WO2021169814A1 - 甲醛转化突变蛋白及其应用 - Google Patents
甲醛转化突变蛋白及其应用 Download PDFInfo
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- WO2021169814A1 WO2021169814A1 PCT/CN2021/076379 CN2021076379W WO2021169814A1 WO 2021169814 A1 WO2021169814 A1 WO 2021169814A1 CN 2021076379 W CN2021076379 W CN 2021076379W WO 2021169814 A1 WO2021169814 A1 WO 2021169814A1
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
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- B01J23/00—Catalysts comprising metals or metal oxides or hydroxides, not provided for in group B01J21/00
- B01J23/02—Catalysts comprising metals or metal oxides or hydroxides, not provided for in group B01J21/00 of the alkali- or alkaline earth metals or beryllium
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
- C12P7/26—Ketones
- C12P7/28—Acetone-containing products
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- C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
- C12Y114/11—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11)
- C12Y114/11023—Flavonol synthase (1.14.11.23)
Definitions
- the invention belongs to the technical field of biochemical industry, and specifically relates to a formaldehyde-converted mutein and its application, and also relates to a method for synthesizing 1,3-dihydroxyacetone, pyruvaldehyde or formaldehyde to lactic acid, and also relates to a synthesis method from hydroxyacetone, pyruvaldehyde or formaldehyde to lactic acid. Synthesis method of aldehyde and 1,3-dihydroxyacetone, formaldehyde to glycolic acid.
- 1,3-dihydroxyacetone (1,3-dihydroxyacetone, DHA) is the simplest three-carbon ketose that exists in nature and has a wide range of uses. Not only can it be used as a raw material for cosmetics, it has a good protective effect on the skin, but also can be used as a basis to synthesize polyester compounds.
- 1,3-dihydroxyacetone is also an important metabolic intermediate product, which can be further converted into some high value-added chemicals and fuel molecules through microbial fermentation, such as ethanol, butanol, lactic acid, succinic acid, etc. Synthesize other higher-value sugars, such as erythrulose, sorbose, etc. Therefore, 1,3-dihydroxyacetone is an important chemical synthesis intermediate, which is widely used in cosmetics manufacturing, food research, medicine and chemical synthesis industries.
- the production methods of 1,3-dihydroxyacetone mainly include microbiological methods and chemical methods.
- Microbiological methods mainly use microbial fermentation to convert glycerol into 1,3-dihydroxyacetone.
- the production capacity of microbiological methods is low and requires Strictly control the conditions of microbial culture and strict requirements on the production environment of microorganisms.
- the purification process of 1,3-dihydroxyacetone in the later stage is complicated, high cost, and environmental pollution.
- the chemical method mainly uses a metal catalyst to oxidize glycerin to produce 1,3-dihydroxyacetone, but the chemical method uses a metal catalyst to pollute the environment and is not conducive to environmental protection.
- Lactic acid is an organic acid with a wide range of uses.
- lactic acid can be used as a sour agent, flavoring agent and other food additives; in the field of medicine, lactate (iron, calcium, sodium, etc.) can be used to supplement the human body Metal elements; in the cosmetics field, lactic acid can be added to skin care products as a humectant; in the textile field, lactic acid can be used to treat textiles; in the chemical field, lactic acid can be used as an intermediate compound to synthesize green organic solvent lactate, also Polylactic acid can be synthesized. Polylactic acid has biodegradability, biocompatibility, good elasticity and excellent physical and chemical properties, making it an ideal polymer material in the future. Due to its wide range of uses and high demand, the synthesis of lactic acid is particularly important.
- the main methods of lactic acid production are chemical synthesis and microbial fermentation, in addition to glycerol conversion.
- the chemical synthesis method firstly uses acetaldehyde and hydrocyanic acid to synthesize lactonitrile through high-pressure reaction, and then the lactonitrile is hydrolyzed by sulfuric acid to generate lactic acid.
- the lactic acid produced by this method is of high purity, since acetaldehyde and highly toxic hydrocyanic acid are used in the raw materials, there is a greater risk of pollution and production.
- the microbial fermentation method uses sugar, starch, and lignocellulose as raw materials to synthesize lactic acid through biotransformation by various bacteria or fungi.
- Microbial fermentation methods use starch or sugar as raw materials for fermentation, and subsequent sterilization and downstream lactic acid separation and purification will lead to higher production costs of lactic acid.
- problems such as inability to continuous production and unstable product properties. These problems are the reasons that limit the large-scale application of microbial fermentation.
- glycerol is mainly reacted under high temperature conditions under the action of inorganic alkali (NaOH, CaO) to obtain lactic acid.
- NaOH, CaO inorganic alkali
- the problem with this method is that the reaction process requires a higher temperature and a high-temperature alkaline environment Higher requirements for equipment.
- Glycolic acid is a kind of hydroxy carboxylic acid with important industrial value, which plays an important role in textile industry, food industry, pharmaceutical industry and so on.
- glycolic acid can be used as a dye tanning agent; in the food industry, glycolic acid can be used as a fragrance and preservative; in the pharmaceutical industry, glycolic acid can be used as a skin protectant.
- glycolic acid can also be used in industrial and household cleaners and adhesives. It can also be converted into a biodegradable polymer with good mechanical properties-polyglycolic acid. It can also be used together with lactic acid to produce medically important products. Copolymer applied. Due to its application in multiple industries, the market demand for glycolic acid is increasing. It is reported that the market demand has reached 40,000 tons as early as 2014.
- glycolic acid is mainly synthesized by chemical methods using fossils as raw materials, specifically, formaldehyde is obtained by carbonylation under high temperature and high pressure.
- this method has severe reaction conditions and high energy consumption.
- microbial fermentation to produce glycolic acid.
- Most of the microbial fermentation methods use glucose, arabinose, and xylose as raw materials.
- the cost of raw materials is relatively high. Problems such as the imbalance of the reducing power of the bacterial cells, and strict fermentation conditions are required during the cultivation process.
- one-carbon compounds such as formic acid, methanol, formaldehyde, etc.
- One-carbon compounds can be used to synthesize basic organic chemical raw materials, fuels and other high value-added chemicals. .
- one-carbon compounds Because of its cheap and easy-to-obtain characteristics, one-carbon compounds have become the most promising compounds for the preparation of high-value compounds from petroleum, and have important scientific significance and development value in the fields of medicine, food, and chemicals.
- the utilization and conversion of one-carbon compounds can also significantly reduce the negative impact on the ecological environment caused by people in the process of using fossil fuels and synthetic materials.
- Formaldehyde can be converted from other one-carbon compounds and then converted into intermediate substances for biological use. At the same time, it has the characteristics of wide sources and low prices.
- the synthesis of high-value compounds using formaldehyde as a precursor has important application prospects.
- an object of the present invention is to provide a formaldehyde-converting mutant protein, which is based on the mutant BFD1 of benzoyl formate decarboxylase derived from Pseudomonas putida to perform error-prone PCR and randomize the target gene. Mutations, the screening of a protein with increased activity in catalyzing the synthesis of 1,3-dihydroxyacetone from formaldehyde, and the activity has the characteristics of thermal stability.
- the second object of the present invention is to provide the application of the above-mentioned formaldehyde conversion mutein.
- the third object of the present invention is to provide a synthesis method from 1,3-dihydroxyacetone, pyruvaldehyde or formaldehyde to lactic acid.
- the fourth object of the present invention is to provide a method for converting hydroxyacetaldehyde, 1,3-dihydroxyacetone or formaldehyde into glycolic acid.
- the present invention provides a formaldehyde conversion mutein, which can catalyze the synthesis of 1,3-dihydroxyacetone from formaldehyde, and its amino acid sequence is as follows: S26, L43, F66 corresponding to SEQ ID NO:1 , R86, T87, G109, A204, H281, A322, F397, L407, M460, W463, V467, V473, S525 at least one of the amino acid residues is mutated; or the amino acid sequence of the formaldehyde conversion mutant protein has The mutation site in the mutated amino acid sequence, and an amino acid sequence that has more than 80% homology with the mutated amino acid sequence, preferably more than 90%, 95% or more than 98% homology .
- the amino acid sequence of the above-mentioned formaldehyde conversion mutant protein includes at least one of the following mutation sites: the serine S at position 26 corresponding to SEQ ID NO:1 is mutated to phenylalanine F, the leucine at position 43 Mutation of amino acid L to glutamine Q, mutation of phenylalanine F at position 66 to leucine L, mutation of arginine R at position 86 to cysteine C, and threonine T at position 87 Mutation to alanine A, mutation of glycine G at position 109 to serine S, mutation of alanine A at position 204 to valine V, mutation of histidine H at position 281 to tyrosine Y, 322 The alanine A at position A was mutated to threonine T, the phenylalanine F at position 397 was mutated to either leucine L or serine S, and the leucine L at position 407 was mutated to methi
- the formaldehyde conversion mutein includes corresponding to SEQ ID NO: 1, at least one of the following substitutions or a combination of two or more different positions: S26F, L43Q, F66L, R86C, T87A, G109S, A204V, H281Y, A322T, F397L/F397S, L407M, M460T, W463R, V467A, V473A, S525A.
- the amino acid sequence of the aforementioned formaldehyde conversion mutein at least includes: histidine H at position 281 corresponding to SEQ ID NO:1 has a mutation, for example, a mutation to tyrosine Y.
- the formaldehyde conversion mutant protein may also include at least one other site mutation, such as S26F, L43Q, F66L, T87A, G109S, A204V, A322T, F397L/F397S, L407M, M460T, W463R, V467A, V473A, Any one or a combination of multiple sites in S525A.
- the amino acid sequence of the aforementioned formaldehyde conversion mutein at least includes: a mutation of serine S at position 26 corresponding to SEQ ID NO:1, for example, a mutation to phenylalanine F.
- the formaldehyde conversion mutant protein may also include at least one other site mutation, such as L43Q, F66L, T87A, G109S, A204V, H281Y, A322T, F397L/F397S, M460T, W463R, V467A, V473A, S525A A combination of any one or more of the sites.
- the amino acid sequence of the aforementioned formaldehyde conversion mutein at least includes: a phenylalanine F mutation at position 397 corresponding to SEQ ID NO:1, for example, a mutation to either leucine L or serine S .
- the formaldehyde conversion mutant protein may also include at least one other site mutation, for example, any of S26F, L43Q, F66L, T87A, G109S, A204V, H281Y, A322T, M460T, W463R, V467A, V473A, and S525A. A combination of one or more sites.
- the amino acid sequence of the aforementioned formaldehyde conversion mutein at least includes: a tryptophan W mutation at position 463 corresponding to SEQ ID NO:1, for example, a mutation to arginine R.
- the formaldehyde conversion mutant protein may also include at least one other site mutation, such as S26F, L43Q, F66L, T87A, G109S, A204V, H281Y, A322T, F397L/F397S, M460T, V467A, V473A, S525A. A combination of any one or more of the sites.
- the amino acid sequence of the aforementioned formaldehyde conversion mutant protein includes: histidine H at position 281 corresponding to SEQ ID NO:1 is tyrosine Y, and the mutation of serine S at position 26 to phenylalanine F .
- the formaldehyde conversion mutein may also include at least one other site mutation, such as any one of W463R, L43Q, G109S, F397S/F397L, M460T, S525A, V194I, V467A, F66L, A204V, A322T, or A combination of multiple sites.
- the amino acid sequence of the aforementioned formaldehyde conversion mutein includes mutations at positions S26F, H281Y, and F397S corresponding to SEQ ID NO:1; in another embodiment, the amino acid sequence of the formaldehyde conversion mutein Including mutations corresponding to the S26F, H281Y, and W463R positions of SEQ ID NO:1.
- amino acid sequence of the above-mentioned formaldehyde-converted mutein is specifically any one of the following 1)-18):
- the histidine at position 281 of SEQ ID NO:1 is mutated to tyrosine, and the leucine at position 407 is mutated to methionine, and the other amino acid residues remain unchanged.
- the resulting amino acid sequence (its The nucleotide sequence is SEQ ID NO: 27);
- the present invention also provides a polynucleotide, which is a polynucleotide encoding the above-mentioned formaldehyde conversion mutein.
- the present invention also provides recombinant vectors, expression cassettes, transgenic cell lines and/or recombinant bacteria containing the above-mentioned formaldehyde-transformed mutant protein and/or the above-mentioned polynucleotide.
- the present invention also provides the use of the above-mentioned formaldehyde conversion mutein as a catalyst, such as a catalyst for the catalytic preparation of 1,3-dihydroxyacetone and hydroxyacetaldehyde and/or lactic acid and/or glycolic acid, for example, the preparation of the substrate formaldehyde 1, A catalyst for 3-dihydroxyacetone and/or lactic acid, or a catalyst for preparing 1,3-dihydroxyacetone and hydroxyacetaldehyde from formaldehyde as a substrate, and then preparing glycolic acid.
- a catalyst such as a catalyst for the catalytic preparation of 1,3-dihydroxyacetone and hydroxyacetaldehyde and/or lactic acid and/or glycolic acid, for example, the preparation of the substrate formaldehyde 1, A catalyst for 3-dihydroxyacetone and/or lactic acid, or a catalyst for preparing 1,3-dihydroxyacetone and hydroxyacetaldehyde from formaldehyde as a
- the present invention also provides the use of recombinant vectors, expression cassettes, transgenic cell lines and/or recombinant bacteria containing the above polynucleotides as catalysts, for example for the catalytic preparation of 1,3-dihydroxyacetone and/or lactic acid and/or glycolic acid
- catalysts for example, a catalyst for the preparation of 1,3-dihydroxyacetone and/or lactic acid from the substrate formaldehyde, or a catalyst for the preparation of 1,3-dihydroxyacetone and hydroxyacetaldehyde from the substrate formaldehyde to produce glycolic acid.
- the formaldehyde conversion mutein of the present invention catalyzes formaldehyde to prepare the target compound lactic acid, it includes: contacting the formaldehyde conversion mutein with formaldehyde to catalyze the production of 1,3-dihydroxyacetone; further, an alkali metal hydroxide may be optionally added And/or alkaline earth metal hydroxides to catalyze the formation of lactic acid from 1,3-dihydroxyacetone.
- the recombinant vector, transgenic cell line or recombinant strain containing the above-mentioned formaldehyde-converting mutein and/or the polynucleotide catalyzes the production of the target compound lactic acid from formaldehyde
- it includes:
- the recombinant vector, transgenic cell line or recombinant strain of the polynucleotide is contacted with formaldehyde to catalyze the production of 1,3-dihydroxyacetone; further, an alkali metal hydroxide and/or alkaline earth metal hydroxide can be optionally added , Catalyzes 1,3-dihydroxyacetone to produce lactic acid.
- the formaldehyde conversion mutein of the present invention catalyzes formaldehyde to prepare the target compound glycolic acid, it includes: contacting the formaldehyde conversion mutein with formaldehyde to catalyze the production of 1,3-dihydroxyacetone and/or hydroxyacetaldehyde; further, adding an oxidizing agent , Oxidize 1,3-dihydroxyacetone and/or hydroxyacetaldehyde to glycolic acid.
- the method when the recombinant vector, transgenic cell line or recombinant strain containing the above-mentioned formaldehyde conversion mutein and/or the polynucleotide catalyzes the preparation of the target compound glycolic acid from formaldehyde, the method includes: converting the formaldehyde conversion mutein and/or The recombinant vector, transgenic cell line or recombinant strain of the polynucleotide is contacted with formaldehyde to catalyze the production of 1,3-dihydroxyacetone and/or hydroxyacetaldehyde; an oxidant is added, and 1,3-dihydroxyacetone and/or Hydroxyacetaldehyde is oxidized to glycolic acid.
- the oxidant may be sodium chlorite, sodium hypochlorite, sodium persulfate or hydrogen peroxide.
- the present invention provides a method for preparing lactic acid, which includes the following steps: 1,3-dihydroxyacetone or pyruvaldehyde is used as a substrate, and an alkali metal hydroxide and/or alkaline earth metal hydroxide is used to catalyze the substrate to produce lactic acid.
- the alkali metal hydroxide may be sodium hydroxide or potassium hydroxide
- the alkaline earth metal hydroxide may be calcium hydroxide or barium hydroxide.
- the molar ratio of the alkali metal hydroxide or alkaline earth metal hydroxide to the substrate 1,3-dihydroxyacetone or pyruvaldehyde may be (1-100):1, for example (2-80 ):1.
- the molar ratio of the alkali metal hydroxide to the substrate 1,3-dihydroxyacetone or pyruvaldehyde may be (5 ⁇ 80):1, for example (10 ⁇ 60):1, (15 ⁇ 50) :1, (20-40):1;
- the molar ratio of the alkaline earth metal hydroxide to the substrate 1,3-dihydroxyacetone or pyruvaldehyde may be (1-20):1, for example (2-10) :1, (2 ⁇ 6):1.
- the reaction of the alkali metal hydroxide and/or alkaline earth metal hydroxide catalyzed by the substrate 1,3-dihydroxyacetone or pyruvaldehyde to produce lactic acid is not carried out under inert gas protection conditions. In other words, the reaction can be carried out in an atmospheric environment.
- the reaction of the alkali metal hydroxide and/or alkaline earth metal hydroxide catalyzed by the substrate 1,3-dihydroxyacetone or pyruvaldehyde to produce lactic acid can be carried out in the range of 10-50°C, preferably It is carried out in the range of 20 to 30°C, for example at room temperature.
- the time for the alkali metal hydroxide and/or alkaline earth metal hydroxide to catalyze the reaction of the substrate 1,3-dihydroxyacetone or pyruvaldehyde to produce lactic acid can be 6 to 80 hours, for example, 12 ⁇ 60 hours, 24 ⁇ 48 hours.
- the substrate 1,3-dihydroxyacetone can use formaldehyde as a substrate, and formaldehyde conversion protein is used to catalyze the synthesis of 1,3-dihydroxyacetone; wherein the formaldehyde conversion protein has the ability to catalyze the conversion of formaldehyde to The function of 1,3-dihydroxyacetone.
- formaldehyde-converting mutein when used to catalyze the synthesis of 1,3-dihydroxyacetone from formaldehyde, the following steps are included: contacting the formaldehyde-converting mutein with formaldehyde to catalyze the formation of 1,3-dihydroxyacetone from the formaldehyde.
- the "formaldehyde conversion protein" used to catalyze the synthesis of 1,3-dihydroxyacetone from formaldehyde refers to a protein that can catalyze the synthesis of 1,3-dihydroxyacetone from formaldehyde, as long as it has the function of catalyzing the conversion of formaldehyde to 1,3-dihydroxyacetone That is, its amino acid sequence and source are not particularly limited. As an example but not limitation, it may be, for example, benzoylformate decarboxylases (BFD) derived from Pseudomonas putida and Pseudomonas fluorescens biovar (Pseudomonas fluorescens biovar I).
- BFD benzoylformate decarboxylases
- BAL Benzaldehyde lyase
- BAL Benzaldehyde lyase
- amino acid mutations to obtain proteins with the above-mentioned functions.
- benzaldehyde lyase or a mutant thereof can be used, and any of the formaldehyde-converting mutant proteins described above can also be used.
- the substrate 1,3-dihydroxyacetone can use formaldehyde as a substrate, using a recombinant vector, a transgenic cell line, or a recombinant vector containing the formaldehyde-converted protein and/or polynucleotide encoding the protein
- the recombinant strain catalyzes the synthesis of 1,3-dihydroxyacetone from formaldehyde.
- the method when catalyzing the synthesis of 1,3-dihydroxyacetone from formaldehyde, includes the following steps: contacting the recombinant vector, transgenic cell line or recombinant strain containing the formaldehyde conversion protein and/or the polynucleotide with formaldehyde to catalyze It produces 1,3-dihydroxyacetone.
- the alkali metal hydroxide and/or alkaline earth metal hydroxide catalyzes the reaction of the substrate 1,3-dihydroxyacetone or pyruvaldehyde to produce lactic acid in the following manner: slow the solution containing the substrate It is added to an aqueous solution containing an alkali metal hydroxide or an aqueous suspension of an alkaline earth metal hydroxide.
- slow addition or “slow addition” is opposite to "direct mixing” or “pouring”.
- “Slow addition” or “slow addition” can be carried out in accordance with the usual methods in the art.
- the slow addition rate of the substrate 1,3-dihydroxyacetone or pyruvaldehyde can be less than or equal to 10mol per hour, that is, the addition rate is less than or equal to 10mol/h; for example, the addition rate can be less than Or equal to 5mol/h, less than or equal to 2.5mol/h, less than or equal to 1mol/h.
- the addition rate can be slower, for example, the addition rate can be even less than or equal to 100 mmol/h or less, but it should not exceed the above upper limit rate.
- the rate of slow addition can be carried out in the usual manner in the art, and can be added in small amounts multiple times, or can be added at a substantially uniform speed, instead of pouring the solution containing the substrate into the alkali metal hydrogen at one time.
- slow addition can be carried out in the usual manner in the art, and can be added in small amounts multiple times, or can be added at a substantially uniform speed, instead of pouring the solution containing the substrate into the alkali metal hydrogen at one time. In an aqueous solution of an oxide or an aqueous suspension of an alkaline earth metal hydroxide.
- the concentration of the substrate in the substrate-containing solution is not particularly limited, and can be 1 to 10000 mM.
- the above-mentioned formaldehyde-converted protein by formaldehyde, or the recombinant protein containing the formaldehyde-converted protein and/or the polynucleotide can be used directly.
- Carriers, transgenic cell lines or recombinant strains catalyze the synthesis of 1,3-dihydroxyacetone in the reaction solution.
- the present invention also provides a method for preparing glycolic acid, which includes the following steps: (1) Converting the above-mentioned formaldehyde into a mutein or a recombinant vector, a transgenic cell line or a recombinant vector containing the formaldehyde-converting mutein and/or the polynucleotide The recombinant strain is contacted with the substrate formaldehyde to catalyze the production of 1,3-dihydroxyacetone and/or hydroxyacetaldehyde; (2) adding an oxidant to oxidize 1,3-dihydroxyacetone and/or hydroxyacetaldehyde to glycolic acid.
- the oxidizing agent in step (2) may be sodium chlorite, sodium hypochlorite, sodium persulfate or hydrogen peroxide.
- step (2) is performed under the condition of pH 3-10.
- the formaldehyde conversion mutein obtained by molecular transformation of the formaldehyde conversion protein greatly improves the efficiency of the production of 1,3-dihydroxyacetone and/or hydroxyacetaldehyde by the condensation of formaldehyde, and provides raw materials for the synthesis of lactic acid and glycolic acid.
- the prior art document CN105777523A discloses a method for preparing lactic acid from sugars under mild conditions.
- the catalyst used is alkali metal or alkaline earth metal hydroxide.
- the sugars include glucose, dihydroxyacetone, pyruvaldehyde, etc., but the method requires It must be carried out under the protection of inert gas, otherwise it is difficult to achieve the substrate conversion rate and lactic acid yield.
- the present invention greatly improves the catalytic conversion of 1,3-dihydroxyacetone or pyruvaldehyde to lactic acid in alkali metal hydroxides and alkaline earth metal hydroxides by optimizing the reaction method by slowly adding a solution containing a substrate. It does not need to be carried out under the protection of inert gas. It can be carried out in an atmospheric environment. It does not require high temperature and high pressure. It can be carried out under normal temperature and pressure. The method is simpler, the cost is lower, and the lactic acid yield is Very high.
- the inventor analyzes and believes that the reason why the document CN105777523A must be carried out under the protection of inert gas may be due to the inhibition of side reactions; in the case of direct mixing of the solution containing the substrate and the solution containing the alkali metal or alkaline earth metal hydroxide catalyst, if Without inert gas protection, the reaction is directly exposed to the air. The presence of oxygen will increase the by-products of the reaction and reduce the yield of lactic acid. In the method of the present invention, the substrate is slowly added, so that a small amount of substrate is quickly converted into product lactic acid in the catalyst system, and side reactions are avoided. Therefore, under the condition of not using inert gas for protection, it is still It achieves a high yield of lactic acid.
- the method of using alkali metal hydroxides and alkaline earth metal hydroxides to catalyze the conversion of 1,3-dihydroxyacetone into lactic acid can also be combined with the method of biocatalyzing formaldehyde to synthesize 1,3-dihydroxyacetone to create a New way to synthesize lactic acid from formaldehyde.
- the conversion from formaldehyde to lactic acid can be realized by catalyzing the two-step process. This pathway has a short path and is carried out under normal temperature and pressure, which has a good application prospect.
- the present invention generates hydroxyacetaldehyde and 1,3-dihydroxyacetone by catalyzing the condensation of formaldehyde to formaldehyde-converting protein, and simultaneously uses an oxidizing agent to oxidize hydroxyacetaldehyde and 1,3-dihydroxyacetone to generate glycolic acid, thereby creating a new formaldehyde synthesis ethanol Sour pathway.
- the annual production volume of formaldehyde is huge, about 20 million tons, which can be produced by methods such as partial oxidation of methanol, hydrogenation of carbon dioxide, partial hydrogenation of carbon monoxide and oxidation of methane. Therefore, the present invention is of great significance to the resource utilization of one-carbon compounds and even carbon dioxide.
- the method for synthesizing glycolic acid provided by the present invention can realize the conversion from formaldehyde to glycolic acid through a two-step reaction of formaldehyde conversion protein catalysis and oxidation of an oxidant (sodium chlorite, sodium hypochlorite, sodium persulfate or hydrogen peroxide).
- an oxidant sodium chlorite, sodium hypochlorite, sodium persulfate or hydrogen peroxide.
- formaldehyde conversion protein in the present invention refers to a protein that can catalyze the synthesis of 1,3-dihydroxyacetone and/or hydroxyacetaldehyde from formaldehyde, as long as it has the ability to catalyze the conversion of formaldehyde to 1,3-dihydroxyacetaldehyde.
- the function of acetone and/or hydroxyacetaldehyde is sufficient, and its amino acid sequence and source are not particularly limited.
- BFD benzoylformate decarboxylases
- BAL Benzaldehyde lyase
- amino acids in the present invention are represented by one-letter or three-letter codes and have the following meanings: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Asp (aspartame) Amino acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamic acid); G: Gly (glycine); H: His (histidine); L: Leu (leucine); K: Lys (lysine); M: Met (methionine); F: Phe (phenylalanine); S: Ser (serine); T: Thr (threonine) ); W: Trp (tryptophan); Y: Tyr (tyrosine); V: Val (valine).
- homoology has the conventional meaning in the art, and refers to the “identity” between two nucleic acid or amino acid sequences, and the percentage represents the comparison obtained after the best alignment. The statistically significant percentage of identical nucleotide or amino acid residues between two sequences, and the differences between the two sequences are randomly distributed over their entire length.
- mutant and variant and variant can be used interchangeably, and “modification” or “mutation” can be used interchangeably.
- These expressions refer to the amino acids of the unmodified or modified protein.
- the formaldehyde conversion mutein of SEQ ID NO:1 as the starting sequence, or based on such a protein contains changes at one or more positions, namely substitutions, insertions and/or deletions, and still Retain its activity.
- Mutant proteins can be obtained by various techniques known in the art.
- exemplary techniques for modifying the DNA sequence encoding the wild-type protein include, but are not limited to, directed mutagenesis, random mutagenesis, and construction of synthetic oligonucleotides.
- substitution with regard to amino acid positions or residues means that the amino acid at a specific position has been replaced by another amino acid. Substitutions can be conservative or non-conservative.
- the mutations are described according to their mutations on specific residues, and their positions are determined by the comparison of the amino acid sequence SEQ ID NO:1 as the starting protein or the reference sequence SEQ ID NO:1. In the context of the present invention, it also relates to any variants carrying these same mutations on functionally equivalent residues.
- corresponding to means that after two sequences are aligned for homology or sequence identity, one sequence corresponds to a designated position in the other sequence.
- corresponding to SEQ ID NO:1 means that the position of the mutation site is determined by comparing with SEQ ID NO:1. Therefore, for example, for “corresponding to the amino acid residue at position 40 of the amino acid sequence shown in SEQ ID NO:1", if 6 ⁇ His is added to the nitrogen end of any amino acid sequence shown in SEQ ID NO:1 Tag, then the 40th position in the obtained mutant corresponding to the amino acid sequence shown in SEQ ID NO:1 may be the 46th position in the mutant.
- SEQ ID NO: 1 only means that SEQ ID NO: 1 is used as the basis for determining the location of the mutation site, but it does not mean that the mutant protein of the present invention can only be modified by modifying SEQ ID NO. :1 shown in the amino acid sequence.
- those skilled in the art can obtain the formaldehyde conversion mutein of the present invention with the amino acid sequence of any formaldehyde conversion enzyme known in the art as the starting sequence, as long as the obtained formaldehyde conversion mutein is compared with SEQ ID NO:1 , Mutation occurs at the site involved in the present invention and maintains the catalytic activity of the formaldehyde converting enzyme, that is, within the scope of the present invention, no other sites except the mutation site are required to be completely consistent with SEQ ID NO:1 .
- the mutation or substitution of the present invention is performed to obtain the formaldehyde conversion mutein of the present invention.
- sequence of SEQ ID NO: 1 with homology is used as the starting sequence to perform the mutation or substitution of the present invention to obtain the formaldehyde conversion mutein of the present invention.
- the sequence of SEQ ID NO: 1 is homologous It is the same as S26, L43, F66, R86, T87, G109, A204, H281, A322, F397, M460, W463, V467, V473, S525 of SEQ ID NO:1, so that the mutation or Substitution, but the other positions can be the same or different.
- the homology between the starting sequence and SEQ ID NO:1 is more than 80%, for example, more than 85%, more than 90%, or more than 95%, so that the specific amino acid corresponding to SEQ ID NO:1 can be determined Site.
- the wild-type formaldehyde converting enzyme (amino acid sequence SEQ ID NO: 23, nucleotide sequence SEQ ID NO: 24) is used as the starting sequence, and the process is still performed at the corresponding position corresponding to SEQ ID NO: 1.
- the mutation or substitution of the present invention obtains the corresponding formaldehyde-converted mutant protein.
- the "departure protein” or “departure sequence” in the present invention refers to the formaldehyde protease or its amino acid sequence before the modification or mutation of the present invention.
- the form of "XaY” is used herein to represent amino acid mutations or substitutions, where a represents the position of the amino acid in SEQ ID NO:1, X represents the wild-type amino acid type at position a in SEQ ID NO:1, and Y represents SEQ ID NO:1 The type of amino acid after the mutation at position a.
- “H281Y” indicates alignment with SEQ ID NO:1, and the histidine H corresponding to the 281st position of SEQ ID NO:1 is replaced by tyrosine Y.
- Figure 1 The product 1,3-dihydroxyacetone detected by HPLC in Example 7.
- Figure 4 Structure diagram of formaldehyde-converted mutant protein.
- Fig. 5 HPLC detection of the chromatogram of the production of lactic acid by 1,3-dihydroxyacetone catalyzed by alkali metal hydroxide in the slow dripping mode in Example 11.
- Fig. 6 HPLC detection of the chromatogram of the production of lactic acid from 1,3-dihydroxyacetone catalyzed by the alkaline earth metal hydroxide in the slow dripping mode in Example 11.
- Fig. 7 HPLC detection of alkaline (earth) metal hydroxide catalyzed by pyruvaldehyde to produce lactic acid in Example 12.
- Figure 8 HPLC detection of oxidizing hydroxyacetaldehyde to produce glycolic acid.
- Figure 9 HPLC detects the oxidation of 1,3-dihydroxyacetone to glycolic acid by an oxidant.
- Figure 10 HPLC detection of sodium chlorite oxidation of formaldehyde conversion protein catalyzed the conversion of hydroxyacetaldehyde and 1,3-dihydroxyacetone into glycolic acid.
- the formaldehyde conversion mutant protein is a mutant BFD1 of benzoylformate decarboxylases (BFD) derived from Pseudomonas putida (hereinafter referred to as the original BFD1 in the present invention), and its nucleotide sequence is SEQ ID NO: 2, add restriction enzyme cut sites NdeI and XhoI at both ends of the nucleotide sequence, and use T4DNA ligase to ligate to the multiple cloning site region of plasmid pET28a to construct recombinant plasmid pET28a-bfd1.
- BFD benzoylformate decarboxylases
- pET28a-bfd1 as a template to design primers (upstream primer: 5'-CCGCGCGGCAGCCATATG-3' (SEQ ID NO: 21) downstream primer: 5'-GGTGGTGGTGGTGGTGCTCGAGTTATT-3' (SEQ ID NO: 22)), which is error-prone PCR, random mutation of the target gene.
- upstream primer 5'-CCGCGCGGCAGCCATATG-3' (SEQ ID NO: 21)
- downstream primer 5'-GGTGGTGGTGGTGGTGCTCGAGTTATT-3' (SEQ ID NO: 22)
- the target gene is recovered by glue, the mutated target gene is connected to the vector pET28a, and the connected vector is transformed into E. coli BL21Gold (DE3), and cultured in a 37°C incubator until a single clone is grown.
- a single clone was picked into a 96-well plate of LB medium containing kanamycin sulfate antibiotics, cultured in a shaker overnight, and the above mutants were replicated to contain isopropyl groups.
- - ⁇ -D-thiogalactoside (IPTG) is cultured in 96-well plates of LB medium for a certain period of time. After the culture is completed, the cells are collected by centrifugation, and then washed with 200 ⁇ L of potassium phosphate buffer (50 mM K 2 HPO 4 and KH 2 PO 4 , 5 mM MgSO 4 , pH 7.4) to collect the cells.
- the cells were resuspended in 50 ⁇ L potassium phosphate buffer, and then an equal volume of formaldehyde solution (potassium phosphate buffer containing 50mM or 30mM or 20mM or 200mM formaldehyde and 1mM thiamine pyrophosphate (TPP)) was added, and reacted at 30°C for 3h.
- formaldehyde solution potassium phosphate buffer containing 50mM or 30mM or 20mM or 200mM formaldehyde and 1mM thiamine pyrophosphate (TPP)
- the obtained strain has a higher catalytic efficiency, which is the beneficial mutant strain, and then through gene sequencing, the corresponding mutation site and amino acid are found. After three rounds of mutant library screening, eighteen mutants with significantly improved enzyme activity and stable activity were finally obtained. Through gene sequencing, the names of the eighteen mutants and the corresponding mutant amino acids are shown in Table 1.
- the inoculation needle picks the positive bacteria of the original BFD1 and the 18 mutants and inoculates them in 5mL LB medium, cultivates them overnight at 37°C, and then inoculates them in 25mL LB medium with 1% (V/V) inoculum at 37°C, 200r/min culture. When OD 600 reached 0.6, 0.1 mM IPTG was added to induce expression at 30°C. After the induction is completed, the above-mentioned cultured mutant cells are collected into centrifuge tubes, and then the cells are resuspended in potassium phosphate buffer and washed. After centrifugation, store the above-mentioned bacteria in a refrigerator at -80°C.
- Example 3 The bacterial cells in Example 3 were taken out, resuspended in potassium phosphate buffer, ultrasonically broken in an ice bath, and centrifuged to collect the supernatant. Take 50 ⁇ L of cell resuspension or supernatant and mix with 50 ⁇ L of formaldehyde solution, and react at 30°C for 3h. After the reaction is complete, take out 90 ⁇ L of the reaction solution and add 60 ⁇ L of tool enzyme buffer 1, and then add 50 ⁇ L of tool enzyme buffer 2. Absorb light at 410nm and detect for 20min. Calculate the enzyme activity of the original BFD1 and the mutant whole cell, the percentage of the mutant enzyme activity relative to the original BFD1 is shown in the following table (the original BFD1 and the original strain whole cell enzyme activity is 100%).
- Example 3 The bacterial cells in Example 3 were taken out, resuspended in potassium phosphate buffer, ultrasonically broken in an ice bath, and centrifuged to collect the supernatant. Take 50 ⁇ L of cell supernatant and heat it at 55°C for 30min. After heating, add 50 ⁇ L of 50mM formaldehyde solution and react at 30°C for 3h. After the reaction is complete, take out 90 ⁇ L of reaction solution and add 60 ⁇ L of tool enzyme buffer 1, and then add 50 ⁇ L of tool Enzyme buffer 2, detection under the condition of 410nm absorption for 20min. After heating the cell supernatant, the activity of the mutant is better than the original BFD1 thermal stability. The relative remaining activities of the original BFD1 and its mutants are shown in Table 7 (take the relative remaining activity of the original BFD1 as 100%).
- Example 3 The original BFD1 and mutant protein V8 cells collected in Example 3 were resuspended in 25 mL potassium phosphate buffer, sonicated in an ice bath, and the supernatant was collected by centrifugation. The expressed mutants were subjected to Ni 2+ chromatography column. Purified by affinity chromatography, eluted with imidazole and desalted by a desalting column, and the purified protein is stored in potassium phosphate buffer for later use.
- the protein concentration of the original BFD1 and mutant V8 purified in Example 6 was diluted to 15 mg/mL with potassium phosphate buffer.
- HPLC detection conditions Column: Aminex HPX-87H, 300mm ⁇ 7.8mm (Bio-Rad) ; Mobile phase: 50% 0.005MH 2 SO 4 solution, 50% H 2 O; UV absorption wavelength: 210 nm; Flow rate: 0.5 mL/min; Column temperature: 65° C.; Injection volume: 5 ⁇ L.
- the HPLC detection results are shown in Figure 1.
- the analysis shows that the original BFD1 and mutant V8 (the amino acid sequence is shown in SEQ ID NO: 25, and the nucleotide sequence is shown in SEQ ID NO: 10) can catalyze the formation of formaldehyde 1 ,3-Dihydroxyacetone.
- Example 8 Alkali metal hydroxides and alkaline earth metal hydroxides catalyze the conversion of 1,3-dihydroxyacetone to lactic acid produced by the catalyzed mutant protein
- Example 7 The reaction solution in Example 7 was centrifuged with an ultrafiltration tube to remove the enzyme in the reaction system. The 17.5mL solution was slowly added to 17.5mL 2M NaOH or KOH solution, and reacted at room temperature for 24 hours. After the reaction, diluted sulfuric acid was added to adjust the pH of the solution. The value is between 1.0-2.0. Pass the 0.22 ⁇ m organic filter membrane, HPLC detects the target product lactic acid, HPLC detection conditions: Chromatographic column: Aminex HPX-87H, 300mm ⁇ 7.8mm (Bio-Rad); mobile phase: 0.010M H 2 SO 4 ; differential detector; flow rate :0.5mL/min; Column temperature: 35°C; Injection volume: 20 ⁇ L.
- the HPLC detection results are shown in Figure 2.
- the analysis shows that alkali metal hydroxides (sodium hydroxide, potassium hydroxide) can further catalyze the conversion of 1,3-dihydroxyacetone produced by the mutant V8 catalyzed by formaldehyde into lactic acid.
- Example 7 The reaction solution in Example 7 was centrifuged with an ultrafiltration tube to remove the enzymes in the reaction system, and 17.5 mL of the solution was slowly added to 17.5 mL of calcium hydroxide (250 mM) suspension, and reacted at room temperature for 24 hours. After the reaction, it was added Dilute sulfuric acid adjusts the pH of the solution to between 1.0-2.0.
- HPLC detection conditions Column: Aminex HPX-87H, 300mm ⁇ 7.8mm (Bio-Rad) Mobile phase: 50% 0.005M H 2 SO 4 solution, 50% H 2 O, ultraviolet absorption wavelength: 210nm, flow rate: 0.5mL/min, column temperature: 65°C, injection volume: 5 ⁇ L.
- the HPLC detection result is shown in Figure 3.
- the analysis shows that the alkaline earth metal hydroxide (calcium hydroxide) can further catalyze the 1,3-dihydroxyacetone produced by the mutant V8 to catalyze the conversion of formaldehyde into lactic acid.
- a single point mutation is carried out in the sequence of the wild-type protein (amino acid sequence is SEQ ID NO: 23, nucleotide sequence is SEQ ID NO: 24). After the mutation is completed and the sequencing is successful, the above mutants are plated.
- a total of 17 strains of the above mutant positive bacteria and the original BFD1 wild-type protein strain were respectively inoculated into 5mL LB medium, cultured overnight at 37°C, and then inoculated into 25mL LB medium with 1% (V/V) inoculum In the medium, culture at 37°C and 200 r/min. When OD 600 reached 0.6, 0.1 mM IPTG was added to induce expression at 30°C. After the induction, the above-mentioned cultured mutant cells were collected into centrifuge tubes, and then resuspended in potassium phosphate buffer and washed the cells. After centrifugation, store the above-mentioned bacteria in a refrigerator at -80°C.
- the cells were taken out, resuspended in potassium phosphate buffer, sonicated in an ice bath, and centrifuged to collect the supernatant. Take 50 ⁇ L of supernatant and mix with 50 ⁇ L of 50mM formaldehyde solution and react at 30°C for 3h. After the reaction is complete, take out 90 ⁇ L of the reaction solution and add 60 ⁇ L of tool enzyme buffer 1, and then add 50 ⁇ L of tool enzyme buffer 2, under the condition of 410nm light absorption Check for 20min.
- the present invention analyzes the crystal structure of mutant V8 (PDB ID: 6M2Y), and through structural analysis, it is concluded that the combined mutations H281Y and S26F form a Pipa interaction (Figure 4), which is a pair of formaldehyde transformation mutations containing H281Y and S26F mutations. The increase in protein activity is of great help.
- Example 11 Comparison of the yield of lactic acid catalyzed by the slow addition method and the direct mixing method alkali metal hydroxide and alkaline earth metal hydroxide
- HPLC detection conditions for the product liquid obtained by alkali metal hydroxide catalysis are as follows: Chromatographic column: Aminex HPX-87H, 300mm ⁇ 7.8mm (Bio-Rad); mobile phase: 0.010M H 2 SO 4 ; differential detector; flow rate: 0.5mL/min; column temperature: 35°C; injection volume: 20 ⁇ L.
- HPLC detection conditions for the product liquid catalyzed by alkaline earth metal hydroxides are as follows: Chromatographic column: Aminex HPX-87H, 300mm ⁇ 7.8mm (Bio-Rad); mobile phase: 0.0025M H 2 SO 4 ; UV 210nm; flow rate: 0.5 mL/min; column temperature: 65°C; injection volume: 5 ⁇ L.
- FIGs 5 and 6 show the HPLC detection results of slow dripping.
- Alkali metal hydroxides NaOH, KOH
- alkaline earth metal hydroxides Ba(OH) 2 , Ca( Both OH) 2
- the calculated yield of lactic acid is shown in Table 11.
- the yield of 1,3-dihydroxyacetone to lactic acid catalyzed by calcium hydroxide was 25.22%
- the yield of 1,3-dihydroxyacetone to lactic acid catalyzed by barium hydroxide was 50.68%
- that of sodium hydroxide The yields of 1,3-dihydroxyacetone to lactic acid catalyzed by potassium hydroxide and potassium hydroxide were 99.84% and 99.74%, respectively.
- Example 12 Alkali metal hydroxides and alkaline earth metal hydroxides catalyze pyruvaldehyde to produce lactic acid
- HPLC detection conditions Chromatographic column: Aminex HPX-87H, 300mm ⁇ 7.8mm (Bio-Rad); mobile phase: 0.0025M H 2 SO 4 ; UV 210nm; flow rate: 0.5 mL/min; column temperature: 65°C; sample volume : 5 ⁇ L.
- the HPLC detection results are shown in Figure 7.
- the analysis shows that both alkali metal hydroxides (NaOH) and alkaline earth metal hydroxides (Ba(OH) 2 ) can catalyze the formation of lactic acid from pyruvaldehyde, and the peak time and lactic acid detected by HPLC The peak times of the standards are the same.
- the calculated yield of lactic acid is shown in Table 12.
- alkali metal hydroxide NaOH
- Alkaline earth alkaline earth metal hydroxide Ba(OH) 2
- Pyruvaldehyde generates lactic acid
- yield of lactic acid 86.46%.
- the formaldehyde conversion mutant protein is derived from Pseudomonas putida (pseudomonas putida) benzoylformate decarboxylases (b enzoylformate decarboxylases, BFD) mutant V1 (abbreviated in the context of the present invention: V1), and its nucleotide sequence is SEQ ID NO: 3 adds restriction sites NdeI and XhoI at both ends of the nucleotide sequence, and ligates it to the multiple cloning site region of plasmid pET28a with T4 DNA ligase to construct a recombinant plasmid pET28a-v1.
- pET28a-v1 as a template to design primers (upstream primer: 5'-CCGCGCGGCAGCCATATG-3' (SEQ ID NO: 21) downstream primer: 5'-GGTGGTGGTGGTGGTGCTCGAGTTATT-3' (SEQ ID NO: 22)), which is error-prone PCR, random mutation of the target gene.
- upstream primer 5'-CCGCGCGGCAGCCATATG-3' (SEQ ID NO: 21)
- downstream primer 5'-GGTGGTGGTGGTGGTGCTCGAGTTATT-3' (SEQ ID NO: 22)
- the target gene is recovered by glue, the mutated target gene is connected to the vector pET28a, and the connected vector is transformed into E. coli BL21Gold (DE3), and cultured in a 37°C incubator until a single clone is grown.
- a single clone was picked into a 96-well plate of LB medium containing kanamycin sulfate antibiotics, cultured in a shaker overnight, and the above mutants were replicated to contain isopropyl groups.
- - ⁇ -D-thiogalactoside (IPTG) is cultured in 96-well plates of LB medium for a certain period of time. After the culture is completed, the cells are collected by centrifugation, and then washed with 200 ⁇ L of sodium phosphate buffer (50 mM Na 2 HPO 4 and NaH 2 PO 4 , 5 mM MgSO 4 , pH 7.4) and collected.
- the bacteria were resuspended in 70 ⁇ L of sodium phosphate buffer, and an equal volume of formaldehyde solution (sodium phosphate buffer containing 200mM or 10mM formaldehyde and 1mM thiamine pyrophosphate (TPP)) was added, and reacted at 30°C for 3h. After the reaction is completed, centrifuge, take out 30 ⁇ L of supernatant and add 150 ⁇ L of diphenylamine reagent (1.5g of diphenylamine dissolved in 100mL of acetic acid, then add 1.5mL of sulfuric acid), heat at 90°C for 30min, after the addition, take out 150 ⁇ L of it in a 96-well plate, Detect the absorbance value at 650nm.
- formaldehyde solution sodium phosphate buffer containing 200mM or 10mM formaldehyde and 1mM thiamine pyrophosphate (TPP)
- TPP thiamine pyrophosphate
- the obtained strain has higher catalytic efficiency, which is the beneficial mutant strain, and then through gene sequencing, the corresponding mutation site and amino acid are found. After three rounds of mutant library screening, twenty mutant proteins with significantly improved enzyme activity were finally obtained. Through gene sequencing, the names of the twenty mutant proteins and the corresponding mutant amino acids are shown in Table 13.
- the inoculation needle picks the positive bacteria of V1 and twenty mutant proteins respectively and inoculates them in 5mL LB medium, cultivated overnight at 37°C, and then inoculates 1% (V/V) inoculum into 25mL LB medium at 37°C. 200r/min culture. When OD 600 reached 0.6, 0.1 mM IPTG was added to induce expression at 30°C. After the induction is completed, the above-mentioned cultured mutant protein bacteria are collected in centrifuge tubes, and then resuspended in sodium phosphate buffer and washed the bacteria. After centrifugation, store the above-mentioned bacteria in a refrigerator at -80°C.
- Example 15 The bacterial cells in Example 15 were taken out, resuspended in sodium phosphate buffer, and ultrasonically broken in an ice bath, and the supernatant was collected by centrifugation. Take 200 ⁇ L of supernatant and 200 ⁇ L of formaldehyde solution and mix it at 30°C for 3h. After the reaction is complete, take out 30 ⁇ L and add 150 ⁇ L of diphenylamine reagent, heat at 90°C for 30min, after the addition, take out 150 ⁇ L in a 96-well plate and detect at 650nm Absorbance values. Take out 90 ⁇ L of the reaction solution and add 60 ⁇ L of Tool Enzyme Buffer 1, and then add 50 ⁇ L of Tool Enzyme Buffer 2, and detect for 20 min under the condition of light absorption at 410 nm.
- the bacterial cells of the mutant protein M3 collected in Example 15 were resuspended in 25 mL sodium phosphate buffer (50mM Na 2 HPO 4 and NaH 2 PO 4 , 5mM MgSO 4 , pH 7.4), sonicated in an ice bath, and collected by centrifugation. After purification, the expressed mutant was purified by affinity chromatography using a Ni 2+ chromatography column, eluted with imidazole and desalted by a desalting column, and the purified protein was stored in sodium phosphate buffer for later use.
- sodium phosphate buffer 50mM Na 2 HPO 4 and NaH 2 PO 4 , 5mM MgSO 4 , pH 7.4
- Example 17 Using sodium phosphate buffer (50mM Na 2 HPO 4 and NaH 2 PO 4 , 5mM MgSO 4 , pH 7.4) the protein of mutant M3 purified in Example 17 (its amino acid sequence is shown in SEQ ID NO: 29, The nucleotide sequence is shown in SEQ ID NO: 28) and the concentration is diluted to 15 mg/mL.
- HPLC detection conditions Chromatographic column: Aminex HPX-87H, 300mm ⁇ 7.8mm (Divot); mobile phase: 0.005M H 2 SO 4 solution; UV detection (210nm) and differential detection; flow rate: 0.5 mL/min; column temperature : 65°C; Sample size: 20 ⁇ L.
- HPLC detection conditions Chromatographic column: Aminex HPX-87H, 300mm ⁇ 7.8mm (Divot); mobile phase: 0.005M H 2 SO 4 solution; UV detection (210nm) and differential detection; flow rate: 0.5 mL/min; column temperature :35°C; Injection volume: 20 ⁇ L.
- HPLC detection results are shown in Figure 8.
- oxidants sodium chlorite, sodium hypochlorite, sodium persulfate and hydrogen peroxide
- HPLC detects the peak time and glycolic acid
- the peak times of the standards are the same.
- the calculated production of glycolic acid is shown in Table 16.
- oxidants sodium chlorite, sodium hypochlorite, sodium sulfite, hydrogen peroxide
- sodium chlorite oxidizes hydroxyacetaldehyde to produce the highest yield of glycolic acid, and the output of glycolic acid is 46.5 mM
- the oxidants sodium hypochlorite, sodium persulfate, and hydrogen peroxide can also oxidize hydroxyacetaldehyde into glycolic acid, with yields of 15.6mM, 6.2mM, and 2.4mM, respectively.
- HPLC detection conditions Chromatographic column: Aminex HPX-87H, 300mm ⁇ 7.8mm (Divot); mobile phase: 0.005M H 2 SO 4 solution; UV detection (210nm) and differential detection; flow rate: 0.5 mL/min; column temperature :35°C; Injection volume: 20 ⁇ L.
- the HPLC detection results are shown in Figure 9.
- the analysis shows that oxidants (sodium chlorite, sodium hypochlorite, hydrogen peroxide) can all catalyze 1,3-dihydroxyacetone to produce glycolic acid, and the peak time detected by HPLC is consistent with the standard of glycolic acid. The peak time of the products is the same.
- the output of glycolic acid is calculated as shown in Table 17.
- oxidants sodium chlorite, sodium hypochlorite and hydrogen peroxide
- sodium hypochlorite oxidizes 1,3-dihydroxyacetone to produce the highest yield of glycolic acid.
- the yield is 22.8mM.
- the oxidants sodium chlorite and hydrogen peroxide can also oxidize 1,3-dihydroxyacetone to produce glycolic acid, and the yields are 17.8mM and 10.3mM, respectively.
- Example 21 Under different pH conditions, sodium chlorite oxidizes hydroxyacetaldehyde to produce glycolic acid
- buffers with different pH values 50 mM citrate buffer at pH 3, 50 mM potassium phosphate buffer at pH 5, 7, and 8, and 50 mM sodium carbonate buffer at pH 10.
- a series of 200 mM sodium chlorite solutions with different pH values and 140 mM hydroxyacetaldehyde solutions with different pH values were prepared using buffers of different pH values. Take 500 ⁇ L of the above-mentioned sodium chlorite solutions of different pH, add an equal volume of 140 mM hydroxyacetaldehyde solution of the same pH, and react at 30°C for 24 hours. After the reaction, the sample was tested by HPLC.
- HPLC detection conditions Chromatographic column: Aminex HPX-87H, 300mm ⁇ 7.8mm (Divot); mobile phase: 0.005M H 2 SO 4 solution; UV detection (210nm) and differential detection; flow rate: 0.5 mL/min; column temperature :35°C; Injection volume: 20 ⁇ L.
- Sodium chlorite solutions with a pH range of 3-10 can oxidize hydroxyacetaldehyde to produce glycolic acid. After calculation, the yield of glycolic acid is shown in Table 18.
- glycolic acid is 51.1mM.
- Example 22 Under different pH conditions, sodium chlorite oxidizes 1,3-dihydroxyacetone to produce glycolic acid
- buffers with different pH values 50 mM citrate buffer at pH 3, 50 mM potassium phosphate buffer at pH 5, 7, and 8, and 50 mM sodium carbonate buffer at pH 10.
- a series of 200mM sodium chlorite solutions with different pH values and 140mM 1,3-dihydroxyacetone solutions with different pH values were prepared using buffers of different pH values. Take 500 ⁇ L of each of the above-mentioned sodium chlorite solutions of different pH, add an equal volume of 140mM 1,3-dihydroxyacetone solution of the same pH, and react at 30°C for 24h. After the reaction, the sample was tested by HPLC.
- HPLC detection conditions Chromatographic column: Aminex HPX-87H, 300mm ⁇ 7.8mm (Divote); Mobile phase: 0.005M H 2 SO 4 solution; UV detection (210nm); Flow rate: 0.5 mL/min; Column temperature: 35°C ; Injection volume: 20 ⁇ L. Under the condition of pH 3-10, sodium chlorite solution can oxidize 1,3-dihydroxyacetone solution to produce glycolic acid. After calculation, the yield of glycolic acid is shown in Table 19.
- glycolic acid Under the condition of pH 3-10, sodium chlorite solution can oxidize 1,3-dihydroxyacetone to produce glycolic acid, and under the condition of pH 8, the highest yield of glycolic acid is 21.7mM.
- Example 23 Different concentrations of sodium chlorite oxidize hydroxyacetaldehyde or 1,3-dihydroxyacetone to produce glycolic acid
- HPLC detection conditions Chromatographic column: Aminex HPX-87H, 300mm ⁇ 7.8mm (Divot); mobile phase: 0.005M H 2 SO 4 solution; UV detection (210nm) and differential detection; flow rate: 0.5 mL/min; column temperature :35°C; Injection volume: 20 ⁇ L.
- Example 24 At different temperatures, sodium chlorite oxidizes 1,3-dihydroxyacetone to produce glycolic acid
- HPLC detection conditions Chromatographic column: Aminex HPX-87H, 300mm ⁇ 7.8mm (Divote); Mobile phase: 0.005M H 2 SO 4 solution; UV detection (210nm) and differential detector; Flow rate: 0.5 mL/min; Column Temperature: 35°C; Injection volume: 20 ⁇ L.
- glycolic acid is calculated as shown in Table 22.
- Example 25 Sodium chlorite oxidizes mixtures of hydroxyacetaldehyde and 1,3-dihydroxyacetone with different molar concentration ratios to produce glycolic acid
- HPLC detection conditions Chromatographic column: Aminex HPX-87H, 300mm ⁇ 7.8mm (Divot); mobile phase: 0.005M H 2 SO 4 solution; UV detection (210nm) and differential detection; flow rate: 0.5 mL/min; column temperature :35°C; Injection volume: 20 ⁇ L.
- the sodium chlorite solution can oxidize the mixed solution of 1,3-dihydroxyacetone and hydroxyacetaldehyde in different molar concentration ratios at different temperatures to produce glycolic acid.
- the output of glycolic acid is calculated as shown in Table 23.
- Example 26 Sodium chlorite oxidizes a mixture of hydroxyacetaldehyde and 1,3-dihydroxyacetone catalyzed by the conversion of formaldehyde to mutein to produce glycolic acid
- Example 18 The reaction solution in Example 18 was centrifuged with an ultrafiltration tube to remove the enzyme in the reaction system.
- the reaction solution of M3 was taken out 500uL to 1.5mL EP tube, and 0.0344g sodium chlorite was added, mixed well and dissolved, the pH of the reaction system It was 7.4, and reacted at 50°C at 1000 rpm for 24 hours. After the reaction, the sample was tested by HPLC.
- HPLC detection conditions Chromatographic column: Aminex HPX-87H, 300mm ⁇ 7.8mm (Divot); mobile phase: 0.005M H 2 SO 4 solution; UV detection (210nm) and differential detection; flow rate: 0.5 mL/min; column temperature :35°C; Injection volume: 20 ⁇ L.
- the HPLC detection results are shown in Figure 10.
- the analysis shows that sodium chlorite can oxidize the hydroxyacetaldehyde and 1,3-dihydroxyacetone that are catalyzed by the conversion of formaldehyde to the mutein to generate glycolic acid.
- the peak time detected by HPLC is consistent with the peak time of the glycolic acid standard.
- the output of glycolic acid is calculated as shown in Table 24.
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Abstract
本发明公开了甲醛转化突变蛋白及其应用,所述突变蛋白可催化甲醛制备1,3-二羟基丙酮,并进一步通过1,3-二羟基丙酮合成乳酸和乙醇酸。还公开了一种合成乳酸和乙醇酸的方法。本发明的突变蛋白能提高甲醛到1,3-二羟基丙酮的催化效率,可以在温和条件下进行。
Description
相关申请的交叉引用
本申请要求以下三件向中国国家知识产权局提交的中国发明专利申请的优先权和权益:2020年2月24日提交的申请号为202010113441.8、发明名称为“甲醛转化突变蛋白及其应用”的在先申请,2020年9月17日提交的申请号为202010983978.X、发明名称为“甲醛转化突变蛋白及其应用”的在先申请,以及2020年9月17日提交的申请号为202010984037.8、发明名称为“一种合成乳酸的方法”的在先申请。所述三件在先申请的全文通过引用的方式纳入本申请中。
本发明属于生物化工技术领域,具体涉及一种甲醛转化突变蛋白及其应用,还涉及一种由1,3-二羟基丙酮、丙酮醛或者甲醛到乳酸的合成方法,还涉及一种由羟基乙醛和1,3-二羟基丙酮、甲醛到乙醇酸的合成方法。
1,3-二羟基丙酮(1,3-dihydroxyacetone,DHA)是自然界存在的最简单的三碳酮糖,用途比较广泛。不仅可以用作化妆品的原料,对皮肤具有很好的保护作用,而且也可以以其为基础合成聚酯类化合物。此外,1,3-二羟基丙酮还是重要的代谢中间产物,通过微生物发酵法可以将其进一步转化为一些高附加值化学品和燃料分子,如乙醇、丁醇、乳酸、琥珀酸等,另外可以合成其他具有更高价值的糖类,如赤藓酮糖,山梨糖等。所以1,3-二羟基丙酮是一个重要的化学合成中间体,广泛应用于化妆品制造、食品研制、医药和化学合成等行业。
目前,1,3-二羟基丙酮的生产方法主要有微生物法和化学法两种,微生物法主要是利用微生物发酵法将甘油转化为1,3-二羟基丙酮,但是微生物方法生产能力低,需要严格控制微生物培养的条件,对微生物的生产环境要求严苛,其次后期1,3-二羟基丙酮的纯化工艺复杂,成本高,污染环境。化学法主要是使用金属催化剂氧化甘油生成1,3-二羟基丙酮,但是化学法使用金属催化剂会对环境造成污染,不利于环境的保护。
乳酸是一种用途广泛的有机酸,在食品工业,乳酸可以被用来作为酸味剂、风味剂等食品添加剂;在医药领域,乳酸盐(铁、钙、钠等)可以用来为人体补充金属元素;在化妆品领域,乳酸可以添加到护肤品中用来作为保湿剂;在纺织领域,乳酸可以用来处理纺织物;在化工领域,乳酸可以作为中间化合物合成绿色有机溶剂乳酸酯,也可以合成聚乳酸。聚乳酸具有生物可降解性、生物相容性、较好的弹性以及优异的物化性,是未来理想的高分子材料。由于其广泛的用途和较高的需求量,乳酸的合成显得尤为重要。
乳酸生产的方法主要有化学合成法和微生物发酵法,此外还有甘油转化法。化学合成法首先利用乙醛和氢氰酸经过高压反应合成乳腈,然后乳腈经硫酸水解生成乳酸。该方法虽然产生的乳酸纯度高,但是由于原料中采用了乙醛和剧毒的氢氰酸等物质,存在较大的污染和生产危险性。微生物发酵法是以糖、淀粉、木质纤维素为原料利用各种细菌或真菌经过生物转化合成乳酸。微生物发酵法以淀粉或糖为原料发酵,其次后续的灭菌和下游乳酸的分离纯 化都会导致乳酸生产成本较高,此外还存在不能连续生产、产品性质不稳定等问题。这些问题都是限制微生物发酵法大规模应用的原因。以甘油为原料合成乳酸,主要是甘油在无机碱(NaOH、CaO)的作用下,在高温条件下反应得到乳酸,该方法存在的问题是:反应过程需要较高的温度,高温的碱性环境对于设备要求较高。
乙醇酸是一种具有重要工业价值的羟基羧酸,在纺织工业,食品工业,制药工业等都具有重要的作用。在纺织工业,乙醇酸可以被用来作为染色鞣剂;在食品工业,乙醇酸可以作为香味剂和防腐剂;在医药工业,乙醇酸则可以用于皮肤保护剂。此外,乙醇酸还可以用在工业及家用清洁剂和粘合剂,还可以转化为具有良好力学性能的生物可降解聚合物-聚乙醇酸,也可以与乳酸一起用于生产在医学上具有重要应用的共聚物。由于其在多个行业的应用,致使乙醇酸市场需求不断增加,有报道称早在2014年其市场需求就已经达到了4万吨。
现在,工业规模生产乙醇酸主要是以化石为原料利用化学法合成,具体是甲醛在高温高压下进行羰基化得来。但是该方法反应条件严苛,能量消耗较大。其次也有研究利用微生物发酵法生产乙醇酸,微生物发酵法大都以葡萄糖、阿拉伯糖、木糖为原料,该方法原料成本较高,微生物发酵生产乙醇酸需要菌体的NADH或NADPH还原力,会造成菌体细胞的还原力不平衡等问题,同时培养过程中需要严格的发酵条件。
近年来,一碳化合物(如:甲酸、甲醇、甲醛等)作为很有应用前景的绿色能源物质得到广泛关注,一碳化合物可以用来合成基本的有机化工原料、燃料和其他高附加值化学品。因其廉价易得的特性,一碳化合物成为替代石油制备高价值化合物最有发展前景的化合物,在医药、食品、化工领域具有重要的科学意义和开发价值。同时一碳化合物的利用和转化也可以显著减少人们在化石燃料利用和合成材料使用过程中对生态环境造成的负面影响。甲醛可以由其他一碳化合物转化而来,进而转化为生物利用的中间物质,同时又兼具来源广泛、价格低廉等特点,以甲醛为前体合成高价值的化合物具有重要的应用前景。
发明内容
为了解决以上问题,本发明的一个目的是提供一种甲醛转化突变蛋白,是从来源于恶臭假单胞菌的苯酰甲酸脱羧酶的突变体BFD1出发,进行易错PCR,对目的基因进行随机突变,筛选到催化甲醛合成1,3-二羟基丙酮活性提高的蛋白,并且该活性具有热稳定性的特点。
本发明第二个目的是提供上述甲醛转化突变蛋白的应用。
本发明第三个目的是提供一种由1,3-二羟基丙酮、丙酮醛或者甲醛到乳酸的合成方法。
本发明第四个目的是提供一种由羟基乙醛、1,3-二羟基丙酮或者甲醛转化为乙醇酸的方法。
本发明采用如下技术方案实现上述目的:
首先,本发明提供了一种甲醛转化突变蛋白,所述甲醛转化突变蛋白能催化甲醛合成1,3-二羟基丙酮,其氨基酸序列是,在对应于SEQ ID NO:1的S26、L43、F66、R86、T87、G109、A204、H281、A322、F397、L407、M460、W463、V467、V473、S525位中至少一个位置处的氨基酸残基发生突变;或者所述甲醛转化突变蛋白的氨基酸序列具有所述发生突变的氨基酸序列中的突变位点,且与所述发生突变的氨基酸序列具有80%以上同源性的氨基酸序列,优选具有90%以上、95%以上或98%以上的同源性。
在一个实施方案中,上述甲醛转化突变蛋白的氨基酸序列至少包括如下突变位点之一:对应于SEQ ID NO:1的第26位的丝氨酸S突变为苯丙氨酸F、第43位的亮氨酸L突变为谷氨酰胺Q、第66位的苯丙氨酸F突变为亮氨酸L、第86位的精氨酸R突变为半胱氨酸C、第87位的苏 氨酸T突变为丙氨酸A、第109位的甘氨酸G突变为丝氨酸S、第204位的丙氨酸A突变为缬氨酸V、第281位的组氨酸H突变为酪氨酸Y、第322位的丙氨酸A突变为苏氨酸T、第397位的苯丙氨酸F突变为亮氨酸L或丝氨酸S中的任一种、第407位的亮氨酸L突变为甲硫氨酸M、第460位的甲硫氨酸M突变为苏氨酸T、第463位的色氨酸W突变为精氨酸R、第467位的缬氨酸V突变为丙氨酸A、第473位的缬氨酸V突变为丙氨酸A、第525位的丝氨酸S突变为丙氨酸A。具体地,所述甲醛转化突变蛋白包括对应于SEQ ID NO:1,发生至少一个如下位点的取代或者是如下二种以上不同位点的组合:S26F、L43Q、F66L、R86C、T87A、G109S、A204V、H281Y、A322T、F397L/F397S、L407M、M460T、W463R、V467A、V473A、S525A。
在一个实施方案中,上述甲醛转化突变蛋白的氨基酸序列至少包括:对应于SEQ ID NO:1的第281位组氨酸H发生突变,例如突变为酪氨酸Y。进一步地,所述甲醛转化突变蛋白还可以包括至少其他一个位点的突变,例如是S26F、L43Q、F66L、T87A、G109S、A204V、A322T、F397L/F397S、L407M、M460T、W463R、V467A、V473A、S525A中的任一个或多个位点的组合。
在一个实施方案中,上述甲醛转化突变蛋白的氨基酸序列至少包括:对应于SEQ ID NO:1的第26位丝氨酸S突变,例如突变为苯丙氨酸F。进一步地,所述甲醛转化突变蛋白还可以包括至少其他一个位点的突变,例如是L43Q、F66L、T87A、G109S、A204V、H281Y、A322T、F397L/F397S、M460T、W463R、V467A、V473A、S525A中的任一个或多个位点的组合。
在一个实施方案中,上述甲醛转化突变蛋白的氨基酸序列至少包括:对应于SEQ ID NO:1的第397位苯丙氨酸F突变,例如突变为亮氨酸L或丝氨酸S中的任一种。进一步地,所述甲醛转化突变蛋白还可以包括至少其他一个位点的突变,例如是S26F、L43Q、F66L、T87A、G109S、A204V、H281Y、A322T、M460T、W463R、V467A、V473A、S525A中的任一个或多个位点的组合。
在一个实施方案中,上述甲醛转化突变蛋白的氨基酸序列至少包括:对应于SEQ ID NO:1的第463位色氨酸W突变,例如突变为精氨酸R。进一步地,所述甲醛转化突变蛋白还可以包括至少其他一个位点的突变,例如是S26F、L43Q、F66L、T87A、G109S、A204V、H281Y、A322T、F397L/F397S、M460T、V467A、V473A、S525A中的任一个或多个位点的组合。
在一个实施方案中,上述甲醛转化突变蛋白的氨基酸序列包括:对应于SEQ ID NO:1的第281位组氨酸H为酪氨酸Y,并且第26位丝氨酸S突变为苯丙氨酸F。进一步地,所述甲醛转化突变蛋白还可以包括至少其他一个位点的突变,例如是W463R、L43Q、G109S、F397S/F397L、M460T、S525A、V194I、V467A、F66L、A204V、A322T、中任一个或多个位点的组合。在一个实施方案中,上述甲醛转化突变蛋白的氨基酸序列包括对应于SEQ ID NO:1的S26F、H281Y、和F397S位点的突变;在又一实施方案中,所述甲醛转化突变蛋白的氨基酸序列包括对应于SEQ ID NO:1的S26F、H281Y、和W463R位点的突变。
作为本发明的示例性方案,上述甲醛转化突变蛋白的氨基酸序列具体为如下1)-18)中任一种:
1)SEQ ID NO:1的第281位组氨酸突变为酪氨酸,其他氨基酸残基保持不变,得到的氨基酸序列(其核苷酸序列为SEQ ID NO:3);
2)SEQ ID NO:1的第86位精氨酸突变为半胱氨酸,其他氨基酸残基保持不变,得到的氨基酸序列(其核苷酸序列为SEQ ID NO:4);
3)SEQ ID NO:1的第281位组氨酸突变为酪氨酸,且将第26位丝氨酸突变为苯丙氨酸,其他氨基酸残基保持不变,得到的氨基酸序列(其核苷酸序列为SEQ ID NO:5);
4)SEQ ID NO:1的第281位组氨酸突变为酪氨酸,且将第397位苯丙氨酸突变为亮氨酸,其他氨基酸残基保持不变,得到的氨基酸序列(其核苷酸序列为SEQ ID NO:6);
5)SEQ ID NO:1的第281位组氨酸突变为酪氨酸,且将第473位缬氨酸突变为丙氨酸,其他氨基酸残基保持不变,得到的氨基酸序列(其核苷酸序列为SEQ ID NO:7);
6)SEQ ID NO:1的第281位组氨酸突变为酪氨酸,且将第26位丝氨酸突变为苯丙氨酸,且将第43位亮氨酸突变为谷氨酰胺,其他氨基酸残基保持不变,得到的氨基酸序列(其核苷酸序列为SEQ ID NO:8);
7)SEQ ID NO:1的第281位组氨酸突变为酪氨酸,且将第26位丝氨酸突变为苯丙氨酸,且将第66位苯丙氨酸突变为亮氨酸,其他氨基酸残基保持不变,得到的氨基酸序列(其核苷酸序列为SEQ ID NO:9);
8)SEQ ID NO:1的第281位组氨酸突变为酪氨酸,且将第26位丝氨酸突变为苯丙氨酸,且将第109位的甘氨酸突变为丝氨酸,其他氨基酸残基保持不变,得到的氨基酸序列(其核苷酸序列为SEQ ID NO:10);
9)SEQ ID NO:1的第281位组氨酸突变为酪氨酸,且将第26位丝氨酸突变为苯丙氨酸,且将第204位丙氨酸突变为缬氨酸,其他氨基酸残基保持不变,得到的氨基酸序列(其核苷酸序列为SEQ ID NO:11);
10)SEQ ID NO:1的第281位组氨酸突变为酪氨酸,且将第26位丝氨酸突变为苯丙氨酸,且将第397位苯丙氨酸突变为丝氨酸,其他氨基酸残基保持不变,得到的氨基酸序列(其核苷酸序列为SEQ ID NO:12);
11)SEQ ID NO:1的第281位组氨酸突变为酪氨酸,且将第26位丝氨酸突变为苯丙氨酸,且将第463位色氨酸突变为精氨酸,其他氨基酸残基保持不变,得到的氨基酸序列(其核苷酸序列为SEQ ID NO:13);
12)SEQ ID NO:1的第281位组氨酸突变为酪氨酸,且将第26位丝氨酸突变为苯丙氨酸,且将第467位缬氨酸突变为丙氨酸,其他氨基酸残基保持不变,得到的氨基酸序列(其核苷酸序列为SEQ ID NO:14);
13)SEQ ID NO:1的第281位组氨酸突变为酪氨酸,且将第87位苏氨酸突变为丙氨酸,且将第322位丙氨酸突变为苏氨酸,其他氨基酸残基保持不变,得到的氨基酸序列(其核苷酸序列为SEQ ID NO:15);
14)SEQ ID NO:1的第281位组氨酸突变为酪氨酸,且将第26位丝氨酸突变为苯丙氨酸,且将第463位色氨酸突变为精氨酸,且将第109位的甘氨酸突变为丝氨酸,其他氨基酸残基保持不变,得到的氨基酸序列(其核苷酸序列为SEQ ID NO:16);
15)SEQ ID NO:1的第281位组氨酸突变为酪氨酸,且将第26位丝氨酸突变为苯丙氨酸,且将第397位苯丙氨酸突变为丝氨酸,且将第109位的甘氨酸突变为丝氨酸,其他氨基酸残基保持不变,得到的氨基酸序列(其核苷酸序列为SEQ ID NO:17);
16)SEQ ID NO:1的第281位组氨酸突变为酪氨酸,且将第26位丝氨酸突变为苯丙氨酸,且将第460位蛋氨酸突变为苏氨酸,且将第525位丝氨酸突变为丙氨酸,其他氨基酸残基保持不变,得到的氨基酸序列(其核苷酸序列为SEQ ID NO:18);
17)SEQ ID NO:1的第281位组氨酸突变为酪氨酸,且将第26位丝氨酸突变为苯丙氨酸,且将第397位苯丙氨酸突变为丝氨酸,且将第109位的甘氨酸突变为丝氨酸,且将第322位丙氨酸突变为苏氨酸,其他氨基酸残基保持不变,得到的氨基酸序列(其核苷酸序列为SEQ ID NO:19);
18)SEQ ID NO:1的第281位组氨酸突变为酪氨酸,且将第26位丝氨酸突变为苯丙氨酸,且将第463位色氨酸突变为精氨酸,且将第109位的甘氨酸突变为丝氨酸,且将第397位的苯丙氨酸突变为丝氨酸,其他氨基酸残基保持不变,得到的氨基酸序列(其核苷酸序列为SEQ ID NO:20);
19)SEQ ID NO:1的第281位组氨酸突变为酪氨酸,且将第109位的甘氨酸突变为丝氨酸,其他氨基酸残基保持不变,得到的氨基酸序列(其核苷酸序列为SEQ ID NO:26);
20)SEQ ID NO:1的第281位组氨酸突变为酪氨酸,且将第407位的亮氨酸突变为甲硫氨酸,其他氨基酸残基保持不变,得到的氨基酸序列(其核苷酸序列为SEQ ID NO:27);
21)SEQ ID NO:1的第281位组氨酸突变为酪氨酸,且将第109位的甘氨酸突变为丝氨酸,且将第407位的亮氨酸突变为甲硫氨酸,其他氨基酸残基保持不变,得到的氨基酸序列(其核苷酸序列为SEQ ID NO:28)。
本发明还提供一种多核苷酸,所述多核苷酸是编码上述甲醛转化突变蛋白的多核苷酸。
本发明还提供含有上述甲醛转化突变蛋白和/或上述多核苷酸的重组载体、表达盒、转基因细胞系和/或重组菌。
本发明还提供上述甲醛转化突变蛋白作为催化剂的用途,例如用于催化制备1,3-二羟基丙酮和羟基乙醛和/或乳酸和/或乙醇酸的催化剂,例如催化底物甲醛制备1,3-二羟基丙酮和/或乳酸的催化剂,或者催化底物甲醛制备1,3-二羟基丙酮和羟基乙醛进而制备乙醇酸的催化剂。
本发明还提供含有上述多核苷酸的重组载体、表达盒、转基因细胞系和/或重组菌作为催化剂的用途,例如用于催化制备1,3-二羟基丙酮和/或乳酸和/或乙醇酸的催化剂,例如催化底物甲醛制备1,3-二羟基丙酮和/或乳酸的催化剂,或者催化底物甲醛制备1,3-二羟基丙酮和羟基乙醛进而制备乙醇酸的催化剂。
在本发明的甲醛转化突变蛋白催化甲醛制备目标化合物乳酸时,包括:将甲醛转化突变蛋白与甲醛接触,催化其生成1,3-二羟基丙酮;进一步地,可任选地加入碱金属氢氧化物和/或碱土金属氢氧化物,催化1,3-二羟基丙酮生成乳酸。
本发明中包含上述甲醛转化突变蛋白和/或所述多核苷酸的重组载体、转基因细胞系或重组菌株在催化甲醛制备目标化合物乳酸时,包括:将包含所述甲醛转化突变蛋白和/或所述多核苷酸的重组载体、转基因细胞系或重组菌株与甲醛接触,催化其生成1,3-二羟基丙酮;进一步地,可任选地加入碱金属氢氧化物和/或碱土金属氢氧化物,催化1,3-二羟基丙酮生成乳酸。
在本发明的甲醛转化突变蛋白催化甲醛制备目标化合物乙醇酸时,包括:将甲醛转化突变蛋白与甲醛接触,催化其生成1,3-二羟基丙酮和/或羟基乙醛;进一步地,加入氧化剂,将1,3-二羟基丙酮和/或羟基乙醛氧化为乙醇酸。
本发明中包含上述甲醛转化突变蛋白和/或所述多核苷酸的重组载体、转基因细胞系或重组菌株在催化甲醛制备目标化合物乙醇酸时,包括:将包含所述甲醛转化突变蛋白和/或所述多核苷酸的重组载体、转基因细胞系或重组菌株与甲醛接触,催化其生成1,3-二羟基丙酮和/或羟基乙醛;加入氧化剂,将1,3-二羟基丙酮和/或羟基乙醛氧化为乙醇酸。
所述氧化剂可以为亚氯酸钠、次氯酸钠、过硫酸钠或过氧化氢。
本发明提供了一种制备乳酸的方法,包括如下步骤:以1,3-二羟基丙酮或丙酮醛为底物,用碱金属氢氧化物和/或碱土金属氢氧化物催化底物产生乳酸。
在一个实施方案中,所述碱金属氢氧化物可以为氢氧化钠、氢氧化钾,碱土金属氢氧化 物可以为氢氧化钙、氢氧化钡。
在一个实施方案中,所述碱金属氢氧化物或碱土金属氢氧化物与底物1,3-二羟基丙酮或丙酮醛的摩尔比可以为(1~100):1,例如(2~80):1。特别地,所述碱金属氢氧化物与底物1,3-二羟基丙酮或丙酮醛的摩尔比可以为(5~80):1,例如(10~60):1,(15~50):1,(20~40):1;所述碱土金属氢氧化物与底物1,3-二羟基丙酮或丙酮醛的摩尔比可以为(1~20):1,例如(2~10):1,(2~6):1。
在一个实施方案中,所述碱金属氢氧化物和/或碱土金属氢氧化物催化底物1,3-二羟基丙酮或丙酮醛产生乳酸的反应不在惰性气体保护条件下进行。换言之,所述反应在大气环境中进行即可。
在一个实施方案中,所述碱金属氢氧化物和/或碱土金属氢氧化物催化底物1,3-二羟基丙酮或丙酮醛产生乳酸的反应可以在10~50℃的范围内进行,优选在20~30℃的范围内进行,例如在室温下进行。
在一个实施方案中,所述碱金属氢氧化物和/或碱土金属氢氧化物催化底物1,3-二羟基丙酮或丙酮醛产生乳酸的反应的时间可以为6~80小时,例如为12~60小时,24~48小时。
在一个实施方案中,所述底物1,3-二羟基丙酮可以以甲醛为底物,用甲醛转化蛋白催化甲醛合成1,3-二羟基丙酮;其中所述甲醛转化蛋白具有催化甲醛转化为1,3-二羟基丙酮的功能。具体地,在用甲醛转化突变蛋白催化甲醛合成1,3-二羟基丙酮时,包括如下步骤:将甲醛转化突变蛋白与甲醛接触,催化甲醛生成1,3-二羟基丙酮。
用于催化甲醛合成1,3-二羟基丙酮的“甲醛转化蛋白”是指能催化甲醛合成1,3-二羟基丙酮的蛋白,只要其具有催化甲醛转化为1,3-二羟基丙酮的功能即可,对其氨基酸序列和来源没有特别限制。仅作为实例但并非限制性地,其可以是例如,来源于恶臭假单胞菌(Pseudomonas putida)的苯酰甲酸脱羧酶(benzoylformate decarboxylases,BFD)和来源于荧光假单胞菌(Pseudomonas fluorescens biovar I)的苯甲醛裂合酶(benzaldehyde lyase,BAL),以及它们经过氨基酸突变后得到具有上述功能的蛋白。例如,可使用苯甲醛裂合酶或其突变体,还可以是上面所述的任一种甲醛转化突变蛋白。
在一个实施方案中,所述底物1,3-二羟基丙酮可以以甲醛为底物,用包含所述甲醛转化蛋白和/或编码所述蛋白的多核苷酸的重组载体、转基因细胞系或重组菌株催化甲醛合成1,3-二羟基丙酮。具体地,在催化甲醛合成1,3-二羟基丙酮时,包括如下步骤:将包含所述甲醛转化蛋白和/或所述多核苷酸的重组载体、转基因细胞系或重组菌株与甲醛接触,催化其生成1,3-二羟基丙酮。
在一个实施方案中,所述碱金属氢氧化物和/或碱土金属氢氧化物催化底物1,3-二羟基丙酮或丙酮醛产生乳酸的反应以如下方式进行:将含有底物的溶液缓慢添加到含有碱金属氢氧化物的水溶液或者碱土金属氢氧化物的水悬浊液中。其中“缓慢添加”或“缓慢加入”与“直接混合”或“倾倒”相对,“缓慢添加”或“缓慢加入”可按照本领域的通常方式进行,可以少量多次地加入,也可以匀速地加入,而不是将含底物的溶液一次性地倾倒入含有碱金属氢氧化物的水溶液或者碱土金属氢氧化物的水悬浊液中。作为优选方案,所述底物1,3-二羟基丙酮或丙酮醛缓慢添加的速率可以为每小时的添加量小于或等于10mol,即添加速率小于或等于10mol/h;例如,添加速率可以小于或等于5mol/h,小于或等于2.5mol/h,小于或等于1mol/h。本领域技术人员可以理解,如果不考虑生产效率的话,添加速率可以更缓慢,例如添加速率甚至可以小于或等于100mmol/h或者更小,但是不宜超过上述上限速率。在本发明教导的上述范围内,本领域技术人员可以根据实际情况确定缓慢添加的速率。本领域技术人员可以 理解“缓慢添加”可按照本领域的通常方式进行,可以少量多次地添加,也可以基本匀速地添加,而不是将含底物的溶液一次性地倾倒入含有碱金属氢氧化物的水溶液或者碱土金属氢氧化物的水悬浊液中。对于含底物的溶液中底物的浓度没有特别限制,可以为1~10000mM,例如可以直接使用经上述甲醛经甲醛转化蛋白、或者包含所述甲醛转化蛋白和/或所述多核苷酸的重组载体、转基因细胞系或重组菌株催化合成1,3-二羟基丙酮所得的反应液进行。
本发明还提供了一种制备乙醇酸的方法,包括如下步骤:(1)将上述甲醛转化突变蛋白或者包含所述甲醛转化突变蛋白和/或所述多核苷酸的重组载体、转基因细胞系或重组菌株与底物甲醛接触,催化生成1,3-二羟基丙酮和/或羟基乙醛;(2)加入氧化剂,将1,3-二羟基丙酮和/或羟基乙醛氧化为乙醇酸。
在一个实施方案中,步骤(2)中所述氧化剂可以为亚氯酸钠、次氯酸钠、过硫酸钠或过氧化氢。
在一个实施方案中,步骤(2)在pH 3-10的条件下进行。
本发明通过对甲醛转化蛋白进行分子改造获得的甲醛转化突变蛋白,大大提高了甲醛缩合产生1,3-二羟基丙酮和/或羟基乙醛的效率,并为乳酸和乙醇酸的合成提供原料。
现有技术文献CN105777523A中公开了一种温和条件下由糖类制备乳酸的方法,所用催化剂为碱金属或碱土金属氢氧化物,糖类包括葡萄糖、二羟基丙酮、丙酮醛等,但是其方法要求必须在惰性气体保护下进行,否则难以实现的底物转化率和乳酸产率。而本发明通过对反应方法的优化,采用缓慢添加含有底物的溶液的方式大大提高了由1,3-二羟基丙酮或丙酮醛在碱金属氢氧化物及碱土金属氢氧化物催化转化为乳酸的产率,并且不必要求在惰性气体保护下进行,在大气环境中进行即可,也不要求高温和高压,在常温常压下进行即可,方法更加简单,成本更低,同时乳酸产率很高。
发明人分析认为,文献CN105777523A中必须要求在惰性气体保护下进行的原因可能在于抑制副反应;在含底物的溶液与含碱金属或碱土金属氢氧化物催化剂的溶液直接混合的情况下,如果不采用惰性气体保护,则反应直接暴露在空气中,氧气的存在会使反应的副产物增多,乳酸的产率就会降低。而本发明的方法中,缓慢地加入底物,使少量的底物在催化剂体系中快速的转化为产物乳酸,避免了副反应的产生,因此,在不使用惰性气体进行保护的条件下,仍然是实现了乳酸的高产率。
本发明的利用碱金属氢氧化物及碱土金属氢氧化物催化1,3-二羟基丙酮转化为乳酸的方法还可以与生物催化甲醛合成1,3-二羟基丙酮的方法相结合,创建了一条新的甲醛合成乳酸的途径。通过催化两步法即可实现由甲醛到乳酸的转化,此途径路径短并且在常温常压下进行,具有较好的应用前景。
本发明通过对甲醛转化蛋白催化甲醛缩合产生羟基乙醛和1,3-二羟基丙酮,同时利用氧化剂氧化羟基乙醛和1,3-二羟基丙酮生成乙醇酸,从而创建一条新的甲醛合成乙醇酸的途径。甲醛每年的生产量巨大,约2000万吨,可由甲醇的部分氧化,二氧化碳的氢化,一氧化碳的部分氢化和甲烷的氧化等方法生产。因此本发明对一碳化合物乃至二氧化碳的资源化利用具有重要意义。
本发明提供的乙醇酸合成的途径,通过甲醛转化蛋白催化和氧化剂(亚氯酸钠、次氯酸钠、过硫酸钠或过氧化氢)氧化两步反应即可实现由甲醛到乙醇酸的转化,此途径路径短并且在常温常压下进行,无需其它苛刻或复杂条件,具有较好的应用前景。
术语和定义
在本发明的上下文中,本发明中“甲醛转化蛋白”是指能催化甲醛合成1,3-二羟基丙酮和/或羟基乙醛的蛋白,只要其具有催化甲醛转化为1,3-二羟基丙酮和/或羟基乙醛的功能即可,对其氨基酸序列和来源没有特别限制。仅作为实例但并非限制性地,其可以是例如,来源于恶臭假单胞菌(Pseudomonas putida)的苯酰甲酸脱羧酶(benzoylformate decarboxylases,BFD)和来源于荧光假单胞菌(Pseudomonas fluorescens biovar I)的苯甲醛裂合酶(benzaldehyde lyase,BAL)。“甲醛转化突变蛋白”是指“甲醛转化蛋白”经过氨基酸突变后得到具有上述功能的蛋白。
本发明中氨基酸由单字母或三字母代码表示,具有如下含义:A:Ala(丙氨酸);R:Arg(精氨酸);N:Asn(天冬酰胺);D:Asp(天冬氨酸);C:Cys(半胱氨酸);Q:Gln(谷氨酰胺);E:Glu(谷氨酸);G:Gly(甘氨酸);H:His(组氨酸);L:Leu(亮氨酸);K:Lys(赖氨酸);M:Met(甲硫氨酸);F:Phe(苯丙氨酸);S:Ser(丝氨酸);T:Thr(苏氨酸);W:Trp(色氨酸);Y:Tyr(酪氨酸);V:Val(缬氨酸)。
本发明中,“同源性”具有本领域常规的含义,是指两个核酸或氨基酸序列之间的“同一性”,其百分比表示在最佳比对(best alignment)后获得的待比较的两个序列之间的相同核苷酸或氨基酸残基的统计学意义的百分比,两个序列之间的差异随机地分布在其整个长度上。
在本发明中,术语“突变体”和“变体”以及“突变蛋白”可以互换使用,“修饰”或“突变”可以互换施用,这些表达是指相对于未修饰或改造蛋白的氨基酸,例如作为出发序列的SEQ ID NO:1的甲醛转化突变蛋白,或来源于此类蛋白的基础上,包含在一个或更多个位置处的改变,即取代、插入和/或缺失,并仍然保留其活性。突变蛋白可以通过本领域已知的各种技术获得。特别地,用于修饰编码野生型蛋白的DNA序列的示例性技术包括但不限于,定向诱变、随机诱变和合成寡核苷酸的构建。
关于氨基酸位置或残基的术语“取代”是指在特定位置处的氨基酸已被其他的氨基酸代替。取代可以是保守的或非保守的。
所述突变根据它们在特定残基上的突变来描述,其位置通过作为出发蛋白的氨基酸序列SEQ ID NO:1比对或参考序列SEQ ID NO:1来确定。在本发明的上下文中,还涉及在功能等同的残基上携带这些相同突变的任何变体。
本文所用的术语“对应于”具有本领域普通技术人员通常理解的意义。具体地说,“对应于”表示两条序列经同源性或序列相同性比对后,一条序列与另一条序列中的指定位置相对应的位置。在本发明中,“对应于SEQ ID NO:1”表示通过与SEQ ID NO:1对比确定突变位点的位置。因此,例如,就“对应于SEQ ID NO:1所示氨基酸序列的第40位的氨基酸残基”而言,如果在SEQ ID NO:1所示任一氨基酸序列的氮端加上6×His标签,那么所得突变体中对应于SEQ ID NO:1所示氨基酸序列的第40位就可能是突变体中的第46位。
本领域技术人员应当理解,“对应于SEQ ID NO:1”仅表示将SEQ ID NO:1作为确定突变位点的位置的基准,但并不代表本发明的突变蛋白仅能通过改造SEQ ID NO:1所示的氨基酸序列所得到。在一个实施方案中,本领域技术人员可以本领域已知的任何甲醛转化酶的氨基酸序列为出发序列得到本发明的甲醛转化突变蛋白,只要所得到的甲醛转化突变蛋白与SEQ ID NO:1对比,在本发明所涉及的位点处发生突变,并保持甲醛转化酶的催化活性,即在本发明的范围内,不需要除突变位点外的其他位点均与SEQ ID NO:1完全一致。在一个实施方案中,以SEQ ID NO:1所示的蛋白为出发序列,进行本发明的突变或取代,得到本发明 的甲醛转化突变蛋白。在一个实施方案中,以SEQ ID NO:1具有同源性的序列为出发序列,进行本发明的突变或取代,得到本发明的甲醛转化突变蛋白,该SEQ ID NO:1的同源性序列与SEQ ID NO:1的S26、L43、F66、R86、T87、G109、A204、H281、A322、F397、M460、W463、V467、V473、S525位点是相同的,以便可以发生本发明的突变或取代,但其他位点可以相同或不同。在一个实施方案中,该出发序列与SEQ ID NO:1的同源性为80%以上,例如85%以上、90%以上或95%以上,以便能够确定对应于SEQ ID NO:1的特定氨基酸位点。在又一个实施方案中,以野生型甲醛转化酶(氨基酸序列SEQ ID NO:23,核苷酸序列SEQ ID NO:24)为出发序列,仍在对应于SEQ ID NO:1的相应位点进行本发明的突变或取代,得到相应的甲醛转化突变蛋白。
本发明中“出发蛋白”或“出发序列”是指进行本发明的修饰或突变之前的甲醛蛋白酶或其氨基酸序列。本文采用“XaY”的形式表示氨基酸的突变或取代,其中a表示SEQ ID NO:1中氨基酸的位置,X表示SEQ ID NO:1中a位置野生型的氨基酸种类,Y表示SEQ ID NO:1中a位置突变后的氨基酸种类。例如,“H281Y”表示与SEQ ID NO:1比对,在对应于SEQ ID NO:1第281位的组氨酸H被酪氨酸Y取代。
图1:实施例7中HPLC检测产物1,3-二羟基丙酮。
图2:实施例8中HPLC检测碱金属氢氧化物催化产生的终产物乳酸。
图3:实施例8中HPLC检测碱土金属氢氧化物催化产生的终产物乳酸。
图4:甲醛转化突变蛋白结构图。
图5:实施例11中HPLC检测碱金属氢氧化物在缓慢滴加方式下催化1,3-二羟基丙酮产生乳酸的图谱。
图6:实施例11中HPLC检测碱土金属氢氧化物在缓慢滴加方式下催化1,3-二羟基丙酮产生乳酸的图谱。
图7:实施例12中HPLC检测碱(土)金属氢氧化物催化丙酮醛产生乳酸的图谱。
图8:HPLC检测氧化剂氧化羟基乙醛生成乙醇酸。
图9:HPLC检测氧化剂氧化1,3-二羟基丙酮生成乙醇酸。
图10:HPLC检测亚氯酸钠氧化甲醛转化蛋白催化甲醛生成的羟基乙醛和1,3-二羟基丙酮转化成乙醇酸。
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解,下列实施例仅为示例性地说明和解释本发明,而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明,以下实施例中使用的原料和试剂均为市售商品,或者可以通过已知方法制备。下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
实施例1 甲醛转化突变蛋白(甲醛转化突变体)构建
甲醛转化突变蛋白是来源于恶臭假单胞菌(Pseudomonas putida)的苯酰甲酸脱羧酶(benzoylformate decarboxylases,BFD)的突变体BFD1(本发明中下文简称:原始BFD1),其核苷酸序列为SEQ ID NO:2,将该核苷酸序列两端加上限制性酶切位点NdeⅠ和XhoⅠ,用T4DNA连接酶连接至质粒pET28a的多克隆位点区域,构建重组质粒pET28a-bfd1。然后以 pET28a-bfd1为模板,设计引物(上游引物:5’-CCGCGCGGCAGCCATATG-3’(SEQ ID NO:21)下游引物:5’-GGTGGTGGTGGTGGTGCTCGAGTTATT-3’(SEQ ID NO:22)),进行易错PCR,对目的基因进行随机突变。PCR完成后,对目的基因进行胶回收,将突变后的目的基因连接在载体pET28a上,再将连接后的载体转化到大肠杆菌BL21Gold(DE3)中,37℃培养箱培养至长出单克隆。
实施例2 甲醛转化突变蛋白的筛选
对实施例1中得到的突变体克隆,挑取单克隆到含有硫酸卡那霉素抗生素的LB培养基的96孔板中,在摇床中过夜培养,将上述突变体复制到含有异丙基-β-D-硫代半乳糖苷(IPTG)的LB培养基的96孔板中培养至一定时间。培养完成后,离心收集菌体,再用200μL磷酸钾缓冲液(50mM K
2HPO
4和KH
2PO
4,5mM MgSO
4,pH 7.4)洗涤并收集菌体。
菌体用50μL磷酸钾缓冲液重悬,再分别加入等体积的甲醛溶液(磷酸钾缓冲液含有50mM或30mM或20mM或200mM甲醛和1mM硫胺素焦磷酸(TPP)),30℃反应3h。反应完成后,离心取90μL上清液于96孔板中,加入60μL的工具酶缓冲液1(0.3mg/mL半乳糖氧化酶,36U/mL辣根过氧化物酶),然后加入50μL的工具酶缓冲液2(3.2mM 2,2'-联氮-双-3-乙基苯并噻唑啉-6-磺酸(ABTS)),在吸收光410nm条件下检测20min。
得到的菌株与原始BFD1比较,催化效率更高的即为有益突变菌株,然后通过基因测序,找到相对应突变的位点及氨基酸。经过三轮突变体库的筛选,最终获得了十八株酶活显著提高,且活性稳定的突变体。通过基因测序,得到所述十八株突变体的名称及对应突变氨基酸见表1。
表1 突变体名称及对应突变氨基酸
实施例3 原始BFD1及其突变体在大肠杆菌中的表达
接种针挑取原始BFD1和18种突变体的阳性菌分别接种于5mLLB培养基中,37℃过夜培养,然后以1%(V/V)接种量接种于25mL LB培养基中,以37℃、200r/min培养。当OD
600达到0.6时,加入0.1mM的IPTG,在30℃下诱导表达。诱导完成后,将上述培养的突变体菌体分别收集到离心管中,然后用磷酸钾缓冲液重悬并清洗菌体。离心后-80℃冰箱保存上述菌体。
实施例4 原始BFD1及其突变体产1,3-二羟基丙酮的活性检测
取出实施例3中的菌体,磷酸钾缓冲液重悬,冰浴超声破菌,离心收集上清。取50μL细胞重悬液或上清液与50μL甲醛溶液混合,于30℃反应3h,反应完成后,取出90μL反应溶液加入60μL的工具酶缓冲液1,然后加入50μL的工具酶缓冲液2,在吸收光410nm条件下检测20min。计算原始BFD1及突变体全细胞酶活力,突变体酶活力相对于原始BFD1的百分比如下表所示(以原始BFD1及出发菌株全细胞酶活力为100%)。
表2 原始BFD1及突变体的全细胞在30mM甲醛浓度下活性
表3 突变体V3及其进一步突变体的全细胞在20mM甲醛浓度下活性
表4 突变体V10及其进一步突变体的细胞破碎上清在20mM甲醛浓度下活性
表5 突变体V11及其进一步突变体的细胞破碎上清在20mM甲醛浓度下活性
表6 突变体V11及其进一步突变体的细胞破碎上清在200mM甲醛浓度下活性
实施例5 原始BFD1及其突变体热稳定性检测
取出实施例3中的菌体,磷酸钾缓冲液重悬,冰浴超声破菌,离心收集上清。取50μL细胞上清液在55℃下加热30min,加热完成后加入50μL 50mM甲醛溶液,于30℃反应3h,反应完成后,取出90μL反应溶液加入60μL的工具酶缓冲液1,然后加入50μL的工具酶缓冲液2,在吸收光410nm条件下检测20min。对细胞上清液加热后,突变体的活性相对于原始BFD1热稳定性较好。原始BFD1及其突变体的相对剩余活性如表7所示(以原始BFD1相对剩余活性为100%)。
表7 原始BFD1及其突变体相对剩余活性
实施例6 原始BFD1转化及其突变体的纯化
实施例3中收集的原始BFD1、突变蛋白V8的菌体,用25mL磷酸钾缓冲液重悬,冰浴超声破菌,离心收集上清,利用Ni
2+层析柱对上述表达的突变体进行亲和层析纯化,用咪唑洗脱后经脱盐柱脱盐,纯化后的蛋白保存在磷酸钾缓冲液中备用。
实施例7 甲醛转化生成1,3-二羟基丙酮的反应
用磷酸钾缓冲液将实施例6中纯化后的原始BFD1和突变体V8的蛋白浓度稀释至15mg/mL。在反应体系中先取一定体积的纯酶,然后加入等量的甲醛溶液(600mM甲醛,1mM TPP,50mM K
2HPO
4和KH
2PO
4,5mM MgSO
4,pH 7.4)),30℃反应1h。反应结束后,取一定体积的反应液加入等体积的乙腈终止反应,离心过0.22μm滤膜,进行HPLC检测,HPLC检测条件:色谱柱:Aminex HPX-87H,300mm×7.8mm(Bio-Rad);流动相:50%0.005M H
2SO
4溶液,50%H
2O;紫外吸收波长:210nm;流速:0.5mL/min;柱温:65℃;进样量:5μL。
HPLC检测结果如图1所示,经分析可知,原始BFD1和突变体V8(其氨基酸序列如SEQ ID NO:25所示,核苷酸序列如SEQ ID NO:10所示)可以催化甲醛生成1,3-二羟基丙酮。
经过计算得出,原始BFD1和突变体V8催化甲醛产生1,3-二羟基丙酮的转化率如下表8所示。
表8 原始BFD1及突变体V8催化甲醛产生1,3-二羟基丙酮的转化率
实施例8 碱金属氢氧化物及碱土金属氢氧化物催化突变蛋白催化产生的1,3-二羟基丙酮到乳酸的转化
对实施例7中的反应溶液使用超滤管离心去除反应体系中的酶,17.5mL的溶液缓慢加入至17.5mL 2M NaOH或KOH溶液,室温下反应24h,反应结束后加入稀硫酸调节溶液的pH值到1.0-2.0之间。过0.22μm的有机滤膜,HPLC检测目标产物乳酸,HPLC检测条件:色谱柱:Aminex HPX-87H,300mm×7.8mm(Bio-Rad);流动相:0.010M H
2SO
4;示差检测器;流速:0.5mL/min;柱温:35℃;进样量:20μL。
HPLC检测结果如图2所示,经分析可知,碱金属氢氧化物(氢氧化钠、氢氧化钾)都可以进一步催化突变体V8催化甲醛产生的1,3-二羟基丙酮转化为乳酸。
对实施例7中的反应溶液使用超滤管离心去除反应体系中的酶,取17.5mL的溶液缓慢加入至17.5ml氢氧化钙(250mM)悬浊液中,室温下反应24h,反应结束后加入稀硫酸调节溶液的pH值到1.0-2.0之间。过0.22μm的有机滤膜,HPLC检测目标产物乳酸,HPLC检测条件:色谱柱:Aminex HPX-87H,300mm×7.8mm(Bio-Rad)流动相:50%0.005M H
2SO
4溶液,50%H
2O,紫外吸收波长:210nm,流速:0.5mL/min,柱温:65℃,进样量:5μL。
HPLC检测结果如图3所示,经分析可知,碱土金属氢氧化物(氢氧化钙)可以进一步催化突变体V8催化甲醛产生的1,3-二羟基丙酮转化为乳酸。
经过计算从甲醛经过突变体V8,及碱金属氢氧化物(氢氧化钠、氢氧化钾)和碱土金属氢氧化物(氢氧化钙)催化产生的乳酸的转化率如下表9所示。
表9 从甲醛到乳酸的转化率
实施例9 对原始BFD1的野生型蛋白及其单个氨基酸位点突变体产1,3-二羟基丙酮的活性检测
对实施例1中筛选得到的氨基酸突变位点S26F、L43Q、F66L、R86C、T87A、G109S、A204V、H281Y、A322T、F397L、F397S、M460T、W463R、V467A、V473A、S525A设计引物并在原始BFD1的野生型蛋白(氨基酸序列为SEQ ID NO:23,核苷酸序列为SEQ ID NO:24)序列中进行单点突变,突变完成并测序成功后,对上述突变体进行划平板。
挑取上述突变体阳性菌及原始BFD1的野生型蛋白株共17株菌分别接种于5mL LB培养基中,37℃过夜培养,然后以1%(V/V)接种量接种于25mL LB培养基中,以37℃、200r/min培养。当OD
600达到0.6时,加入0.1mM的IPTG,在30℃下诱导表达。诱导完成后, 将上述培养的突变体菌体分别收集到离心管中,然后用磷酸钾缓冲液重悬并清洗菌体。离心后-80℃冰箱保存上述菌体。
取出菌体,磷酸钾缓冲液重悬,冰浴超声破菌,离心收集上清。取50μL上清液与50μL50mM甲醛溶液混合,于30℃反应3h,反应完成后,取出90μL反应溶液加入60μL的工具酶缓冲液1,然后加入50μL的工具酶缓冲液2,在吸收光410nm条件下检测20min。计算原始BFD1的野生型蛋白及突变体全细胞酶活力,突变体酶活力相对于原始BFD1的野生型蛋白的百分比如下表所示(以原始BFD1的野生型蛋白酶活力为100%)。
表10 突变体名称及对应突变氨基酸及在50mM甲醛浓度下活性
实施例10 甲醛转化突变蛋白的结构分析
本发明解析了突变体V8的晶体结构(PDB ID:6M2Y),并通过结构分析得出组合突变H281Y和S26F形成了派派相互作用(图4),这对包含H281Y和S26F突变的甲醛转化突变蛋白活性的提高有很大的帮助。
实施例11 缓慢添加方法与直接混合方法碱金属氢氧化物及碱土金属氢氧化物催化乳酸产率的对比
配制8份25mL 100mM的1,3-二羟基丙酮溶液,配制2份25mL 250mM Ba(OH)
2悬浊液,2份25mL 250mM Ca(OH)
2悬浊液,2份25mL 2M NaOH溶液,2份25mL 2M KOH溶液。其中将4份25mL的1,3-二羟基丙酮溶液分别与上述的氢氧化钡悬浊液、氢氧化钙悬浊液、氢氧化钠溶液、氢氧化钾溶液直接混合(即将25mL的1,3-二羟基丙酮溶液一次性倒入碱金属氢氧化物溶液或碱土金属氢氧化物的悬浊液中),然后在室温下反应48h;另外4份25mL的1,3-二羟基丙酮溶液分别缓慢加入(历时5小时)到上述的氢氧化钡悬浊液、氢氧化钙悬浊液、氢氧化钠溶液、氢氧化钾溶液中,在室温下搅拌反应48h。反应结束后调节pH值到1.0-2.0之间,离心过0.22μm滤膜,进行HPLC检测。
对于采用碱金属氢氧化物催化所得产物液的HPLC检测条件如下:色谱柱:Aminex HPX-87H,300mm×7.8mm(Bio-Rad);流动相:0.010M H
2SO
4;示差检测器;流速:0.5mL/min;柱温:35℃;进样量:20μL。
对于采用碱土金属氢氧化物催化所得产物液的HPLC检测条件如下:色谱柱:Aminex HPX-87H,300mm×7.8mm(Bio-Rad);流动相:0.0025M H
2SO
4;紫外210nm;流速:0.5mL/min;柱温:65℃;进样量:5μL。
四种反应体系均生成乳酸,图5和图6示出了缓慢滴加方式的HPLC检测结果,碱金属氢氧化物(NaOH,KOH)和碱土金属氢氧化物(Ba(OH)
2,Ca(OH)
2)均能催化1,3-二羟基丙酮生成乳酸,且HPLC检测出峰时间与乳酸标准品的出峰时间一致。计算得到乳酸的产率如表11所示。
表11 缓慢添加和直接混合两种方式下,碱金属/碱土金属氢氧化物催化1,3-二羟基丙酮到乳酸的产率
由表11中的数据可以看出,与直接混合方式的反应相比,将1,3-二羟基丙酮缓慢添加到碱土金属氢氧化物悬浊液(Ba(OH)
2,Ca(OH)
2)和碱金属氢氧化物(NaOH,KOH)溶液中乳酸的产率明显更高。
使用缓慢添加的方式,氢氧化钙催化1,3-二羟基丙酮到乳酸的产率为25.22%,氢氧化钡催化1,3-二羟基丙酮到乳酸的产率为50.68%,而氢氧化钠和氢氧化钾催化1,3-二羟基丙酮到乳酸的产率分别为99.84%和99.74%。
实施例12 碱金属氢氧化物及碱土金属氢氧化物催化丙酮醛产生乳酸
配制2份25mL 50mM的丙酮醛溶液,25mL 200mM氢氧化钡悬浊液,25mL 2M氢氧化钠溶液。将上述丙酮醛溶液分别缓慢加入(历时3小时)到氢氧化钡悬浊液和氢氧化钠溶液中,室温下搅拌反应48h,反应结束后调节pH值到1.0-2.0之间,离心过0.22μm滤膜,进行HPLC检测。
HPLC检测条件:色谱柱:Aminex HPX-87H,300mm×7.8mm(Bio-Rad);流动相:0.0025M H
2SO
4;紫外210nm;流速:0.5mL/min;柱温:65℃;进样量:5μL。
HPLC检测结果如图7所示,经分析可知,碱金属氢氧化物(NaOH),碱土金属氢氧化物(Ba(OH)
2)均能催化丙酮醛生成乳酸,且HPLC检测出峰时间与乳酸标准品的出峰时间一致。经过计算乳酸的产率如表12所示。
表12 碱金属/碱土金属氢氧化物催化丙酮醛到乳酸的产率
由表12中的数据可以看出,碱金属氢氧化物(NaOH)能够催化丙酮醛生成乳酸,且乳酸的产率为98.54%,碱土碱土金属氢氧化物(Ba(OH)
2)也能够催化丙酮醛生成乳酸,且乳酸的产率为86.46%。
实施例13 甲醛转化突变蛋白(甲醛转化突变体)表达载体的构建
甲醛转化突变蛋白是来源于恶臭假单胞菌(pseudomonas putida)的苯甲酰甲酸脱羧酶(b enzoylformate decarboxylases,BFD)的突变体V1(本发明上下文中简称:V1),其核苷酸序列为SEQ ID NO:3将该核苷酸序列两端加上限制性酶切位点NdeⅠ和XhoⅠ,用T4DNA连接酶连接至质粒pET28a的多克隆位点区域,构建重组质粒pET28a-v1。然后以pET28a-v1为模板,设计引物(上游引物:5’-CCGCGCGGCAGCCATATG-3’(SEQ ID NO:21)下游引物:5’-GGTGGTGGTGGTGGTGCTCGAGTTATT-3’(SEQ ID NO:22)),进行易错PCR,对目的基因进行随机突变。PCR完成后,对目的基因进行胶回收,将突变后的目的基因连接在载体pET28a上,再将连接后的载体转化到大肠杆菌BL21Gold(DE3)中,37℃培养箱培养至长出单克隆。
实施例14 甲醛转化突变蛋白的筛选
对实施例13中得到的突变体克隆,挑取单克隆到含有硫酸卡那霉素抗生素的LB培养基的96孔板中,在摇床中过夜培养,将上述突变体复制到含有异丙基-β-D-硫代半乳糖苷(IPTG)的LB培养基的96孔板中培养至一定时间。培养完成后,离心收集菌体,再用200μL磷酸钠缓冲液(50mM Na
2HPO
4和NaH
2PO
4,5mM MgSO
4,pH 7.4)洗涤并收集菌体。
菌体用70μL磷酸钠缓冲液重悬,再分别加入等体积的甲醛溶液(磷酸钠缓冲液含有200mM或10mM甲醛和1mM硫胺素焦磷酸(TPP)),30℃反应3h。反应完成后离心,取出30μL上清液加入150μL二苯胺试剂(1.5g二苯胺溶于100mL乙酸中,再加入1.5mL硫酸),在90℃加热30min,加入完成后取出150μL于96孔板中,在650nm下检测吸光度值。再取90μL上清液于96孔板中,加入60μL的工具酶缓冲液1(0.3mg/mL半乳糖氧化酶,36U/mL辣根过氧化物酶),然后加入50μL的工具酶缓冲液2(3.2mM 2,2'-联氮-双-3-乙基苯并噻唑啉-6-磺酸(ABTS)),在吸收光410nm条件下检测20min。
得到的菌株与V1比较,催化效率更高的即为有益突变菌株,然后通过基因测序,找到相对应突变的位点及氨基酸。经过三轮突变体库的筛选,最终获得了二十株酶活显著提高的突变蛋白。通过基因测序,得到所述二十株突变蛋白的名称及对应突变氨基酸见表13。
表13 突变蛋白名称及对应突变氨基酸
实施例15 V1及其突变蛋白在大肠杆菌中的表达
接种针挑取V1和二十株突变蛋白的阳性菌分别接种于5mL LB培养基中,37℃过夜培养,然后以1%(V/V)接种量接种于25mL LB培养基中,以37℃ 200r/min培养。当OD
600达到0.6时,加入0.1mM的IPTG,在30℃下诱导表达。诱导完成后,将上述培养的突变蛋白菌体分别收集到离心管中,然后用磷酸钠缓冲液重悬并清洗菌体。离心后-80℃冰箱保存 上述菌体。
实施例16 V1及其突变蛋白活性检测
取出实施例15中的菌体,磷酸钠缓冲液重悬,冰浴超声破菌,离心收集上清。取200μL上清液与200μL甲醛溶液混合,于30℃反应3h,反应完成后,取出30μL加入150μL二苯胺试剂,在90℃加热30min,加入完成后取出150μL于96孔板中,在650nm下检测吸光度值。再取出90μL反应溶液加入60μL的工具酶缓冲液1,然后加入50μL的工具酶缓冲液2,在吸收光410nm条件下检测20min。
计算V1及突变蛋白关于羟基乙醛和1,3-二羟基丙酮的酶活力,突变蛋白酶活力相对于V1的百分比如下表14所示(以V1及出发菌株破碎上清液酶活力为100%)。
表14 V1及突变蛋白的上清液在200mM甲醛浓度下活性
实施例17 突变蛋白M3的纯化
实施例15中收集的突变蛋白M3的菌体,用25mL磷酸钠缓冲液(50mM Na
2HPO
4和NaH
2PO
4,5mM MgSO
4,pH 7.4)重悬,冰浴超声破菌,离心收集上清,利用Ni
2+层析柱对上述表达的突变体进行亲和层析纯化,用咪唑洗脱后经脱盐柱脱盐,纯化后的蛋白保存在磷酸钠缓冲液中备用。
实施例18 甲醛转化生成羟基乙醛和1,3-二羟基丙酮的反应
用磷酸钠缓冲液(50mM Na
2HPO
4和NaH
2PO
4,5mM MgSO
4,pH 7.4)将实施例17中纯化后的突变体M3的蛋白(其氨基酸序列如SEQ ID NO:29所示,核苷酸序列如SEQ ID NO:28所示)浓度稀释至15mg/mL。在反应体系中先取一定体积的纯酶,然后加入等量的甲醛溶液(1200mM甲醛,1mM TPP,50mM Na
2HPO
4和NaH
2PO
4,5mM MgSO
4,pH 7.4),30℃反应3h。反应结束后,进行HPLC检测。
HPLC检测条件:色谱柱:Aminex HPX-87H,300mm×7.8mm(迪沃特);流动相:0.005M H
2SO
4溶液;紫外检测(210nm)和示差检测;流速:0.5mL/min;柱温:65℃;进样量:20μL。
经过计算得出,突变蛋白M3催化甲醛产生羟基乙醛和1,3-二羟基丙酮的产量如下表15所示。
表15 突变蛋白M3催化甲醛产生羟基乙醛和1,3-二羟基丙酮的产量
实施例19 羟基乙醛到乙醇酸的氧化反应
使用pH 7.0的磷酸盐缓冲液配制200mM亚氯酸钠溶液,活性氯为3.2%的次氯酸钠溶液,200mM过氧化氢溶液,200mM过硫酸钠溶液。取上述氧化剂溶液各500μL,分别加入 等体积的140mM的羟基乙醛溶液,在30℃反应24h。反应结束后,样品进行HPLC检测。
HPLC检测条件:色谱柱:Aminex HPX-87H,300mm×7.8mm(迪沃特);流动相:0.005M H
2SO
4溶液;紫外检测(210nm)和示差检测;流速:0.5mL/min;柱温:35℃;进样量:20μL。
HPLC检测结果如图8所示,经分析可知,氧化剂(亚氯酸钠、次氯酸钠、过硫酸钠和过氧化氢)均能将羟基乙醛氧化为乙醇酸,且HPLC检测出峰时间与乙醇酸标准品的出峰时间一致。经过计算乙醇酸的产量如表16所示。
表16 不同氧化剂氧化羟基乙醛生成乙醇酸
四种氧化剂(亚氯酸钠、次氯酸钠、亚硫酸钠、过氧化氢)均能将羟基乙醛氧化为乙醇酸,其中亚氯酸钠氧化羟基乙醛产生最高产量的乙醇酸,乙醇酸的产量为46.5mM,氧化剂次氯酸钠、过硫酸钠、过氧化氢也能氧化羟基乙醛成乙醇酸,产量分别为15.6mM、6.2mM和2.4mM。
实施例20 1,3-二羟基丙酮到乙醇酸的氧化反应
使用pH 7.0的50mM磷酸钾缓冲液配制200mM亚氯酸钠溶液,活性氯为3.2%的次氯酸钠溶液,200mM过氧化氢溶液。取上述氧化剂溶液各500μL,加入等体积的140mM的1,3-二羟基丙酮溶液,在30℃反应24h。反应结束后,样品进行HPLC检测。
HPLC检测条件:色谱柱:Aminex HPX-87H,300mm×7.8mm(迪沃特);流动相:0.005M H
2SO
4溶液;紫外检测(210nm)和示差检测;流速:0.5mL/min;柱温:35℃;进样量:20μL。
HPLC检测结果如图9所示,经分析可知,氧化剂(亚氯酸钠,次氯酸钠,过氧化氢)均能催化1,3-二羟基丙酮生成乙醇酸,且HPLC检测出峰时间与乙醇酸标准品的出峰时间一致。经过计算乙醇酸的产量如表17所示。
表17 不同氧化剂氧化1,3-二羟基丙酮生成乙醇酸
三种氧化剂(亚氯酸钠、次氯酸钠和过氧化氢)均能将1,3-二羟基丙酮氧化为乙醇酸,其中次氯酸钠氧化1,3-二羟基丙酮产生最高产量的乙醇酸,乙醇酸的产量为22.8mM,氧化剂亚氯酸钠和过氧化氢也能氧化1,3-二羟基丙酮生成乙醇酸,产量分别为17.8mM和10.3mM。
实施例21 不同pH条件下,亚氯酸钠氧化羟基乙醛产生乙醇酸
配制不同pH值的缓冲液:pH 3的50mM柠檬酸缓冲液,pH 5、7、8的50mM磷酸钾缓冲液,pH 10的50mM碳酸钠缓冲液。使用不同pH的缓冲液配制一系列不同pH的200mM亚氯酸钠溶液和不同pH值的140mM羟基乙醛溶液。取上述不同pH的亚氯酸钠溶液各500μL,加入等体积的相同pH的140mM的羟基乙醛溶液,在30℃反应24h。反应结束后样品 进行HPLC检测。
HPLC检测条件:色谱柱:Aminex HPX-87H,300mm×7.8mm(迪沃特);流动相:0.005M H
2SO
4溶液;紫外检测(210nm)和示差检测;流速:0.5mL/min;柱温:35℃;进样量:20μL。pH范围3-10的亚氯酸钠溶液均能氧化羟基乙醛产生乙醇酸,经过计算乙醇酸的产量如表18所示。
表18 不同pH条件下亚氯酸钠氧化羟基乙醛生成乙醇酸
在pH 3-10的条件下,亚氯酸钠溶液均能氧化羟基乙醛生乙醇酸,其中在pH 3-8的条件下,能产生较高产量的乙醇酸,在pH 3下产生最高产量的乙醇酸51.1mM。
实施例22不同pH条件下,亚氯酸钠氧化1,3-二羟基丙酮产生乙醇酸
配制不同pH值的缓冲液:pH 3的50mM柠檬酸缓冲液,pH 5、7、8的50mM磷酸钾缓冲液,pH 10的50mM碳酸钠缓冲液。使用不同pH的缓冲液配制一系列不同pH的200mM亚氯酸钠溶液和不同pH值的140mM 1,3-二羟基丙酮溶液。取上述不同pH的亚氯酸钠溶液各500μL,加入等体积的相同pH的140mM 1,3-二羟基丙酮溶液,在30℃反应24h。反应结束后,样品进行HPLC检测。
HPLC检测条件:色谱柱:Aminex HPX-87H,300mm×7.8mm(迪沃特);流动相:0.005M H
2SO
4溶液;紫外检测(210nm);流速:0.5mL/min;柱温:35℃;进样量:20μL。在pH 3-10的条件下,亚氯酸钠溶液均能氧化1,3-二羟基丙酮溶液产生乙醇酸,经过计算乙醇酸的产量如表19所示。
表19 不同pH条件下,亚氯酸钠氧化1,3-二羟基丙酮生成乙醇酸
在pH 3-10的条件下,亚氯酸钠溶液均能氧化1,3-二羟基丙酮产生乙醇酸,其中在pH 8的条件下,产生最高产量的乙醇酸21.7mM。
实施例23 不同浓度的亚氯酸钠氧化羟基乙醛或1,3-二羟基丙酮产生乙醇酸
配制pH 8的50mM磷酸钠缓冲液,再使用该缓冲液配制不同浓度的亚氯酸钠溶液(280mM、350mM、420mM、490mM、560mM、630mM、700mM)和140mM羟基乙醛溶液或140mM 1,3-二羟基丙酮溶液。取上述不同浓度的亚氯酸钠溶液各500μL,加入等体积的140mM羟基乙醛溶液或1,3-二羟基丙酮溶液,在30℃条件下反应24h。反应结束后,样品进行HPLC检测。
HPLC检测条件:色谱柱:Aminex HPX-87H,300mm×7.8mm(迪沃特);流动相:0.005M H
2SO
4溶液;紫外检测(210nm)和示差检测;流速:0.5mL/min;柱温:35℃; 进样量:20μL。
随着反应体系中亚氯酸钠溶液浓度的增加,反应体系中乙醇酸的产量增加,经过计算乙醇酸的产量如表20和表21所示。
表20 不同浓度的亚氯酸钠氧化羟基乙醛产生乙醇酸
表21 不同浓度的亚氯酸钠氧化1,3-二羟基丙酮产生乙醇酸
在亚氯酸钠氧化羟基乙醛的反应体系中,当亚氯酸钠浓度和羟基乙醛浓度为3:1时,乙醇酸的产量达到最高;在亚氯酸钠氧化1,3-二羟基丙酮的反应体系中,当亚氯酸钠和1,3-二羟基丙酮浓度为4:1时,乙醇酸的产量达到最高。
实施例24 在不同温度下,亚氯酸钠氧化1,3-二羟基丙酮产生乙醇酸
配制pH 8的50mM磷酸钠缓冲液,再使用该缓冲液配制560mM亚氯酸钠溶液和140mM 1,3-二羟基丙酮溶液。取500μL亚氯酸钠溶液数份,分别加入等体积的140mM 1,3-二羟基丙酮溶液,然后在25℃、30℃、35℃、40℃、45℃和50℃条件下反应24h。反应结束后,样品进行HPLC检测。
HPLC检测条件:色谱柱:Aminex HPX-87H,300mm×7.8mm(迪沃特);流动相:0.005M H
2SO
4溶液;紫外检测(210nm)和示差检测器;流速:0.5mL/min;柱温:35℃;进样量:20μL。
不同温度下,亚氯酸钠溶液均能氧化1,3-二羟基丙酮溶液产生乙醇酸,经过计算乙醇酸的产量如表22所示。
表22 不同温度下,亚氯酸钠氧化1,3-二羟基丙酮产生乙醇酸
升高反应体系的温度,有助于1,3-二羟基丙酮到乙醇酸的转化。
实施例25 亚氯酸钠氧化不同摩尔浓度比例的羟基乙醛和1,3-二羟基丙酮的混合物产生乙醇酸
配制pH 8的50mM磷酸钠缓冲液,再使用该缓冲液配制800mM亚氯酸钠溶液和总浓度为200mM的不同摩尔浓度比的1,3-二羟基丙酮和羟基乙醛的混合溶液(二者的摩尔浓度比分别为9:1、8:2、7:3、4:6、5:5、6:4、3:7、2:8和1:9)。取500μL亚氯酸钠溶液数份,分别加入等体积的1,3-二羟基丙酮溶液和羟基乙醛的混合溶液,在50℃条件下反应48h。反应结束后,样品进行HPLC检测。
HPLC检测条件:色谱柱:Aminex HPX-87H,300mm×7.8mm(迪沃特);流动相:0.005M H
2SO
4溶液;紫外检测(210nm)和示差检测;流速:0.5mL/min;柱温:35℃;进样量:20μL。不同温度下亚氯酸钠溶液均能氧化不同摩尔浓度比例的1,3-二羟基丙酮和羟基乙醛的混合溶液产生乙醇酸,经过计算乙醇酸的产量如表23所示。
表23 亚氯酸钠氧化不同摩尔浓度比例的1,3-二羟基丙酮和羟基乙醛的混合溶液产生乙醇酸
实施例26 亚氯酸钠氧化由甲醛转化突变蛋白催化产生的羟基乙醛和1,3-二羟基丙酮混合液生成乙醇酸
对实施例18中的反应溶液使用超滤管离心去除反应体系中的酶,M3的反应溶液取出500uL至1.5mL EP管中,加入0.0344g亚氯酸钠,充分混匀溶解,反应体系的pH为7.4,在50℃1000rpm反应24h,反应结束后,样品进行HPLC检测。
HPLC检测条件:色谱柱:Aminex HPX-87H,300mm×7.8mm(迪沃特);流动相:0.005M H
2SO
4溶液;紫外检测(210nm)和示差检测;流速:0.5mL/min;柱温:35℃;进样量:20μL。
HPLC检测结果如图10所示,经分析可知,亚氯酸钠能够氧化由甲醛转化突变蛋白催化甲醛生成的羟基乙醛和1,3-二羟基丙酮生成乙醇酸。且HPLC检测出峰时间与乙醇酸标准品的出峰时间一致。经过计算乙醇酸的产量如表24所示。
表24 亚氯酸钠氧化由甲醛转化突变蛋白催化甲醛生成的羟基乙醛和1,3-二羟基丙酮生 成乙醇酸
以上,对本发明的甲醛转化突变蛋白及其用于催化合成1,3-二羟基丙酮、羟基乙醛、乳酸以及乙醇酸的方法进行了说明。但是,本发明不限定于上述实施方式。凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (11)
- 一种甲醛转化突变蛋白,其特征在于,所述甲醛转化突变蛋白能催化甲醛合成1,3-二羟基丙酮和/或羟基乙醛,其氨基酸序列是在对应于SEQ ID NO:1的S26、L43、F66、R86、T87、G109、A204、H281、A322、F397、L407M、M460、W463、V467、V473、S525位中至少一个位置处的氨基酸残基发生突变;或者所述甲醛转化突变蛋白的氨基酸序列具有所述发生突变的氨基酸序列中的突变位点,且与所述发生突变的氨基酸序列具有80%以上同源性的氨基酸序列,优选具有90%以上、95%以上或98%以上的同源性,所述位置是与SEQ ID NO:1对比确定。
- 根据权利要求1所述的甲醛转化突变蛋白,其特征在于,所述甲醛转化突变蛋白的氨基酸序列至少包括如下突变位点之一或如下位点的任意组合:对应于SEQ ID NO:1的S26F、L43Q、F66L、R86C、T87A、G109S、A204V、H281Y、A322T、F397L、F397S、L407M、M460T、W463R、V467A、V473A、S525A;优选地,所述甲醛转化突变蛋白由如SEQ ID NO:3-20、SEQ ID NO:26-28任一所示的多核苷酸序列编码。
- 根据权利要求1或2所述的甲醛转化突变蛋白,其特征在于,所述甲醛转化突变蛋白的氨基酸序列包括:(1)对应于SEQ ID NO:1的第281位组氨酸H为酪氨酸Y;或(2)对应于SEQ ID NO:1的第281位组氨酸H为酪氨酸Y,并且第26位丝氨酸S突变为苯丙氨酸F。
- 根据权利要求1-3中任一项所述的甲醛转化突变蛋白,其特征在于,所述甲醛转化突变蛋白还包括至少其他一个位点的突变,例如是W463R、L43Q、G109S、F397S、M460T、S525A、V194I、V467A、F66L、A204V、F397S、L407M、G109S、A322T、W463R、G109S、F397S中任一个或多个位点的组合;优选地,所述甲醛转化突变蛋白由如SEQ ID NO:8-14、SEQ ID NO:16-20任一所示的多核苷酸序列编码。
- 一种多核苷酸,其特征在于,所述多核苷酸是编码权利要求1-4任一项所述甲醛转化突变蛋白的多核苷酸。
- 根据权利要求5所示的多核苷酸,其特征在于,所述多核苷酸的核苷酸序列如SEQ ID NO:3-20、SEQ ID NO:26-28任一所示。
- 含有权利要求1-4任一项所述甲醛转化突变蛋白和/或权利要求5或6所述多核苷酸的重组载体、表达盒、转基因细胞系和/或重组菌。
- 权利要求1-4任一项所述甲醛转化突变蛋白或权利要求7所述重组载体、表达盒、转基因细胞系和/或重组菌作为催化剂的用途,例如用于催化制备1,3-二羟基丙酮和/或羟基乙醛乳酸和/或乙醇酸的催化剂,例如催化底物甲醛制备1,3-二羟基丙酮和/或乳酸和/或乙醇酸的催化剂。
- 一种制备乳酸的方法,包括如下步骤:以1,3-二羟基丙酮或丙酮醛为底物,用碱金属氢氧化物和/或碱土金属氢氧化物催化底物产生乳酸;优选地,其中所述碱金属氢氧化物为氢氧化钠或氢氧化钾,碱土金属氢氧化物为氢氧化钙或氢氧化钡;优选地,其中碱金属氢氧化物或碱土金属氢氧化物与底物1,3-二羟基丙酮或丙酮醛的摩 尔比为(1~100):1,例如(2~80):1;特别地,所述碱金属氢氧化物与底物1,3-二羟基丙酮或丙酮醛的摩尔比可以为(5~80):1,例如(10~60):1,(15~50):1,(20~40):1;所述碱土金属氢氧化物与底物1,3-二羟基丙酮或丙酮醛的摩尔比可以为(1~20):1,例如(2~10):1,(2~6):1;优选地,其中所述碱金属氢氧化物和/或碱土金属氢氧化物催化底物1,3-二羟基丙酮或丙酮醛产生乳酸的反应可以在10~50℃的范围内进行,优选在20~30℃的范围内进行,例如在室温下进行;优选地,其中所述碱金属氢氧化物和/或碱土金属氢氧化物催化底物1,3-二羟基丙酮或丙酮醛产生乳酸的反应不在惰性气体保护条件下进行;更优选地,其中所述碱金属氢氧化物和/或碱土金属氢氧化物催化底物1,3-二羟基丙酮或丙酮醛产生乳酸的反应以如下方式进行:将含有底物的溶液缓慢加添加到含有碱金属氢氧化物的水溶液或者碱土金属氢氧化物的水悬浊液中;例如,所述底物1,3-二羟基丙酮或丙酮醛缓慢添加的速率可以为每小时的添加量小于或等于10mol,即添加速率小于或等于10mol/h;例如,添加速率可以小于或等于5mol/h,小于或等于2.5mol/h,小于或等于1mol/h。
- 根据权利要求9的方法,其中所述底物1,3-二羟基丙酮以权利要求1-4任一项所述甲醛转化突变蛋白或权利要求7所述重组载体、表达盒、转基因细胞系和/或重组菌作为催化剂,催化底物甲醛制备得到。
- 一种制备乙醇酸的方法,包括如下步骤:(1)将上述甲醛转化突变蛋白或者包含所述甲醛转化突变蛋白和/或所述多核苷酸的重组载体、转基因细胞系或重组菌株与底物甲醛接触,催化生成1,3-二羟基丙酮和羟基乙醛;以及(2)加入氧化剂,将1,3-二羟基丙酮和羟基乙醛氧化乙醇酸;优选地,步骤(2)中所述氧化剂为亚氯酸钠、次氯酸钠、亚硫酸钠或过氧化氢;优选地,步骤(2)在pH 3-10的条件下进行。
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Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2007228927A (ja) * | 2006-03-02 | 2007-09-13 | Kaneka Corp | グリコール酸の製造方法 |
CN105132400A (zh) * | 2015-07-24 | 2015-12-09 | 中国科学院天津工业生物技术研究所 | 具有催化甲醛合成1,3-二羟基丙酮功能的酶及其制备方法 |
CN105777523A (zh) * | 2016-04-07 | 2016-07-20 | 农业部环境保护科研监测所 | 一种温和条件下由糖类制备乳酸的方法 |
CN106916794A (zh) * | 2017-02-22 | 2017-07-04 | 中国科学院天津工业生物技术研究所 | 催化甲醛合成羟基乙醛的酶及其应用 |
CN107699536A (zh) * | 2017-11-27 | 2018-02-16 | 南京工业大学 | 一种基因工程菌及其在生产d‑1,2,4‑丁三醇中的应用 |
WO2018095973A1 (en) * | 2016-11-24 | 2018-05-31 | Haldor Topsøe A/S | A method and a system for producing glycolic acid and/or glycolate |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB9123354D0 (en) * | 1991-11-04 | 1991-12-18 | Bp Chem Int Ltd | Production of hydroxy carboxylic compounds |
AT503802B1 (de) * | 2006-07-26 | 2008-01-15 | Vtu Engineering Planungs Und B | Verfahren zur herstellung von milchsäure bzw. eines salzes davon |
US9200288B2 (en) * | 2010-04-27 | 2015-12-01 | The Regents Of The University Of California | Production of 1,4-butanediol by recombinant microorganisms |
CN107849522A (zh) * | 2015-07-21 | 2018-03-27 | 多伦多大学管理委员会 | 用于产生1,3‑丁二醇的方法和微生物 |
WO2018038667A1 (en) * | 2016-08-25 | 2018-03-01 | Medivir Ab | Respiratory syncytial virus inhibitors |
CN108118037B (zh) * | 2016-11-28 | 2021-08-31 | 青岛蔚蓝生物集团有限公司 | 一种耐热性提高的葡萄糖氧化酶突变体 |
CN110551701B (zh) * | 2018-05-31 | 2022-08-05 | 中国科学院天津工业生物技术研究所 | 羰基还原酶突变体及其在环戊二酮类化合物还原中的应用 |
-
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Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2007228927A (ja) * | 2006-03-02 | 2007-09-13 | Kaneka Corp | グリコール酸の製造方法 |
CN105132400A (zh) * | 2015-07-24 | 2015-12-09 | 中国科学院天津工业生物技术研究所 | 具有催化甲醛合成1,3-二羟基丙酮功能的酶及其制备方法 |
CN105777523A (zh) * | 2016-04-07 | 2016-07-20 | 农业部环境保护科研监测所 | 一种温和条件下由糖类制备乳酸的方法 |
WO2018095973A1 (en) * | 2016-11-24 | 2018-05-31 | Haldor Topsøe A/S | A method and a system for producing glycolic acid and/or glycolate |
CN106916794A (zh) * | 2017-02-22 | 2017-07-04 | 中国科学院天津工业生物技术研究所 | 催化甲醛合成羟基乙醛的酶及其应用 |
CN107699536A (zh) * | 2017-11-27 | 2018-02-16 | 南京工业大学 | 一种基因工程菌及其在生产d‑1,2,4‑丁三醇中的应用 |
Non-Patent Citations (3)
Title |
---|
ALEJANDRA YEP, MICHAEL J MCLEISH: "Saturation Mutagenesis of Putative Catalytic Residues of Benzoylformate Decarboxylase Provides a Challenge to the Accepted Mechanism", PNAS, vol. 105, no. 15, 15 April 2008 (2008-04-15), pages 5733 - 5738, XP055841259 * |
CUI BO, ZHUO BINGZHAO, LU XIAOYUN, WANG WEN, XIAO DONGGUANG, JIANG HUIFENG: "Enzymatic synthesis of xylulose from formaldehyde", SHENGWU GONGCHENG XUEBAO, CHINA, 25 July 2018 (2018-07-25), China, pages 1128 - 1136, XP055841268, [retrieved on 20210915], DOI: 10.13345/j.cjb.170466 * |
XIAOYUN LU, YUWAN LIU, YIQUN YANG, SHANSHAN WANG, QIAN WANG, XIYA WANG, ZHIHUI YAN, JIAN CHENG, CUI LIU, XUE YANG, HAO LUO, SHENG : "Constructing a synthetic pathway for acetyl-coenzyme A from one-carbon through enzyme design", NATURE COMMUNICATIONS, vol. 10, no. 1, 1 December 2019 (2019-12-01), XP055690259, DOI: 10.1038/s41467-019-09095-z * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115109770A (zh) * | 2022-06-30 | 2022-09-27 | 中国科学院天津工业生物技术研究所 | 苯甲醛裂解酶突变体及在制备1,4-二羟基-2-丁酮中的应用 |
CN115109770B (zh) * | 2022-06-30 | 2023-09-05 | 中国科学院天津工业生物技术研究所 | 苯甲醛裂解酶突变体及在制备1,4-二羟基-2-丁酮中的应用 |
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