3-hexulose-6-phosphate synthase (3-hexulose-6-phosphate synthase; EC 4.1.2.43), ribose 5-phosphate isomerase At least one of the enzymes (ribose-5-phosphate isomerase; EC 5.3.1.6), ribose-phosphate diphosphokinase (EC 2.7.6.1) and nicotinamide phosphoribosyltransferase (EC 2.4.2.12) one;
  • the biological enzyme group includes hexokinase (hexokinase; EC 2.7.1.1), 6-phospho-3-hexuloisomerase ; EC 5.3.1.27), 3-hexulose-6-phosphate synthase (3-hexulose-6-phosphate synthase; EC 4.1.2.43), ribose-5-phosphate isomerase (ribose-5-phosphate isomerase; EC 5.3.1.6 ), at least one of ribose-phosphate diphosphokinase (EC 2.7.6.1) and nicotinamide phosphoribosyltransferase (EC 2.4.2.12).
  • the biological enzyme group includes mannose kinase (mannokinase; EC 2.7.1.7), mannose-6-phosphate isomerase (mannose-6-phosphate isomerase) ; EC 5.3.1.8), hexulose 6-phosphate isomerase (6-phospho-3-hexuloisomerase; EC 5.3.1.27), hexulose 6-phosphate synthase (3-hexulose-6-phosphate synthase; EC 4.1.2.43), ribose-5-phosphate isomerase (EC 5.3.1.6), ribose-phosphate diphosphokinase (EC 2.7.6.1) and nicotinamide phosphoribosyltransferase at least one of;
  • sucrose in the disaccharides as the reaction substrate, which can include white granulated sugar, brown granulated sugar, brown granulated sugar, soft white sugar, monocrystalline rock sugar, polycrystalline rock sugar, brown sugar, brown sugar, borneol, sugar cube, icing sugar, liquid Syrup, etc.; the molecular structure of sucrose is composed of D-glucose and D-fructose in hexoses, which can be hydrolyzed by common physical methods such as heat, acid, alkali, etc., and then degraded from disaccharide to monosaccharide.
  • the enzyme group is converted into ⁇ -nicotinamide mononucleotide/nicotinic acid mononucleotide;
  • reaction substrate which can include maltose and isomaltose; the molecular structure of maltose is composed of two D-glucose in hexoses, and common physical methods such as heat, acid, Alkali, etc.
  • maltose can also be directly converted and synthesized to the product using the enzyme group;
  • This group of enzymes includes maltase (EC 3.2.1.20) and/or isomaltase (EC 3.2.1.10), polyphosphate-glucose phosphotransferase (EC 2.7.1.63), glucose- 6-phosphate isomerase (glucose-6-phosphate isomerase; EC 5.3.1.9), hexokinase (hexokinase; EC 2.7.1.1), 6-phospho-3-hexuloisomerase; EC 5.3.1.27), 3-hexulose-6-phosphate synthase (EC 4.1.2.43), ribose-5-phosphate isomerase (EC 5.3.
  • raffinose in trisaccharides Take raffinose in trisaccharides as the reaction substrate; the molecular structure of raffinose is composed of galactose, D-glucose and D-fructose in hexoses; the enzyme group contains ⁇ -galactosidase (alpha-galactosidase; EC 3.2.1.22), one or more enzymes of the class sucrases, polyphosphate-glucose phosphotransferase (EC 2.7.1.63), glucose-6- Phosphate-6-phosphate isomerase (EC 5.3.1.9), hexokinase (EC 2.7.1.1), 6-phospho-3-hexuloisomerase (EC 5.3) .1.27), hexulose-6-phosphate synthase (3-hexulose-6-phosphate synthase; EC 4.1.2.43), ribose-5-phosphate isomerase (ribose-5-phosphate
  • this polysaccharide can be first used physical methods commonly used such as heat, Acids, alkalis, etc. are hydrolyzed to monosaccharides and then converted to ⁇ -nicotinamide mononucleotide/nicotinic acid mononucleotide using the above-mentioned biological enzyme group; the polysaccharides can also be used including amylases (Amylase; EC 3.2.1.1-3), Pullulanase (Pullulanase; EC 3.2.1.41), etc.
  • one or more of the sucrases may be enzymes comprising invertase (invertase; EC 3.2.1.26), isomaltase (EC 3.2.1.10), sucrose alpha-glucosidase (sucrose) alpha-glucosidase; EC 3.2.1.48), etc., as long as the activity of the enzyme can catalyze the degradation of polysaccharides to D-glucose and/or D-fructose of hexoses.
  • invertase invertase
  • isomaltase EC 3.2.1.10
  • sucrose alpha-glucosidase sucrose alpha-glucosidase
  • EC 3.2.1.48 sucrose alpha-glucosidase
  • the method of the present invention may further comprise the step of adding an inert material for absorbing or decomposing formaldehyde into the reaction substrate.
  • the inert material can be a material that only physically or chemically changes with formaldehyde and does not have any effect on the biological enzyme reaction, preferably zeolite, alumina, activated carbon, lime, filter membrane and the like.
  • the reaction conditions in the method of the present invention may include a temperature of 25-40°C, preferably 30-39°C, more preferably 35-38°C.
  • the pH of the reaction system is 6.0-8.5, preferably pH 7.0-8.0, more preferably pH 7.5-7.8.
  • the present invention also provides a method for preparing a biological product, comprising the following steps:
  • reaction substrate comprising a hexose source and nicotinic acid or a derivative thereof, in the presence of a phosphate donor, a mononucleotide of nicotinic acid or a derivative thereof is catalyzed by a biological enzyme reaction;
  • the biological product may be selected from the group consisting of derivatives of nicotinamide riboside or any salt thereof, derivatives of oxidized nicotinamide adenine dinucleotide or any salt thereof, and derivatives of reduced nicotinamide adenine dinucleotide or any salt thereof.
  • the method of the present invention comprises using nicotinamide and nicotinic acid in combination, and after the enzymatic reaction, ⁇ -nicotinamide mononucleotide and nicotinic acid mononucleotide can be simultaneously produced.
  • the ⁇ -nicotinamide mononucleotide and/or nicotinic acid mononucleotide produced by the method of the present invention can be used as an intermediate to further convert and synthesize the nicotinic acid by a biological enzyme method or a chemical synthesis method.
  • the immobilized cells and the immobilized enzyme can be placed in an immobilization reaction device to carry out the immobilization reaction.
  • the immobilization reaction can be carried out according to the steps described in Chinese patent application CN106032520A.
  • the immobilized reaction device may include a columnar reactor with an inlet and an outlet, a reaction regulating tank, a high-flow water pump, a pH regulating device and a pH probe, and a temperature regulating device and a temperature probe.
  • the method of the present invention may include: firstly, using polymerase chain reaction to synthesize recombinant enzyme protein from the bacterial species to which the biological enzyme belongs, and express it in Escherichia coli HB101, and then crush the bacterial body with a cell crusher After centrifugation, the enzyme supernatant was obtained and the immobilized enzyme/cell was prepared with its broken liquid or supernatant enzyme liquid; 100ml of reaction solution was prepared in the reaction tank, and 1.21g of tris, 90mg of D-glucose was added.
  • reaction solution Maintain at 37°C and pH 7.5 and add 20g of zeolite to stir, at the same time add 1ml polyphosphate-glucose phosphotransferase supernatant enzyme solution, 1ml glucose-6-phosphate isomerase supernatant enzyme solution mix, 2ml hexose 6- Phosphoisomerase supernatant enzyme solution, 2ml hexulose 6-phosphate synthase supernatant enzyme solution, 2ml ribose 5-phosphate isomerase supernatant enzyme solution, 0.5ml phosphoribosyl diphosphate kinase supernatant enzyme solution
  • the concentration of ⁇ -nicotinamide mononucleotide reached 2mM after 3 hours of reaction, while the concentration in the 4-hour sample did not change much.
  • the enzymatic reaction can be terminated, and the zeolite is filtered with a medium-speed filter paper to obtain a solution containing ⁇ -nicotinamide mononucleotide.
  • reaction 1 solution configuration was carried out in the reaction tank, and 1.21g of tris(hydroxymethylaminomethane), 90mg of D-fructose, 406mg of magnesium chloride hexahydrate, 667mg of polyphosphate, 242mg of nicotinamide and 605mg of adenosine triphosphate disodium salt were added.
  • 80ml of pure water was adjusted to pH 7.5-7.8 with 0.1M hydrochloric acid/sodium hydroxide; the reaction solution was maintained at 37°C and pH 7.5, and 20g of zeolite was added for stirring, and 1ml of hexokinase supernatant enzyme solution and 1ml of polyphosphoric acid were added at the same time.
  • Reaction regulation tank from Gene Harbor (Hong Kong) Biotechnology Co., Ltd., BR-1L;
  • Adjustable flow suction pump purchased from SURGEFLO company, FL-32;
  • pH control device from Gene Harbor (Hong Kong) Biotechnology Co., Ltd., AR-1;
  • LB culture medium purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.;
  • IPTG purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.
  • Ampicillin purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.;
  • Glucose-6-phosphate sodium salt purchased from Merck, USA;
  • Fructose-6-phosphate disodium salt purchased from Merck, USA;
  • Hexose 6-phosphate from Gene Harbor (Hong Kong) Biotechnology Co., Ltd.;
  • Ribulose 5-phosphate sodium salt purchased from Merck, USA;
  • Ribose 5-phosphate disodium salt purchased from Merck, USA;
  • Mannose 6-phosphate sodium salt purchased from Merck, USA;
  • Phosphoribose diphosphate pentasodium salt purchased from Merck, USA;
  • Adenosine monophosphate disodium salt purchased from Merck, USA;
  • Adenosine diphosphate sodium salt purchased from Merck, USA;
  • Adenosine purchased from Merck, USA;
  • Magnesium chloride hexahydrate purchased from Merck, USA;
  • Sodium polyphosphate purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.;
  • Adenosine triphosphate disodium salt purchased from Merck, USA;
  • D-glucose monohydrate purchased from Merck, USA;
  • D-Fructose purchased from Merck, USA;
  • D-Mannose purchased from Merck, USA;
  • Niacinamide purchased from Merck, USA;
  • Niacin purchased from Merck, USA;
  • PCR primers were designed based on the DNA sequence STZ38851.1 (SEQ3) encoding polyphosphate-glucose phosphotransferase in the genome of Mycobacteroides abscessus, specifically:
  • a single species of the above-mentioned strains was selected and inoculated into 4mL LB medium (containing 100ug/ml ampicillin), cultivated for 16 hours in a shaker at 37°C and 200rpm as primary seeds, and received 100mL LB medium by 1% inoculation ratio after completion. (containing 100ug/ml ampicillin), cultured at 37°C, 200rpm shaker for 10 hours as secondary seeds, and then received 100L fermentation in 60L LB medium (containing 100ug/ml ampicillin) at a 1% inoculation ratio Cultivated in jars.
  • the initial fermentation conditions were 37°C, 200 rpm, pH 7.0.
  • the obtained Escherichia coli cells containing polyphosphate-glucose phosphotransferase were prepared into a supernatant enzyme solution; the preparation method of the supernatant enzyme solution was to add sodium phosphate buffer (PBS100mM pH 7.5) to every 1 g of cells to beat, and then use a pressure type
  • the cell crusher is crushed at the setting of 700-800MPa to obtain the cell crushing liquid, and the supernatant liquid is obtained by centrifugation with the tube centrifuge at the setting of 10,000rpm and 100L/hr.
  • Each 1ml of the supernatant enzyme liquid contains 0.2g cells.
  • SDS-PAGE gel electrophoresis test confirmed the synthesis of polyphosphate-glucose phosphotransferase.
  • PCR primers were designed based on the DNA sequence KXG93161.1 (SEQ6) encoding glucose-6-phosphate isomerase in the genome of Escherichia coli, specifically:
  • Glucose-6-phosphate isomerase sequence design PCR primers, specifically
  • a single species of the above-mentioned strains was selected and inoculated into 4mL LB medium (containing 100ug/ml ampicillin), cultivated for 16 hours in a shaker at 37°C and 200rpm as primary seeds, and received 100mL LB medium by 1% inoculation ratio after completion. (containing 100ug/ml ampicillin), cultivated for 10 hours in a shaker at 37°C and 200rpm as secondary seeds, and then connected to a 100L fermenter of 60L LB medium (containing 100ug/ml ampicillin) at a 1% inoculation ratio after completion cultivated in.
  • the initial fermentation conditions were 37°C, 200 rpm, pH 7.0.
  • the obtained Escherichia coli cells containing glucose-6-phosphate isomerase were prepared into a supernatant enzyme solution: the preparation method of the supernatant enzyme solution was to add sodium phosphate buffer (PBS 100mM pH 7.5) to every 1 g of cells to beat, and then use The pressure cell crusher was crushed at the setting of 700-800MPa to obtain the cell crushing liquid and centrifuged with a tube centrifuge at the setting of 10,000rpm and 100L/hr to get the supernatant liquid, each 1ml supernatant enzyme liquid Contains 0.2g of cells. As shown in Figure 2, SDS-PAGE gel electrophoresis test confirmed the synthesis of glucose-6-phosphate isomerase.
  • the enzyme activity of the cells was detected according to their enzymatic reaction.
  • the method was to add the supernatant enzyme solution containing 1 mg of total protein to 1 ml of the reaction solution (200 mM PBS pH 7.5, 5 mM glucose-6-phosphate sodium salt), and at celsius
  • the reaction was carried out under the temperature control of 37 degrees for 5 minutes, and after completion, the fructose-6-phosphate content in the sample produced in the enzymatic reaction was analyzed by high performance liquid chromatography in Annex 1.
  • the enzyme activity of the supernatant enzyme solution is about 0.24 nmol/min/mg.
  • PCR primers were designed based on the DNA sequence EEZ28043.1 (SEQ9) encoding hexokinase in the genome of Bacteroides fragilis, specifically:
  • a single species of the above-mentioned strains was selected and inoculated into 4mL LB medium (containing 100ug/ml ampicillin), cultivated for 16 hours in a shaker at 37°C and 200rpm as primary seeds, and received 100mL LB medium by 1% inoculation ratio after completion. (containing 100ug/ml ampicillin), cultivated for 10 hours in a shaker at 37°C and 200rpm as secondary seeds, and then connected to a 100L fermenter of 60L LB medium (containing 100ug/ml ampicillin) at a 1% inoculation ratio after completion cultivated in.
  • the initial fermentation conditions were 37°C, 200 rpm, pH 7.0.
  • Fermentation was carried out for 9 hours, IPTG was added to a final concentration of 1 mM, and the fermentation was completed at 20 hours.
  • the fermentation broth was centrifuged at 12,500 rpm for 10 minutes at 4° C. to obtain 1.55 kg of E. coli cells containing hexokinase.
  • the obtained hexokinase-containing Escherichia coli cells were prepared into a supernatant enzyme solution: the preparation method of the supernatant enzyme solution was to add sodium phosphate buffer (PBS 100mM pH 7.5) to each 1 g of cells for beating, and then use a pressure cell disruptor.
  • PBS 100mM pH 7.5 sodium phosphate buffer
  • the enzymatic activity of the cells was detected according to their enzymatic reaction.
  • the method was to add 1 mg of total protein to 1 ml of the reaction solution (200 mM PBS pH 7.5, 10 mM D-fructose, 5 mM adenosine triphosphate disodium salt, 20 mM magnesium chloride hexahydrate).
  • the supernatant enzyme solution was subjected to a reaction at a temperature of 37 degrees Celsius for 5 minutes.
  • the fructose-6-phosphate content in the sample produced in the enzyme reaction was analyzed by the high-performance liquid chromatography method in Annex 1. According to the above method, the enzyme activity of the enzyme solution is about 0.18 nmol/min/mg.
  • PCR primers were designed based on the DNA sequence AOR96716.1 (SEQ12) encoding hexulose 6-phosphate isomerase in the genome of Bacillus subtilis, specifically:
  • a single species of the above-mentioned strains was selected and inoculated into 4mL LB medium (containing 100ug/ml ampicillin), cultivated for 16 hours in a shaker at 37°C and 200rpm as primary seeds, and received 100mL LB medium by 1% inoculation ratio after completion. (containing 100ug/ml ampicillin), cultivated for 10 hours in a shaker at 37°C and 200rpm as secondary seeds, and then connected to a 100L fermenter of 60L LB medium (containing 100ug/ml ampicillin) at a 1% inoculation ratio after completion cultivated in.
  • the initial fermentation conditions were 37°C, 200 rpm, pH 7.0.
  • the obtained Escherichia coli cells containing hexose 6-phosphate isomerase are prepared into a supernatant enzyme solution: the preparation method of the supernatant enzyme solution is to add sodium phosphate buffer (PBS 100mM pH 7.5) to every 1 g of cells to beat the pulp, Use a pressure cell crusher at the setting of 700-800MPa to crush the cell fluid and centrifuge it with a tube centrifuge at 10,000rpm and 100L/hr to get the supernatant, each 1ml of the supernatant enzyme solution Contains 0.2g of cells. As shown in Figure 4, SDS-PAGE gel electrophoresis test confirmed the synthesis of ketohexose 6-phosphate isomerase.
  • PCR primers were designed based on the DNA sequence ARW30002.1 (SEQ15) encoding hexulose 6-phosphate synthase in the genome of Bacillus subtilis subsp.subtilis, specifically:
  • the DNA of Bacillus subtilis subsp.subtilis is used as the substrate, and the above primers are used for PCR amplification to obtain the hexose 6-phosphate synthase gene, and the restriction endonucleases BamHI and EcoRI are used to process the PCR
  • the product was ligated into pET-21a to give pET-3H6PS.
  • the recombinant expression vector was transformed into Escherichia coli HB101 to obtain a recombinant expression strain of hexose 6-phosphate synthase.
  • a single species of the above-mentioned strains was selected and inoculated into 4mL LB medium (containing 100ug/ml ampicillin), cultivated for 16 hours in a shaker at 37°C and 200rpm as primary seeds, and received 100mL LB medium by 1% inoculation ratio after completion. (containing 100ug/ml ampicillin), cultivated for 10 hours in a shaker at 37°C and 200rpm as secondary seeds, and then connected to a 100L fermenter of 60L LB medium (containing 100ug/ml ampicillin) at a 1% inoculation ratio after completion cultivated in.
  • the initial fermentation conditions were 37°C, 200 rpm, pH 7.0.
  • Fermentation was carried out for 9 hours, IPTG was added to a final concentration of 1 mM, and the fermentation was completed at 20 hours.
  • the fermentation broth was centrifuged at 12,500 rpm for 10 minutes at 4° C. to obtain 1.91 kg of Escherichia coli cells containing hexulose 6-phosphate synthase.
  • the obtained E. coli cells containing ketohexose 6-phosphate synthase were prepared into a supernatant enzyme solution.
  • the preparation method of the supernatant enzyme solution is to add sodium phosphate buffer (PBS 100mM pH 7.5) to every 1 g of cells to beat, and then use a pressure cell disruptor to break the cell broken solution at a setting of 700-800 MPa to obtain a cell broken solution.
  • PBS 100mM pH 7.5 sodium phosphate buffer
  • the cells were tested for enzyme activity according to their enzymatic reaction.
  • the method was to add supernatant enzyme containing 1 mg of total protein to 1 ml of reaction solution (200 mM PBS pH 7.5, 5 mM hexose 6-phosphate, 20 mM magnesium chloride hexahydrate).
  • the reaction was carried out under the temperature control of 37 degrees Celsius for 5 minutes.
  • the ribulose 5-phosphate content in the sample produced in the enzymatic reaction was analyzed by high performance liquid chromatography in Annex 1. According to the above method, the enzyme activity of the supernatant enzyme solution is about 0.07 nmol/min/mg.
  • PCR primers were designed based on the DNA sequence QNS47947.1 (SEQ18) encoding ribose 5-phosphate isomerase in the genome of Escherichia coli, specifically:
  • Escherichia coli Escherichia coli
  • PCR amplification with the above-mentioned primers to obtain the ribose 5-phosphate isomerase gene
  • restriction endonucleases BamHI and EcoRI to process the PCR product and connect it to pET- In 21a
  • pET-R5PI is obtained.
  • the recombinant expression vector was transformed into Escherichia coli HB101 to obtain a recombinant expression strain of ribose 5-phosphate isomerase.
  • a single species of the above-mentioned strains was selected and inoculated into 4mL LB medium (containing 100ug/ml ampicillin), cultivated for 16 hours in a shaker at 37°C and 200rpm as primary seeds, and received 100mL LB medium by 1% inoculation ratio after completion. (containing 100ug/ml ampicillin), cultivated for 10 hours in a shaker at 37°C and 200rpm as secondary seeds, and then connected to a 100L fermenter of 60L LB medium (containing 100ug/ml ampicillin) at a 1% inoculation ratio after completion cultivated in.
  • the initial fermentation conditions were 37°C, 200 rpm, pH 7.0.
  • the obtained Escherichia coli cells containing ribose 5-phosphate isomerase are prepared into a supernatant enzyme solution: the preparation method of the supernatant enzyme solution is to add sodium phosphate buffer (PBS 100mM pH 7.5) to every 1 g of cells to beat, using pressure
  • the cell crusher was crushed at the setting of 700-800MPa to obtain the cell crushing liquid, and the supernatant liquid was obtained by centrifugation with a tube centrifuge at the setting of 10,000rpm and 100L/hr. Contains 0.2g of cells.
  • SDS-PAGE gel electrophoresis test confirmed the synthesis of ribose 5-phosphate isomerase.
  • the enzymatic activity of the cells was detected according to their enzymatic reaction.
  • the method was to add 1 mg of total protein to 1 ml of reaction solution (200 mM PBS pH 7.5, 5 mM sodium ribulose 5-phosphate, 20 mM magnesium chloride hexahydrate).
  • the clear enzyme solution was subjected to a 5-minute reaction under the temperature control of 37 degrees Celsius.
  • the ribose 5-phosphate content in the sample produced in the enzyme reaction was analyzed by high-performance liquid chromatography in Annex 1. According to the above method, the enzyme activity of the supernatant enzyme solution is about 0.16 nmol/min/mg.
  • PCR primers were designed based on the DNA sequence KXG95745.1 (SEQ21) encoding phosphoribosyl diphosphate kinase in the genome of Escherichia coli, specifically:
  • PCR primers were designed for the phosphoribosyl diphosphate kinase sequence, specifically
  • Escherichia coli Escherichia coli
  • carry out PCR amplification with the above-mentioned primers to obtain the phosphoribosyl diphosphate kinase gene utilize restriction endonucleases BamHI and EcoRI to process the PCR product and connect it to pET-21a , to obtain pET-R5PI.
  • the recombinant expression vector was transformed into Escherichia coli HB101 to obtain a recombinant expression strain of phosphoribosyl diphosphate kinase.
  • a single species of the above-mentioned strains was selected and inoculated into 4mL LB medium (containing 100ug/ml ampicillin), cultivated for 16 hours in a shaker at 37°C and 200rpm as primary seeds, and received 100mL LB medium by 1% inoculation ratio after completion. (containing 100ug/ml ampicillin), cultivated for 10 hours in a shaker at 37°C and 200rpm as secondary seeds, and then connected to a 100L fermenter of 60L LB medium (containing 100ug/ml ampicillin) at a 1% inoculation ratio after completion cultivated in.
  • the initial fermentation conditions were 37°C, 200 rpm, pH 7.0.
  • the obtained Escherichia coli cells containing phosphoribosyl diphosphate kinase are prepared into supernatant enzyme solution: the preparation method of supernatant enzyme solution is to add sodium phosphate buffer (PBS 100mM pH 7.5) to every 1g of cells to beat, and then use pressure cells
  • the crusher is crushed at the setting of 700-800MPa to obtain the cell broken liquid and centrifuged with the tube centrifuge at the setting of 10,000rpm and 100L/hr to get the supernatant liquid, each 1ml of the supernatant enzyme liquid contains 0.2 g cells.
  • SDS-PAGE gel electrophoresis test confirmed the synthesis of phosphoribosylbisphosphate kinase.
  • Cells were assayed for their enzymatic activity according to their enzymatic reaction.
  • the method was to add 1 mg of a reaction solution (200 mM PBS pH 7.5, 10 mM ribose 5-phosphate disodium salt, 5 mM adenosine triphosphate disodium salt, 20 mM magnesium chloride hexahydrate) to 1 ml of the reaction solution.
  • the supernatant enzyme solution with the total protein content was subjected to a 5-minute reaction at a temperature of 37 degrees Celsius. After completion, the content of phosphoribosyl diphosphate produced in the enzyme reaction in the sample was analyzed by high-performance liquid chromatography in Annex 1. According to the above method, the enzyme activity of the supernatant enzyme solution is about 0.14 nmol/min/mg.
  • PCR primers were designed based on the DNA sequence RKE26735.1 (SEQ24) encoding nicotinamide phosphoribosyltransferase in the genome of Rhodococcus pyridinivorans, specifically:
  • pET-R5PI The recombinant expression vector was transformed into Escherichia coli HB101 to obtain a recombinant expression strain of nicotinamide phosphoribosyltransferase.
  • a single species of the above-mentioned strains was selected and inoculated into 4mL LB medium (containing 100ug/ml ampicillin), cultivated at 37°C, 200rpm shaker for 16 hours as primary seeds, and received 100mL LB medium by 1% inoculation ratio after completion. (containing 100ug/ml ampicillin), cultivated for 10 hours at 37°C and 200rpm in a shaker as secondary seeds, and then connected to a 100L fermenter of 60L LB medium (containing 100ug/ml ampicillin) at a 1% inoculation ratio after completion cultivated in.
  • the initial fermentation conditions were 37°C, 200 rpm, pH 7.0.
  • the obtained Escherichia coli cells containing nicotinamide phosphoribosyltransferase are prepared into a supernatant enzyme solution: the preparation method of the supernatant enzyme solution is to add sodium phosphate buffer (PBS 100mM pH 7.5) to every 1 g of cells to beat, using a pressure formula
  • the cell crusher is crushed under the setting of 700-800MPa to obtain the cell crushed liquid and centrifuged with a tube centrifuge at the setting of 10,000rpm and 100L/hr to get the supernatant liquid.
  • Each 1ml of the supernatant enzyme liquid contains 0.2g cells.
  • SDS-PAGE gel electrophoresis test confirmed the synthesis of nicotinamide phosphoribosyltransferase.
  • Cells were assayed for their enzymatic activity according to their enzymatic reaction.
  • the method was to add 1 mg of total protein to 1 ml of reaction solution (200 mM PBS pH 7.5, 5 mM phosphoribosyl diphosphate pentasodium salt, 10 mM nicotinamide, 20 mM magnesium chloride hexahydrate).
  • the amount of supernatant enzyme solution was carried out for 5 minutes under the temperature control of 37 degrees Celsius. After completion, the content of ⁇ -nicotinamide mononucleotide produced in the enzyme reaction in the sample was measured by high performance liquid chromatography in Annex 2. analyze. According to the above method, the enzyme activity of the supernatant enzyme solution is about 0.09 nmol/min/mg.
  • PCR primers were designed based on the DNA sequence BAC76403.1 (SEQ27) encoding polyphospho-AMP phosphotransferase in the genome of Acinetobacter johnsonii, specifically:
  • the polyphospho-AMP phosphotransferase gene was amplified by PCR with the above-mentioned primers, and the PCR products were treated with restriction endonucleases BamHI and EcoRI and were Ligation into pET-21a gave pET-PAP1.
  • the recombinant expression vector was transformed into Escherichia coli HB101 to obtain a recombinant expression strain of polyphosphoric acid-AMP phosphotransferase.
  • a single species of the above-mentioned strains was selected and inoculated into 4mL LB medium (containing 100ug/ml ampicillin), cultivated for 16 hours in a shaker at 37°C and 200rpm as primary seeds, and received 100mL LB medium by 1% inoculation ratio after completion. (containing 100ug/ml ampicillin), cultivated for 10 hours in a shaker at 37°C and 200rpm as secondary seeds, and then connected to a 100L fermenter of 60L LB medium (containing 100ug/ml ampicillin) at a 1% inoculation ratio after completion cultivated in.
  • the initial fermentation conditions were 37°C, 200 rpm, pH 7.0.
  • the obtained Escherichia coli cells containing polyphosphoric acid-AMP phosphotransferase were prepared into a supernatant enzyme solution: the preparation method of the supernatant enzyme solution was to add sodium phosphate buffer (PBS100mM pH 7.5) to each 1g of cells for beating, using pressure The cell crusher was crushed at the setting of 700-800MPa to obtain the cell crushing liquid, and the supernatant liquid was obtained by centrifugation with a tube centrifuge at the setting of 10,000rpm and 100L/hr. Contains 0.2g of cells. As shown in Figure 9, SDS-PAGE gel electrophoresis test confirmed the synthesis of polyphospho-AMP phosphotransferase.
  • Preparation 10 Preparation of adenylate kinase (EC 2.7.4.3)
  • PCR primers were designed based on the DNA sequence KIG05708.1 (SEQ30) encoding adenylate kinase in the genome of the phosphaloxide degrading bacteria (Burkholderia sp. MR1), specifically:
  • phosphatidic acid degrading bacteria (Burkholderia sp.MR1) as the substrate, carry out PCR amplification with the above-mentioned primers to obtain the adenylate kinase gene, utilize the restriction endonucleases BamHI and EcoRI to process the PCR product and connect it into pET-21a, resulting in pET-AK.
  • the recombinant expression vector was transformed into Escherichia coli HB101 to obtain a recombinant expression strain of adenylate kinase.
  • a single species of the above-mentioned strains was selected and inoculated into 4mL LB medium (containing 100ug/ml ampicillin), cultivated for 16 hours in a shaker at 37°C and 200rpm as primary seeds, and received 100mL LB medium by 1% inoculation ratio after completion. (containing 100ug/ml ampicillin), cultivated for 10 hours in a shaker at 37°C and 200rpm as secondary seeds, and then connected to a 100L fermenter of 60L LB medium (containing 100ug/ml ampicillin) at a 1% inoculation ratio after completion cultivated in.
  • the initial fermentation conditions were 37°C, 200 rpm, pH 7.0.
  • Fermentation was carried out for 9 hours, IPTG was added to a final concentration of 1 mM, and the fermentation was completed at 20 hours.
  • the fermentation broth was centrifuged at 12,500 rpm for 10 minutes at 4° C. to obtain 1.33 kg of E. coli cells containing adenylate kinase.
  • the obtained E. coli cells containing adenylate kinase were prepared into a supernatant enzyme solution: the preparation method of the supernatant enzyme solution was to add sodium phosphate buffer (PBS 100mM pH 7.5) to each 1 g of cells for beating, and then use pressure cell disruption.
  • PBS 100mM pH 7.5 sodium phosphate buffer
  • the device was crushed at 700-800MPa to obtain cell fragmentation liquid, and centrifuged with a tube centrifuge at 10,000rpm and 100L/hr to get the supernatant liquid, each 1ml of supernatant enzyme liquid contained 0.2g cells .
  • SDS-PAGE gel electrophoresis test confirmed the synthesis of adenylate kinase.
  • the enzymatic activity of the cells was detected according to their enzymatic reaction.
  • the method was to add the enzyme containing 1 mg of total protein to 1 ml of the reaction solution (200 mM PBS pH 7.5, 5 mM adenosine, 10 mM adenosine triphosphate disodium salt, 20 mM magnesium chloride hexahydrate).
  • the reaction was carried out under the temperature control of 37 degrees Celsius for 5 minutes.
  • the high performance liquid chromatography in Annex 2 was used to analyze the adenosine monophosphate produced in the enzymatic reaction in the sample.
  • the enzyme activity of the enzyme solution is about 0.48nmol/min/mg.
  • PCR primers were designed based on the DNA sequence ENO92539.1 (SEQ33) encoding polyphosphokinase in the genome of Thauera sp. 28, specifically:
  • the DNA of Thauera sp. 28 was used as the substrate, and the polyphosphokinase gene was amplified by PCR with the above primers, and the PCR products were treated with restriction enzymes BamHI and EcoRI and ligated. into pET-21a, resulting in pET-PPK2.
  • the recombinant expression vector was transformed into Escherichia coli HB101 to obtain a recombinant expression strain of polyphosphokinase.
  • a single species of the above-mentioned strains was selected and inoculated into 4mL LB medium (containing 100ug/ml ampicillin), cultivated for 16 hours in a shaker at 37°C and 200rpm as primary seeds, and received 100mL LB medium by 1% inoculation ratio after completion. (containing 100ug/ml ampicillin), cultivated for 10 hours in a shaker at 37°C and 200rpm as secondary seeds, and then connected to a 100L fermenter of 60L LB medium (containing 100ug/ml ampicillin) at a 1% inoculation ratio after completion cultivated in.
  • the initial fermentation conditions were 37°C, 200 rpm, pH 7.0.
  • Fermentation was carried out for 9 hours, IPTG was added to a final concentration of 1 mM, and the fermentation was completed at 20 hours.
  • the fermentation broth was centrifuged at 12,500 rpm for 10 minutes at 4° C. to obtain 1.11 kg of E. coli cells containing polyphosphokinase.
  • the obtained Escherichia coli cells containing polyphosphokinase were prepared into a supernatant enzyme solution: the preparation method of the supernatant enzyme solution was to add sodium phosphate buffer (PBS 100mM pH 7.5) to each 1g of cells for beating, and then use pressure cell disruption.
  • PBS 100mM pH 7.5 sodium phosphate buffer
  • the device was crushed at 700-800MPa to obtain cell fragmentation liquid, and centrifuged with a tube centrifuge at 10,000rpm and 100L/hr to get the supernatant liquid, each 1ml of supernatant enzyme liquid contained 0.2g cells .
  • SDS-PAGE gel electrophoresis test confirmed the synthesis of polyphosphokinase.
  • Cells were assayed for enzymatic activity according to their enzymatic reaction, which was to add 1 mg of total
  • the protein content of the supernatant enzyme solution was subjected to a 5-minute reaction at a temperature of 37 degrees Celsius. After completion, the content of adenosine triphosphate produced in the enzyme reaction in the sample was analyzed by high-performance liquid chromatography in Annex 1. According to the above method, the enzyme activity of the supernatant enzyme solution is about 0.03 nmol/min/mg.
  • PCR primers were designed based on the DNA sequence AWV22863.1 (SEQ36) encoding mannokinase in the genome of Roseomonas mucosa, specifically:
  • a single species of the above-mentioned strains was selected and inoculated into 4mL LB medium (containing 100ug/ml ampicillin), cultivated for 16 hours in a shaker at 37°C and 200rpm as primary seeds, and received 100mL LB medium by 1% inoculation ratio after completion. (containing 100ug/ml ampicillin), cultivated for 10 hours in a shaker at 37°C and 200rpm as secondary seeds, and then connected to a 100L fermenter of 60L LB medium (containing 100ug/ml ampicillin) at a 1% inoculation ratio after completion cultivated in.
  • the initial fermentation conditions were 37°C, 200 rpm, pH 7.0.
  • Fermentation was carried out for 9 hours, IPTG was added to a final concentration of 1 mM, and the fermentation was completed at 20 hours.
  • the fermentation broth was centrifuged at 12,500 rpm for 10 minutes at 4° C. to obtain 1.36 kg of E. coli cells containing mannokinase.
  • the obtained Escherichia coli cells containing mannokinase were prepared into an enzyme solution.
  • the preparation method of the enzyme solution is to add sodium phosphate buffer (PBS 100mM pH 7.5) to each 1g of cells and beat, and then use a pressure cell disruptor to break the cell breaker at a setting of 700-800MPa to obtain a cell breakage liquid and centrifuge it with a tube.
  • PBS 100mM pH 7.5 sodium phosphate buffer
  • the machine was centrifuged at 10,000 rpm and 100 L/hr to take the supernatant, and each 1 ml of the enzyme solution contained 0.2 g of cells.
  • SDS-PAGE gel electrophoresis test confirmed the synthesis of mannokinase.
  • the cells were tested for enzyme activity according to their enzymatic reaction.
  • the method was to add enzyme containing 1 mg of total protein to 1 ml of reaction solution (200 mM PBS pH 7.5, 5 mM mannose, 10 mM adenosine triphosphate disodium salt, 20 mM magnesium chloride hexahydrate).
  • the reaction was carried out under the temperature control of 37 degrees Celsius for 5 minutes.
  • the mannose 6-phosphate produced in the enzyme reaction in the sample was analyzed by high performance liquid chromatography.
  • the enzyme activity of the enzyme solution is about 0.14 nmol/min/mg.
  • PCR primers were designed based on the DNA sequence BCA74065.1 (SEQ39) encoding mannose-6-phosphate isomerase in the genome of Escherichia coli, specifically:
  • Escherichia coli Escherichia coli
  • carry out PCR amplification with the above-mentioned primers to obtain the mannose-6-phosphate isomerase gene utilize restriction endonucleases BamHI and EcoRI to process the PCR product and connect it to In pET-21a, pET-M6PI was obtained.
  • the recombinant expression vector was transformed into Escherichia coli HB101 to obtain a recombinant expression strain of mannose-6-phosphate isomerase.
  • a single species of the above-mentioned strains was selected and inoculated into 4mL LB medium (containing 100ug/ml ampicillin), cultivated for 16 hours in a shaker at 37°C and 200rpm as primary seeds, and received 100mL LB medium by 1% inoculation ratio after completion. (containing 100ug/ml ampicillin), cultivated for 10 hours in a shaker at 37°C and 200rpm as secondary seeds, and then connected to a 100L fermenter of 60L LB medium (containing 100ug/ml ampicillin) at a 1% inoculation ratio after completion cultivated in.
  • the initial fermentation conditions were 37°C, 200 rpm, pH 7.0.
  • the obtained Escherichia coli cells containing mannose-6-phosphate isomerase are prepared into a supernatant enzyme solution: the preparation method of the supernatant enzyme solution is to add sodium phosphate buffer (PBS 100mM pH7.5) to every 1 g of cells and after beating , use a pressure cell crusher to crush the cell crushed liquid at the setting of 700-800MPa, and centrifuge the supernatant with a tube centrifuge at the setting of 10,000rpm and 100L/hr, every 1ml of enzyme solution Contains 0.2g of cells. As shown in Figure 13, SDS-PAGE gel electrophoresis test confirmed the synthesis of mannose-6-phosphate isomerase.
  • PBS 100mM pH7.5 sodium phosphate buffer
  • the enzyme activity of cells was detected by adding an enzyme solution containing 1 mg of total protein to 1 ml of reaction solution (200 mM PBS pH 7.5, 10 mM mannose 6-phosphate sodium salt, 20 mM magnesium chloride hexahydrate). , under the temperature control of 37 degrees Celsius, the reaction was carried out for 5 minutes, and after completion, the fructose 6-phosphate produced in the enzyme reaction in the sample was analyzed by high performance liquid chromatography. According to the above method, the enzyme activity of the enzyme solution is about 0.21nmol/min/mg.
  • PCR primers were designed based on the DNA sequence BAV00088.1 (SEQ42) encoding maltase in the genome of Aurantimicrobium minutum, specifically:
  • a single species of the above-mentioned strains was selected and inoculated into 4mL LB medium (containing 100ug/ml ampicillin), cultivated for 16 hours in a shaker at 37°C and 200rpm as primary seeds, and received 100mL LB medium by 1% inoculation ratio after completion. (containing 100ug/ml ampicillin), cultivated for 10 hours in a shaker at 37°C and 200rpm as secondary seeds, and then connected to a 100L fermenter of 60L LB medium (containing 100ug/ml ampicillin) at a 1% inoculation ratio after completion cultivated in.
  • the initial fermentation conditions were 37°C, 200 rpm, pH 7.0.
  • Fermentation was carried out for 9 hours, IPTG was added to a final concentration of 1 mM, and the fermentation was completed at 20 hours.
  • the fermentation broth was centrifuged at 12,500 rpm for 10 minutes at 4°C to obtain 0.78 kg of E. coli cells containing maltase.
  • the obtained Escherichia coli cells containing maltase are prepared into supernatant enzyme solution: the preparation method of supernatant enzyme solution is to add sodium phosphate buffer (PBS 100mM pH 7.5) to every 1 g of cells to beat, and then use a pressure cell disruptor to make the solution.
  • PBS 100mM pH 7.5 sodium phosphate buffer
  • the enzymatic activity of the cells was detected.
  • the method was to add an enzyme solution containing 1 mg of total protein to 1 ml of reaction solution (200 mM PBS pH 7.5, 5 mM maltose, 20 mM magnesium chloride hexahydrate) at 37 degrees Celsius.
  • the reaction was carried out under temperature control for 5 minutes, and after completion, the glucose produced in the enzymatic reaction in the sample was analyzed by high performance liquid chromatography.
  • the enzyme activity of the enzyme solution is about 0.08 nmol/min/mg.
  • PCR primers were designed based on the DNA sequence QQJ91524.1 (SEQ45) encoding sucrase in the genome of Enterococcus faecium, specifically:
  • a single species of the above-mentioned strains was selected and inoculated into 4mL LB medium (containing 100ug/ml ampicillin), cultivated for 16 hours in a shaker at 37°C and 200rpm as primary seeds, and received 100mL LB medium by 1% inoculation ratio after completion. (containing 100ug/ml ampicillin), cultivated for 10 hours in a shaker at 37°C and 200rpm as secondary seeds, and then connected to a 100L fermenter of 60L LB medium (containing 100ug/ml ampicillin) at a 1% inoculation ratio after completion cultivated in.
  • the initial fermentation conditions were 37°C, 200 rpm, pH 7.0.
  • sucrase-containing Escherichia coli cells were prepared into a supernatant enzyme solution: the preparation method of the supernatant enzyme solution was to add sodium phosphate buffer (PBS 100mM pH 7.5) to every 1 g of cells for beating, and then use a pressure cell disruptor to remove the slurry.
  • PBS 100mM pH 7.5 sodium phosphate buffer
  • the cells were tested for enzyme activity according to their enzymatic reaction.
  • the method was to add an enzyme solution containing 1 mg of total protein to 1 ml of a reaction solution (200 mM PBS pH 7.5, 5 mM sucrose, 20 mM magnesium chloride hexahydrate) at 37 degrees Celsius.
  • the reaction was carried out under temperature control for 5 minutes, and after completion, the glucose produced in the enzymatic reaction in the sample was analyzed by high performance liquid chromatography.
  • the enzyme activity of the enzyme solution is about 0.06nmol/min/mg.
  • PCR primers were designed based on the DNA sequence AVB07708.1 (SEQ48) encoding 5'-nucleotidase in the genome of Salmonella enterica, specifically:
  • the 5'-nucleotidase gene was obtained by PCR amplification with the above-mentioned primers, and the PCR products were processed with restriction enzymes BamHI and EcoR I and connected. into pET-21a, resulting in pET-USHA.
  • the recombinant expression vector was transformed into Escherichia coli HB101 to obtain a 5'-nucleotidase recombinant expression strain.
  • a single colony of the above-mentioned strains was selected and inoculated into 4mL LB medium (containing 100 ⁇ g/ml ampicillin), cultivated for 16 hours in a shaker at 37°C and 200rpm as primary seeds, and received 100mL LB medium by 1% inoculation ratio after completion. (containing 100 ⁇ g/ml ampicillin), cultured at 37°C, 200 rpm shaker for 10 hours as secondary seeds, and then received 60L LB medium (containing 100 ⁇ g/ml ampicillin) at a 1% inoculation ratio after completion. Cultured in a 100L fermenter. The initial fermentation conditions were 37°C, 200 rpm, pH 7.0.
  • Fermentation was carried out for 9 hours by adding IPTG to a final concentration of 1 mM and the fermentation was completed at 20 hours.
  • the fermentation broth was centrifuged at 12,500 rpm for 10 minutes at 4°C to obtain 1.55 kg of E. coli cells containing 5'-nucleotidase.
  • the obtained 5'-nucleotidase-containing Escherichia coli cells were prepared into a supernatant enzyme solution: the preparation method of the supernatant enzyme solution was to add sodium phosphate buffer (PBS 100mM pH 7.5) to each 1g of cells to make a slurry, then use pressure
  • the cell crusher was crushed at the setting of 700-800MPa to obtain the cell crushing liquid, and the supernatant liquid was obtained by centrifugation with the tube centrifuge at the setting of 10,000rpm and 100L/hr.
  • Each 1ml of enzyme solution contained 0.2g cell.
  • SDS-PAGE gel electrophoresis test confirmed the synthesis of 5'-nucleotidase.
  • the enzyme activity of the supernatant enzyme solution is detected according to its enzymatic reaction, and the enzyme activity of the enzyme solution is detected according to its enzymatic reaction.
  • the enzyme solution containing 1 mg of total protein was added to the enzyme solution, and the reaction was carried out under the temperature control of 37 degrees Celsius for 5 minutes. analyze. According to the above method, the enzyme activity of the enzyme solution is about 0.62 nmol/min/mg.
  • Embodiment 1 use D-glucose (edible glucose) to carry out enzymatic reaction with the enzyme supernatant of enzyme combination to prepare ⁇ -nicotinamide mononucleotide
  • D-glucose edible glucose
  • Embodiment 2 use D-fructose to carry out enzymatic reaction with the supernatant enzyme liquid of enzyme combination to prepare ⁇ -nicotinamide mononucleotide
  • the enzyme supernatants belonging to the D-fructose combination, the core combination and the auxiliary combination were prepared according to the preparation example and mixed in the proportions in Table 3 to obtain 100 ml of the mixed supernatant enzyme solution for use.
  • 1.2g nicotinamide and 1.2g adenosine triphosphate disodium salt add 600ml pure water, start the external stirring device until all raw materials are completely dissolved, adjust the pH value of the solution to pH 7.5 with 0.1M hydrochloric acid/sodium hydroxide, and use pure Dilute the volume of water to 1L and keep it warm until the temperature of the solution is stable at 37°C, add 100ml of the above-prepared supernatant enzyme solution and 20g of zeolite simultaneously under stirring at 10-2-rpm, and use a pH control device to monitor the reaction process in real time. The pH of the sample was changed and adjusted with 0.1M hydrochloric acid/sodium hydroxide solution.
  • Embodiment 3 use D-glucose (edible glucose) to carry out the enzymatic reaction of mixed enzyme group with immobilized enzyme to prepare ⁇ -nicotinamide mononucleotide
  • D-glucose edible glucose
  • the mixed immobilized enzyme was prepared on the solid-phase carrier according to the dosage ratio of the supernatant enzyme solution in each combination in Table 5; the shapes of the carriers were all strips: 25cm long, 5cm wide , thickness 5mm, the following table shows the weight of each immobilized enzyme product is 32.6g.
  • Enzyme The ratio of the total protein weight of each supernatant enzyme solution to the total weight of immobilized enzyme polyphosphate-glucose phosphotransferase 8 Glucose-6-Phosphate Isomerase 6 ketohexose 6-phosphate isomerase 16 ketohexose 6-phosphate synthase 16 ribose 5-phosphate isomerase 10 phosphoribosyl diphosphate kinase 4 nicotinamide phosphoribosyltransferase 4 polyphosphokinase 2 adenylate kinase 6 polyphosphate-AMP phosphotransferase 6
  • the immobilized enzyme carrier prepared above was installed in an immobilized enzyme reactor.
  • the reactor is a cylinder made of plexiglass, with a height of 7 cm and a radius of 4.5 cm.
  • Use a knife to neatly shave off about 3 cm of the head and tail ends of the above-mentioned carrier with a slope of 45°, and roll it tightly into a homogeneous cylinder with a height of 5 cm and a radius of 4.5 cm, and the weight is 8.8 g.
  • Fig. 1 of CN106032520A in which the capacity of the reaction control tank is 2L; the high flow pump is an adjustable flow suction pump with a flow rate of 0.5L/min; The pH of the sodium hydroxide solution is regulated, and the flow rate of the dosing pump is 1ml per minute.
  • immobilized enzymes can increase the amount of enzyme used without increasing the liquid solubility, which has a great effect on increasing the reaction speed and improving the conversion rate. At the same time, The immobilized enzyme can be easily reused many times, and the reaction solution does not require multiple purification steps due to the large amount of protein.
  • Embodiment 4 simultaneously use D-glucose (edible glucose) and D-fructose to carry out enzymatic reaction with the enzyme supernatant of mixed enzyme combination to prepare ⁇ -nicotinamide mononucleotide
  • Embodiment 5 use maltose to carry out enzymatic reaction with the supernatant enzyme liquid of enzyme combination to prepare ⁇ -nicotinamide mononucleotide
  • Adjust with 0.1M hydrochloric acid/sodium hydroxide solution take samples every 60min and analyze the content of ⁇ -nicotinamide mononucleotide according to the method in Annex 2; after 12hr of reaction, the content of ⁇ -nicotinamide mononucleotide The content reached 2.4 mM (see Table 10 below).
  • the zeolite is removed by filtering with medium-speed filter paper to obtain a ⁇ -nicotinamide mononucleotide solution.
  • Embodiment 6 use sucrose to carry out enzymatic reaction with the supernatant enzyme liquid of enzyme combination to prepare ⁇ -nicotinamide mononucleotide
  • Enzyme The total protein weight of the supernatant enzyme solution of each enzyme and the total mixed supernatant enzyme solution
  • Adjust with 0.1M hydrochloric acid/sodium hydroxide solution take samples every 60min and analyze the content of ⁇ -nicotinamide mononucleotide according to the method in Annex 2; after 12 hours of reaction, the content of ⁇ -nicotinamide mononucleotide Levels reached 2.1 mM (see Table 12 below).
  • the zeolite is removed by filtering with medium-speed filter paper to obtain a ⁇ -nicotinamide mononucleotide solution.
  • Embodiment 7 use D-glucose (edible glucose) to carry out enzymatic reaction with the enzyme supernatant of enzyme combination to prepare ⁇ -nicotinic acid mononucleotide
  • D-glucose edible glucose
  • Embodiment 8 use D-glucose (edible glucose) to carry out enzymatic reaction with the enzyme supernatant of enzyme combination to prepare ⁇ -nicotinamide riboside
  • Comparative Example 1 Use conventional biological enzyme method to prepare ⁇ -nicotinamide mononucleotide by enzymatic reaction with ribose as substrate
  • ⁇ -nicotinamide mononucleotide by the conventional biological enzymatic method using ribose as a substrate, it is necessary to use more adenosine triphosphate disodium salt as the supply and energy of the phosphate group than the method of the present invention. Therefore, in the reaction of the same amount of adenosine triphosphate, The method of the present invention can produce more ⁇ -nicotinamide mononucleotide in a shorter time, and is better than the conventional biological enzyme method in production time and cost-effectiveness.
  • Annex 2 Analysis of nucleoside and mononucleotide content of nicotinic acid or its derivatives (e.g. ⁇ -nicotinamide mononucleotide, nicotinamide adenine dinucleotide, nicotinamide riboside, nicotinic acid) by HPLC conditions for mononucleotide, nicotinic adenine dinucleotide, nicotinic riboside, etc.)

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Abstract

提供了一种制备烟酸或其衍生物的单核苷酸的方法，包括采用包含己糖源和烟酸或其衍生物的反应底物，在磷酸供体的存在下，经生物酶催化反应生成烟酸或其衍生物的单核苷酸，并采用该单核苷酸作为中间体制备烟酸或其衍生物的核苷等生物产物。

Description

烟酸或其衍生物的单核苷酸及其生物产物的制备方法 技术领域
本发明涉及生物科技领域，具体而言，本发明属于生化领域，特别是涉及烟酸或其衍生物的单核苷酸的制备方法以及利用烟酸或其衍生物的单核苷酸作为中间体制备各种生物产物的方法。
背景技术
烟酸或其衍生物的核苷和单核苷酸是合成各种身体需要的生物产物的重要中间体。其中，一个代表性的例子是β-烟酰胺单核苷酸(NMN)。β-烟酰胺单核苷酸是一种生物中都需要的烟酰胺腺苷二核苷酸中间体，在身体中通过烟酰胺单核苷酸腺苷转移酶在各器官细胞中转化成烟酰胺腺苷二核苷酸。生物中均需要烟酰胺腺苷二核苷酸的存在，是维持生命的必要物质和不可缺少的辅酶：细胞中氢离子的生化反应、细胞核中的能量代谢调控以至维护生理时钟的机制中烟酰胺腺苷二核苷酸都参与其中并且其身体中的水平对上述生理作用有着决定性的影响，如此同时，烟酰胺腺苷二核苷酸肩负了基因修复和稳定端粒体的功能，增强身体的免疫能力和促进增生肌肉中的微丝血管等作用。
哈佛大学教授David Sinclair首先发现身体中烟酰胺腺苷二核苷酸的水平与身体的健康和衰老有着紧密的关联：人体随着年岁增长而身体中烟酰胺腺苷二核苷酸的水平会下降，继而减低新陈代谢并引发相关的疾病，并提出提升烟酰胺腺苷二核苷酸的水平有助身体新陈代谢从而抵抗衰老所带来的影响，并认为使用β-烟酰胺单核苷酸能达至相关的目的。华盛顿大学教授今井真一郎在小鼠上引证β-烟酰胺单核苷酸能提升烟酰胺腺苷二核苷酸的水平并在体能上表现出逆转衰老的效果，加上国内外的研究圴指出提升烟酰胺腺苷二核苷酸水平的重要性，大众对β-烟酰胺单核苷酸加深了认识。
β-烟酰胺单核苷酸是天然物质，可以在日常膳食中吸取，以牛油果和西兰花中的蕴含量较高，但只透过饮食摄取每天身体所需的分量，则需要进食数十 公斤的蔬果方能达标，恐怕难以实现。因此，服用β-烟酰胺单核苷酸作为烟酰胺腺苷二核苷酸补充剂更为合适。
烟酸或其衍生物的核苷和单核苷酸(例如β-烟酰胺单核苷酸)可以从酵母菌中提取或利用化学和传统酶法合成。酵母菌中的烟酸或其衍生物的核苷和单核苷酸(例如β-烟酰胺单核苷酸)含量极微，需要透过多个步骤才能提取到纯度较低的产物，因此售价高昂并且质量较低，并非适合普罗大众使用，而化学方法有着与提取法相同的缺点，因此这两种方法逐被淘汰。
酶法合成有着产能较高和节能的优点，并能生产出纯度较高的产品，将成为生产烟酸或其衍生物的核苷和单核苷酸(例如β-烟酰胺单核苷酸)的主流工艺。传统酶法合成烟酸或其衍生物的核苷和单核苷酸(例如β-烟酰胺单核苷酸)以核糖为初始原材料进行酶法反应。核糖的需求每年迅速增长，除可用于烟酸或其衍生物的核苷和单核苷酸(例如β-烟酰胺单核苷酸)的生产外，更经常使用在膳食补充剂、食品饮料、功能营养领域，其中近年核糖更应用于改良新兴“人造肉”的风味，是生产该潮流食品中不可或缺的成分。核糖的需求远比其供应高，做成近年价格上涨和供应不稳的情况。
全球人口老化，在老年护理的开支和需求连年具增，大多数已发展和发展中的国家在该问题上亦已束手无策；烟酸或其衍生物的核苷和单核苷酸(例如β-烟酰胺单核苷酸)的使用正能改善上述方面的压力，延缓和逆转衰老符合普罗大众的愿望并切合社会上的需要的双赢机会。因此，烟酸或其衍生物的核苷和单核苷酸(例如β-烟酰胺单核苷酸)的需求将按年上升，核糖的供求将更为严峻，以致价格和供应都不稳，不利烟酸或其衍生物的核苷和单核苷酸的生产，对用家和社会都做成巨大损失，因此有必要开发以使用多元化和可持续性的烟酸或其衍生物的核苷和单核苷酸的酶法制备工艺。
发明内容
如上所述，烟酸或其衍生物的核苷和单核苷酸为生物中生成各种生物产物(特别是烟酰胺腺苷二核苷酸)的前体，科学界近年对该物质已经进行了深入的研究。提供一种烟酸或其衍生物的单核苷酸及其生物产物的改进方法可以为该物质进行量化生产，对该领域上的研究提供便利。
因此，本发明提供了一种新型的烟酸或其衍生物的单核苷酸及其生物产物的工业量化制备方法以及利用烟酸或其衍生物的单核苷酸作为中间体制备各种生物产物(特别是烟酸或其衍生物的核苷)的方法。
具体而言，本发明提供了：
1.一种制备烟酸或其衍生物的单核苷酸的方法，包括下列步骤：采用包含己糖源和烟酸或其衍生物的反应底物，在磷酸供体的存在下，经生物酶催化反应生成烟酸或其衍生物的单核苷酸。
其中所述烟酸或其衍生物的单核苷酸选自β-烟酰胺单核苷酸和烟酸单核苷酸中的至少一者。
其中所述己糖源来自具有六个碳原子的单糖、能够生成己糖的多糖或其混合物。
其中所述的单糖选自D-葡萄糖、D-甘露糖、D-半乳糖、D-果糖或其混合物中的任意一者，优选为D-葡萄糖、D-甘露糖和D-果糖，更优选为D-葡萄糖和D-果糖。
其中所述的己糖源来自通过糖苷键连接多种己糖单元的多糖，优选为蔗糖、麦芽糖、菊粉、棉子糖、麦芽糊精、淀粉或其混合物，更优选为蔗糖、麦芽糖和棉子糖，更优选为蔗糖和麦芽糖。
其中所述生物酶为单独的生物酶或者包含多种生物酶的生物酶组。
其中所述反应的条件包括：温度为25-40℃，优选为30-39℃，更优选为35-38℃；并且/或者反应体系的pH为6.0-8.5，优选为pH 7.0-8.0，更优选为pH7.5-7.8。
其中所述反应底物还包含辅助离子，辅助离子包含金属离子、氯离子、镁离子、钙离子、钾离子、钠离子、锌离子、氟离子、硫离子、碳酸根类离子、亚硫酸根类离子以及含磷类离子中的至少一者，优选钠离子、镁离子、钾离子、碳酸根类离子、亚硫酸根类离子和含磷类离子中的至少一者。
其中所述磷酸供体选自ATP或其盐、ADP或其盐、AMP或其盐、CTP或其盐、GTP或其盐、UTP或其盐、ITP或其盐和多聚磷酸或其盐中的至少一者，优选ATP或其盐、ADP或其盐、AMP或其盐和多聚磷酸或其盐中的至少一者。
其中所述磷酸供体可以为多聚磷酸或ATP或其盐，所述己糖源来自D-葡萄糖，并且所述生物酶为生物酶组，该生物酶组包含聚磷酸-葡萄糖磷酸转移酶(EC 2.7.1.63)、葡萄糖-6-磷酸异构酶(EC 5.3.1.9)、己酮糖6-磷酸异构酶(EC 5.3.1.27)、己酮糖6-磷酸合酶(EC 4.1.2.43)、核糖5-磷酸异构酶(EC 5.3.1.6)、磷酸核糖二磷酸激酶(EC 2.7.6.1)和烟酰胺磷酸核糖转移酶(EC 2.4.2.12)；其中生物酶组中的聚磷酸-葡萄糖磷酸转移酶以多聚磷酸和D-葡萄糖为底物转化合成至葡萄糖-6-磷酸和多聚磷酸(n-1)，生物酶组中的葡萄糖-6-磷酸异构酶以葡萄糖-6-磷酸为底物转化合成至果糖-6-磷酸，生物酶组中的己酮糖6-磷酸异构酶以果糖-6-磷酸为底物转化合成至阿拉伯糖-3-己酮糖-6-磷酸酯，生物酶组中的己酮糖6-磷酸合酶以阿拉伯糖-3-己酮糖-6-磷酸酯为底物转化合成至核酮糖-5-磷酸和甲醛，生物酶组中的核糖5-磷酸异构酶核酮糖-5-磷酸为底物转化合成至核糖-5-磷酸，生物酶组中的磷酸核糖二磷酸激酶以核糖-5-磷酸和磷酸供体为底物转化合成至5-磷酰核糖-1-焦磷酸和AMP，生物酶组中的烟酰胺磷酸核糖转移酶以烟酰胺和5-磷酰核糖-1-焦磷酸为底物转化合成至β-烟酰胺单核苷酸和焦磷酸，或以烟酸为底物转化合成至烟酸单核苷酸和焦磷酸。
其中所述己糖源来自D-葡萄糖，并且所述生物酶为生物酶组，该生物酶组包含葡萄糖异构酶(EC 5.3.1.5)、己糖激酶(EC 2.7.1.1)、葡萄糖-6-磷酸异构酶(EC 5.3.1.9)、己酮糖6-磷酸异构酶(EC 5.3.1.27)、己酮糖6-磷酸合酶(EC 4.1.2.43)、核糖5-磷酸异构酶(EC 5.3.1.6)、磷酸核糖二磷酸激酶(EC 2.7.6.1)和烟酰胺磷酸核糖转移酶(EC 2.4.2.12)。
其中所述己糖源来自D-果糖，并且所述生物酶为生物酶组，该生物酶组包含己糖激酶(EC 2.7.1.1)、己酮糖6-磷酸异构酶(EC 5.3.1.27)、己酮糖6-磷酸合酶(EC 4.1.2.43)、核糖5-磷酸异构酶(EC 5.3.1.6)、磷酸核糖二磷酸激酶(EC 2.7.6.1)和烟酰胺磷酸核糖转移酶(EC 2.4.2.12)。
其中所述己糖源来自D-甘露糖，并且所述生物酶为生物酶组，该生物酶组包含甘露糖激酶(EC 2.7.1.7)、甘露糖-6-磷酸异构酶(EC 5.3.1.8)、己酮糖6-磷酸异构酶(6EC 5.3.1.27)、己酮糖6-磷酸合酶(EC 4.1.2.43)、核糖5-磷酸异构酶(EC 5.3.1.6)、磷酸核糖二磷酸激酶(EC 2.7.6.1)和烟酰胺磷酸核糖转移酶。
其中所述己糖源来自蔗糖、双糖麦芽糖、棉子糖、麦芽糊精和淀粉中的至少一者并且其中在生物酶催化反应之前，将所述己糖源用选自热、酸、碱中 的至少一种方法水解蔗糖转化为选自D-葡萄糖、半乳糖、D-果糖和D-甘露糖中的至少一种单糖，然后将所述单糖投入反应底物中；或者
其中所述己糖源为蔗糖、双糖麦芽糖类、棉子糖、麦芽糊精和淀粉中的至少一者，并且直接使用生物酶组将包含所述己糖源和烟酸或其衍生物的核苷和反应底物转化为β-烟酰胺单核苷酸，其中所述生物酶组包含淀粉酶类(EC 3.2.1.1-3)、普鲁兰酶(EC 3.2.1.41)、麦芽糖酶(EC 3.2.1.20)、异麦芽糖酶(EC 3.2.1.10)、α-半乳糖苷酶(EC 3.2.1.22)、蔗糖酶(EC 3.2.1.26)、、蔗糖α-葡糖苷酶(EC 3.2.1.48)、聚磷酸-葡萄糖磷酸转移酶(EC 2.7.1.63)、葡萄糖-6-磷酸异构酶(EC 5.3.1.9)、己糖激酶(EC 2.7.1.1)、己酮糖6-磷酸异构酶(EC 5.3.1.27)、己酮糖6-磷酸合酶(EC 4.1.2.43)、核糖5-磷酸异构酶(EC 5.3.1.6)、磷酸核糖二磷酸激酶(EC 2.7.6.1)和烟酰胺磷酸核糖转移酶(EC 2.4.2.12)中的至少一者。
其中，所述蔗糖包含白砂糖、黄砂糖、赤砂糖、绵白糖、单晶体冰糖、多晶体冰糖、红糖、黑糖、冰片糖、方糖、糖霜、液体糖浆中的至少一者；并且
所述双糖麦芽糖包含选自麦芽糖和异麦芽糖中的至少一者。
其中，所述生物酶包含ATP再生酶，ATP再生酶包含聚磷酸-AMP磷酸转移酶(EC 2.7.4.B2)、多聚磷酸激酶(EC 2.7.4.1)、腺苷激酶(EC 2.7.1.20)中的至少一者；并且其中所述方法还包括ATP再生步骤，该步骤包括：聚磷酸-AMP磷酸转移酶以多聚磷酸和AMP为底物转化生成ADP，多聚磷酸激酶以多聚磷酸和AMP/ADP为底物转化生成ADP/ATP，以及腺苷激酶以两分子的ADP为底物转化生成ATP和AMP。
其中，多聚磷酸激酶(EC 2.7.4.1)包括II类和III类中的两组酶，其中使用II类的多聚磷酸激酶以多聚磷酸和ADP为底物转化生成ATP，使用III类的多聚磷酸激酶以多聚磷酸和AMP/ADP为底物转化生成ADP/ATP；在使用II类的多聚磷酸激酶时由AMP转化合成至ATP时还包括使用聚磷酸-AMP磷酸转移酶和/或腺苷激酶，并且在使用III类的多聚磷酸激酶时，所述方法还包括单独使用该酶由AMP转化合成至ADP和ATP。
其中，所述反应底物还包含多聚磷酸、腺苷和腺嘌呤中的至少一者，所述磷酸供体为ATP，所述生物酶包含ATP再生酶，并且ATP再生酶包含腺苷酸激酶(EC 2.7.4.3)、腺嘌呤磷酸核糖转移酶(EC 2.4.2.7)中的至少一者，
其中所述方法还包括ATP再生步骤，该步骤包括：腺苷酸激酶先以一分子的腺苷和ATP为底物转化生成AMP和ADP，然后使用所述ATP再生酶组以多聚磷酸为主要底物将AMP和ADP转化生成两分子的ATP或一分子的ADP和ATP；腺嘌呤磷酸核糖转移酶则以一分子的腺嘌呤和5-磷酰核糖-1-焦磷酸为底物转化生成一分子的AMP和磷酸，然后使用ATP再生酶组以多聚磷酸为主要底物将AMP转化生成ADP和ATP。
其中，所述反应底物还包含烟酰胺和烟酸中的至少一者，并且所述单核苷酸为β-烟酰胺单核苷酸和烟酸单核苷酸中的至少一者。
其中所述方法还包括在反应底物中添加出用于吸咐或分解甲醛的隋性物料的步骤，优选地所述惰性物料为沸石。
其中所生物酶组为重组酶，该重组酶在所属的菌种进行合成并于大肠杆菌HB101中进行表达和提取；
优选地，所述生物酶为选自细胞破碎液、上清酶液、纯酶和以任何方式制备的固定化酶/细胞中的至少一种形式。
2.一种制备生物产物的方法，包括下列步骤：
(i)采用包含己糖源和烟酸或其衍生物的反应底物，在磷酸供体的存在下，经生物酶催化反应生成烟酸或其衍生物的单核苷酸；以及
(ii)利用生成的烟酸或其衍生物的单核苷酸作为中间体制备所述生物产物。
其中所述生物产物选自烟酰胺核苷或其任何盐分的衍生物、氧化型烟酰胺腺嘌呤二核苷酸或其任何盐分的衍生物、还原型烟酰胺腺嘌呤二核苷酸或其任何盐分的衍生物、氧化型烟酰胺腺嘌呤二核苷酸磷酸或其任何盐分的衍生物、还原型烟酰胺腺嘌呤二核苷酸磷酸或其任何盐分的衍生物、烟酸核苷或其任何盐分的衍生物、烟酸腺嘌呤二核苷酸或其任何盐分的衍生物和烟酸腺嘌呤二核苷酸磷酸或其任何盐分的衍生物中的至少一者。
本发明具有以下优点和积极效果：
1.本发明的方法采用新型的酶法制备或生产烟酸或其衍生物的单核苷酸以及采用该单核苷酸作为中间体制备烟酸或其衍生物的核苷等生物产物，克服了提取法和化学合成法的缺点。
2.本发明利用生物酶制备烟酸或其衍生物的单核苷酸及其生物产物除了继承该生产方式的优点外面，更比传统酶法工艺的成本更相宜，而且其中的制备方法与生物中的烟酸或其衍生物的核苷和单核苷酸(例如β-烟酰胺单核苷酸/烟酸单核苷酸)的生成相呼应，其产物较化学生产更适合生物所使用。
3.本发明选择各种的己糖源替代核糖作为底物原料之一制备β-烟酰胺单核苷酸和烟酸单核苷酸，通过各种酶组合的使用以进行β-烟酰胺单核苷酸/烟酸单核苷酸的生产，使用不同的己糖和产生己糖的多糖进行生产时只需使用相配的生物酶组合而不需要作大规模的生产设备改动，有利于灵活工业生产操作。
4.己糖源可以优选为D-葡萄糖和D-果糖：这两种己糖都是流通全球并且价格低廉，是代替核糖为原材料的理想选择，并且各种生物酶的配合上更可以使用更多糖类，其中蔗糖、淀粉、麦芽糖等也在日常生活中十分普遍，因此本发明的制备方法的多样化和可持续性是传统酶法无可比拟的。
5.本发明的制备方法还可以使用ATP再生酶或其组合：ATP再生酶或其组合可以循环使用反应体系中的ATP，进一步降低生产成本，同时酶法反应中的ADP和AMP皆为副产物，使用该酶组转化合成该副产物至底物对β-烟酰胺单核苷酸/的烟酸单核苷酸生产更为有利。ATP再生酶或其组合则使该制备方法更多样化，以腺苷和腺嘌呤辅助ATP的使用能降低ATP的使用量，降低生产成本的同时可减少传统酶法生产中对ATP用量的依赖。
6.本发明的制备方法还可以使用隋性物料去除制备方法中所产生的甲醛，隋性物料可以为沸石或具有相等效果的材料如活性炭；去除副产物能对烟酸或其衍生物的单核苷酸及其各种生物产物的生产更为有利，提升酶法反应的产能。
附图简要说明
图1示出聚磷酸葡糖磷酸转移酶的SDS-PAGE凝胶电泳结果图，其中
列1为聚磷酸葡糖磷酸转移酶细胞破碎液；
列2为聚磷酸葡糖磷酸转移酶酶上清液以及
列3为预染蛋白分子量标准(PageRuler TM Prestained Protein Ladder，10到180kDa,Thermo Fisher Scientific)。
图2示出葡萄糖-6-磷酸异构酶的SDS-PAGE凝胶电泳结果图，其中
列2为葡萄糖-6-磷酸异构酶细胞破碎液；列3为葡萄糖-6-磷酸异构酶上清液以及列1为预染蛋白分子量标准(PageRuler TM Prestained Protein Ladder,10到180kDa,Thermo Fisher Scientific)。
图3示出己糖激酶的SDS-PAGE凝胶电泳结果图，其中
列1:己糖激酶细胞破碎液；
列2:己糖激酶酶上清液；以及
列3:预染蛋白分子量标准(PageRuler TM Prestained Protein Ladder,10到180kDa,Thermo Fisher Scientific)。
图4示出6-磷酸己酮糖异构酶的SDS-PAGE凝胶电泳结果图，其中
列1为6-磷酸己酮糖异构酶细胞破碎液；
列2为6-磷酸己酮糖异构酶上清液以及
列3为预染蛋白分子量标准(PageRuler TM Prestained Protein Ladder,10 to 180kDa,Thermo Fisher Scientific)。
图5示出己酮糖6-磷酸合酶的SDS-PAGE凝胶电泳结果图，其中
列1:己酮糖6-磷酸合酶细胞破碎液；
列2:己酮糖6-磷酸合酶酶上清液；
列3:预染蛋白分子量标准(PageRuler TM Prestained Protein Ladder,10至180kDa,Thermo Fisher Scientific)。
图6示出核糖5-磷酸异构酶的SDS-PAGE凝胶电泳结果图，其中
列1:预染蛋白分子量标准(PageRuler TM Prestained Protein Ladder,10 to 180kDa,Thermo Fisher Scientific)；
列2:核糖5-磷酸异构酶细胞破碎液；
列3:核糖5-磷酸异构酶酶上清液。
图7示出磷酸核糖二磷酸激酶的SDS-PAGE凝胶电泳结果图，其中
列1:预染蛋白分子量标准(PageRuler TM Prestained Protein Ladder,10 to 180kDa,Thermo Fisher Scientific)
列2:磷酸核糖二磷酸激酶细胞破碎液；以及
列3:磷酸核糖二磷酸激酶酶上清液。
图8示出烟酰胺磷酸核糖转移酶的SDS-PAGE凝胶电泳结果图，其中
列1:预染蛋白分子量标准(PageRuler TM Prestained Protein Ladder,10到180kDa,Thermo Fisher Scientific)
列2:烟酰胺磷酸核糖转移酶细胞破碎液
列3:烟酰胺磷酸核糖转移酶酶上清液。
图9示出多聚磷酸-AMP磷酸转移酶的SDS-PAGE凝胶电泳结果图，其中
列1:预染蛋白分子量标准(PageRuler TM Prestained Protein Ladder,10到180kDa,Thermo Fisher Scientific)
列2:多聚磷酸-AMP磷酸转移酶细胞破碎液；
列3:多聚磷酸-AMP磷酸转移酶酶上清液。
图10示出腺苷酸激酶的SDS-PAGE凝胶电泳结果图，其中
列1:预染蛋白分子量标准(PageRuler TM Prestained Protein Ladder,10到180kDa,Thermo Fisher Scientific)
列2:腺苷酸激酶细胞破碎液
列3:腺苷酸激酶酶上清液。
图11示出多聚磷酸激酶的SDS-PAGE凝胶电泳结果图，其中
列1:预染蛋白分子量标准(PageRuler TM Prestained Protein Ladder,10到180kDa,Thermo Fisher Scientific)；
列2:多聚磷酸激酶细胞破碎液；
列3:多聚磷酸激酶酶上清液。
图12示出甘露糖激酶细胞的SDS-PAGE凝胶电泳结果图，其中
列1:甘露糖激酶细胞破碎液；
列2:甘露糖激酶酶上清液；
列3:预染蛋白分子量标准(PageRuler TM Prestained Protein Ladder,10到180kDa,Thermo Fisher Scientific)。
图13示出甘露糖-6-磷酸异构酶的SDS-PAGE凝胶电泳结果图，其中
列1:预染蛋白分子量标准(PageRuler TM Prestained Protein Ladder,10 to 180kDa,Thermo Fisher Scientific)；
列2:甘露糖-6-磷酸异构酶细胞破碎液；以及
列3:甘露糖-6-磷酸异构酶酶上清液。
图14示出聚麦芽糖酶的SDS-PAGE凝胶电泳结果图，其中
列1:预染蛋白分子量标准(PageRuler TM Prestained Protein Ladder,10到180kDa,Thermo Fisher Scientific)
列2:麦芽糖酶细胞破碎液
列3:麦芽糖酶酶上清液。
图15示出蔗糖酶的SDS-PAGE凝胶电泳结果图，其中
列1:预染蛋白分子量标准(PageRuler TM Prestained Protein Ladder，10到180kDa,Thermo Fisher Scientific)；
列2:蔗糖酶细胞破碎液；以及
列3:蔗糖酶酶上清液。
图16示出5’-核苷酸酶的SDS-PAGE凝胶电泳结果图，其中
列1:预染蛋白分子量标准(PageRuler TM Prestained Protein Ladder,10到180kDa,Thermo Fisher Scientific)；
列2:5’-核苷酸酶细胞破碎液；以及
列3:5’-核苷酸酶酶上清液。
具体实施方式
以下通过具体实施方式的描述对本发明作进一步说明，但这并非是对本发明的限制，本领域技术人员根据本发明的基本思想，可以做出各种修改或改进，但是只要不脱离本发明的基本思想，均在本发明的范围之内。
本发明人开发了一种方制备烟酸或其衍生物的单核苷酸的方法，包括下列步骤：采用包含己糖源和烟酸或其衍生物的反应底物，在磷酸供体的存在下，经生物酶催化反应生成烟酸或其衍生物的单核苷酸。以己糖源(例如多糖、D-葡萄糖和D-果糖等单糖)为烟酸或其衍生物的单核苷酸的生产工艺的原材料，打破传统酶法工艺上使用核糖或更稀有和昂贵的化合物为原材料的做法。
本发明同时开启了工艺上的便利：在两种己糖为原材料的同时，使用不同的己糖时只需使用或者更换相配合的酶组合进行生产而其中的生产设备无需 更换并且可以互通使用，确保了工业上的可行性。本发明人认为，烟酸或其衍生物的单核苷酸(例如β-烟酰胺单核苷酸)或以其作为中间体制备的各种生物产物的长远发展必须建基于制备方法上可提供的多样性和可持续性，而这两方面皆与原材料的供应和价格息息相关。传统酶法工艺只能倚靠核糖为单一原材料，做法与上述两大重点背道而驰。本发明人还意识到现有核糖的供求在技术上的量产发展远远追不上应用的步伐，若再加上烟酸或其衍生物的单核苷酸(例如β-烟酰胺单核苷酸)或以其作为中间体制备的各种生物产物的需求，情况在可预见的将来会更加严峻，直接打击烟酸或其衍生物的单核苷酸(例如β-烟酰胺单核苷酸)或以其作为中间体制备的各种生物产物在社会上的普及。如此同时，工艺上缺乏多样性和可持续性对投资生产烟酸或其衍生物的核苷和单核苷酸(例如β-烟酰胺单核苷酸)带来不确定的因素，对工业生产存在隐忧。
本发明人开发的方法，在解决当前的问题时同时将成本降低，比传统工艺上需要使用的三磷酸腺苷(ATP)的用量减少，再配合辅助组合中以多聚磷酸再生三磷酸腺苷进一步压定成本，创造更有利的条件。
具体来说，本发明的方法采用包含己糖源和烟酸或其衍生物的反应底物，在磷酸供体的存在下，经生物酶催化反应生成烟酸或其衍生物的单核苷酸。
烟酸或其衍生物可以为烟酸和烟酰胺。因此，烟酸或其衍生物的单核苷酸及其生物产物可以为β-烟酰胺单核苷酸、烟酸单核苷酸、氧化型烟酰胺腺嘌呤二核苷酸或其任何盐分的衍生物、还原型烟酰胺腺嘌呤二核苷酸或其任何盐分的衍生物、氧化型烟酰胺腺嘌呤二核苷酸磷酸或其任何盐分的衍生物、还原型烟酰胺腺嘌呤二核苷酸磷酸或其任何盐分的衍生物、烟酸核苷或其任何盐分的衍生物、烟酸单核苷酸、烟酸腺嘌呤二核苷酸或其任何盐分的衍生物和烟酸腺嘌呤二核苷酸磷酸或其任何盐分的衍生物，优选β-烟酰胺单核苷酸。烟酸和烟酰胺的结构是本领域已知的，并且可以通过市场方便地获得。
如上所述，本发明的一个显著优点是己糖源(例如多糖、D-葡萄糖和D-果糖等单糖)为烟酸或其衍生物的单核苷酸及其生物产物的生产工艺的原材料，这代替了传统酶法工艺上使用核糖或更稀有和昂贵的化合物为原材料的做法。己糖源可以来自具有六个碳原子的单糖、能够生成己糖的多糖或其混合物。
具有六个碳原子的单糖可以选自D-葡萄糖、D-甘露糖、D-半乳糖、D-果糖或其混合物中的任意一者，优选为D-葡萄糖、D-甘露糖和D-果糖，更优选为D-葡萄糖和D-果糖。
己糖源也可以来自通过糖苷键连接多种己糖单元的多糖，只要该多糖类能够被生物酶的活性催化降解至己糖单糖(如D-葡萄糖和/或D-果糖)即可。多糖可以为蔗糖、麦芽糖、菊粉、棉子糖、麦芽糊精、淀粉或其混合物，更优选为蔗糖、麦芽糖和棉子糖，最优选为蔗糖和麦芽糖。
蔗糖可以包含白砂糖、黄砂糖、赤砂糖、绵白糖、单晶体冰糖、多晶体冰糖、红糖、黑糖、冰片糖、方糖、糖霜、液体糖浆中的至少一者；并且麦芽糖可以包含选自麦芽糖和异麦芽糖中的至少一者。
在生物酶催化反应之前，可以通过选自热、酸和碱中的至少一种方法水解多糖转化为选自D-葡萄糖、半乳糖、D-果糖和D-甘露糖中的至少一种单糖，然后将所述单糖投入反应底物中。
磷酸供体是指在酶法反应中能够提供磷酸的任何化合物。磷酸供体可以选自ATP或其盐、ADP或其盐、AMP或其盐、CTP或其盐、GTP或其盐、UTP或其盐、ITP或其盐和不同磷酸链长度所组成的多聚磷酸或其盐中的至少一者。
在一个实施方案中，反应底物还可以包含辅助离子和多聚磷酸或其盐中的至少一者。多聚磷酸或其盐优选多聚磷酸的钠盐。多聚磷酸的聚合度可为3-20,000；优选地，多聚磷酸聚合度可为3-7,000，更优选为3-75。
任选地，辅助离子可以包含金属离子、氯离子、镁离子、钙离子、钾离子、钠离子、锌离子、氟离子、硫离子、碳酸根类离子、亚硫酸根类离子以及含磷类离子中的至少一者，优选钠离子、镁离子、钾离子、碳酸根类离子、亚硫酸根类离子和含磷类离子中的至少一者。辅助离子可为其无机盐或有机盐的状态，优选为六水氯化镁、氯化钠、氯化锰、硫酸镁和碳酸钾中的至少一者，更优选为六水氯化镁、氯化钠和碳酸钾中的至少一者。
此外，本领域公知的是，反应底物还可以包含其他添加剂，例如，pH调节剂，如缓冲液/盐，优选为磷酸钠缓冲液、磷酸钾缓冲液和三羟甲基氨基甲烷缓冲液、更优选为磷酸钠缓冲液和三羟甲基氨基甲烷缓冲液。pH调节剂的浓度可以为0.001M-1M，优选为0.01M-0.5M，更优选为0.05M-0.3M。
在一个实施方案中，生物酶为单独的生物酶或者包含多种生物酶的生物酶组。
在一个实施方案中，生物酶组为重组酶，该重组酶在所属的菌种进行合成并于载体中进行表达和提取。
优选地，所述生物酶为选自细胞破碎液、上清酶液、纯酶和固定化酶/细胞中的至少一种形式。
任选的，载体可以包括大肠杆菌(例如大肠杆菌HB101)、酵母菌。这样，可以使用包含重组酶的细胞或其碎片作为所述生物酶。例如，细胞可以为大肠杆菌细胞、酵母菌细胞。载体可以包括大肠杆菌、酵母菌、芽胞杆菌等生物科学中常用的方式来进行重组酶的表达。生物酶或生物酶组可以在细胞、破碎液、上清液或纯化酶的液态中使用，或以任何方式和其对应载体制成固定化细胞或固定化酶进行酶催化反应。
根据本发明的一个实施方案，可以根据底物的组成选择合适或者相配合的生物酶或者生物酶组。
例如，当反应底物含有烟酰胺或烟酸、多聚磷酸和D-葡萄糖，并且磷酸供体为ATP时，生物酶组包含聚磷酸-葡萄糖磷酸转移酶(EC 2.7.1.63)、葡萄糖-6-磷酸异构酶(EC 5.3.1.9)、己酮糖6-磷酸异构酶(EC 5.3.1.27)、己酮糖6-磷酸合酶(EC 4.1.2.43)、核糖5-磷酸异构酶(EC 5.3.1.6)、磷酸核糖二磷酸激酶(EC 2.7.6.1)和烟酰胺磷酸核糖转移酶(EC 2.4.2.12)中的至少一者。在这种情况下，具体的反应过程包括：生物酶组中的聚磷酸-葡萄糖磷酸转移酶以多聚磷酸和D-葡萄糖为底物转化合成至葡萄糖-6-磷酸和多聚磷酸(n-1)，生物酶组中的葡萄糖-6-磷酸异构酶以葡萄糖-6-磷酸为底物转化合成至果糖-6-磷酸，生物酶组中的己酮糖6-磷酸异构酶以果糖-6-磷酸为底物转化合成至阿拉伯糖-3-己酮糖-6-磷酸酯，生物酶组中的己酮糖6-磷酸合酶以阿拉伯糖-3-己酮糖-6-磷酸酯为底物转化合成至核酮糖-5-磷酸和甲醛，生物酶组中的核糖5-磷酸异构酶核酮糖-5-磷酸为底物转化合成至核糖-5-磷酸，生物酶组中的磷酸核糖二磷酸激酶以核糖-5-磷酸和磷酸供体为底物转化合成至5-磷酰核糖-1-焦磷酸和AMP，生物酶组中的烟酰胺磷酸核糖转移酶以烟酰胺和5-磷酰核糖-1-焦磷酸为底物转化合成至β-烟酰胺单核苷酸和焦磷酸，或以烟酸为底物转化合成至烟酸单核苷酸和焦磷酸。
如果己糖源来自D-果糖，生物酶组可以包含己糖激酶(EC 2.7.1.1)、己酮糖6-磷酸异构酶(EC 5.3.1.27)、己酮糖6-磷酸合酶(EC 4.1.2.43)、核糖5-磷酸异构酶(EC 5.3.1.6)、磷酸核糖二磷酸激酶(EC 2.7.6.1)和烟酰胺磷酸核糖转移酶(EC 2.4.2.12)中的至少一者。
如果己糖源来自D-葡萄糖，生物酶组可以包含葡萄糖异构酶(EC 5.3.1.5)、己糖激酶(EC 2.7.1.1)、己酮糖6-磷酸异构酶(EC 5.3.1.27)、己酮糖6-磷酸合酶(EC 4.1.2.43)、核糖5-磷酸异构酶(EC 5.3.1.6)、磷酸核糖二磷酸激酶(EC 2.7.6.1)和烟酰胺磷酸核糖转移酶(EC 2.4.2.12)中的至少一者。
如果己糖源来自D-甘露糖，生物酶组可以包含甘露糖激酶(EC 2.7.1.7)、甘露糖-6-磷酸异构酶(EC 5.3.1.8)、己酮糖6-磷酸异构酶(6EC 5.3.1.27)、己酮糖6-磷酸合酶(EC 4.1.2.43)、核糖5-磷酸异构酶(EC 5.3.1.6)、磷酸核糖二磷酸激酶(EC 2.7.6.1)和烟酰胺磷酸核糖转移酶中的至少一者。
当己糖源为蔗糖、双糖麦芽糖类、棉子糖、麦芽糊精和淀粉中的至少一者，可以直接使用生物酶组将包含所述己糖源和烟酸或其衍生物的核苷和反应底物转化为β-烟酰胺单核苷酸/单核苷酸。在这种情况下，生物酶组包含淀粉酶类(EC 3.2.1.1-3)、普鲁兰酶(EC 3.2.1.41)、麦芽糖酶(EC 3.2.1.20)、异麦芽糖酶(EC 3.2.1.10)、α-半乳糖苷酶(EC 3.2.1.22)、蔗糖酶(EC 3.2.1.26)、、蔗糖α-葡糖苷酶(EC 3.2.1.48)、聚磷酸-葡萄糖磷酸转移酶(EC 2.7.1.63)、葡萄糖-6-磷酸异构酶(EC 5.3.1.9)、己糖激酶(EC 2.7.1.1)、己酮糖6-磷酸异构酶(EC 5.3.1.27)、己酮糖6-磷酸合酶(EC 4.1.2.43)、核糖5-磷酸异构酶(EC 5.3.1.6)、磷酸核糖二磷酸激酶(EC 2.7.6.1)和烟酰胺磷酸核糖转移酶(EC 2.4.2.12)中的至少一者。
根据一个优选的实施方案，生物酶还可以包含ATP再生酶，ATP再生酶可以包含聚磷酸-AMP磷酸转移酶(EC 2.7.4.B2)、多聚磷酸激酶(EC 2.7.4.1)、腺苷激酶(EC 2.7.1.20)中的至少一者。在这种情况下，本发明的方法还可以包括ATP再生步骤，该步骤包括：聚磷酸-AMP磷酸转移酶以多聚磷酸和AMP为底物转化生成ADP，多聚磷酸激酶以多聚磷酸和AMP/ADP为底物转化生成ADP/ATP，以及腺苷激酶以两分子的ADP为底物转化生成ATP和AMP。
任选地，多聚磷酸激酶(EC 2.7.4.1)包括II类和III类中的两组酶，其中使用II类的多聚磷酸激酶以多聚磷酸和ADP为底物转化生成ATP，使用III类的多聚磷酸激酶则可以以多聚磷酸和AMP/ADP为底物转化生成ADP/ATP；在使用II类的多聚磷酸激酶时由AMP转化合成至ATP时还包括使用聚磷酸-AMP磷酸转移酶和/或腺苷激酶，并且在使用III类的多聚磷酸激酶时，本发明的方法还可以包括单独使用该酶由AMP转化合成至ADP和ATP。
本发明的反应底物还可以包含腺苷和腺嘌呤中的至少一者，并且ATP再生酶包含腺苷酸激酶(EC 2.7.4.3)、腺嘌呤磷酸核糖转移酶(EC 2.4.2.7)中的至少一者，
根据一个优选的实施方案，本发明的方法还可以包括ATP再生步骤，该步骤包括：腺苷酸激酶先以一分子的腺苷和ATP为底物转化生成AMP和ADP，然后使用所述ATP再生酶组以多聚磷酸为主要底物将AMP和ADP转化生成两分子的ATP；腺嘌呤磷酸核糖转移酶则以一分子的腺嘌呤和5-磷酰核糖-1-焦磷酸为底物转化生成一分子的AMP和磷酸，然后使用ATP再生酶组以多聚磷酸为主要底物将AMP转化生成ATP。
本发明的方法还可以包括下列的任意一个步骤或其组合：
1.以己糖类中的D-葡萄糖为反应底物，生物酶组包含聚磷酸-葡萄糖磷酸转移酶(polyphosphate-glucose phosphotransferase；EC 2.7.1.63)、葡萄糖-6-磷酸异构酶(glucose-6-phosphate isomerase；EC 5.3.1.9)、己酮糖6-磷酸异构酶(6-phospho-3-hexuloisomerase；EC 5.3.1.27)、己酮糖6-磷酸合酶(3-hexulose-6-phosphate synthase；EC 4.1.2.43)、核糖5-磷酸异构酶(ribose-5-phosphate isomerase；EC 5.3.1.6)、磷酸核糖二磷酸激酶(ribose-phosphate diphosphokinase；EC 2.7.6.1)和烟酰胺磷酸核糖转移酶(nicotinamide phosphoribosyltransferase；EC 2.4.2.12)中的至少一者；其中酶组中的聚磷酸-葡萄糖磷酸转移酶以多聚磷酸和D-葡萄糖为底物转化合成至葡萄糖-6-磷酸和多聚磷酸(n-1)；酶组中的葡萄糖-6-磷酸异构酶以葡萄糖-6-磷酸为底物转化合成至果糖-6-磷酸；酶组中的己酮糖6-磷酸异构酶以果糖-6-磷酸为底物转化合成至阿拉伯糖-3-己酮糖-6-磷酸酯；酶组中的己酮糖6-磷酸合酶以阿拉伯糖-3-己酮糖-6-磷酸酯为底物转化合成至核酮糖-5-磷酸和甲醛；酶组中的核糖5-磷酸异构酶核酮糖-5-磷酸为底物转化合成至核糖-5-磷酸；酶组中的磷酸核糖二磷酸激酶以核糖-5-磷酸和ATP为底物转化 合成至5-磷酰核糖-1-焦磷酸和AMP；酶组中的烟酰胺磷酸核糖转移酶以烟酰胺和5-磷酰核糖-1-焦磷酸为底物转化合成至β-烟酰胺单核苷酸和焦磷酸，或以烟酸为底物转化合成至烟酸单核苷酸和焦磷酸；
2.以己糖类中的D-葡萄糖为反应底物，生物酶组包含葡萄糖异构酶(glucose isomerase；EC 5.3.1.5)、己糖激酶(hexokinase；EC 2.7.1.1)、己酮糖6-磷酸异构酶(6-phospho-3-hexuloisomerase；EC 5.3.1.27)、己酮糖6-磷酸合酶(3-hexulose-6-phosphate synthase；EC 4.1.2.43)、核糖5-磷酸异构酶(ribose-5-phosphate isomerase；EC 5.3.1.6)、磷酸核糖二磷酸激酶(ribose-phosphate diphosphokinase；EC 2.7.6.1)和烟酰胺磷酸核糖转移酶(nicotinamide phosphoribosyltransferase；EC 2.4.2.12)中的至少一者；
3.以己糖类中的D-果糖为反应底物，其中生物酶组包含己糖激酶(hexokinase；EC 2.7.1.1)、己酮糖6-磷酸异构酶(6-phospho-3-hexuloisomerase；EC 5.3.1.27)、己酮糖6-磷酸合酶(3-hexulose-6-phosphate synthase；EC 4.1.2.43)、核糖5-磷酸异构酶(ribose-5-phosphate isomerase；EC 5.3.1.6)、磷酸核糖二磷酸激酶(ribose-phosphate diphosphokinase；EC 2.7.6.1)和烟酰胺磷酸核糖转移酶(nicotinamide phosphoribosyltransferase；EC 2.4.2.12)中的至少一者。
4.以己糖类中的D-甘露糖为反应底物，其中生物酶组包含甘露糖激酶(mannokinase；EC 2.7.1.7)、甘露糖-6-磷酸异构酶(mannose-6-phosphate isomerase；EC 5.3.1.8)、己酮糖6-磷酸异构酶(6-phospho-3-hexuloisomerase；EC 5.3.1.27)、己酮糖6-磷酸合酶(3-hexulose-6-phosphate synthase；EC 4.1.2.43)、核糖5-磷酸异构酶(ribose-5-phosphate isomerase；EC 5.3.1.6)、磷酸核糖二磷酸激酶(ribose-phosphate diphosphokinase；EC 2.7.6.1)和烟酰胺磷酸核糖转移酶中的至少一者；
5.以双糖类中的蔗糖类为反应底物，其中可以包括白砂糖、黄砂糖、赤砂糖、绵白糖、单晶体冰糖、多晶体冰糖、红糖、黑糖、冰片糖、方糖、糖霜、液体糖浆等；蔗糖的分子结构是由己糖类中的D-葡萄糖和D-果糖组成，可以先使用常用的物理方法如热、酸、碱等进行水解由双糖降解至单糖后使用上述的生物酶组进行转化合成至β-烟酰胺单核苷酸/烟酸单核苷酸；
6.以双糖类中的蔗糖类为反应底物并直接使用生物酶组转化合成至β-烟酰胺单核苷酸/烟酸单核苷酸；该生物酶组包含蔗糖酶类(sucrase)中的一种或多 种的酶、聚磷酸-葡萄糖磷酸转移酶(polyphosphate-glucose phosphotransferase；EC 2.7.1.63)、葡萄糖-6-磷酸异构酶(glucose-6-phosphate isomerase；EC 5.3.1.9)、己糖激酶(hexokinase；EC 2.7.1.1)、己酮糖6-磷酸异构酶(6-phospho-3-hexuloisomerase；EC 5.3.1.27)、己酮糖6-磷酸合酶(3-hexulose-6-phosphate synthase；EC 4.1.2.43)、核糖5-磷酸异构酶(ribose-5-phosphate isomerase；EC 5.3.1.6)、磷酸核糖二磷酸激酶(ribose-phosphate diphosphokinase；EC 2.7.6.1)和烟酰胺磷酸核糖转移酶(nicotinamide phosphoribosyltransferase；EC 2.4.2.12)；
7.以双糖麦芽糖类为反应底物，其中可以包括麦芽糖和异麦芽糖；麦芽糖类的分子结构是由己糖类中的两个D-葡萄糖组成，可以先使用常用的物理方法如热、酸、碱等进行水解由双糖降解至单糖后使用上述的酶组进行转化合成至β-烟酰胺单核苷酸/烟酸单核苷酸；麦芽糖类还可以直接使用酶组转化合成至产物；该酶组包含麦芽糖酶(maltase；EC 3.2.1.20)和/或异麦芽糖酶(isomaltase；EC 3.2.1.10)、聚磷酸-葡萄糖磷酸转移酶(polyphosphate-glucose phosphotransferase；EC 2.7.1.63)、葡萄糖-6-磷酸异构酶(glucose-6-phosphate isomerase；EC 5.3.1.9)、己糖激酶(hexokinase；EC 2.7.1.1)、己酮糖6-磷酸异构酶(6-phospho-3-hexuloisomerase；EC 5.3.1.27)、己酮糖6-磷酸合酶(3-hexulose-6-phosphate synthase；EC 4.1.2.43)、核糖5-磷酸异构酶(ribose-5-phosphate isomerase；EC 5.3.1.6)、磷酸核糖二磷酸激酶(ribose-phosphate diphosphokinase；EC 2.7.6.1)和烟酰胺磷酸核糖转移酶(nicotinamide phosphoribosyltransferase；EC 2.4.2.12)中的至少一者；
8.以三糖类中的棉子糖为反应底物；棉子糖的分子结构是由己糖类中的半乳糖、D-葡萄糖和D-果糖组成；该酶组包含α-半乳糖苷酶(alpha-galactosidase；EC 3.2.1.22)、蔗糖酶类(sucrase)中的一种或多种的酶、聚磷酸-葡萄糖磷酸转移酶(polyphosphate-glucose phosphotransferase；EC 2.7.1.63)、葡萄糖-6-磷酸异构酶(glucose-6-phosphate isomerase；EC 5.3.1.9)、己糖激酶(hexokinase；EC 2.7.1.1)、己酮糖6-磷酸异构酶(6-phospho-3-hexuloisomerase；EC 5.3.1.27)、己酮糖6-磷酸合酶(3-hexulose-6-phosphate synthase；EC 4.1.2.43)、核糖5-磷酸异构酶(ribose-5-phosphate isomerase；EC 5.3.1.6)、磷酸核糖二磷酸激酶(ribose-phosphate diphosphokinase；EC 2.7.6.1)和烟酰胺磷酸核糖转移酶(nicotinamide phosphoribosyltransferase；EC 2.4.2.12)中的至少一者；或者
9.以常见的多糖类如麦芽糊精和淀粉等在其结构上全部或部分以D-葡萄糖和/或D-果糖组成为底物，该多糖可以是先使用常用的物理方法如热、酸、碱等进行水解至单糖继而使用上述的生物酶组转化合成至β-烟酰胺单核苷酸/烟酸单核苷酸；该多糖类还可以使用包括淀粉酶类(Amylase；EC 3.2.1.1-3)、普鲁兰酶类(Pullulanase；EC 3.2.1.41)等水解多糖类至单糖并单独或混合使用上述的的酶组转化合成至β-烟酰胺单核苷酸/烟酸单核苷酸。
在一个方面中，蔗糖酶中的一种或多种的酶可以是包含蔗糖酶(invertase；EC 3.2.1.26)、异麦芽糖酶(isomaltase；EC 3.2.1.10)、蔗糖α-葡糖苷酶(sucrose alpha-glucosidase；EC 3.2.1.48)等，只要该酶的活性能催化多糖类降解至己糖类的D-葡萄糖和/或D-果糖即可。
作为一个优选实施方案，本发明的方法还可以包括在反应底物中添加出用于吸咐或分解甲醛的隋性物料的步骤。优选地，惰性物料可以是只与甲醛发生物理或化学上的变化并不对生物酶反应具有任何影响的物料，优选沸石、三氧化铝、活性炭、石灰、滤膜等。
在一个实施方案中，本发明的方法中的反应条件可以包括：温度为25-40℃，优选为30-39℃，更优选为35-38℃。反应体系的pH为6.0-8.5，优选为pH 7.0-8.0，更优选为pH7.5-7.8。
如上所述，烟酸或其衍生物的单核苷酸可以用作各种有价值的生物产物的中间体或者前体。因此，本发明还提供了一种制备生物产物的方法，包括下列步骤：
(i)采用包含己糖源和烟酸或其衍生物的反应底物，在磷酸供体的存在下，经生物酶催化反应生成烟酸或其衍生物的单核苷酸；以及
(ii)利用生成的烟酸或其衍生物的单核苷酸作为中间体制备所述生物产物。
生物产物可以选自烟酰胺核苷或其任何盐分的衍生物、氧化型烟酰胺腺嘌呤二核苷酸或其任何盐分的衍生物、还原型烟酰胺腺嘌呤二核苷酸或其任何盐分的衍生物、氧化型烟酰胺腺嘌呤二核苷酸磷酸或其任何盐分的衍生物、还原型烟酰胺腺嘌呤二核苷酸磷酸或其任何盐分的衍生物、烟酸核苷或其任何盐分的衍生物、烟酸腺嘌呤二核苷酸或其任何盐分的衍生物和烟酸腺嘌呤二核苷酸磷酸或其任何盐分的衍生物中的至少一者。
在一个实施方案中，本发明的方法包括将烟酰胺和烟酸混合使用，并且在酶反应后可同时产得β-烟酰胺单核苷酸和烟酸单核苷酸。
在一个实施方案中，本发明制备的方法生产的β-烟酰胺单核苷酸和/或烟酸单核苷酸可以使用为中间体以生物酶法或化学合成化的方法进一步转化合成得到烟酸核糖、烟酸腺苷二核苷酸、磷酸烟酸腺苷二核苷酸、烟酰胺核糖、还原型和氧化型烟酰胺腺苷二核苷酸和还原型和氧化型磷酸烟酸腺苷二核苷酸中的至少一者。
为了进行本发明的反应，可以将所述的固定化细胞和固定化酶置于固定化反应装置中，以进行固定化反应。例如，可按照中国专利申请CN106032520A记载的步骤进行所述固定化反应。
固定化反应装置可以包括具有入口和出口的柱状反应器、反应调控罐、高流量水泵、pH调控装置与pH探头、温度调控装置与温度探头。
在一个具体例子中，本发明的方法可以包括：首先以多聚酶链式反应从生物酶所属的菌种进行重组酶蛋白合成并于大肠杆菌HB101中进行表达，将该菌体以细胞破碎机进行破碎和离心后取得酶上清液并且以其破碎液或上清酶液制备固定化酶/细胞；在反应罐中进行100ml反应溶液配置，当中加入1.21g三羟甲基氨基甲烷、90mg D-葡萄糖(食用葡萄糖)、406mg的六水氯化镁、667mg的多聚磷酸盐、242mg烟酰胺和605mg三磷酸腺苷二钠盐，加入80ml纯水后以0.1M盐酸/氢氧化钠调节至pH 7.5-7.8；反应溶液维持在37℃和pH 7.5并加入20g沸石进行搅拌，同时加入1ml聚磷酸-葡萄糖磷酸转移酶上清酶液、1ml葡萄糖-6-磷酸异构酶上清酶液混合、2ml己酮糖6-磷酸异构酶上清酶液、2ml己酮糖6-磷酸合酶上清酶液、2ml核糖5-磷酸异构酶上清酶液、0.5ml磷酸核糖二磷酸激酶上清酶液和0.5ml烟酰胺磷酸核糖转移酶上清酶液和0.5ml多聚磷酸激酶上清酶液、0.25ml腺苷酸激酶上清酶液和0.25ml聚磷酸-AMP磷酸转移酶上清酶液，以附件2的高效液相色谱分析反应液中β-烟酰胺单核苷酸的浓度变化，反应3小时后β-烟酰胺单核苷酸的浓度达至2mM，而4小时的样本中浓度变化不大，可以结束酶反应，以中速滤纸将沸石过滤后得含有β-烟酰胺单核苷酸溶液。
当以D-果糖为的方法的原材料时，反应溶液的配置和组合有变化外，反应设备则保持不变。反应罐中进行100ml反应1溶液配置，当中加入1.21g三 羟甲基氨基甲烷，90mg D-果糖、406mg的六水氯化镁、667mg的多聚磷酸盐、242mg烟酰胺和605mg三磷酸腺苷二钠盐，加入80ml纯水后以0.1M盐酸/氢氧化钠调节至pH 7.5-7.8；反应溶液维持在37℃和pH 7.5并加入20g沸石进行搅拌，同时加入1ml己糖激酶上清酶液和1ml多聚磷酸激酶上清酶液混合、2ml己酮糖6-磷酸异构酶上清酶液、2ml己酮糖6-磷酸合酶上清酶液、2ml核糖5-磷酸异构酶上清酶液、0.5ml磷酸核糖二磷酸激酶上清酶液和0.5ml烟酰胺磷酸核糖转移酶上清酶液和0.5ml多聚磷酸激酶上清酶液、0.25ml腺苷酸激酶上清酶液和0.25ml聚磷酸-AMP磷酸转移酶上清酶液，以附件2的高效液相色谱分析反应液中β-烟酰胺单核苷酸的浓度变化，反应3小时后β-烟酰胺单核苷酸的浓度达至1.8mM，而4小时的样本中浓度变化不大，可以结束酶反应，以中速滤纸将沸石过滤后得含有β-烟酰胺单核苷酸溶液。
以下通过例子的方式进一步解释或说明本发明内容，但这些例子不应被理解为对本发明保护范围的限制。
例子
以下例子中未注明具体条件的，均按常规条件或制造商建议的条件进行。除非特别说明，否则所述百分比为重量百分比。
下列例子中所用材料和设备的描述如下：
反应调控罐：来自基因港(香港)生物科技有限公司，BR-1L；
可调节流量式吸水泵：购自SURGEFLO公司，FL-32；
酸碱度调控装置：来自基因港(香港)生物科技有限公司，AR-1；
LB培养基:购自上海阿拉丁生化科技股份有限公司；
IPTG:购自上海阿拉丁生化科技股份有限公司；
氨苄青霉素:购自上海阿拉丁生化科技股份有限公司；
葡萄糖-6-磷酸钠盐:购自Merck,USA；
果糖-6-磷酸二钠盐：购自Merck,USA；
己酮糖6-磷酸:来自基因港(香港)生物科技有限公司；
核酮糖5-磷酸钠盐:购自Merck,USA；
核糖5-磷酸二钠盐:购自Merck,USA；
甘露糖6-磷酸钠盐:购自Merck,USA；
磷酸核糖二磷酸五钠盐:购自Merck,USA；
单磷酸腺苷二钠盐：购自Merck,USA；
二磷酸腺苷钠盐：购自Merck,USA；
腺苷:购自Merck,USA；
六水氯化镁：购自Merck,USA；
多聚磷酸纳：购自上海阿拉丁生化科技股份有限公司；
三羟甲基氨基甲烷：购自Merck,USA；
三磷酸腺苷二钠盐：购自Merck,USA；
D-葡萄糖一水：购自购自Merck,USA；
D-果糖:购自购自Merck,USA；
D-甘露糖:购自Merck,USA；
麦芽糖:购自Merck,USA；
蔗糖:购自Merck,USA；
烟酰胺：购自Merck,USA；
烟酸：购自Merck,USA；
制备例1：制备聚磷酸-葡萄糖磷酸转移酶(EC 2.7.1.63)
基于Mycobacteroides abscessus基因组中的编码聚磷酸-葡萄糖磷酸转移酶的DNA序列STZ38851.1(SEQ3)设计PCR引物，具体为
上游引物PGPT1：
5’-CTGACC GGATCCATGACAGCCACCGACTCCGCACCG-3'(SEQ1)
下游引物PGPT2：
5'-TATGCG GAATTCCTAAGGCGAAACGCCAGCGTGTGC-3'(SEQ2)
以分枝杆菌(Mycobacteroides abscessus)的DNA为底物，以上述引物进行PCR扩增得聚磷酸-葡萄糖磷酸转移酶基因，利用限制性内切酶BamH I和EcoRI处理PCR产物并将其连接至pET-21a中，得到pET-PGPT。将此重组表达载体转化至大肠杆菌HB101中，得到聚磷酸-葡萄糖磷酸转移酶重组表达菌株。
将上述菌株挑选单一种落接种到4mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养16小时作为初级种子，完成后按1％接种比例接到100mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养10小时作为二级种子，完成后按1％接种比例接到60L LB培养基(含100 ug/ml氨苄青霉素)的100L发酵罐中培养。发酵初始条件为37℃、200rpm、pH7.0。发酵进行至9小时加入IPTG至最终浓度为1mM，发酵20小时结束。发酵液在4℃下以12,500rpm离心10分钟，得含聚磷酸-葡萄糖磷酸转移酶的大肠杆菌细胞1.22kg。将所得含聚磷酸-葡萄糖磷酸转移酶的大肠杆菌细胞配制成上清酶液；上清酶液的配制方法为每1g的细胞中加入磷酸钠缓冲液(PBS100mM pH 7.5)打浆后，使用压力式细胞破碎器以700-800MPa的设定下进行破碎得细胞破碎液并以管式离心机在10，000rpm和100L/hr的设定下进行离心取上清液，每1ml上清酶液中含0.2g细胞。如图1所示，SDS-PAGE凝胶电泳测试证实了聚磷酸-葡萄糖磷酸转移酶的合成。
根据其酶法反应对细胞进行酶活力检测，该方法为在1ml的反应溶液(200mM PBS pH 7.5,10mM多聚磷酸盐(n＝3-20)，5mM D-葡萄糖)中加入含有1mg总蛋白量的上清酶液，在摄氏37度的温控下进行5分钟反应，完成后以附件1的高效液相色谱方法对样本中在酶反应中所产生的进行葡萄糖-6-磷酸含量分析。根据上述方法本上清酶液的酶活为约0.22nmol/min/mg。
制备例2：制备葡萄糖-6-磷酸异构酶(EC 5.3.1.9)
基于大肠肝菌(Escherichia colis)基因组中的编码葡萄糖-6-磷酸异构酶的DNA序列KXG93161.1(SEQ6)设计PCR引物，具体为
葡萄糖-6-磷酸异构酶序列设计PCR引物，具体为
上游引物G6PI1：
5’-CTGACC GGATCCATGAAAAACATCAATCCAACGCAG-3'(SEQ4)
下游引物G6PI2：
5'-TATGCG GAATTCTTAACCGCGCCACGCTTTATAGCG-3'(SEQ5)
以大肠肝菌(Escherichia colis)的DNA为底物，以上述引物进行PCR扩增得葡萄糖-6-磷酸异构酶基因，利用限制性内切酶BamH I和EcoRI处理PCR产物并将其连接至pET-21a中，得到pET-G6PI。将此重组表达载体转化至大肠杆菌HB101中，得到葡萄糖-6-磷酸异构酶重组表达菌株。
将上述菌株挑选单一种落接种到4mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养16小时作为初级种子，完成后按1％接种比 例接到100mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养10小时作为二级种子，完成后按1％接种比例接到60L LB培养基(含100ug/ml氨苄青霉素)的100L发酵罐中培养。发酵初始条件为37℃、200rpm、pH7.0。发酵进行至9小时加入IPTG至最终浓度为1mM，发酵20小时结束。发酵液在4℃下以12,500rpm离心10分钟，得含葡萄糖-6-磷酸异构酶的大肠杆菌细胞1.07kg。将所得含葡萄糖-6-磷酸异构酶的大肠杆菌细胞配制成上清酶液：上清酶液的配制方法为每1g的细胞中加入磷酸钠缓冲液(PBS 100mM pH 7.5)打浆后，使用压力式细胞破碎器以700-800MPa的设定下进行破碎得细胞破碎液并以管式离心机在10，000rpm和100L/hr的设定下进行离心取上清液，每1ml上清酶液中含0.2g细胞。如图2所示，SDS-PAGE凝胶电泳测试证实了葡萄糖-6-磷酸异构酶的合成。
根据其酶法反应对细胞进行酶活力检测，该方法为在1ml的反应溶液(200mM PBS pH 7.5，5mM葡萄糖-6-磷酸钠盐)中加入含有1mg总蛋白量的上清酶液，在摄氏37度的温控下进行5分钟反应，完成后以附件1的高效液相色谱对样本中在酶反应中所产生的进行果糖-6-磷酸含量分析。根据上述方法本上清酶液的酶活约为0.24nmol/min/mg。
制备例3：制备己糖激酶(EC 2.7.1.1)
基于脆弱拟杆菌(Bacteroides fragilis)基因组中的编码己糖激酶的DNA序列EEZ28043.1(SEQ9)设计PCR引物，具体为
己糖激酶序列设计PCR引物，具体为
上游引物FHK-1：
5’-CTGACC GGATCCATGGAGAAGAATATTTTCAAACTG-3'(SEQ7)
下游引物FHK-2：
5'-TATGCG GAATTCTCAGGATAATGCCGCAATCCCCGT-3'(SEQ8)
以脆弱拟杆菌(Bacteroides fragilis)的DNA为底物，以上述引物进行PCR扩增得己糖激酶基因，利用限制性内切酶BamH I和EcoRI处理PCR产物并将其连接至pET-21a中，得到pET-FHK。将此重组表达载体转化至大肠杆菌HB101中，得到己糖激酶重组表达菌株。
将上述菌株挑选单一种落接种到4mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养16小时作为初级种子，完成后按1％接种比例接到100mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养10小时作为二级种子，完成后按1％接种比例接到60L LB培养基(含100ug/ml氨苄青霉素)的100L发酵罐中培养。发酵初始条件为37℃、200rpm、pH7.0。发酵进行至9小时加入IPTG至最终浓度为1mM，发酵20小时结束。发酵液在4℃下以12,500rpm离心10分钟，得含己糖激酶的大肠杆菌细胞1.55kg。将所得含己糖激酶的大肠杆菌细胞配制成上清酶液：上清酶液的配制方法为每1g的细胞中加入磷酸钠缓冲液(PBS 100mM pH 7.5)打浆后，使用压力式细胞破碎器以700-800MPa的设定下进行破碎得细胞破碎液并以管式离心机在10，000rpm和100L/hr的设定下进行离心取上清液，每1ml上清酶液中含0.2g细胞。如图3所示，SDS-PAGE凝胶电泳测试证实了己糖激酶的合成。
根据其酶法反应对细胞进行酶活力检测，该方法为在1ml的反应溶液(200mM PBS pH 7.5，10mM D-果糖，5mM三磷酸腺苷二钠盐，20mM六水氯化镁)中加入含有1mg总蛋白量的上清酶液，在摄氏37度的温控下进行5分钟反应，完成后以附件1的高效液相色谱方法对样本中在酶反应中所产生的进行果糖-6-磷酸含量分析。根据上述方法本酶液的酶活约为0.18nmol/min/mg。
制备例4：制备己酮糖6-磷酸异构酶(EC 5.3.1.27)
基于枯草芽孢杆菌(Bacillus subtilis)基因组中的编码己酮糖6-磷酸异构酶的DNA序列AOR96716.1(SEQ12)设计PCR引物，具体为
己酮糖6-磷酸异构酶序列设计PCR引物，具体为
上游引物6P3HI-1：
5’-CTGACC GGATCCATGAAAACGACTGAATACGTAGCG-3'(SEQ10)
下游引物6P3HI-2：
5'-TATGCG GAATTCCTATTCAAGGTTTGCGTGGTGAGT-3'(SEQ11)
以枯草芽孢杆菌(Bacillus subtilis)的DNA为底物，以上述引物进行PCR扩增得己酮糖6-磷酸异构酶基因，利用限制性内切酶BamH I和EcoRI处理PCR产物并将其连接至pET-21a中，得到pET-6P3HI。将此重组表达载体转化至大肠杆菌HB101中，得到己酮糖6-磷酸异构酶重组表达菌株。
将上述菌株挑选单一种落接种到4mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养16小时作为初级种子，完成后按1％接种比例接到100mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养10小时作为二级种子，完成后按1％接种比例接到60L LB培养基(含100ug/ml氨苄青霉素)的100L发酵罐中培养。发酵初始条件为37℃、200rpm、pH7.0。发酵进行至9小时加入IPTG至最终浓度为1mM，发酵20小时结束。发酵液在4℃下以12,500rpm离心10分钟，得含己酮糖6-磷酸异构酶的大肠杆菌细胞1.57kg。将所得含己酮糖6-磷酸异构酶的大肠杆菌细胞配制成上清酶液：上清酶液的配制方法为每1g的细胞中加入磷酸钠缓冲液(PBS 100mM pH 7.5)打浆后，使用压力式细胞破碎器以700-800MPa的设定下进行破碎得细胞破碎液并以管式离心机在10,000rpm和100L/hr的设定下进行离心取上清液，每1ml上清酶液中含0.2g细胞。如图4所示，SDS-PAGE凝胶电泳测试证实了己酮糖6-磷酸异构酶的合成。
根据其酶法反应对细胞进行酶活力检测，该方法为在1ml的反应溶液(200mM PBS pH 7.5，5mM果糖-6-磷酸二钠盐，20mM六水氯化镁)中加入含有1mg总蛋白量的上清酶液，在摄氏37度的温控下进行5分钟反应，完成后以附件1的高效液相色谱对样本中在酶反应中所产生的进行己酮糖6-磷酸含量分析。根据上述方法本上清酶液的酶活约为0.12nmol/min/mg。
制备例5：制备己酮糖6-磷酸合酶(EC 4.1.2.43)
基于枯草芽孢杆菌枯草亚种(Bacillus subtilis subsp.subtilis)基因组中的编码己酮糖6-磷酸合酶的DNA序列ARW30002.1(SEQ15)设计PCR引物，具体为
己酮糖6-磷酸合酶序列设计PCR引物，具体为
上游引物3H6PS-1：
5’-CTGACC GGATCCATGGAATTACAGCTTGCATTAGAC-3'(SEQ13)
下游引物3H6PS-2：
5'-TATGCG GAATTCTTATCCTTGGACAATCAGCTGCTT-3'(SEQ14)
以枯草芽孢杆菌枯草亚种(Bacillus subtilis subsp.subtilis)的DNA为底物，以上述引物进行PCR扩增得己酮糖6-磷酸合酶基因，利用限制性内切酶BamH I和EcoRI处理PCR产物并将其连接至pET-21a中，得到pET-3H6PS。将此重组表达载体转化至大肠杆菌HB101中，得到己酮糖6-磷酸合酶重组表达菌株。
将上述菌株挑选单一种落接种到4mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养16小时作为初级种子，完成后按1％接种比例接到100mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养10小时作为二级种子，完成后按1％接种比例接到60L LB培养基(含100ug/ml氨苄青霉素)的100L发酵罐中培养。发酵初始条件为37℃、200rpm、pH7.0。发酵进行至9小时加入IPTG至最终浓度为1mM，发酵20小时结束。发酵液在4℃下以12,500rpm离心10分钟，得含己酮糖6-磷酸合酶的大肠杆菌细胞1.91kg。将所得含己酮糖6-磷酸合酶的大肠杆菌细胞配制成上清酶液。上清酶液的配制方法为每1g的细胞中加入磷酸钠缓冲液(PBS 100mM pH 7.5)打浆后，使用压力式细胞破碎器以700-800MPa的设定下进行破碎得细胞破碎液并以管式离心机在10,000rpm和100L/hr的设定下进行离心取上清液，每1ml上清酶液中含0.2g细胞。如图5所示，SDS-PAGE凝胶电泳测试证实了己酮糖6-磷酸合酶的合成。
根据其酶法反应对细胞进行酶活力检测，该方法为在1ml的反应溶液(200mM PBS pH 7.5，5mM己酮糖6-磷酸，20mM六水氯化镁)中加入含有1mg总蛋白量的上清酶液，在摄氏37度的温控下进行5分钟反应，完成后以附件1的高效液相色谱对样本中在酶反应中所产生的进行核酮糖5-磷酸含量分析。根据上述方法本上清酶液的酶活约为0.07nmol/min/mg。
制备例6：制备核糖5-磷酸异构酶(EC 5.3.1.6)
基于大肠杆菌(Escherichia coli)基因组中的编码核糖5-磷酸异构酶的DNA序列QNS47947.1(SEQ18)设计PCR引物，具体为
上游引物R5PI-1：
5’-CTGACC GGATCCATGACGCAGGATGAATTGAAAAAA-3'(SEQ16)
下游引物R5PI-2：
5'-TATGCG GAATTCTCATTTCACAATGGTTTTGACACC-3'(SEQ17)
以大肠杆菌(Escherichia coli)的DNA为底物，以上述引物进行PCR扩增得核糖5-磷酸异构酶基因，利用限制性内切酶BamH I和EcoRI处理PCR产物并将其连接至pET-21a中，得到pET-R5PI。将此重组表达载体转化至大肠杆菌HB101中，得到核糖5-磷酸异构酶重组表达菌株。
将上述菌株挑选单一种落接种到4mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养16小时作为初级种子，完成后按1％接种比例接到100mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养10小时作为二级种子，完成后按1％接种比例接到60L LB培养基(含100ug/ml氨苄青霉素)的100L发酵罐中培养。发酵初始条件为37℃、200rpm、pH7.0。发酵进行至9小时加入IPTG至最终浓度为1mM，发酵20小时结束。发酵液在4℃下以12,500rpm离心10分钟，得含核糖5-磷酸异构酶的大肠杆菌细胞1.02kg。将所得含核糖5-磷酸异构酶的大肠杆菌细胞配制成上清酶液:上清酶液的配制方法为每1g的细胞中加入磷酸钠缓冲液(PBS 100mM pH 7.5)打浆后，使用压力式细胞破碎器以700-800MPa的设定下进行破碎得细胞破碎液并以管式离心机在10，000rpm和100L/hr的设定下进行离心取上清液，每1ml上清酶液中含0.2g细胞。如图5所示，SDS-PAGE凝胶电泳测试证实了核糖5-磷酸异构酶的合成。
根据其酶法反应对细胞进行酶活力检测，该方法为在1ml的反应溶液(200mM PBS pH 7.5，5mM核酮糖5-磷酸钠盐，20mM六水氯化镁)中加入含有1mg总蛋白量的上清酶液，在摄氏37度的温控下进行5分钟反应，完成后以附件1的高效液相色谱对样本中在酶反应中所产生的进行核糖5-磷酸含量分析。根据上述方法本上清酶液的酶活约为0.16nmol/min/mg。
制备例7：制备磷酸核糖二磷酸激酶(EC 2.7.6.1)
基于大肠杆菌(Escherichia coli)基因组中的编码磷酸核糖二磷酸激酶的DNA序列KXG95745.1(SEQ21)设计PCR引物，具体为
磷酸核糖二磷酸激酶序列设计PCR引物，具体为
上游引物PPDK-1：
5’-CTGACC GGATCCATGCCTGAGGTTCTTCTCGTGCCT-3'(SEQ19)
下游引物PPDK-2：
5'-TATGCG GAATTCTTAGTGTTCGAACATGGCAGAGAT-3'(SEQ20)
以大肠杆菌(Escherichia coli)的DNA为底物，以上述引物进行PCR扩增得磷酸核糖二磷酸激酶基因，利用限制性内切酶BamH I和EcoRI处理PCR产物并将其连接至pET-21a中，得到pET-R5PI。将此重组表达载体转化至大肠杆菌HB101中，得到磷酸核糖二磷酸激酶重组表达菌株。
将上述菌株挑选单一种落接种到4mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养16小时作为初级种子，完成后按1％接种比例接到100mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养10小时作为二级种子，完成后按1％接种比例接到60L LB培养基(含100ug/ml氨苄青霉素)的100L发酵罐中培养。发酵初始条件为37℃、200rpm、pH7.0。发酵进行至9小时加入IPTG至最终浓度为1mM，发酵20小时结束。发酵液在4℃下以12,500rpm离心10分钟，得含磷酸核糖二磷酸激的大肠杆菌细胞1.67kg。将所得含磷酸核糖二磷酸激酶的大肠杆菌细胞配制成上清酶液:上清酶液的配制方法为每1g的细胞中加入磷酸钠缓冲液(PBS 100mM pH 7.5)打浆后，使用压力式细胞破碎器以700-800MPa的设定下进行破碎得细胞破碎液并以管式离心机在10，000rpm和100L/hr的设定下进行离心取上清液，每1ml上清酶液中含0.2g细胞。如图7所示，SDS-PAGE凝胶电泳测试证实了磷酸核糖二磷酸激酶的合成。
根据其酶法反应对细胞进行酶活力检测，该方法为在1ml的反应溶液(200mM PBS pH 7.5，10mM核糖5-磷酸二钠盐，5mM三磷酸腺苷二钠盐，20mM六水氯化镁)中加入含有1mg总蛋白量的上清酶液，在摄氏37度的温控下进行5分钟反应，完成后以附件1的高效液相色谱对样本中在酶反应中所产生的进行磷酸核糖二磷酸含量分析。根据上述方法本上清酶液的酶活约为0.14nmol/min/mg。
制备例8：制备烟酰胺磷酸核糖转移酶(EC 2.4.2.12)
基于吡啶菌(Rhodococcus pyridinivorans)基因组中的编码烟酰胺磷酸核糖转移酶的DNA序列RKE26735.1(SEQ24)设计PCR引物，具体为
烟酰胺磷酸核糖转移酶序列设计PCR引物，具体为
上游引物NAMPT-1：
5’-CTGACC GGATCCATGCCCACTATCTCGGGATTCGAC-3'(SEQ22)
下游引物NAMPT-2：
5'-TATGCG GAATTCTCAGGCAGGCACCGCGGCGCTGTC-3'(SEQ23)
以吡啶菌(Rhodococcus pyridinivorans)的DNA为底物，以上述引物进行PCR扩增得烟酰胺磷酸核糖转移酶基因，利用限制性内切酶BamH I和EcoRI处理PCR产物并将其连接至pET-21a中，得到pET-R5PI。将此重组表达载体转化至大肠杆菌HB101中，得到烟酰胺磷酸核糖转移酶重组表达菌株。
将上述菌株挑选单一种落接种到4mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养16小时作为初级种子，完成后按1％接种比例接到100mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养10小时作为二级种子，完成后按1％接种比例接到60L LB培养基(含100ug/ml氨苄青霉素)的100L发酵罐中培养。发酵初始条件为37℃、200rpm、pH7.0。发酵进行至9小时加入IPTG至最终浓度为1mM，发酵20小时结束。发酵液在4℃下以12,500rpm离心10分钟，得含烟酰胺磷酸核糖转移酶的大肠杆菌细胞1.22kg。将所得含烟酰胺磷酸核糖转移酶的大肠杆菌细胞配制成上清酶液:上清酶液的配制方法为每1g的细胞中加入磷酸钠缓冲液(PBS 100mM pH 7.5)打浆后，使用压力式细胞破碎器以700-800MPa的设定下进行破碎得细胞破碎液并以管式离心机在10，000rpm和100L/hr的设定下进行离心取上清液，每1ml上清酶液中含0.2g细胞。如图8所示，SDS-PAGE凝胶电泳测试证实了烟酰胺磷酸核糖转移酶的合成。
根据其酶法反应对细胞进行酶活力检测，该方法为在1ml的反应溶液(200mM PBS pH 7.5，5mM磷酸核糖二磷酸五钠盐，10mM烟酰胺，20mM六水氯化镁)中加入含有1mg总蛋白量的上清酶液，在摄氏37度的温控下进行5分钟反应，完成后以附件2的高效液相色谱对样本中在酶反应中所产生的进行β-烟酰胺单核苷酸含量分析。根据上述方法本上清酶液的酶活约为0.09nmol/min/mg。
制备例9：制备辅助组合的多聚磷酸-AMP磷酸转移酶(EC 2.7.4.B2)
基于约氏不动杆菌(Acinetobacter johnsonii)基因组中的编码多聚磷酸-AMP磷酸转移酶的DNA序列BAC76403.1(SEQ27)设计PCR引物，具体为
多聚磷酸-AMP磷酸转移酶序列设计PCR引物，具体为
上游引物PAP1-1：
5’-CTGACC GGATCCATGGATACAGAAACGATCGCCAGT-3'(SEQ25)
下游引物PAP1-2：
5'-TATGCG GAATTCTTAATCCGTGTCGCGATCCGCTTT-3'(SEQ26)
以约氏不动杆菌(Acinetobacter johnsonii)的DNA为底物，以上述引物进行PCR扩增得多聚磷酸-AMP磷酸转移酶基因，利用限制性内切酶BamH I和EcoRI处理PCR产物并将其连接至pET-21a中，得到pET-PAP1。将此重组表达载体转化至大肠杆菌HB101中，得到多聚磷酸-AMP磷酸转移酶重组表达菌株。
将上述菌株挑选单一种落接种到4mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养16小时作为初级种子，完成后按1％接种比例接到100mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养10小时作为二级种子，完成后按1％接种比例接到60L LB培养基(含100ug/ml氨苄青霉素)的100L发酵罐中培养。发酵初始条件为37℃、200rpm、pH7.0。发酵进行至9小时加入IPTG至最终浓度为1mM，发酵20小时结束。发酵液在4℃下以12,500rpm离心10分钟，得含多聚磷酸-AMP磷酸转移酶的大肠杆菌细胞1.01kg。将所得含多聚磷酸-AMP磷酸转移酶的大肠杆菌细胞配制成上清酶液：上清酶液的配制方法为每1g的细胞中加入磷酸钠缓冲液(PBS100mM pH 7.5)打浆后，使用压力式细胞破碎器以700-800MPa的设定下进行破碎得细胞破碎液并以管式离心机在10，000rpm和100L/hr的设定下进行离心取上清液，每1ml上清酶液中含0.2g细胞。如图9所示，SDS-PAGE凝胶电泳测试证实了多聚磷酸-AMP磷酸转移酶的合成。
根据其酶法反应对细胞进行酶活力检测，该方法为在1ml的反应溶液(200mM PBS pH 7.5，5mM磷酸核糖二磷酸，10mM单磷酸腺苷二钠盐，5mM多聚磷酸盐，20mM六水氯化镁)中加入含有1mg总蛋白量的上清酶液，在摄氏37度的温控下进行5分钟反应，完成后以附件2的高效液相色谱对样本中在酶反应中所产生的进行二磷酸腺苷分析。根据上述方法本酶液的酶活约为0.12nmol/min/mg。
制备例10：制备腺苷酸激酶(EC 2.7.4.3)
基于杀螟硫磷降解菌(Burkholderia sp.MR1)基因组中的编码腺苷酸激酶的DNA序列KIG05708.1(SEQ30)设计PCR引物，具体为
腺苷酸激酶序列设计PCR引物，具体为
上游引物AK-1：
5’-CTGACC GGATCCATGCGTTTGATCCTGTTGGGCGCG-3'(SEQ28)
下游引物AK-2：
5'-TATGCG GAATTCTTACTTCAATGCCTCGAACACGCG-3'(SEQ29)
以杀螟硫磷降解菌(Burkholderia sp.MR1)的DNA为底物，以上述引物进行PCR扩增得腺苷酸激酶基因，利用限制性内切酶BamH I和EcoRI处理PCR产物并将其连接至pET-21a中，得到pET-AK。将此重组表达载体转化至大肠杆菌HB101中，得到腺苷酸激酶重组表达菌株。
将上述菌株挑选单一种落接种到4mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养16小时作为初级种子，完成后按1％接种比例接到100mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养10小时作为二级种子，完成后按1％接种比例接到60L LB培养基(含100ug/ml氨苄青霉素)的100L发酵罐中培养。发酵初始条件为37℃、200rpm、pH7.0。发酵进行至9小时加入IPTG至最终浓度为1mM，发酵20小时结束。发酵液在4℃下以12,500rpm离心10分钟，得含腺苷酸激酶的大肠杆菌细胞1.33kg。将所得含腺苷酸激酶的大肠杆菌细胞配制成上清酶液：上清酶液的配制方法为每1g的细胞中加入磷酸钠缓冲液(PBS 100mM pH 7.5)打浆后，使用压力式细胞破碎器以700-800MPa的设定下进行破碎得细胞破碎液并以管式离心机在10,000rpm和100L/hr的设定下进行离心取上清液，每1ml上 清酶液中含0.2g细胞。如图10所示，SDS-PAGE凝胶电泳测试证实了腺苷酸激酶的合成。
根据其酶法反应对细胞进行酶活力检测，该方法为在1ml的反应溶液(200mM PBS pH 7.5，5mM腺苷，10mM三磷酸腺苷二钠盐，20mM六水氯化镁)中加入含有1mg总蛋白量的酶液，在摄氏37度的温控下进行5分钟反应，完成后以附件2的高效液相色谱对样本中在酶反应中所产生的进行單磷酸腺苷分析。根据上述方法本酶液的酶活约为0.48nmol/min/mg。
制备例11：制备多多聚磷酸激酶(EC 2.7.4.1)
基于陶厄氏菌属细菌(Thauera sp.28)基因组中的编码多多聚磷酸激酶的DNA序列ENO92539.1(SEQ33)设计PCR引物，具体为
多多聚磷酸激酶序列设计PCR引物，具体为
上游引物PPK2-1：
5’-CTGACC GGATCCATGCCCCAGTTCAATCGCACCTCC-3'(SEQ31)
下游引物PPK2-2：
5'-TATGCG GAATTCTCAGCTGCTGGCTGCGAGCTCATT-3'(SEQ32)
以陶厄氏菌属细菌(Thauera sp.28)的DNA为底物，以上述引物进行PCR扩增得多多聚磷酸激酶基因，利用限制性内切酶BamH I和EcoRI处理PCR产物并将其连接至pET-21a中，得到pET-PPK2。将此重组表达载体转化至大肠杆菌HB101中，得到多多聚磷酸激酶重组表达菌株。
将上述菌株挑选单一种落接种到4mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养16小时作为初级种子，完成后按1％接种比例接到100mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养10小时作为二级种子，完成后按1％接种比例接到60L LB培养基(含100ug/ml氨苄青霉素)的100L发酵罐中培养。发酵初始条件为37℃、200rpm、pH7.0。发酵进行至9小时加入IPTG至最终浓度为1mM，发酵20小时结束。发酵液在4℃下以12,500rpm离心10分钟，得含多多聚磷酸激酶的大肠杆菌细胞1.11kg。将所得含多多聚磷酸激酶的大肠杆菌细胞配制成上清酶液：上清酶液的配制方法为每1g的细胞中加入磷酸钠缓冲液(PBS 100mM pH 7.5)打浆后，使用压力式细胞破碎器以700-800MPa的设定下进行破碎得细胞破碎液 并以管式离心机在10,000rpm和100L/hr的设定下进行离心取上清液，每1ml上清酶液中含0.2g细胞。如图11所示，SDS-PAGE凝胶电泳测试证实了多多聚磷酸激酶的合成。
根据其酶法反应对细胞进行酶活力检测，该方法为在1ml的反应溶液(200mM PBS pH 7.5，10mM二磷酸腺苷钠盐，5mM多聚磷酸盐，20mM六水氯化镁)中加入含有1mg总蛋白量的上清酶液，在摄氏37度的温控下进行5分钟反应，完成后以附件1的高效液相色谱对样本中在酶反应中所产生的进行三磷酸腺苷含量分析。根据上述方法本上清酶液的酶活约为0.03nmol/min/mg。
制备例12：制备甘露糖激酶(EC 2.7.1.7)
基于玫瑰单胞菌(Roseomonas mucosa)基因组中的编码甘露糖激酶的DNA序列AWV22863.1(SEQ36)设计PCR引物，具体为
甘露糖激酶序列设计PCR引物，具体为
上游引物MK-1：
5’-CTGACC GGATCCATGCGCATCAAGCTTGGCGTGGAT-3'(SEQ34)
下游引物MK-2：
5'-TATGCG GAATTCTCAGCCGCGGTCCCAGAGCCGGGC-3'(SEQ35)
以玫瑰单胞菌(Roseomonas mucosa)的DNA为底物，以上述引物进行PCR扩增得甘露糖激酶基因，利用限制性内切酶BamH I和EcoRI处理PCR产物并将其连接至pET-21a中，得到pET-MK。将此重组表达载体转化至大肠杆菌HB101中，得到甘露糖激酶重组表达菌株。
将上述菌株挑选单一种落接种到4mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养16小时作为初级种子，完成后按1％接种比例接到100mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养10小时作为二级种子，完成后按1％接种比例接到60L LB培养基(含100ug/ml氨苄青霉素)的100L发酵罐中培养。发酵初始条件为37℃、200rpm、pH7.0。发酵进行至9小时加入IPTG至最终浓度为1mM，发酵20小时结束。发酵液在4℃下以12,500rpm离心10分钟，得含甘露糖激酶的大肠杆菌细胞1.36kg。将所得含甘露糖激酶的大肠杆菌细胞配制成酶液。酶液的配制方法为 每1g的细胞中加入磷酸钠缓冲液(PBS 100mM pH 7.5)打浆后，使用压力式细胞破碎器以700-800MPa的设定下进行破碎得细胞破碎液并以管式离心机在10，000rpm和100L/hr的设定下进行离心取上清液，每1ml酶液中含0.2g细胞。如图12所示，SDS-PAGE凝胶电泳测试证实了甘露糖激酶的合成。
根据其酶法反应对细胞进行酶活力检测，该方法为在1ml的反应溶液(200mM PBS pH 7.5，5mM甘露糖，10mM三磷酸腺苷二钠盐，20mM六水氯化镁)中加入含有1mg总蛋白量的酶液，在摄氏37度的温控下进行5分钟反应，完成后以高效液相色谱对样本中在酶反应中所产生的进行甘露糖6-磷酸分析。根据上述方法本酶液的酶活约为0.14nmol/min/mg。
制备例13：制备甘露糖-6-磷酸异构酶(EC 5.3.1.8)
基于大肠杆菌(Escherichia coli)基因组中的编码甘露糖-6-磷酸异构酶的DNA序列BCA74065.1(SEQ39)设计PCR引物，具体为
甘露糖-6-磷酸异构酶序列设计PCR引物，具体为
上游引物M6PI-1：
5’-CTGACC GGATCCATGCAAAAACTCATTAACTCAGTG-3'(SEQ37)
下游引物M6PI-2：
5'-TATGCG GAATTCTTACAGCTTGTTGTAAACACGCGC-3'(SEQ38)
以大肠杆菌(Escherichia coli)的DNA为底物，以上述引物进行PCR扩增得甘露糖-6-磷酸异构酶基因，利用限制性内切酶BamH I和EcoRI处理PCR产物并将其连接至pET-21a中，得到pET-M6PI。将此重组表达载体转化至大肠杆菌HB101中，得到甘露糖-6-磷酸异构酶重组表达菌株。
将上述菌株挑选单一种落接种到4mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养16小时作为初级种子，完成后按1％接种比例接到100mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养10小时作为二级种子，完成后按1％接种比例接到60L LB培养基(含100ug/ml氨苄青霉素)的100L发酵罐中培养。发酵初始条件为37℃、200rpm、pH7.0。发酵进行至9小时加入IPTG至最终浓度为1mM，发酵20小时结束。发酵液在4℃下以12,500rpm离心10分钟，得含甘露糖-6-磷酸异构酶的大肠杆菌细胞0.78kg。将所得含甘露糖-6-磷酸异构酶的大肠杆菌细胞配制成上清酶 液:上清酶液的配制方法为每1g的细胞中加入磷酸钠缓冲液(PBS 100mM pH7.5)打浆后，使用压力式细胞破碎器以700-800MPa的设定下进行破碎得细胞破碎液并以管式离心机在10，000rpm和100L/hr的设定下进行离心取上清液，每1ml酶液中含0.2g细胞。如图13所示，SDS-PAGE凝胶电泳测试证实了甘露糖-6-磷酸异构酶的合成。
根据其酶法反应对细胞进行酶活力检测，该方法为在1ml的反应溶液(200mM PBS pH 7.5，10mM甘露糖6-磷酸钠盐，20mM六水氯化镁)中加入含有1mg总蛋白量的酶液，在摄氏37度的温控下进行5分钟反应，完成后以高效液相色谱对样本中在酶反应中所产生的进行果糖6-磷酸分析。根据上述方法本酶液的酶活约为0.21nmol/min/mg。
制备例14：制备麦芽糖酶(EC 3.2.1.20)
基于Aurantimicrobium minutum基因组中的编码麦芽糖酶的DNA序列BAV00088.1(SEQ42)设计PCR引物，具体为
麦芽糖酶序列设计PCR引物，具体为
上游引物MALT-1：
5’-CTGACC GGATCCATGGTTGAAAAAGAGTGGTGGCGT-3'(SEQ40)
下游引物MALT-2：
5'-TATGCG GAATTCCTAGACCAACCACACCGCAGTAGC-3'(SEQ41)
以Aurantimicrobium minutum的DNA为底物，以上述引物进行PCR扩增得麦芽糖酶酶基因，利用限制性内切酶BamH I和EcoRI处理PCR产物并将其连接至pET-21a中，得到pET-MALT。将此重组表达载体转化至大肠杆菌HB101中，得到麦芽糖酶重组表达菌株。
将上述菌株挑选单一种落接种到4mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养16小时作为初级种子，完成后按1％接种比例接到100mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养10小时作为二级种子，完成后按1％接种比例接到60L LB培养基(含100ug/ml氨苄青霉素)的100L发酵罐中培养。发酵初始条件为37℃、200rpm、pH7.0。发酵进行至9小时加入IPTG至最终浓度为1mM，发酵20小时结束。 发酵液在4℃下以12,500rpm离心10分钟，得到含麦芽糖酶的大肠杆菌细胞0.78kg。将所得含麦芽糖酶的大肠杆菌细胞配制成上清酶液:上清酶液的配制方法为每1g的细胞中加入磷酸钠缓冲液(PBS 100mM pH 7.5)打浆后，使用压力式细胞破碎器以700-800MPa的设定下进行破碎得细胞破碎液并以管式离心机在10，000rpm和100L/hr的设定下进行离心取上清液，每1ml酶液中含0.2g细胞。如图14所示，SDS-PAGE凝胶电泳测试证实了麦芽糖酶的合成。
根据其酶法反应对细胞进行酶活力检测，该方法为在1ml的反应溶液(200mM PBS pH 7.5，5mM麦芽糖，20mM六水氯化镁)中加入含有1mg总蛋白量的酶液，在摄氏37度的温控下进行5分钟反应，完成后以高效液相色谱对样本中在酶反应中所产生的进行葡萄糖分析。根据上述方法本酶液的酶活约为0.08nmol/min/mg。
制备例15：制备蔗糖酶(EC 3.2.1.26)
基于屎肠球菌(Enterococcus faecium)基因组中的编码蔗糖酶的DNA序列QQJ91524.1(SEQ45)设计PCR引物，具体为
麦芽糖酶序列设计PCR引物，具体为
上游引物SUC-1：
5’-CTGACC GGATCCATGGTTGAAAAAGAGTGGTGGCGT-3'(SEQ43)
下游引物SUC-2：
5'-TATGCG GAATTCCTAGACCAACCACACCGCAGTAGC-3'(SEQ44)
以屎肠球菌(Enterococcus faecium)的DNA为底物，以上述引物进行PCR扩增得麦芽糖酶酶基因，利用限制性内切酶BamH I和EcoRI处理PCR产物并将其连接至pET-21a中，得到pET-SUC。将此重组表达载体转化至大肠杆菌HB101中，得到蔗糖酶酶重组表达菌株。
将上述菌株挑选单一种落接种到4mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养16小时作为初级种子，完成后按1％接种比例接到100mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养10小时作为二级种子，完成后按1％接种比例接到60L LB培养基(含100ug/ml氨苄青霉素)的100L发酵罐中培养。发酵初始条件为37℃、200rpm、 pH7.0。发酵进行至9小时加入IPTG至最终浓度为1mM，发酵20小时结束。发酵液在4℃下以12,500rpm离心10分钟，得到含蔗糖酶的大肠杆菌细胞0.78kg。将所得含蔗糖酶的大肠杆菌细胞配制成上清酶液:上清酶液的配制方法为每1g的细胞中加入磷酸钠缓冲液(PBS 100mM pH 7.5)打浆后，使用压力式细胞破碎器以700-800MPa的设定下进行破碎得细胞破碎液并以管式离心机在10，000rpm和100L/hr的设定下进行离心取上清液，每1ml酶液中含0.2g细胞。如图15所示，SDS-PAGE凝胶电泳测试证实了蔗糖酶的合成。
根据其酶法反应对细胞进行酶活力检测，该方法为在1ml的反应溶液(200mM PBS pH 7.5，5mM蔗糖，20mM六水氯化镁)中加入含有1mg总蛋白量的酶液，在摄氏37度的温控下进行5分钟反应，完成后以高效液相色谱对样本中在酶反应中所产生的进行葡萄糖分析。根据上述方法本酶液的酶活约为0.06nmol/min/mg。
制备例16：制备5’-核苷酸酶(EC 3.1.3.5)
基于肠道沙门氏菌(Salmonella enterica)基因组中的编码5’-核苷酸酶的DNA序列AVB07708.1(SEQ48)设计PCR引物，具体为
上游引物NUCL1：
5’-CTGACC GGATCCATGAAAGTAAAACTGCTTGCTGCC-3'(SEQ46)
下游引物NUCL2：
5’-TATGCG GAATTCTTACTTCTTCACATCCGCAACGCG-3'(SEQ47)
以肠道沙门氏菌(Salmonella enterica)的基因组DNA为底物，以上述引物进行PCR扩增得5’-核苷酸酶基因，利用限制性内切酶BamH I和EcoR I处理PCR产物并将其连接至pET-21a中，得到pET-USHA。将此重组表达载体转化至大肠杆菌HB101中，得到5’-核苷酸酶重组表达菌株。
将上述菌株挑选单一菌落接种到4mL LB培养基(含100μg/ml氨苄青霉素)中，在37℃、200rpm摇床中培养16小时作为初级种子，完成后按1％接种比例接到100mL LB培养基(含100μg/ml氨苄青霉素)中，在37℃、200rpm摇床中培养10小时作为二级种子，完成后按1％接种比例接到容纳有60L LB培养基(含100μg/ml氨苄青霉素)的100L发酵罐中培养。发酵初始条件为37℃、200rpm、pH7.0。发酵进行至9小时加入IPTG至最终浓度为1mM，发酵20小 时结束。发酵液在4℃下以12,500rpm离心10分钟，得含5’-核苷酸酶的大肠杆菌细胞1.55kg。将所得含5’-核苷酸酶的大肠杆菌细胞配制成上清酶液：上清酶液的配制方法为每1g的细胞中加入磷酸钠缓冲液(PBS 100mM pH 7.5)打浆后，使用压力式细胞破碎器以700-800MPa的设定下进行破碎得细胞破碎液并以管式离心机在10,000rpm和100L/hr的设定下进行离心取上清液，每1ml酶液中含0.2g细胞。如图16所示，SDS-PAGE凝胶电泳测试证实了5’-核苷酸酶的合成。
根据其酶法反应对该上清酶液进行酶活力检测，根据其酶法反应对该酶液进行酶活力检测，该方法为在1ml的反应溶液(200mM PBS pH 7.5,10mMβ-烟酰胺单核苷酸)中加入含有1mg总蛋白量的酶液，在摄氏37度的温控下进行5分钟反应，完成后以高效液相色谱对样本中在酶反应中所产生的烟酰胺核苷进行含量分析。根据上述方法本酶液的酶活约为0.62nmol/min/mg。
实施例1:使用D-葡萄糖(食用葡萄糖)以酶组合的酶上清液进行酶法反应制备β-烟酰胺单核苷酸
按制备例制备各种所属的酶上清液并按下表1的比例进行混合成100ml备用。
表1
Figure PCTCN2021094844-appb-000001
Figure PCTCN2021094844-appb-000002
使用2L反应器配制反应溶液，在烧杯内按下述的次序加入12.1g三羟甲基氨基甲烷、990mg食用葡萄糖、4.1g的六水氯化镁、12.2g多聚磷酸盐(n＝3-20)、1.2g烟酰胺和1.2g三磷酸腺苷二钠盐并加入600ml纯水，启动外置搅拌装置至所有原材料完全溶解，以0.1M的盐酸/氢氧化钠调节溶液的酸碱值至pH7.5，以纯水定容到1L并保温至溶液的温度稳定在37℃，在10-20rpm的搅拌下同时加入100ml上述制备的上清酶液和20g的沸石，使用酸碱度调控装置实时监控反应过程中的pH变化并以0.1M盐酸/氢氧化钠溶液作调节，每60min取样并以附件2的方法分析β-烟酰胺单核苷酸含量分析的高效液相分析的方法对该样进行β-烟酰胺单核苷酸含量分析；反应在3hr后，β-烟酰胺单核苷酸的含量已达至3.3mM(见下表2)。反应溶液从反应器中排出后使用中速滤纸过滤取去沸石后得β-烟酰胺单核苷酸溶液。
表2
Figure PCTCN2021094844-appb-000003
实施例2:使用D-果糖以酶组合的上清酶液进行酶法反应制备β-烟酰胺单核苷酸
按制备例制备D-果糖组合、核心组合和辅助组合中所属的酶上清液并按下表3的比例进行混合得混合上清酶液各100ml备用。
表3
Figure PCTCN2021094844-appb-000004
Figure PCTCN2021094844-appb-000005
使用2L反应器配制反应溶液，在反应器内按下述的次序加入12.1g三羟甲基氨基甲烷，900mgD-果糖、4.1g六水氯化镁、12.2g多聚磷酸盐(n＝3-20)、1.2g烟酰胺和1.2g三磷酸腺苷二钠盐并加入600ml纯水，启动外置搅拌装置至所有原材料完全溶解，以0.1M的盐酸/氢氧化钠调节溶液的酸碱值至pH 7.5，以纯水定容到1L并并保温至溶液的温度稳定在37℃，在10-2-rpm的搅拌下同时加入100ml上述制备的上清酶液和20g的沸石，使用酸碱度调控装置实时监控反应过程中的pH变化并以0.1M盐酸/氢氧化钠溶液作调节，每60min取样并以附件2的方法对该样进行β-烟酰胺单核苷酸含量分析；反应在3hr后，β-烟酰胺单核苷酸的含量达至3.4mM(参见下表4)。反应溶液从反应器中排出后使用中速滤纸过滤取去沸石后得β-烟酰胺单核苷酸溶液。
表4
反应时间(min) β-烟酰胺单核苷酸(mM)
60 1.7
120 3.2
180 3.4
实施例3:使用D-葡萄糖(食用葡萄糖)以固定化酶进行混合酶组的酶法反应制备β-烟酰胺单核苷酸
根据中国专利CN1982445B的实施例3的方法，在固相载体上按下表5的各组合中的上清酶液用量比例制备混合固定化酶；载体的形状皆为条形：长25cm、宽5cm、厚5mm，下表为各固定化酶成品的重量为32.6g。
表5
生物酶 各上清酶液的总蛋白重量与固定化酶总重量的比例
聚磷酸-葡萄糖磷酸转移酶 8
葡萄糖-6-磷酸异构酶 6
己酮糖6-磷酸异构酶 16
己酮糖6-磷酸合酶 16
核糖5-磷酸异构酶 10
磷酸核糖二磷酸激酶 4
烟酰胺磷酸核糖转移酶 4
多聚磷酸激酶 2
腺苷酸激酶 6
聚磷酸-AMP磷酸转移酶 6
将上述制得的固定化酶载体安装于固定化酶反应器中。该反应器为有机玻璃制成的圆柱体，高7cm、半径4.5cm。用刀将上述载体头尾端约3cm以斜度45°整齐削去，紧握卷成高5cm、半径4.5cm的均质圆柱体，重量为8.8g。将该圆柱体插入反应器中，使其松紧程度符合中国发明专利申请CN106032520A中松紧程度符合表1中所述的3级标准，并且使其侧壁与反应器的内壁之间不留空隙。完成安装后，按CN106032520A图1进行其它配备装置的安装程序，其中反应调控罐容量为2L；高流量水泵是可调节流量式吸水泵，流速为0.5L/分钟；酸碱度调控装置采用0.1M盐酸/氢氧化钠溶液进行pH调控，其加液泵的流量为每分钟1ml。
在反应调控罐中按下述的次序加入12.1g三羟甲基氨基甲烷、990mg食用葡萄糖、4.1g六水氯化镁、12.2g多聚磷酸盐(n＝3-20)、1.2g烟酰胺和1.2g三磷酸腺苷二钠盐并加入600ml纯水，启动外置搅拌装置至所有原材料完全溶解，以0.1M的盐酸/氢氧化钠调节溶液的酸碱值至pH 7.5，以纯水定溶至1L并并保温至溶液的温度稳定在37℃后加入沸石，将反应调控罐连接高流量水泵和装有混合各组口固定化酶的反应器中并启动反应，每30min取样一次，以附件2的高效液相色谱分析当中β-烟酰胺单核苷酸在反应溶液中的含量变化，经1.5hr的反应后，β-烟酰胺单核苷酸的浓度已达至3.3mM(见下表6)。反应溶液从反应器中排出后使用中速滤纸过滤取去沸石后得β-烟酰胺单核苷酸溶液。采用固定化酶有多个便利工业生产和降低成本的好处：固定化酶可以在不增加液体溶量的情况下增加酶的使用量，对增加反应速度和提升转化率作用巨大，与此同时，固定化酶可以简便地多次重用，而且反应溶液中不会因含有大量蛋白而需要作多步的纯化工序。
表6
反应时间(min) β-烟酰胺单核苷酸(mM)
30 1.4
60 3.1
90 3.3
实施例4:同时使用D-葡萄糖(食用葡萄糖)和D-果糖以混合酶组合的酶上清液进行酶法反应制备β-烟酰胺单核苷酸
按实施例1-12制备所属的酶上清液并按下表7的比例进行混合得100ml上清酶液备用。
表7
Figure PCTCN2021094844-appb-000006
Figure PCTCN2021094844-appb-000007
使用2L反应器配制反应溶液，在反应器内按下述的次序加入12.1g三羟甲基氨基甲烷，495mg食用葡萄糖、450mg D-果糖、4.1g六水氯化镁、12.2g多聚磷酸盐(n＝3-20)、1.2g烟酰胺和1.2g三磷酸腺
Figure PCTCN2021094844-appb-000008
二钠盐并加入600ml纯水，启动外置搅拌装置至所有原材料完全溶解，以0.1M的盐酸/氢氧化钠调节溶液的酸碱值至pH 7.5，以纯水定溶至1L并并保温至溶液的温度稳定在37℃，在10-20rpm的搅拌下加入100ml的上清酶液和20g的沸石，使用酸碱度调控装置实时监控反应过程中的pH变化并以0.1M盐酸/氢氧化钠溶液作调节，每60min取样并以附件2的方法对该样进行β-烟酰胺单核苷酸含量分析。如表8所示，反应在3hr后，β-烟酰胺单核苷酸的含量达至2.3mM而在4hr时没有明显的增幅。反应溶液从反应器中排出后使用中速滤纸过滤取去沸石后得β-烟酰胺单核苷酸溶液。
表8
反应时间(min) β-烟酰胺单核苷酸(mM)
60 0.9
120 1.7
180 2.3
240 2.4
实施例5:使用麦芽糖以酶组合的上清酶液进行酶法反应制备β-烟酰胺单核苷酸
按制备例制备各种所属的酶上清液并按下表9的比例进行混合成100ml综合酶上清液备用。
表9
Figure PCTCN2021094844-appb-000009
使用2L反应器配制反应溶液，在反应器内按下述的次序加入12.1g三羟甲基氨基甲烷，495mg麦芽糖、4.1g六水氯化镁、12.2g多聚磷酸盐(n＝3-20)、1.2g烟酰胺和1.2g三磷酸腺苷二钠盐并加入600ml纯水，启动外置搅拌装置至所有原材料完全溶解，以0.1M的盐酸/氢氧化钠调节溶液的酸碱值至pH 7.5，以纯水定容到1L并并保温至溶液的温度稳定在37℃，在10-2-rpm的搅拌下加 入100ml综合酶上清液和20g的沸石，使用酸碱度调控装置实时监控反应过程中的pH变化并以0.1M盐酸/氢氧化钠溶液作调节，每60min取样并以附件2的方法对该样进行β-烟酰胺单核苷酸含量分析；反应在12hr后，β-烟酰胺单核苷酸的含量达至2.4mM(参见下表10)。反应溶液从反应器中排出后使用中速滤纸过滤取去沸石后得β-烟酰胺单核苷酸溶液。
表10
反应时间(hr) β-烟酰胺单核苷酸(mM)
1 0
2 0
3 0.2
4 0.28
5 0.4
6 0.6
7 0.8
8 1.4
9 1.6
10 1.8
11 2.2
12 2.4
实施例6:使用蔗糖以酶组合的上清酶液进行酶法反应制备β-烟酰胺单核苷酸
按制备例制备各种所属的酶上清液并按下表11的比例进行混合成100ml综合酶上清液备用。
表11
生物酶 各种酶的上清酶液的总蛋白重量与总混合上清酶液的总
  蛋白重量对比
聚磷酸-葡萄糖磷酸转移酶 1
葡萄糖-6-磷酸异构酶 1
6-磷酸己酮糖异构酶 4
己酮糖6-磷酸合酶 4
核糖5-磷酸异构酶 4
磷酸核糖二磷酸激酶 1
烟酰胺磷酸核糖转移酶 1
多聚磷酸激酶 2
腺苷酸激酶 1
聚磷酸-AMP磷酸转移酶 1
蔗糖酶 4
使用2L反应器配制反应溶液，在反应器内按下述的次序加入12.1g三羟甲基氨基甲烷，495mg蔗糖、4.1g六水氯化镁、12.2g多聚磷酸盐(n＝3-20)、1.2g烟酰胺和1.2g三磷酸腺苷二钠盐并加入600ml纯水，启动外置搅拌装置至所有原材料完全溶解，以0.1M的盐酸/氢氧化钠调节溶液的酸碱值至pH 7.5，以纯水定容到1L并并保温至溶液的温度稳定在37℃，在10-2-rpm的搅拌下加入100ml综合酶上清液和20g的沸石，使用酸碱度调控装置实时监控反应过程中的pH变化并以0.1M盐酸/氢氧化钠溶液作调节，每60min取样并以附件2的方法对该样进行β-烟酰胺单核苷酸含量分析；反应在12hr后，β-烟酰胺单核苷酸的含量达至2.1mM(参见下表12)。反应溶液从反应器中排出后使用中速滤纸过滤取去沸石后得β-烟酰胺单核苷酸溶液。
表12
反应时间(hr) β-烟酰胺单核苷酸(mM)
1 0
2 0
3 0
4 0.2
5 0.5
6 0.7
7 0.9
8 1.2
9 1.5
10 1.9
11 2.0
12 2.1
实施例7:使用D-葡萄糖(食用葡萄糖)以酶组合的酶上清液进行酶法反应制备β-烟酸单核苷酸
按制备例1-2和5-10制备各种所属的酶上清液并按下表13的比例进行混合成100ml综合酶上清液备用。
表13
Figure PCTCN2021094844-appb-000010
Figure PCTCN2021094844-appb-000011
使用2L反应器配制反应溶液，在烧杯内按下述的次序加入12.1g三羟甲基氨基甲烷、990mg食用葡萄糖、4.1g的六水氯化镁、12.2g多聚磷酸盐(n＝3-20)、1.2g烟酸和1.2g三磷酸腺苷二钠盐并加入600ml纯水，启动外置搅拌装置至所有原材料完全溶解，以0.1M的盐酸/氢氧化钠调节溶液的酸碱值至pH7.5，以纯水定容到1L并保温至溶液的温度稳定在37℃，在10-20rpm的搅拌下同时加入100ml上述制备的上清酶液和20g的沸石，使用酸碱度调控装置实时监控反应过程中的pH变化并以0.1M盐酸/氢氧化钠溶液作调节，每60min取样并以附件2对该样进行β-烟酰胺单核苷酸含量分析；反应在3hr后，β-烟酰胺单核苷酸的含量已达至3.1mM(见下表14)。反应溶液从反应器中排出后使用中速滤纸过滤取去沸石后得β-烟酰胺单核苷酸溶液。
表14
反应时间(min) β-烟酰胺单核苷酸(mM)
60 1.4
120 2.7
180 3.1
实施例8:使用D-葡萄糖(食用葡萄糖)以酶组合的酶上清液进行酶法反应制备β-烟酰胺核苷
按制备例制备各种所属的酶上清液并按下表15的比例进行混合成100ml综合酶上清液备用。
表15
Figure PCTCN2021094844-appb-000012
Figure PCTCN2021094844-appb-000013
使用2L反应器配制反应溶液，在烧杯内按下述的次序加入12.1g三羟甲基氨基甲烷、990mg食用葡萄糖、4.1g的六水氯化镁、12.2g多聚磷酸盐(n＝3-20)、1.2g烟酰胺和1.2g三磷酸腺苷二钠盐并加入600ml纯水，启动外置搅拌装置至所有原材料完全溶解，以0.1M的盐酸/氢氧化钠调节溶液的酸碱值至pH7.5，以纯水定容到1L并保温至溶液的温度稳定在37℃，在10-20rpm的搅拌下同时加入100ml上述制备的上清酶液和20g的沸石，使用酸碱度调控装置实时监控反应过程中的pH变化并以0.1M盐酸/氢氧化钠溶液作调节，每60min取样并以附件2对该样进行β-烟酰胺核苷含量分析；反应在3hr后，β-烟酰胺单核苷酸的含量已达至3.5mM(见下表16)。反应溶液从反应器中排出后使用中速滤纸过滤取去沸石后得β-烟酰胺单核苷酸溶液。
表16
Figure PCTCN2021094844-appb-000014
Figure PCTCN2021094844-appb-000015
比较例1:使用常规生物酶法以核糖作为底物进行酶法反应制备β-烟酰胺单核苷酸
使用2L反应器配制反应溶液，在烧杯内按下述的次序加入12.1g三羟甲基氨基甲烷、990mg核糖、4.1g的六水氯化镁、1.21g烟酰胺和12.1g三磷酸腺苷二钠盐并加入600ml纯水，启动外置搅拌装置至所有原材料完全溶解，以0.1M的盐酸/氢氧化钠调节溶液的酸碱值至pH 7.5，以纯水定容到1L并保温至溶液的温度稳定在37℃，使用酸碱度调控装置实时监控反应过程中的pH变化并以0.1M盐酸/氢氧化钠溶液作调节，在10-20rpm的搅拌下按次序加入20mL的核糖5-磷酸激酶上清酶液，每30min取样并以附件1对该样进行核糖5-磷酸的含量检测；反应在60min的样本中能检测得到核糖5-磷酸的合成，此时可同时加入40ml的磷酸核糖二磷酸激酶上清酶液和40mL的烟酰胺磷酸核糖转移酶，每30min取样并以附件2对该样进行β-烟酰胺单核苷酸的含量检测；反应在180min的样本中开始检测得到β-烟酰胺单核苷酸的含量，在反应540min后，β-烟酰胺单核苷酸的含量已达至1.3mM(见下表17)并没有再显注增长，反应随之结束，反应溶液从反应器中排出。
以核糖为底物的常规生物酶法制备β-烟酰胺单核苷酸，需要比本发明的方法多用三磷酸腺苷二钠盐以作为磷酸组的供应和能量，因此在同等份量的三磷酸腺苷的反应中，本发明的方法可以在更短时间生产较多的β-烟酰胺单核苷酸，在生产时间和成本效益上较常规生物酶法更优。
表17
Figure PCTCN2021094844-appb-000016
本发明不受上述具体文字描述的限制，本发明可在权利要求书所概括的范围内做各种修改或改变。这些改变均在本发明要求保护的范围之内。
附件1:以高效液相分析糖类和其衍生物的含量的条件
Figure PCTCN2021094844-appb-000017
色谱流出曲线:梯度
时间(min) 流速(ml/min) ％流动相A ％流动相B
0 1.0 100 0
40 1.0 0 100
50 1.0 0 100
60 1.2 100 0
80 1.0 100 0
附件2:以高效液相分析烟酸或其衍生物的核苷和单核苷酸含量(例如β-烟酰胺单核苷酸、烟酰胺腺嘌呤二核苷酸、烟酰胺核苷、烟酸单核苷酸、烟酸腺嘌呤二核苷酸、烟酸核苷等)的条件
高效液相色谱分析方法
Figure PCTCN2021094844-appb-000018
时间(min) 流速(ml/min) ％流动相A ％流动相B
0 0.8 100 0
1 0.8 100 0
13 0.8 90.5 9.5
16 0.8 85 15
18 0.8 61 39
26 0.8 61 39
28 0.8 50 50
31 0.8 50 50
32 0.8 100 0
33 0.8 100 0
35 1.0 100 0
38.5 1.0 100 0
39.5 0.8 100 0

Claims (23)

  1. 一种制备烟酸或其衍生物的单核苷酸的方法，包括下列步骤：采用包含己糖源和烟酸或其衍生物的反应底物，在磷酸供体的存在下，经生物酶催化反应生成烟酸或其衍生物的单核苷酸。
  2. 根据权利要求1所述的方法，其中所述烟酸或其衍生物的单核苷酸选自β-烟酰胺单核苷酸和烟酸单核苷酸中的至少一者。
  3. 根据权利要求1-2中任意一项所述的方法，其中所述己糖源来自具有六个碳原子的单糖、能够生成己糖的多糖或其混合物。
  4. 根据权利要求3所述的方法，其中所述的单糖选自D-葡萄糖、D-甘露糖、D-半乳糖、D-果糖或其混合物中的任意一者，优选为D-葡萄糖、D-甘露糖和D-果糖，更优选为D-葡萄糖和D-果糖。
  5. 根据权利要求3所述的方法，其中所述的己糖源来自通过糖苷键连接多种己糖单元的多糖，优选为蔗糖、麦芽糖、菊粉、棉子糖、麦芽糊精、淀粉或其混合物，更优选为蔗糖、麦芽糖和棉子糖，更优选为蔗糖和麦芽糖。
  6. 根据权利要求1-5中任意一项所述的方法，其中所述生物酶为单独的生物酶或者包含多种生物酶的生物酶组。
  7. 根据权利要求1-6中任意一项所述的方法，其中所述反应的条件包括：温度为25-40℃，优选为30-39℃，更优选为35-38℃；并且/或者反应体系的pH为6.0-8.5，优选为pH 7.0-8.0，更优选为pH7.5-7.8。
  8. 根据权利要求1-7中任意一项所述的方法，其中所述反应底物还包含辅助离子，辅助离子包含金属离子、氯离子、镁离子、钙离子、钾离子、钠 离子、锌离子、氟离子、硫离子、碳酸根类离子、亚硫酸根类离子以及含磷类离子中的至少一者，优选钠离子、镁离子、钾离子、碳酸根类离子、亚硫酸根类离子和含磷类离子中的至少一者。
  9. 根据权利要求1-8中任意一项所述的方法，其中所述磷酸供体选自ATP或其盐、ADP或其盐、AMP或其盐、CTP或其盐、GTP或其盐、UTP或其盐、ITP或其盐和多聚磷酸或其盐中的至少一者，优选ATP或其盐、ADP或其盐、AMP或其盐和多聚磷酸或其盐中的至少一者。
  10. 根据权利要求1-9中任意一项所述的方法，其中所述磷酸供体可以为多聚磷酸或ATP或其盐，所述己糖源来自D-葡萄糖，并且所述生物酶为生物酶组，该生物酶组包含聚磷酸-葡萄糖磷酸转移酶(EC 2.7.1.63)、葡萄糖-6-磷酸异构酶(EC 5.3.1.9)、己酮糖6-磷酸异构酶(EC 5.3.1.27)、己酮糖6-磷酸合酶(EC 4.1.2.43)、核糖5-磷酸异构酶(EC 5.3.1.6)、磷酸核糖二磷酸激酶(EC 2.7.6.1)和烟酰胺磷酸核糖转移酶(EC 2.4.2.12)；其中生物酶组中的聚磷酸-葡萄糖磷酸转移酶以多聚磷酸和D-葡萄糖为底物转化合成至葡萄糖-6-磷酸和多聚磷酸(n-1)，生物酶组中的葡萄糖-6-磷酸异构酶以葡萄糖-6-磷酸为底物转化合成至果糖-6-磷酸，生物酶组中的己酮糖6-磷酸异构酶以果糖-6-磷酸为底物转化合成至阿拉伯糖-3-己酮糖-6-磷酸酯，生物酶组中的己酮糖6-磷酸合酶以阿拉伯糖-3-己酮糖-6-磷酸酯为底物转化合成至核酮糖-5-磷酸和甲醛，生物酶组中的核糖5-磷酸异构酶核酮糖-5-磷酸为底物转化合成至核糖-5-磷酸，生物酶组中的磷酸核糖二磷酸激酶以核糖-5-磷酸和磷酸供体为底物转化合成至5-磷酰核糖-1-焦磷酸和AMP，生物酶组中的烟酰胺磷酸核糖转移酶以烟酰胺和5-磷酰核糖-1-焦磷酸为底物转化合成至β-烟酰胺单核苷酸和焦磷酸，或以烟酸为底物转化合成至烟酸单核苷酸和焦磷酸。
  11. 根据权利要求1-9中任意一项所述的方法，其中所述己糖源来自D-葡萄糖，并且所述生物酶为生物酶组，该生物酶组包含葡萄糖异构酶(EC 5.3.1.5)、己糖激酶(EC 2.7.1.1)、葡萄糖-6-磷酸异构酶(EC 5.3.1.9)、己酮糖6-磷酸异构酶(EC 5.3.1.27)、己酮糖6-磷酸合酶(EC 4.1.2.43)、核糖5-磷酸异构酶 (EC 5.3.1.6)、磷酸核糖二磷酸激酶(EC 2.7.6.1)和烟酰胺磷酸核糖转移酶(EC 2.4.2.12)。
  12. 根据权利要求1-9中任意一项所述的方法，其中所述己糖源来自D-果糖，并且所述生物酶为生物酶组，该生物酶组包含己糖激酶(EC 2.7.1.1)、己酮糖6-磷酸异构酶(EC 5.3.1.27)、己酮糖6-磷酸合酶(EC 4.1.2.43)、核糖5-磷酸异构酶(EC 5.3.1.6)、磷酸核糖二磷酸激酶(EC 2.7.6.1)和烟酰胺磷酸核糖转移酶(EC 2.4.2.12)。
  13. 根据权利要求1-9中任意一项所述的方法，其中所述己糖源来自D-甘露糖，并且所述生物酶为生物酶组，该生物酶组包含甘露糖激酶(EC 2.7.1.7)、甘露糖-6-磷酸异构酶(EC 5.3.1.8)、己酮糖6-磷酸异构酶(6 EC 5.3.1.27)、己酮糖6-磷酸合酶(EC 4.1.2.43)、核糖5-磷酸异构酶(EC 5.3.1.6)、磷酸核糖二磷酸激酶(EC 2.7.6.1)和烟酰胺磷酸核糖转移酶。
  14. 根据权利要求1-9中任意一项所述的方法，其中所述己糖源来自蔗糖、双糖麦芽糖、棉子糖、麦芽糊精和淀粉中的至少一者并且其中在生物酶催化反应之前，将所述己糖源用选自热、酸、碱中的至少一种方法水解蔗糖转化为选自D-葡萄糖、半乳糖、D-果糖和D-甘露糖中的至少一种单糖，然后将所述单糖投入反应底物中；或者
    其中所述己糖源为蔗糖、双糖麦芽糖类、棉子糖、麦芽糊精和淀粉中的至少一者，并且直接使用生物酶组将包含所述己糖源和烟酸或其衍生物的核苷和反应底物转化为β-烟酰胺单核苷酸，其中所述生物酶组包含淀粉酶类(EC 3.2.1.1-3)、普鲁兰酶(EC 3.2.1.41)、麦芽糖酶(EC 3.2.1.20)、异麦芽糖酶(EC 3.2.1.10)、α-半乳糖苷酶(EC 3.2.1.22)、蔗糖酶(EC 3.2.1.26)、、蔗糖α-葡糖苷酶(EC 3.2.1.48)、聚磷酸-葡萄糖磷酸转移酶(EC 2.7.1.63)、葡萄糖-6-磷酸异构酶(EC 5.3.1.9)、己糖激酶(EC 2.7.1.1)、己酮糖6-磷酸异构酶(EC 5.3.1.27)、己酮糖6-磷酸合酶(EC 4.1.2.43)、核糖5-磷酸异构酶(EC 5.3.1.6)、磷酸核糖二磷酸激酶(EC 2.7.6.1)和烟酰胺磷酸核糖转移酶(EC 2.4.2.12)中的至少一者。
  15. 根据权利要求14所述的方法，其中，所述蔗糖包含白砂糖、黄砂糖、赤砂糖、绵白糖、单晶体冰糖、多晶体冰糖、红糖、黑糖、冰片糖、方糖、糖霜、液体糖浆中的至少一者；并且
    所述双糖麦芽糖包含选自麦芽糖和异麦芽糖中的至少一者。
  16. 根据权利要求1-15中任意一项所述的方法，其中，所述生物酶包含ATP再生酶，ATP再生酶包含聚磷酸-AMP磷酸转移酶(EC 2.7.4.B2)、多聚磷酸激酶(EC 2.7.4.1)、腺苷激酶(EC 2.7.1.20)中的至少一者；并且其中所述方法还包括ATP再生步骤，该步骤包括：聚磷酸-AMP磷酸转移酶以多聚磷酸和AMP为底物转化生成ADP，多聚磷酸激酶以多聚磷酸和AMP/ADP为底物转化生成ADP/ATP，以及腺苷激酶以两分子的ADP为底物转化生成ATP和AMP。
  17. 根据权利要求16所述的方法，其中，多聚磷酸激酶(EC 2.7.4.1)包括II类和III类中的两组酶，其中使用II类的多聚磷酸激酶以多聚磷酸和ADP为底物转化生成ATP，使用III类的多聚磷酸激酶以多聚磷酸和AMP/ADP为底物转化生成ADP和ATP；在使用II类的多聚磷酸激酶时由AMP转化合成至ATP时还包括使用聚磷酸-AMP磷酸转移酶和/或腺苷激酶，并且在使用III类的多聚磷酸激酶时，所述方法还包括单独使用该酶由AMP转化合成至ATP。
  18. 根据权利要求1-17中任意一项所述的方法，其中，所述反应底物还包含多聚磷酸、腺苷和腺嘌呤中的至少一者，所述磷酸供体为ATP，所述生物酶包含ATP再生酶，并且ATP再生酶包含腺苷酸激酶(EC 2.7.4.3)、腺嘌呤磷酸核糖转移酶(EC 2.4.2.7)中的至少一者，
    其中所述方法还包括ATP再生步骤，该步骤包括：腺苷酸激酶先以一分子的腺苷和ATP为底物转化生成AMP和ADP，然后使用所述ATP再生酶以多聚磷酸为主要底物将AMP和ADP转化生成两分子的ATP或一分子的ADP和ATP；腺嘌呤磷酸核糖转移酶则以一分子的腺嘌呤和5-磷酰核糖-1-焦磷酸为底 物转化生成一分子的AMP和磷酸，然后使用ATP再生酶组以多聚磷酸为主要底物将AMP转化生成ADP和ATP。
  19. 根据权利要求1-18中任意一项所述的方法，其中，所述反应底物还包含烟酰胺和烟酸中的至少一者，并且所述单核苷酸为β-烟酰胺单核苷酸和烟酸单核苷酸中的至少一者。
  20. 根据权利要求1-19中任意一项所述的方法，其中所述方法还包括在反应底物中添加出用于吸咐或分解甲醛的隋性物料的步骤，优选地所述惰性物料为沸石。
  21. 根据权利要求1-20中任意一项所述的方法，其中所生物酶组为重组酶，该重组酶在所属的菌种进行合成并于大肠杆菌HB101中进行表达和提取；
    优选地，所述生物酶为选自细胞破碎液、上清酶液、纯酶和以任何方式制备的固定化酶/细胞中的至少一种形式。
  22. 一种制备生物产物的方法，包括下列步骤：
    (i)采用包含己糖源和烟酸或其衍生物的反应底物，在磷酸供体的存在下，经生物酶催化反应生成烟酸或其衍生物的单核苷酸；以及
    (ii)利用生成的烟酸或其衍生物的单核苷酸作为中间体制备所述生物产物。
  23. 根据权利要求22所述的方法，其中所述生物产物选自烟酰胺核苷或其任何盐分的衍生物、氧化型烟酰胺腺嘌呤二核苷酸或其任何盐分的衍生物、还原型烟酰胺腺嘌呤二核苷酸或其任何盐分的衍生物、氧化型烟酰胺腺嘌呤二核苷酸磷酸或其任何盐分的衍生物、还原型烟酰胺腺嘌呤二核苷酸磷酸或其任何盐分的衍生物、烟酸核苷或其任何盐分的衍生物、烟酸腺嘌呤二核苷酸或其任何盐分的衍生物和烟酸腺嘌呤二核苷酸磷酸或其任何盐分的衍生物中的至少一者。
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