CN112852890A - Biological synthesis method of polyhydroxy diketone and hydroxy furanone compound - Google Patents
Biological synthesis method of polyhydroxy diketone and hydroxy furanone compound Download PDFInfo
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Abstract
The invention provides a method for carrying out aldol condensation reaction, which comprises the step of catalyzing substrate alpha-hydroxy ketone and methylglyoxal to carry out aldol condensation reaction by using a fructose-6-phosphate aldolase or transaldolase catalyst or a whole-cell catalyst containing the enzyme; wherein the substrate alpha-hydroxy ketone can be hydroxy acetaldehyde, dihydroxyacetone, hydroxy acetone or 1-hydroxy-2-butanone. The invention also provides a preparation method of the hydroxyfuranone compound, which is characterized in that the product of the aldol condensation reaction is cyclized in an alkaline disodium hydrogen phosphate environment to generate the hydroxyfuranone compound. The method disclosed by the invention is mild in reaction and environment-friendly, and avoids the use of organic and metal catalysts; synthesizing polyhydroxy furanone precursor based on the aldol condensation reaction catalyzed by enzyme, and the conversion rate is high; the reaction steps are few, and a multi-step separation and purification process is avoided. The invention also provides the use of a fructose-6-phosphate aldolase or transaldolase for catalyzing an aldol condensation reaction.
Description
Technical Field
The invention belongs to the technical field of biosynthesis, and particularly relates to a method for catalytically synthesizing hydroxyfuranone compounds by using fructose-6-phosphate aldolase.
Background
The hydroxy furanone perfume belongs to the top-grade perfume with the most value, the most extensive application and the largest yield, and has the reputation of the king of perfume. The two most representative compounds are: (1) furanone, 2, 5-dimethyl-4-hydroxy-3-furanone, is mainly present in the flavor components of pineapple in nature, commonly known as pineapple ketone; sweet caramel aroma, fruity; and (2) soy sauce ketone, 2-methyl-5-ethyl-4-hydroxy-3-furanone, which is mainly present in soy sauce in nature, has high-intensity sweet caramel aroma, and can improve the delicate flavor and caramel-type flavor of soy sauce. The two kinds of hydroxyl furanones are used as flavoring agents and widely applied to the fields of food, tobacco, beverage and the like.
The content of the hydroxyl furanone spices in the nature is low, the price of natural products is high, and the hydroxyl furanone spices are mainly produced by a chemical synthesis method at present. A domestic furanone producer, Dalianjin chrysanthemum spice, Inc., develops a method for synthesizing furanone by using 3, 4-hexanedione as a raw material and performing substitution, hydrolysis and cyclization by using bromine water, wherein the total yield is 46% (CN 200910188283.6); the foreign furanone producer developed a method for preparing furanone by condensation and cyclization by taking methylglyoxal as a raw material, and the reaction route is short and relatively competitive (US 5009753); in addition, researchers have developed a method for preparing furanones from lactic acid, pyruvate and 3, 4-diacetyl-2, 5-hexanedione (CN201610964204.6, cn201910195433. x). The chemical method for preparing the furanone has the defects of long reaction steps, low yield, serious environmental pollution, high cost and the like. The biological fermentation technology is used for synthesizing the spice compound, the product is good in fragrance, resources can be saved, sustainable development is facilitated, and the method is a trend of future development. Researchers screen and obtain zygosaccharomyces rouxii, ledebur yeast and pichia guillieri, furanone and soy sauce ketone can be prepared by a fermentation method, however, expensive fructose 1, 6-diphosphate and rhamnose are required to be added into a culture medium as raw materials, the yield is low and is not higher than 800mg/L, the production cost is high, and the method is not suitable for large-scale production.
Therefore, the development of a synthetic method of hydroxyfuranone compounds with high production efficiency, environmental optimization and low cost is urgently needed.
Disclosure of Invention
Fructose-6-phosphate aldolase (FSA), generally refers to an enzyme that can reversibly catalyze the binding of Dihydroxyacetone (DHA) and glyceraldehyde-3-phosphate (G3P) to form fructose-6-phosphate (Schurmann and Sprenger, J.biol.chem.,2001,276(14), 11055-11061); transaldolase (Transaldolase) generally refers to an enzyme that reversibly catalyzes the synthesis of erythrulose 4-phosphate and fructose 6-phosphate from sedoheptulose 7-phosphate and glycerate 3-phosphate. The inventors have found that fructose-6-phosphate aldolase and transaldolase may also catalyze aldol condensation reactions between hydroxyketones and methylglyoxal to produce hydroxydiketones, which may further be used to produce furanones. Specifically, the inventors have found that fructose-6-phosphate aldolase or transaldolase can catalyze the aldol condensation reaction between hydroxyacetaldehyde and methylglyoxal to produce 2, 3-dihydroxy-4-keto-valeraldehyde; can catalyze the aldol condensation reaction between dihydroxyacetone and methylglyoxal to generate 1,3, 4-trihydroxy-2, 5-hexanedione; can catalyze the aldol condensation reaction between hydroxyacetone and methylglyoxal to generate 3, 4-dihydroxy-2, 5-hexanedione; can catalyze the aldol condensation reaction between 1-hydroxy-2-butanone and methylglyoxal to generate 3, 4-dihydroxy-2, 5-heptanedione. The 3, 4-dihydroxy-2, 5-hexanedione can be cyclized in an alkaline disodium hydrogen phosphate environment to generate furanone, namely 2, 5-dimethyl-4-hydroxy-3-furanone (commonly known as bromelain); the 3, 4-dihydroxy-2, 5-heptanedione can be cyclized in an alkaline disodium hydrogen phosphate environment to produce soy sauce ketone, i.e. 2-methyl-5-ethyl-4-hydroxy-3-furanone.
It is an object of the present invention to provide the use of a fructose-6-phosphate aldolase or transaldolase enzyme catalyst or a whole-cell catalyst comprising cells of a fructose-6-phosphate aldolase or transaldolase enzyme for catalyzing an aldol condensation reaction.
In some embodiments, the fructose-6-phosphate aldolase or transaldolase may catalyze the aldol condensation reaction of a substrate, an alpha-hydroxyketone, with methylglyoxal.
In some preferred embodiments, the substrate α -hydroxyketone may be hydroxyacetaldehyde, dihydroxyacetone, hydroxyacetone, or 1-hydroxy-2-butanone.
In some preferred embodiments, the substrates are hydroxyacetaldehyde and methylglyoxal, and the resulting product is 2, 3-dihydroxy-4-keto-valeraldehyde.
In some preferred embodiments, the substrate is dihydroxyacetone and methylglyoxal, and the resulting product is 1,3, 4-trihydroxy-2, 5-hexanedione.
In some preferred embodiments, the substrates are hydroxyacetone and methylglyoxal, and the resulting product is 3, 4-dihydroxy-2, 5-hexanedione.
In some preferred embodiments, the substrate is 1-hydroxy-2-butanone and methylglyoxal, and the product is 3, 4-dihydroxy-2, 5-heptanedione.
In the context of the present invention, the fructose-6-phosphate aldolase may be a fructose-6-phosphate aldolase derived from any species, including but not limited to fructose-6-phosphate aldolases derived from Escherichia or Shigella, such as fructose-6-phosphate aldolase derived from Escherichia coli. In some preferred embodiments, the amino acid sequence of the fructose-6-phosphate aldolase of the present invention is (a), (b), or (c) as follows: (a) a polypeptide consisting of an amino acid sequence shown in SEQ ID No. 1; (b) a polypeptide which is obtained by substituting, deleting or adding 1-10 amino acids, such as 1-9 amino acids, 1-8 amino acids, 1-7 amino acids, 1-6 amino acids, 1-5 amino acids, 1-4 amino acids, 1-3 amino acids or 1-2 amino acids, from the amino acid sequence in (a) and has the function of catalyzing aldol condensation reaction between hydroxyketone and methylglyoxal; (c) a polypeptide which has more than 90%, such as more than 95%, preferably more than 96%, more than 97%, more than 98% or more than 99% identity with the amino acid sequence in (a) and has the function of catalyzing aldol condensation reaction between hydroxy-ketone and methylglyoxal.
In the context of the present invention, the transaldolase includes, but is not limited to, transaldolase derived from Listeria innocua, Listeria elispot, and Clostridium. In some preferred embodiments, the amino acid sequence of the transaldolase according to the invention is (a), (b) or (c) as follows: (a) polypeptide consisting of an amino acid sequence shown in SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO. 4; (b) a polypeptide which is obtained by substituting, deleting or adding 1-10 amino acids, such as 1-9 amino acids, 1-8 amino acids, 1-7 amino acids, 1-6 amino acids, 1-5 amino acids, 1-4 amino acids, 1-3 amino acids or 1-2 amino acids, from the amino acid sequence in (a) and has the function of catalyzing aldol condensation reaction between hydroxyketone and methylglyoxal; (c) a polypeptide which has more than 90%, such as more than 95%, preferably more than 96%, more than 97%, more than 98% or more than 99% identity with the amino acid sequence in (a) and has the function of catalyzing aldol condensation reaction between alpha-hydroxy ketone and methylglyoxal.
In the context of the present invention, the term "identity" may also be referred to as sequence identity, and refers to the degree of similarity between two amino acid sequences, typically calculated as the percentage of amino acid residues that are identical at the corresponding positions after alignment of the amino acid sequences of two polypeptides or proteins. The identity between two amino acid sequences can be determined by sequence analysis software or programs known in the art, e.g., BLAST, FASTA, and the like.
In the context of the present invention, the fructose-6-phosphate aldolase or transaldolase enzyme catalyst may be a purified fructose-6-phosphate aldolase or transaldolase, may be a crude enzyme solution comprising a fructose-6-phosphate aldolase or transaldolase, or may be an enzyme preparation comprising a fructose-6-phosphate aldolase or transaldolase. The fructose-6-phosphate aldolase or transaldolase may be obtained by extraction and purification from a microorganism naturally producing the enzyme, such as Escherichia coli, or may be obtained by extraction and purification from a recombinant host cell expressing an exogenous fructose-6-phosphate aldolase or transaldolase gene. The crude enzyme solution may be an intermediate product in the extraction and purification process of fructose-6-phosphate aldolase or transaldolase, for example, a crude enzyme solution containing fructose-6-phosphate aldolase or transaldolase and other cellular impurities but still having the function of catalyzing the aldol condensation reaction between hydroxyketone and methylglyoxal. The enzyme preparation may contain suitable adjuvants or carriers.
In the context of the present invention, the cell containing fructose-6-phosphate aldolase or transaldolase, i.e., a host cell expressing fructose-6-phosphate aldolase or transaldolase, which host cell has the function of catalyzing the aldol condensation reaction of alpha-hydroxyketone and methylglyoxal, wherein the host cell includes, but is not limited to, microbial cells, including microorganisms such as bacteria, fungi, and the like, such as Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, lactic acid bacteria, Saccharomyces cerevisiae, and the like; higher eukaryotic cells such as mammalian cells are also included. Preferably, the host cell is E.coli, for example E.coli BL21(DE 3).
The cell containing fructose-6-phosphate aldolase or transaldolase may be obtained by introducing a gene encoding fructose-6-phosphate aldolase or transaldolase into a host cell and screening the host cell for expression of fructose-6-phosphate aldolase or transaldolase. The gene encoding fructose-6-phosphate aldolase or transaldolase may be present on an expression vector in the host cell, for example, the gene encoding fructose-6-phosphate aldolase or transaldolase may be cloned on an expression vector (e.g., a plasmid), and the expression vector may be transferred into the host cell by calcium-induced competent transformation or by electroporation. The gene encoding the fructose-6-phosphate aldolase or transaldolase may also be integrated into the genome of the host cell, for example by homologous recombination. Other methods for transferring a gene expressing a foreign protein into a host cell to express the foreign protein in the host cell are also well known to those skilled in the art.
Another object of the present invention is to provide a method for performing an aldol condensation reaction comprising the step of catalyzing the aldol condensation reaction of a substrate, alpha-hydroxyketone, and methylglyoxal using a fructose-6-phosphate aldolase or transaldolase catalyst.
According to the invention, the substrate α -hydroxyketone may be hydroxyacetaldehyde, dihydroxyacetone, hydroxyacetone or 1-hydroxy-2-butanone.
In some preferred embodiments, the substrates are hydroxyacetaldehyde and methylglyoxal, and the resulting product is 2, 3-dihydroxy-4-keto-valeraldehyde.
In some preferred embodiments, the substrate is dihydroxyacetone and methylglyoxal, and the resulting product is 1,3, 4-trihydroxy-2, 5-hexanedione.
In some preferred embodiments, the substrates are hydroxyacetone and methylglyoxal, and the resulting product is 3, 4-dihydroxy-2, 5-hexanedione.
In a preferred embodiment, the enzyme-catalyzed reaction system comprises hydroxyacetone in an amount of 10 to 100mM, more preferably 50 mM; methylglyoxal in an amount of 10-100mM, more preferably 50 mM; the concentration of fructose-6-phosphate aldolase or transaldolase is 0.1-100U/mL, preferably 1-50U/mL, more preferably 1-20U/mL, for example 2-10U/mL; the pH of the buffer is 6-8, preferably 7.5; the catalytic reaction temperature is 20-37 ℃, and the more preferable temperature is 30 ℃; the reaction time is 2 to 24 hours, and more preferably 24 hours.
In some preferred embodiments, the substrates are 1-hydroxy-2-butanone and methylglyoxal, and the resulting product is 3, 4-dihydroxy-2, 5-heptanedione.
In a preferred embodiment, the enzyme-catalyzed reaction system comprises 1-hydroxy-2-butanone in an amount of 10-100mM, more preferably 50 mM; methylglyoxal in an amount of 10-100mM, more preferably 50 mM; the enzyme concentration of fructose-6-phosphate aldolase or transaldolase is 0.1-100U/mL, preferably 1-50U/mL, more preferably 1-20U/mL, for example 2-10U/mL; the pH of the buffer is 6-8, preferably 7.5; the catalytic reaction temperature is 20-37 ℃, and the more preferable temperature is 30 ℃; the reaction time is 2 to 24 hours, and more preferably 24 hours.
It is a further object of the present invention to provide a method for performing an aldol condensation reaction comprising the step of catalyzing the aldol condensation reaction of a substrate, an alpha-hydroxy ketone, and methylglyoxal using a whole cell catalyst, wherein the whole cell catalyst is a cell comprising fructose-6-phosphate aldolase or transaldolase.
According to the invention, the substrate α -hydroxyketone may be hydroxyacetaldehyde, dihydroxyacetone, hydroxyacetone or 1-hydroxy-2-butanone.
In some preferred embodiments, the substrates are hydroxyacetaldehyde and methylglyoxal, and the resulting product is 2, 3-dihydroxy-4-keto-valeraldehyde.
In some preferred embodiments, the substrate is dihydroxyacetone and methylglyoxal, and the resulting product is 1,3, 4-trihydroxy-2, 5-hexanedione.
In some preferred embodiments, the substrates are hydroxyacetone and methylglyoxal, and the resulting product is 3, 4-dihydroxy-2, 5-hexanedione.
In some preferred embodiments, the whole cell catalytic reaction system comprises hydroxyacetone in an amount of 50 to 1000mM, more preferably 500 mM; methylglyoxal content of 50-1000mM, more preferably methylglyoxal content of 500 mM; cell concentration (OD)600) The concentration (OD) of the cells is 10 to 200, and the preferable concentration is600) Is 60; the pH of the buffer is 6-8, preferably 7.5; the catalytic reaction temperature is 20-37 ℃, and the more preferable temperature is 30 ℃; the reaction time is 2 to 24 hours, and more preferably 4 hours.
In some preferred embodiments, the substrates are 1-hydroxy-2-butanone and methylglyoxal, and the resulting product is 3, 4-dihydroxy-2, 5-heptanedione.
In a preferred embodiment, the whole-cell catalytic reaction system comprises 1-hydroxy-2-butanone in an amount of 50-1000mM, more preferably in an amount of 500 mM; methylglyoxal content of 50-1000mM, more preferably methylglyoxal content of 300 mM; cell concentration (OD)600) The concentration (OD) of the cells is 10 to 200, and the preferable concentration is600) Is 60; the pH of the buffer is 6-8, preferably 7.5; the catalytic reaction temperature is 20-37 ℃, and the more preferable temperature is 30 ℃; the reaction time is 2 to 24 hours, and more preferably 24 hours.
The fourth purpose of the invention is to provide a preparation method of hydroxyl furanone compounds, which comprises the following steps:
step (1) aldol condensation reaction: conducting the aldol condensation reaction according to the second or third object; and
step (2) cyclization reaction: cyclizing the reaction liquid containing the aldol condensation reaction product obtained in the step (1) in an alkaline disodium hydrogen phosphate environment to generate a hydroxyfuranone compound.
In some preferred embodiments, the substrate of step (1) is hydroxyacetone and methylglyoxal, the product of the aldol condensation reaction obtained is 3, 4-dihydroxyhexanedione, and the hydroxyfuranone compound obtained in step (2) is 2, 5-dimethyl-4-hydroxy-3-furanone (i.e., furanone).
In a preferred embodiment, in the step (2), Na is added to the reaction liquid containing 3, 4-dihydroxyhexanedione2HPO4The final concentration is 20-300g/L, the reaction temperature is 30-90 ℃, the reaction is carried out for 2-24 hours, and Na is more preferable2HPO4The concentration was 300g/L, the reaction temperature was 70 ℃ and the reaction time was 24 hours.
In some preferred embodiments, the substrate of step (1) is 1-hydroxy-2-butanone and methylglyoxal, the product of the aldol condensation reaction obtained is 3, 4-dihydroxy-2, 5-heptanedione, and the hydroxyfuranone compound obtained in step (2) is 2-methyl-5-ethyl-4-hydroxy-3-furanone (i.e., soy sauce ketone).
In a preferred embodiment, in the step (2), Na is added to the reaction liquid containing 3, 4-dihydroxy-2, 5-heptanedione2HPO4The final concentration is 20-300g/L, the reaction temperature is 30-90 ℃, the reaction is carried out for 2-24 hours, and Na is more preferable2HPO4The concentration was 300g/L, the reaction temperature was 70 ℃ and the reaction time was 24 hours.
Advantageous effects
Compared with the prior production method of the hydroxyl furanone compound, the method has the following advantages:
(1) the reaction is mild, the environment is friendly, and organic and metal catalysts are avoided;
(2) the conversion rate is high, the polyhydroxy furanone precursor is synthesized by the aldol condensation reaction based on enzyme catalysis, and the conversion rate is higher than 80%;
(3) the reaction steps are few, and a multi-step separation and purification process is avoided.
Drawings
FIG. 1 shows the HPLC analysis results of the whole-cell catalyzed hydroxyacetone and methylglyoxal synthesis of 3, 4-dihydroxy-2, 5-hexanedione of example 5.
FIG. 2 shows the results of HPLC analysis for preparing furanone in example 6.
FIG. 3 shows the HPLC analysis results of the whole-cell catalyzed synthesis of 3, 4-dihydroxy-2, 5-heptanedione from 1-hydroxy-2-butanone and methylglyoxal of example 8.
Detailed Description
The present invention will be described in further detail with reference to examples.
The percentage concentrations mentioned in the present invention and examples are, unless otherwise specified, mass/mass (W/W, unit g/100g) percentage concentrations, mass/volume (W/V, unit g/100mL) percentage concentrations or volume/volume (V/V, unit mL/100mL) percentage concentrations.
The methods used in the following examples are conventional unless otherwise specified, and specific procedures can be found in: molecular Cloning: A Laboratory Manual (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).
Materials or reagents having the same names used in the respective examples are the same unless otherwise specified. The various biological material access approaches described in the examples are provided for the purpose of specific disclosure, and should not be construed as limiting the source of biological material in practicing the invention. In fact, the sources of the biological materials used are wide and any biological material that can be obtained without violating the law and ethics can be used instead as suggested in the examples.
The primers and genes used in the present invention were synthesized by Jiangsu Jinzhi Biotechnology GmbH.
The embodiments are provided in order to provide detailed embodiments and specific procedures, which will help understanding of the present invention, but the scope of the present invention is not limited to the following embodiments.
EXAMPLE 1 expression and purification of fructose-6-phosphate Aldolase
1. Construction of recombinant Strain containing fructose-6-phosphate Aldolase
A primer is designed according to a fructose-6-phosphate aldolase gene (SEQ ID NO:5) derived from Escherichia coli MG1655 in Genbank, a fragment derived from the fructose-6-phosphate aldolase gene (FSA) is amplified by taking an Escherichia coli MG1655 genome as a template, and the FSA fragment is connected to an expression vector pET21 by adopting an enzyme digestion connection method to obtain a vector plasmid containing the fructose-6-phosphate aldolase gene FSA, which is named as pET 21-FSA.
2. The recombinant plasmid pET21-FSA is chemically transformed into Escherichia coli BL21(DE3) to obtain Escherichia coli recombinant strain 21 FSA.
3. Respectively culturing and inducing the Escherichia coli recombinant strain 21FSA, selecting an LB culture medium to culture the Escherichia coli recombinant strain, adding IPTG, and inducing for about 20 h.
4. Collecting and concentrating the Escherichia coli recombinant strain 21FSA, centrifuging the induced recombinant strain liquid, collecting thalli, and purifying the target protein by adopting an ultrasonic disruption method and a nickel column affinity purification method.
Example 2 expression and purification of transaldolase
1. The gene sequence (SEQ ID NO.6) derived from the harmless Listeria transaldolase, the gene sequence (SEQ ID NO.7) derived from the Listeria elispot transaldolase and the gene sequence (SEQ ID NO.8) derived from the Clostridium transaldolase were obtained based on database search, and Jiangsu Jinzhi Biotechnology Limited was entrusted to complete gene synthesis.
2. The primers were designed to amplify the PCR fragment using the above gene as template, and the PCR fragment was constructed by restriction enzyme ligation and expressed in pET21 to obtain a recombinant expression vector, and the recombinant plasmid was transferred to Escherichia coli BL21(DE3) for expression, and the expression and purification method was as in example 1.
Example 3 functional characterization of fructose-6-phosphate Aldolase and Transaldolase
The method for measuring the enzymatic activities of fructose-6-phosphate aldolase and transaldolase comprises the steps of establishing a 200 mu L reaction system, adding 20mM hydroxyacetone, 10mM methylglyoxal, 50mM ethanolamine buffer solution, 37 ℃, pH7.5, reacting for 30min, and detecting the consumption of the hydroxyacetone by adopting high performance liquid chromatography, wherein the enzymatic activity is defined as the quantity of the acetone consuming hydroxyl per milligram of protein per minute. It was found by measurement that the fructose-6-phosphate aldolase activity was 1.2U/mg, the transaldolase activity derived from a harmless Listeria was 2.1U/mg, the transaldolase activity derived from Listeria elispot was 1.8U/mg, and the transaldolase activity derived from Clostridium was 0.8U/mg.
Establishing a 1mL reaction system in which the fructose-6-phosphate aldolase obtained in example 1 or the three transaldolases obtained in example 2 were added in an amount of 0.5U/mL and the concentration of α -hydroxyketone (hydroxyacetaldehyde or dihydroxyacetone or hydroxyacetone or 1-hydroxy-2-butanone) was 20 mM; methylglyoxal concentration 10 mM; the ethanolamine buffer concentration was 50mM, 37 ℃ and pH7.5, and the reaction was carried out for 24 hours. After the reaction is finished, the sample is boiled for 5min, centrifuged for 20min at 14000rmp and filtered by a 0.22 mu m microporous filter membrane, and the filtrate is subjected to high performance liquid analysis. The HPLC analysis was performed under the following conditions: the instrument is an Agilent high performance liquid chromatograph 1200, and the analysis column comprises: Sugar-Pak, mobile phase: ultrapure water, flow rate: 0.4mL/min, column temperature: 80 ℃, detector: a differential refractive index detector, the loading was 10. mu.L.
The results show that both fructose-6-phosphate aldolase and transaldolase can catalyze the aldol reaction between alpha-hydroxyketone and methylglyoxal, i.e. the aldol reaction between hydroxyacetaldehyde and methylglyoxal to produce the product 2, 3-dihydroxy-4-keto-pentanal, and the aldol reaction between dihydroxyacetone and methylglyoxal to produce the product 1,3, 4-trihydroxy-2, 5-hexanedione; catalyzing aldol condensation reaction between hydroxyacetone and methylglyoxal to generate a product 3, 4-dihydroxy-2, 5-hexanedione; catalyzing aldol condensation reaction between 1-hydroxy-2-butanone and methylglyoxal to generate the product 3, 4-dihydroxy-2, 5-heptanedione.
EXAMPLE 4 enzymatic preparation of 3, 4-dihydroxy-2, 5-hexanedione
A reaction system was set up in which the substrate hydroxyacetone was 50mM, the methylglyoxal was 50mM, the fructose-6-phosphate aldolase obtained in example 1 or the transaldolase derived from Listeria innocua or Listeria evans or Clostridium obtained in example 2 was 2U/mL, the ethanolamine buffer concentration was 50mM, pH7.5, the reaction conditions were controlled at 30 ℃ and, after 24 hours of reaction, the sample was subjected to High Performance Liquid Chromatography (HPLC) analysis (HPLC conditions were the same as in example 1). After 24 hours of reaction, fructose-6-phosphate aldolase catalyzes hydroxyacetone and methylglyoxal to obtain 45mM of 3, 4-dihydroxy-2, 5-hexanedione with the conversion rate of 90 percent; transaldolase derived from harmless listeria catalyzes hydroxyacetone and methylglyoxal to obtain 43mM of 3, 4-dihydroxy-2, 5-hexanedione with a conversion rate of 86%, transaldolase derived from listeria elispot catalyzes hydroxyacetone and methylglyoxal to obtain 41mM of 3, 4-dihydroxy-2, 5-hexanedione with a conversion rate of 82%, transaldolase derived from clostridium catalyzes hydroxyacetone and methylglyoxal to obtain 40mM of 3, 4-dihydroxy-2, 5-hexanedione with a conversion rate of 80%.
EXAMPLE 5 Whole-cell catalytic preparation of 3, 4-dihydroxy-2, 5-hexanedione
(1) Preparation of Whole cell catalyst
First, as described in example 1 or 2, a recombinant strain of Escherichia coli containing fructose-6-phosphate aldolase or a transaldolase gene derived from Listeria innocua or Listeria elsdenii or Clostridium is cultured and induced, and then, the recombinant strain of Escherichia coli is collected and concentrated, and a bacterial solution of the induced recombinant strain is collected and the bacterial solution is concentrated with triethanolamine buffer as a whole cell catalyst.
(2) Whole-cell catalysis hydroxyacetone and methylglyoxal synthesis 3, 4-dihydroxy-2, 5-hexanedione
A reaction system was set up in which the substrate hydroxyacetone concentration was 600mM, the methylglyoxal concentration was 500mM, and the whole-cell catalyst concentration (OD)600) 60, ethanolamine buffer concentration of 100mM, pH7.5, reaction conditions of 30 ℃ were controlled, and after 4 hours of reaction, HPLC analysis was carried out on the sample (HPLC conditions were the same as in example 1). As a result, the yield of 3, 4-dihydroxy-2, 5-hexanedione using the whole-cell catalyst containing fructose-6-phosphate aldolase was 440mM, and the conversion was 88% (FIG. 1); the yield of 3, 4-dihydroxy-2, 5-hexanedione obtained with the whole-cell catalyst containing transaldolase derived from Listeria innocua was 460mM, the conversion was 92%, the yield of 3, 4-dihydroxy-2, 5-hexanedione obtained with the whole-cell catalyst containing transaldolase derived from Listeria elii was 450mM, the conversion was 90%, the yield obtained with the whole-cell catalyst containing transaldolase derived from ClostridiumThe yield of 3, 4-dihydroxy-2, 5-hexanedione obtained with the whole-cell catalyst of (1) was 420mM, and the conversion was 81%.
EXAMPLE 6 preparation of furanone (2, 5-dimethyl-4-hydroxy-3-furanone)
To the reaction solution containing 3, 4-dihydroxy-2, 5-hexanedione obtained in example 5 by catalysis with the whole-cell catalyst containing fructose-6-phosphate aldolase was added Na2HPO4The final concentration is 300g/L, the reaction temperature is 70 ℃, the reaction is carried out for 24 hours, and after the reaction is finished, the sample is subjected to high performance liquid chromatography analysis. The HPLC analysis was performed under the following conditions: the instrument is an Agilent high performance liquid chromatograph 1200, and the analysis column comprises: Sugar-Pak, mobile phase: ultrapure water, flow rate: 0.4mL/min, column temperature: 80 ℃, detector: a differential refractive index detector, the loading was 10. mu.L. The results show a conversion of 50% (fig. 2).
EXAMPLE 7 enzymatic preparation of 3, 4-dihydroxy-2, 5-heptanedione
A reaction system was established in which the substrate 1-hydroxy-2-butanone was 50mM, methylglyoxal was 50mM, the fructose-6-phosphate aldolase obtained in example 1 or the transaldolase derived from a harmless Listeria monocytogenes or Listeria elispot or Clostridium obtained in example 2 was 2U/mL, the ethanolamine buffer concentration was 50mM, pH7.5, the reaction conditions were controlled at 30 ℃ and, after 24 hours of reaction, the sample was subjected to HPLC analysis. After 24 hours of reaction, the obtained 3, 4-dihydroxy-2, 5-heptanedione is more than 40mM, the conversion rate is more than 80%, wherein the conversion rate of fructose-6-phosphate aldolase is 84%, the conversion rate of transaldolase derived from listeria innocua is 81%, the conversion rate of transaldolase derived from listeria elii is 80%, and the conversion rate of transaldolase derived from clostridium is 80%.
EXAMPLE 8 Whole-cell catalytic preparation of 3, 4-dihydroxy-2, 5-heptanedione
(1) The whole-cell catalyst was prepared as described in example 5.
(2) The whole cell catalyzes 1-hydroxy-2-butanone and methylglyoxal to synthesize 3, 4-dihydroxy-2, 5-heptanedione.
Establishing a reaction system in which the concentration of the substrate 1-hydroxy-2-butanone is300mM, methylglyoxal concentration 200mM, whole cell catalyst concentration (OD)600) The concentration of ethanolamine buffer was 80 mM, pH7.5, the reaction conditions were controlled at 30 ℃ and after 24 hours of reaction, HPLC analysis was carried out on the sample (HPLC conditions were the same as in example 5). As a result, the yield of 3, 4-dihydroxy-2, 5-heptanedione using the whole-cell catalyst containing fructose-6-phosphate aldolase was 160mM, and the conversion was 80% (FIG. 3); the yield of 3, 4-dihydroxy-2, 5-heptanedione using the whole-cell catalyst containing transaldolase derived from listeria innocua was 140mM, the conversion rate was 70%, the yield of 3, 4-dihydroxy-2, 5-heptanedione using the whole-cell catalyst containing transaldolase derived from listeria elidis was 142mM, the conversion rate was 71%, the yield of 3, 4-dihydroxy-2, 5-heptanedione using the whole-cell catalyst containing transaldolase derived from clostridium sp was 146mM, and the conversion rate was 73%.
EXAMPLE 9 preparation of Soy sauce Ketone (2-methyl-5-ethyl-4-hydroxy-3-Furanone)
To the reaction solution containing 3, 4-dihydroxy-2, 5-heptanedione obtained by catalysis in example 8 using the whole-cell catalyst containing fructose-6-phosphate aldolase was added Na2HPO4The final concentration is 300g/L, the reaction temperature is 70 ℃, the reaction is carried out for 24 hours, and after the reaction is finished, the sample is subjected to high performance liquid chromatography analysis. The HPLC analysis was performed under the following conditions: the instrument is an Agilent high performance liquid chromatograph 1200, and the analysis column comprises: Sugar-Pak, mobile phase: ultrapure water, flow rate: 0.4mL/min, column temperature: 80 ℃, detector: a differential refractive index detector, the loading was 10. mu.L. The results show a conversion of 50%.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. Use of a fructose-6-phosphate aldolase or transaldolase catalyst or a whole-cell catalyst comprising cells of a fructose-6-phosphate aldolase or transaldolase enzyme for catalyzing an aldol condensation reaction;
preferably, the fructose-6-phosphate aldolase or transaldolase catalyst or the whole-cell catalyst of the cell containing fructose-6-phosphate aldolase or transaldolase can catalyze the aldol condensation reaction of the substrate alpha-hydroxyketone with methylglyoxal;
more preferably, the substrate α -hydroxyketone may be hydroxyacetaldehyde, dihydroxyacetone, hydroxyacetone, or 1-hydroxy-2-butanone;
for example, the substrates are hydroxyacetaldehyde and methylglyoxal, and the resulting product is 2, 3-dihydroxy-4-keto-valeraldehyde;
for example, the substrate is dihydroxyacetone and methylglyoxal, and the resulting product is 1,3, 4-trihydroxy-2, 5-hexanedione;
for example, the substrate is hydroxyacetone and methylglyoxal, and the resulting product is 3, 4-dihydroxy-2, 5-hexanedione;
for example, the substrate is 1-hydroxy-2-butanone and methylglyoxal, and the product is 3, 4-dihydroxy-2, 5-heptanedione.
2. A method of conducting an aldol condensation reaction comprising the step of catalyzing an aldol condensation reaction of a substrate, an alpha-hydroxyketone, and methylglyoxal using a fructose-6-phosphate aldolase or transaldolase catalyst;
preferably, the substrate alpha-hydroxyketone is hydroxyacetaldehyde, dihydroxyacetone, hydroxyacetone or 1-hydroxy-2-butanone;
preferably, the substrate is hydroxyacetaldehyde and methylglyoxal, and the resulting product is 2, 3-dihydroxy-4-keto-valeraldehyde; or the substrate is dihydroxyacetone and methylglyoxal, and the obtained product is 1,3, 4-trihydroxy-2, 5-hexanedione.
3. The method of claim 2, wherein the substrates are hydroxyacetone and methylglyoxal, and the resulting product is 3, 4-dihydroxy-2, 5-hexanedione;
preferably, the enzyme catalysis reaction system contains hydroxyacetone with the content of 10-100mM, and more preferably, the content of the hydroxyacetone is 50 mM; methylglyoxal in an amount of 10-100mM, more preferably 50 mM; the concentration of fructose-6-phosphate aldolase or transaldolase is 0.1-100U/mL, preferably 1-50U/mL, more preferably 1-20U/mL, for example 2-10U/mL; the pH of the buffer is 6-8, preferably 7.5; the catalytic reaction temperature is 20-37 ℃, and the more preferable temperature is 30 ℃; the reaction time is 2 to 24 hours, more preferably 24 hours;
or, the substrate is 1-hydroxy-2-butanone and methylglyoxal, and the obtained product is 3, 4-dihydroxy-2, 5-heptanedione;
preferably, the enzyme catalysis reaction system contains 1-hydroxy-2-butanone in the content of 10-100mM, and more preferably, the content of 1-hydroxy-2-butanone is 50 mM; methylglyoxal in an amount of 10-100mM, more preferably 50 mM; the concentration of fructose-6-phosphate aldolase or transaldolase is 0.1-100U/mL, preferably 1-50U/mL, more preferably 1-20U/mL, for example 2-10U/mL; the pH of the buffer is 6-8, preferably 7.5; the catalytic reaction temperature is 20-37 ℃, and the more preferable temperature is 30 ℃; the reaction time is 2 to 24 hours, and more preferably 24 hours.
4. A method for performing an aldol condensation reaction comprising the step of catalyzing an aldol condensation reaction of a substrate, α -hydroxyketone, and methylglyoxal using a whole-cell catalyst, wherein the whole-cell catalyst is a host cell expressing a fructose 6-phosphate aldolase or transaldolase, the host cell having the function of catalyzing the aldol condensation reaction of the α -hydroxyketone and methylglyoxal;
preferably, the host cell is a bacterial cell, a fungal cell or a mammalian cell; more preferably, the host cell is E.coli.
5. The method of claim 4, wherein the substrate a-hydroxyketone is hydroxyacetaldehyde, dihydroxyacetone, hydroxyacetone, or 1-hydroxy-2-butanone;
preferably, the substrate is hydroxyacetaldehyde and methylglyoxal, and the resulting product is 2, 3-dihydroxy-4-keto-valeraldehyde; or the substrate is dihydroxyacetone and methylglyoxal, and the obtained product is 1,3, 4-trihydroxy-2, 5-hexanedione.
6. The method of claim 4, wherein the substrates are hydroxyacetone and methylglyoxal, and the resulting product is 3, 4-dihydroxy-2, 5-hexanedione;
preferably, the whole cell catalytic reaction system contains hydroxyacetone in an amount of 50 to 1000mM, and more preferably contains hydroxyacetone in an amount of 500 mM; methylglyoxal content of 50-1000mM, more preferably methylglyoxal content of 500 mM; the cell concentration is preferably 10 to 200 as measured at OD600, more preferably 60 as measured at OD 600; the pH of the buffer is 6-8, preferably 7.5; the catalytic reaction temperature is 20-37 ℃, and the more preferable temperature is 30 ℃; the reaction time is 2 to 24 hours, more preferably 4 hours;
or, the substrate is 1-hydroxy-2-butanone and methylglyoxal, and the obtained product is 3, 4-dihydroxy-2, 5-heptanedione;
preferably, the whole-cell catalytic reaction system contains 1-hydroxy-2-butanone in an amount of 50-1000mM, and more preferably, the content of 1-hydroxy-2-butanone is 500 mM; methylglyoxal content of 50-1000mM, more preferably methylglyoxal content of 300 mM; the cell concentration is preferably 10 to 200 as measured at OD600, more preferably 60 as measured at OD 600; the pH of the buffer is 6-8, preferably 7.5; the catalytic reaction temperature is 20-37 ℃, and the more preferable temperature is 30 ℃; the reaction time is 2 to 24 hours, and more preferably 24 hours.
7. A preparation method of hydroxyfuranone compounds comprises the following steps:
step (1) aldol condensation reaction: performing the aldol condensation reaction of any one of claims 2-6; and
step (2) cyclization reaction: cyclizing the reaction liquid containing the aldol condensation reaction product obtained in the step (1) in an alkaline disodium hydrogen phosphate environment to generate a hydroxyfuranone compound.
8. The method according to claim 7, wherein the substrate in step (1) is hydroxyacetone and methylglyoxal, the product of the aldol condensation reaction is 3, 4-dihydroxyhexanedione, and the hydroxyfuranone compound in step (2) is 2, 5-dimethyl-4-hydroxy-3-furanone;
preferably, in the step (2), Na is added to the reaction solution containing 3, 4-dihydroxyhexanedione2HPO4The final concentration is 20-300g/L, the reaction temperature is 30-90 ℃, and the reaction lasts 2-24 hours; more preferably, Na2HPO4The concentration was 300g/L, the reaction temperature was 70 ℃ and the reaction time was 24 hours.
9. The method according to claim 7, wherein the substrate in step (1) is 1-hydroxy-2-butanone and methylglyoxal, the product of the aldol condensation reaction is 3, 4-dihydroxy-2, 5-heptanedione, and the hydroxyfuranone compound in step (2) is 2-methyl-5-ethyl-4-hydroxy-3-furanone;
preferably, in the step (2), Na is added to the reaction solution containing 3, 4-dihydroxy-2, 5-heptanedione2HPO4The final concentration is 20-300g/L, the reaction temperature is 30-90 ℃, and the reaction lasts 2-24 hours; more preferably, Na2HPO4The concentration was 300g/L, the reaction temperature was 70 ℃ and the reaction time was 24 hours.
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