CN112852793A - Synthesis method of chiral 1, 3-dihydroxy-1-aryl acetone compound - Google Patents
Synthesis method of chiral 1, 3-dihydroxy-1-aryl acetone compound Download PDFInfo
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Abstract
The invention discloses a method for generating optical pure chiral 1, 3-dihydroxy-1-substituted aryl acetone by catalyzing a reaction of hydroxy pyruvic acid and benzaldehyde with different substituents by a biocatalyst. In order to obtain the hydroxy pyruvic acid with low cost, the D-amino acid oxidase is used for converting the D-serine without separation, and the method has the remarkable characteristics of mild reaction condition, good stereoselectivity, no pollution and the like.
Description
Technical Field
The invention belongs to the field of biological catalysis, and relates to a method for catalyzing hydroxy pyruvic acid and benzaldehyde with different substituents to react by using a biological catalyst pyruvate decarboxylase to generate an optical homochiral 1, 3-dihydroxy-1-aryl acetone compound.
Background
The 1,3-dihydroxy ketone compound is an important intermediate for synthesizing a plurality of substances, chiral amino alcohol, rare sugar, steroid and the like can be obtained by utilizing the hydroxy ketone compound, and the compounds have great application potential in the aspects of functional foods, medicines, medicaments and synthetic chemistry.
The current methods for synthesizing 1, 3-dihydroxy-1-aryl acetone are mainly divided into two major types, chemical method and enzymatic method. According to literature reports, Mark E.B. Smith et al achieved the synthesis of 1, 3-dihydroxy-1-phenylacetone using 3- (4-morpholinyl) -propanesulfonic acid as a solvent to promote the formation of the C-C bond between the acceptor and donor aldehydes (Smith MEB, Smities K, Senuss T, Dalby PA, Hailes HC. the First practical of the Transketolaser reaction. European Journal of Organic chemistry.2006; 2006(5): 1121-3.); celin Richter et al used Dihydroxyfumaric Acid as a substrate to synthesize 1, 3-dihydroxy-1-arylacetone (Richter C, Berndt F, Kunde T, Mahrwald R. Decarboxlative case Reactions of dihydrofumic Acid: A Preparative Approach to the gyloxylate Scenario. org Lett.2016; 18(12): 2950-3.); in addition, Kirsty Smith et al obtain 1, 3-dihydroxy-1-phenylacetone racemate and derivatives thereof through multi-step chemical reactions, providing substrates for the synthesis of hydroxyamines of a single configuration (Smith K, Smith MEB, Kaulmann U, Galman JL, Ward JM, Hailes HC. Stereoselectivity of an ω -transaminase-mediated amino of 1,3-dihydroxy-1-phenylpropane-2-one. tetrahedron: asymmetry.2009; 20(5): 570-4.). The chiral hydroxyketone synthesized by the chemical method has low yield, the metal catalyst has high price and is difficult to industrially produce, and in addition, the chemical synthesis method has complex steps and relates to the processes of group protection, product purification and recovery and the like. The biocatalysis has the characteristics of low price, reusability and environmental friendliness, and has the advantages of high-efficiency chemical selectivity, regioselectivity, enantioselectivity and the like, so that the biocatalysis is widely applied to synthesis of hydroxyketone compounds.
Enzymatic synthesis can be achieved using transketolase and pyruvate decarboxylase. Transketolases belong to the thiamine pyrophosphate (ThDP) dependent enzymes, catalyzing the reversible transfer of a carbonyl group between a donor aldehyde and an acceptor aldehyde. A study has shown that Marion Lorilla et al not only achieve the enzymatic synthesis of a series of short-chain ketoses by a tandem transaminase and a Transketolase (Lorilla ore M, De Sousa M, Bruna F, Heuson E, Gefflaut T, De Berardinis V, et al. one-point, Two-step carbohydrate synthesis of native ray-erythro (3S,4S) ketose by ligation a thermal synthesis of native chemistry.2017; 19(2):425-35.) but also obtain Rare sugars lacking in nature by Transketolase (Lorilla ore M, Dumoulin R, L' environment M, ramblin A, therapy V, therapy L, thermal analysis of L.2019. native carbohydrate synthesis of N.2019. Nature. Many studies have been made on the synthesis of aliphatic hydroxy ketones by transketolase, but the application of this enzyme to aromatic aldehyde substrates has been less studied. Studies have shown that Panwajee Payongsri et al improve the activity of transketolase on carboxybenzaldehyde substrates by site-directed saturation mutagenesis and provide technical support for the synthesis of chloramphenicol analogs (Panwajee Payongsri, John Strafford, Andrew MacMurray, Helen C.Hailesb and Paul A.Dalby.random substrate and enzyme engineering of transgenic for aromatics.organic & Biomolecular chemistry.2012(10): 9021-9); james L.Galman et al extend the substrate spectrum of transketolase by using aromatic and heterocyclic aldehydes as receptor substrates for transketolase (Galman J L, Steadman D, Bacon S, et al, α' -dihydroketolase formation using aromatic and heteroaromatic aldehydes with organic carbohydrates enzymes [ J ] Chemical Communications,2010,46(40): 7608-7610.). However, the substrate spectrum of the current preketolase is still narrow, and the low activity is also a main factor limiting the development and application of the preketolase.
Pyruvate decarboxylase also belongs to the ThDP-dependent enzymes Torsten Sehl and
rother et al achieved the biosynthesis of aromatic hydroxyketone-PAC and its derivatives using Pyruvate Decarboxylase and the products were of a single configuration (Rother E Gocke D, Kolter G, Gerhards T, Berthold CL, Gauchenova E, Knoll M, et al.S-Selective Mixed ligation by Structure Design-Based Design of the Pyruvate Decarboxylase from Acetobacter passamultiple Chem.2011; 3(10): 1587. 1596.).At present, no research report for synthesizing the dihydroxyketone compound by catalyzing hydroxyl pyruvic acid by pyruvate decarboxylase is available.
Disclosure of Invention
The invention provides pyruvate decarboxylase and a method for efficiently synthesizing 1, 3-dihydroxy-1-aryl acetone, namely, the method utilizes the pyruvate decarboxylase as a catalyst and takes Hydroxypyruvate (HPA) and aromatic aldehyde as substrates to prepare chiral 1, 3-dihydroxy-1-aromatic acetone compounds (as shown in chemical formula 1).
The amino acid sequence of the pyruvate decarboxylase is SEQ ID NO: 1, and said pyruvate decarboxylase has the ability to convert Hydroxypyruvate (HPA) and aromatic aldehydes into 1, 3-dihydroxy-1-arylacetone. The pyruvate decarboxylase is derived from Macrococcus hajekii.
Because the price of the hydroxyl pyruvic acid is expensive, the D-amino acid oxidase is utilized to obtain the hydroxyl pyruvic acid from D-serine, and the gene sequence for producing the D-amino acid oxidase comes from literature reports, and the amino acid sequence is SEQ ID NO: 2.
the vector used by the genetic engineering bacteria for producing the pyruvate decarboxylase is pET series plasmids.
The carrier used by the genetic engineering bacteria for producing the D-amino acid oxidase is pET series plasmid.
The genetic engineering bacteria for producing pyruvate decarboxylase is Escherichia coli BL21 series.
The genetic engineering bacteria for producing the D-amino acid oxidase are Escherichia coli BL21 series.
The pyruvate decarboxylase and D-amino acid oxidase used for producing the chiral aromatic hydroxyketone compound may be cultures of the above pyruvate decarboxylase genetically engineered bacteria and D-amino acid oxidase genetically engineered bacteria, or may be bacterial cells obtained by centrifuging a culture medium or processed products thereof. Wherein the processed product refers to extract obtained from thallus, broken solution or separated product obtained by separating and/or purifying pyruvate decarboxylase and D-amino acid oxidase. The most used in the present invention is bacterial cells obtained by centrifuging a culture medium.
The invention also relates to a method for generating corresponding chiral aromatic hydroxy ketone by using whole cells to catalyze the conversion of benzaldehyde substrates with different substituents, which comprises the following steps:
respectively culturing the gene engineering bacteria of the pyruvate decarboxylase and the D-amino acid oxidase for a certain time, adding an inducer IPTG for culturing for a period of time, and centrifugally collecting bacteria. Resuspending the cells in a buffer solution, and adding 100mM D-Serine and 1mM MgCl2·6H2O and 0.1mM ThDP, and adding catalase (15KU), incubating at 25 deg.C and 200rpm for 1h, and adding 20mM of aromatic aldehyde substrates with different substituents (dissolved in 20% DMSO) for 24 h. After the reaction, the protein was removed by centrifugation, the supernatant was extracted with ethyl acetate, and the product was purified by silica gel column chromatography after removing the organic solvent.
The buffer solution used in the reaction is an aqueous solution having a pH of 6.0 to 8.0 and a temperature of 20 to 40 ℃, preferably a pH of 7.0 and a temperature of 25 ℃.
The substrate concentration according to the present invention is not limited, and is usually 5mM-40mM, and 20mM is most preferably selected in consideration of the conversion rate of the substrate and the yield of the product.
The biocatalyst used in the present invention is whole cells, which are used in an amount of 5-50g/L, preferably 30 g/L.
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FIG. 11, 3-dihydroxy-1-propiophenone hydrogen spectrum
Detailed Description
The following examples are further illustrated for the purpose of better understanding the present invention, but are not to be construed as limiting the invention.
Example 1: construction and culture of genetically engineered bacteria
The specific construction and culture method of the gene engineering bacteria for producing pyruvate decarboxylase comprises the following steps: pyruvate decarboxylase nucleic acid sequence from Macrococcus hajekii SEQ ID NO: 3Carrying out gene synthesis, constructing into pET series vectors, and carrying out heterologous expression in host bacteria escherichia coli. The strain preserved at-80 ℃ was thawed, streaked on a plate, and cultured overnight in a 37 ℃ incubator. Selecting single colony on the plate, inoculating into 20mL LB culture medium containing corresponding antibiotic, culturing for about 12h to obtain seed solution, inoculating into 700mL LB culture medium containing corresponding antibiotic according to 1% of inoculum size, and culturing on a shaker at 37 deg.C and 200rpm to OD600When the concentration was about 0.6 to 0.8, IPTG was added to the mixture at a final concentration of 0.1mmol/L to induce the cells at 25 ℃ for 12 hours, and the cells were collected by centrifugation at 6000 rpm.
The specific construction and culture method of the genetic engineering bacteria for producing the D-amino acid oxidase comprises the following steps: the document reports that the D-amino acid oxidase nucleic acid sequence derived from Rhodotorula toruloides NP11 is SEQ ID NO: 4, synthesizing the gene, constructing the gene into a pET series vector, and performing heterologous expression through host bacteria Escherichia coli. The strain preserved at-80 ℃ was thawed, streaked on a plate, and cultured overnight in a 37 ℃ incubator. Selecting single colony on the plate, inoculating into 20mL LB culture medium containing corresponding antibiotic, culturing for about 12h to obtain seed solution, inoculating into 700mL LB culture medium containing corresponding antibiotic according to 1% of inoculum size, and culturing on a shaker at 37 deg.C and 200rpm to OD600When the concentration was about 0.6 to 0.8, IPTG was added to the mixture at a final concentration of 0.1mmol/L to induce the cells at 25 ℃ for 12 hours, and the cells were collected by centrifugation at 6000 rpm.
Example 2: pyruvate decarboxylase catalyzes benzaldehyde and hydroxyacetonic acid to prepare 1,3-dihydroxy-1-phenyl acetone.
(1) 0.26g of hydroxypyruvate (25mM), 0.01g of ThDP (0.1mM), 3.00g of a pyruvate decarboxylase gene-engineering bacterium and 20mM of benzaldehyde (dissolved in 20% DMSO) were added to 80mL of water, the pH of the reaction system was adjusted to 8.0, and the reaction was carried out at 25 ℃ for 24 hours. After the reaction, the protein was removed by centrifugation, the supernatant was extracted with ethyl acetate, and the completion of the extraction was confirmed by thin layer chromatography. And (3) removing the organic solvent by rotary evaporation to obtain a crude product, and detecting the substrate conversion rate and the product yield by using a gas phase and detecting the ratio of the product to the byproduct isomer by using a liquid phase HPLC. The detection result is as follows: the pyruvate decarboxylase catalyzes to obtain an R configuration product, the ee value is more than 99 percent, the conversion rate of the benzaldehyde substrate is 61 percent, and the ratio of the product (1) to the byproduct isomer (2) is 98: 2. The R-configuration NMR data are shown in FIG. 1. The product and by-product isomer structural formulas are as follows:
(2) 0.26g of hydroxypyruvate (25mM), 0.01g of ThDP (0.1mM), 3.00g of a pyruvate decarboxylase gene-engineering bacterium and 20mM of benzaldehyde (dissolved in 20% DMSO) were added to 80mL of water to adjust the pH of the reaction system to 7.5, and the reaction was carried out at 25 ℃ for 24 hours. After the reaction, the protein was removed by centrifugation, the supernatant was extracted with ethyl acetate, and the completion of the extraction was confirmed by thin layer chromatography. And (3) removing the organic solvent by rotary evaporation to obtain a crude product, and detecting the substrate conversion rate and the product yield by using a gas phase and detecting the ratio of the product to the byproduct isomer by using a liquid phase HPLC. The detection result is as follows: the pyruvate decarboxylase catalyzes to obtain an R configuration product, the ee value is more than 99 percent, the conversion rate of the benzaldehyde substrate is 73 percent, and the ratio of the product (1) to the byproduct isomer (2) is 98: 2.
(3) 0.26g of hydroxypyruvate (25mM), 0.01g of ThDP (0.1mM), 3.00g of a pyruvate decarboxylase gene-engineering bacterium and 20mM of benzaldehyde (dissolved in 20% DMSO) were added to 80mL of water, the pH of the reaction system was adjusted to 7.0, and the reaction was carried out at 25 ℃ for 24 hours. After the reaction, the protein was removed by centrifugation, the supernatant was extracted with ethyl acetate, and the completion of the extraction was confirmed by thin layer chromatography. And (3) removing the organic solvent by rotary evaporation to obtain a crude product, and detecting the substrate conversion rate and the product yield by using a gas phase and detecting the ratio of the product to the byproduct isomer by using a liquid phase HPLC. The detection result is as follows: the pyruvate decarboxylase catalyzes to obtain an R configuration product, the ee value is more than 99 percent, the conversion rate of the benzaldehyde substrate is 96 percent, and the ratio of the product (1) to the byproduct isomer (2) is 98: 2.
(4) 0.26g of hydroxypyruvate (25mM), 0.01g of ThDP (0.1mM), 3.00g of a pyruvate decarboxylase gene-engineering bacterium and 20mM of benzaldehyde (dissolved in 20% DMSO) were added to 80mL of water, the pH of the reaction system was adjusted to 6.5, and the reaction was carried out at 25 ℃ for 24 hours. After the reaction, the protein was removed by centrifugation, the supernatant was extracted with ethyl acetate, and the completion of the extraction was confirmed by thin layer chromatography. And (3) removing the organic solvent by rotary evaporation to obtain a crude product, and detecting the substrate conversion rate and the product yield by using a gas phase and detecting the ratio of the product to the byproduct isomer by using a liquid phase HPLC. The detection result is as follows: the pyruvate decarboxylase catalyzes to obtain an R configuration product, the ee value is more than 99 percent, the conversion rate of the benzaldehyde substrate is 78 percent, and the ratio of the product (1) to the byproduct isomer (2) is 98: 2.
(5) 0.26g of hydroxypyruvate (25mM), 0.01g of ThDP (0.1mM), 3.00g of a pyruvate decarboxylase gene-engineering bacterium and 20mM of benzaldehyde (dissolved in 20% DMSO) were added to 80mL of water, the pH of the reaction system was adjusted to 6.0, and the reaction was carried out at 25 ℃ for 24 hours. After the reaction, the protein was removed by centrifugation, the supernatant was extracted with ethyl acetate, and the completion of the extraction was confirmed by thin layer chromatography. And (3) removing the organic solvent by rotary evaporation to obtain a crude product, and detecting the substrate conversion rate and the product yield by using a gas phase and detecting the ratio of the product to the byproduct isomer by using a liquid phase HPLC. The detection result is as follows: the pyruvate decarboxylase catalyzes to obtain an R configuration product, the ee value is more than 99 percent, the conversion rate of the benzaldehyde substrate is 62 percent, and the ratio of the product (1) to the byproduct isomer (2) is 98: 2.
Example 3: pyruvate decarboxylase and D-amino acid oxidase gene engineering bacteria are used for preparing 1, 3-dihydroxy-1-aryl acetone.
2.00g D-amino acid oxidase gene engineering bacteria, 3.00g pyruvate decarboxylase gene engineering bacteria, 1.05g D-spring (100mM), 0.02g MgCl2·6H2O (1mM), 0.01g ThDP (0.1mM), 60mg catalase (15KU) was added to 80mL of water, the reaction was adjusted to pH 7.0, incubated at 25 ℃ and 200rpm for 1 hour, and then reacted for 24 hours with 20mM benzaldehyde (dissolved in 20% DMSO). After the reaction, the protein was removed by centrifugation, the supernatant was extracted with ethyl acetate, and the completion of the extraction was confirmed by thin layer chromatography. And (3) removing the organic solvent by rotary evaporation to obtain a crude product, and detecting the substrate conversion rate and the product yield by using a gas phase and detecting the ratio of the product to the byproduct isomer by using a liquid phase HPLC. The detection result is as follows: obtaining R configuration product, ee value by pyruvate decarboxylase and D-amino acid oxidase catalysis>99% with a benzaldehyde substrate conversion of 96%, wherein the ratio of product (1) to by-product isomer (2) is 98: 2. Purifying the crude product by using a silica gel column, wherein the mobile phase comprises 10-20% of ethyl acetate and petroleum ether, and obtaining the aromatic hydroxy ketone product. The product and by-product isomer structural formulas are as follows:
example 4: pyruvate decarboxylase catalyzes the substitution of benzaldehyde and hydroxyacetonic acid to prepare 1,3-dihydroxy-1- (substituted phenyl) acetone.
(1) 0.26g of hydroxypyruvate (25mM), 0.01g of ThDP (0.1mM), and 3.00g of pyruvate decarboxylase-engineered bacterium were added to 80mL of water, the pH of the reaction system was adjusted to 7.0, and 20mM of p-fluorobenzaldehyde (dissolved in 20% DMSO) was added thereto at 25 ℃ to react for 24 hours. After the reaction, the protein was removed by centrifugation, the supernatant was extracted with ethyl acetate, and the completion of the extraction was confirmed by thin layer chromatography. And (3) removing the organic solvent by rotary evaporation to obtain a crude product, and detecting the substrate conversion rate and the product yield by using a gas phase and detecting the ratio of the product to the byproduct isomer by using a liquid phase HPLC. The detection result is as follows: the pyruvate decarboxylase catalyzes to obtain an R configuration product, the ee value is more than 99 percent, the conversion rate of a p-fluorobenzaldehyde substrate is 97 percent, and the proportion of the product (3) and the byproduct isomer (4) is more than 99: 1.
(2) 0.26g of hydroxypyruvate (25mM), 0.01g of ThDP (0.1mM), and 3.00g of pyruvate decarboxylase-engineered bacteria were added to 80mL of water, the pH of the reaction system was adjusted to 7.0, and 20mM of p-methylthiobenzaldehyde (dissolved in 20% DMSO) was added thereto at 25 ℃ to react for 24 hours. After the reaction, the protein was removed by centrifugation, the supernatant was extracted with ethyl acetate, and the completion of the extraction was confirmed by thin layer chromatography. And (3) removing the organic solvent by rotary evaporation to obtain a crude product, and detecting the substrate conversion rate and the product yield by using a gas phase and detecting the ratio of the product to the byproduct isomer by using a liquid phase HPLC. The detection result is as follows: the pyruvate decarboxylase is catalyzed to obtain an R configuration product, the ee value is more than 99 percent, the conversion rate of the p-methylthiobenzaldehyde substrate is 68 percent, and the ratio of the product (5) to the byproduct isomer (6) is more than 99: 1.
(3) 0.26g of hydroxypyruvate (25mM), 0.01g of ThDP (0.1mM), and 3.00g of pyruvate decarboxylase-engineered bacteria were added to 80mL of water, the pH of the reaction system was adjusted to 7.0, and 20mM of p-bromobenzaldehyde (dissolved in 20% DMSO) was added thereto at 25 ℃ to react for 24 hours. After the reaction, the protein was removed by centrifugation, the supernatant was extracted with ethyl acetate, and the completion of the extraction was confirmed by thin layer chromatography. And (3) removing the organic solvent by rotary evaporation to obtain a crude product, and detecting the substrate conversion rate and the product yield by using a gas phase and detecting the ratio of the product to the byproduct isomer by using a liquid phase HPLC. The detection result is as follows: catalyzing by pyruvate decarboxylase to obtain an R configuration product, wherein the ee value is more than 99%, the conversion rate of a p-bromobenzaldehyde substrate is 94%, and the proportion of the product (7) to the byproduct isomer (8) is 96: 4.
(4) 0.26g of hydroxypyruvate (25mM), 0.01g of ThDP (0.1mM), and 3.00g of pyruvate decarboxylase-engineered bacteria were added to 80mL of water, the pH of the reaction system was adjusted to 7.0, and 20mM of p-chlorobenzaldehyde (dissolved in 20% DMSO) was added thereto at 25 ℃ to react for 24 hours. After the reaction, the protein was removed by centrifugation, the supernatant was extracted with ethyl acetate, and the completion of the extraction was confirmed by thin layer chromatography. And (3) removing the organic solvent by rotary evaporation to obtain a crude product, and detecting the substrate conversion rate and the product yield by using a gas phase and detecting the ratio of the product to the byproduct isomer by using a liquid phase HPLC. The detection result is as follows: catalyzing by pyruvate decarboxylase to obtain an R configuration product, wherein the ee value is more than 99%, the conversion rate of a p-chlorobenzaldehyde substrate is 96%, and the proportion of a product (9) to a byproduct isomer (10) is 91: 9.
(5) 0.26g of hydroxypyruvate (25mM), 0.01g of ThDP (0.1mM), and 3.00g of pyruvate decarboxylase-engineered bacteria were added to 80mL of water, the pH of the reaction system was adjusted to 7.0, and 20mM of p-tolualdehyde (dissolved in 20% DMSO) was added thereto at 25 ℃ to react for 24 hours. After the reaction, the protein was removed by centrifugation, the supernatant was extracted with ethyl acetate, and the completion of the extraction was confirmed by thin layer chromatography. And (3) removing the organic solvent by rotary evaporation to obtain a crude product, and detecting the substrate conversion rate and the product yield by using a gas phase and detecting the ratio of the product to the byproduct isomer by using a liquid phase HPLC. The detection result is as follows: the pyruvate decarboxylase is catalyzed to obtain an R configuration product, the ee value is more than 99 percent, the conversion rate of the p-tolualdehyde substrate is 94 percent, and the proportion of the product (11) and the byproduct isomer (12) is more than 99: 1.
(6) 0.26g of hydroxypyruvate (25mM), 0.01g of ThDP (0.1mM), and 3.00g of pyruvate decarboxylase-engineered bacterium were added to 80mL of water, the pH of the reaction system was adjusted to 7.0, and 20mM of o-chlorobenzaldehyde (dissolved in 20% DMSO) was added thereto at 25 ℃ to react for 24 hours. After the reaction, the protein was removed by centrifugation, the supernatant was extracted with ethyl acetate, and the completion of the extraction was confirmed by thin layer chromatography. And (3) removing the organic solvent by rotary evaporation to obtain a crude product, and detecting the substrate conversion rate and the product yield by using a gas phase and detecting the ratio of the product to the byproduct isomer by using a liquid phase HPLC. The detection result is as follows: catalyzing by pyruvate decarboxylase to obtain an R configuration product, wherein the ee value is more than 99%, the conversion rate of an o-chlorobenzaldehyde substrate is 98%, and the proportion of a product (13) to a byproduct isomer (14) is 90: 10.
(7) 0.26g of hydroxypyruvate (25mM), 0.01g of ThDP (0.1mM), and 3.00g of pyruvate decarboxylase-engineered bacterium were added to 80mL of water, the pH of the reaction system was adjusted to 7.0, and 20mM o-fluorobenzaldehyde (dissolved in 20% DMSO) was added thereto at 25 ℃ to react for 24 hours. After the reaction, the protein was removed by centrifugation, the supernatant was extracted with ethyl acetate, and the completion of the extraction was confirmed by thin layer chromatography. And (3) removing the organic solvent by rotary evaporation to obtain a crude product, and detecting the substrate conversion rate and the product yield by using a gas phase and detecting the ratio of the product to the byproduct isomer by using a liquid phase HPLC. The detection result is as follows: catalyzing by pyruvate decarboxylase to obtain an R configuration product, wherein the ee value is more than 99%, the conversion rate of an o-fluorobenzaldehyde substrate is 98%, and the proportion of a product (15) to a byproduct isomer (16) is 98: 2.
(8) 0.26g of hydroxypyruvate (25mM), 0.01g of ThDP (0.1mM), and 3.00g of pyruvate decarboxylase-engineered bacteria were added to 80mL of water, and the reaction system was adjusted to pH 7.0 at 25 ℃ and reacted with 20mM of m-tolualdehyde (dissolved in 20% DMSO) for 24 hours. After the reaction, the protein was removed by centrifugation, the supernatant was extracted with ethyl acetate, and the completion of the extraction was confirmed by thin layer chromatography. And (3) removing the organic solvent by rotary evaporation to obtain a crude product, and detecting the substrate conversion rate and the product yield by using a gas phase and detecting the ratio of the product to the byproduct isomer by using a liquid phase HPLC. The detection result is as follows: catalyzing by pyruvate decarboxylase to obtain an R configuration product, wherein the ee value is more than 99%, the conversion rate of a m-tolualdehyde substrate is 99%, and the proportion of a product (17) to a byproduct isomer (18) is 98: 2.
example 5: pyruvate decarboxylase and D-amino acid oxidase gene engineering bacteria are used for preparing 1, 3-dihydroxy-1-substituted aryl acetone.
(1) 2.00g D-amino acid oxidase gene engineering bacteria, 3.00g pyruvate decarboxylase gene engineering bacteria, 1.05g D-spring (100mM), 0.02g MgCl2·6H2O (1mM), 0.01g ThDP (0.1mM), 60mg catalase (15KU) was added to 80mL of water, the reaction was adjusted to pH 7.0, incubated at 25 ℃ for 1 hour at 200rpm, and then reacted for 24 hours with 20mM p-fluorobenzaldehyde (dissolved in 20% DMSO). After the reaction is finished, centrifuging to remove protein, extracting supernatant by using ethyl acetate, and detecting the result as follows: the conversion rate of the p-tolualdehyde substrate is 97 percent, the pyruvate decarboxylase and the D-amino acid oxidase are catalyzed to obtain an R configuration product, and the ee value is>99% in which the ratio of the product (3) and the by-product isomer (4) is>99:1。
(2) 2.00g D-amino acid oxidase gene engineering bacteria, 3.00g CKetoacid decarboxylase genetically engineered bacterium, 1.05g D-spring (100mM), 0.02g MgCl2·6H2O (1mM), 0.01g ThDP (0.1mM), 60mg catalase (15KU) was added to 80mL of water, the reaction system was adjusted to pH 7.0, incubated at 25 ℃ for 1 hour at 200rpm, and then reacted for 24 hours with 20mM p-methylthiobenzaldehyde (dissolved in 20% DMSO). After the reaction is finished, centrifuging to remove protein, extracting supernatant by using ethyl acetate, and detecting the result as follows: the conversion rate of the p-methylthiobenzaldehyde substrate is 68 percent, and the conversion rate of the p-methylthiobenzaldehyde substrate is catalyzed by pyruvate decarboxylase and D-amino acid oxidase to obtain an R configuration product with ee value>99% in which the ratio of the product (5) and the by-product isomer (6) is>99:1。
(3) 2.00g D-amino acid oxidase gene engineering bacteria, 3.00g pyruvate decarboxylase gene engineering bacteria, 1.05g D-spring (100mM), 0.02g MgCl2·6H2O (1mM), 0.01g ThDP (0.1mM), 60mg catalase (15KU) was added to 80mL of water, the reaction was adjusted to pH 7.0, incubated at 25 ℃ for 1 hour at 200rpm, and then reacted for 24 hours with 20mM p-bromobenzaldehyde (dissolved in 20% DMSO). After the reaction is finished, centrifuging to remove protein, extracting supernatant by using ethyl acetate, and detecting the result as follows: the conversion rate of the bromobenzaldehyde substrate is 94 percent, and the pyruvate decarboxylase and the D-amino acid oxidase are catalyzed to obtain an R configuration product with ee value>99% wherein the ratio of product (7) and byproduct isomer (8) is 96: 4.
(4) 2.00g D-amino acid oxidase gene engineering bacteria, 3.00g pyruvate decarboxylase gene engineering bacteria, 1.05g D-spring (100mM), 0.02g MgCl2·6H2O (1mM), 0.01g ThDP (0.1mM), 60mg catalase (15KU) was added to 80mL of water, the reaction was adjusted to pH 7.0, incubated at 25 ℃ for 1 hour at 200rpm, and then reacted for 24 hours with 20mM p-chlorobenzaldehyde (dissolved in 20% DMSO). After the reaction is finished, centrifuging to remove protein, extracting supernatant by using ethyl acetate, and detecting the result as follows: the conversion rate of the p-chlorobenzaldehyde substrate is 96 percent, the pyruvate decarboxylase and the D-amino acid oxidase are catalyzed to obtain an R configuration product, and the ee value is>99% wherein the ratio of product (9) and byproduct isomer (10) is 91: 9.
(5) 2.00g D-amino acid oxidase gene engineering bacteria, 3.00g pyruvate decarboxylase gene engineering bacteria, 1.05g D-spring (100mM), 0.02g MgCl2·6H2O(1mM)0.01g of ThDP (0.1mM), 60mg of catalase (15KU) was added to 80mL of water, the reaction system was incubated at 25 ℃ for 1 hour with pH 7.0 and 200rpm, and then reacted for 24 hours with 20mM p-tolualdehyde (dissolved in 20% DMSO). After the reaction is finished, centrifuging to remove protein, extracting supernatant by using ethyl acetate, and detecting the result as follows: the conversion rate of p-tolualdehyde substrate is 94%, and the R configuration product is obtained by the catalysis of pyruvate decarboxylase and D-amino acid oxidase, and the ee value is>99% in which the ratio of the product (11) and the by-product isomer (12) is>99:1。
(6) 2.00g D-amino acid oxidase gene engineering bacteria, 3.00g pyruvate decarboxylase gene engineering bacteria, 1.05g D-spring (100mM), 0.02g MgCl2·6H2O (1mM), 0.01g ThDP (0.1mM), 60mg catalase (15KU) was added to 80mL of water, the reaction was adjusted to pH 7.0, incubated at 25 ℃ for 1 hour at 200rpm, and then reacted for 24 hours with 20mM O-chlorobenzaldehyde (dissolved in 20% DMSO). After the reaction is finished, centrifuging to remove protein, extracting supernatant by using ethyl acetate, and detecting the result as follows: the conversion rate of the o-chlorobenzaldehyde substrate is 98 percent, and the R configuration product is obtained under the catalysis of pyruvate decarboxylase and D-amino acid oxidase, and the ee value is>99% wherein the ratio of product (13) and byproduct isomer (14) is 90: 10.
(7) 2.00g D-amino acid oxidase gene engineering bacteria, 3.00g pyruvate decarboxylase gene engineering bacteria, 1.05g D-spring (100mM), 0.02g MgCl2·6H2O (1mM), 0.01g ThDP (0.1mM), 60mg catalase (15KU) was added to 80mL of water, the reaction was adjusted to pH 7.0, incubated at 25 ℃ for 1 hour at 200rpm, and then reacted for 24 hours with 20mM O-fluorobenzaldehyde (dissolved in 20% DMSO). After the reaction is finished, centrifuging to remove protein, extracting supernatant by using ethyl acetate, and detecting the result as follows: the conversion rate of the o-fluorobenzaldehyde substrate is 98 percent, and an R configuration product is obtained under the catalysis of pyruvate decarboxylase and D-amino acid oxidase, and the ee value is>99% wherein the ratio of product (15) and byproduct isomer (16) is 98: 2.
(8) 2.00g D-amino acid oxidase gene engineering bacteria, 3.00g pyruvate decarboxylase gene engineering bacteria, 1.05g D-spring (100mM), 0.02g MgCl2·6H2O (1mM), 0.01g of ThDP (0.1mM), 60mg of catalase (15KU) were added to 80mL of water, the pH of the reaction system was adjusted to 7.0, 25 ℃,after incubation at 200rpm for 1h, 20mM m-tolualdehyde (in 20% DMSO) was added and reacted for 24 h. After the reaction is finished, centrifuging to remove protein, extracting supernatant by using ethyl acetate, and detecting the result as follows: the conversion rate of the m-methyl benzaldehyde substrate is 99 percent, the pyruvate decarboxylase and the D-amino acid oxidase are catalyzed to obtain an R configuration product, and the ee value>99% wherein the ratio of product (17) and byproduct isomer (18) is 98: 2.
sequence listing
<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences
<120> method for synthesizing chiral 1, 3-dihydroxy-1-aryl acetone compound
<130> 1
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 546
<212> PRT
<213> Macrococcus hajekii
<400> 1
Met Lys Arg Arg Val Gly Gln Tyr Leu Ile Asp Gln Ile Ser Val Tyr
1 5 10 15
Gly Val Asp Gln Ile Phe Gly Val Pro Gly Asp Phe Asn Leu Ala Phe
20 25 30
Leu Asp Asp Ile Val Ser His Glu Gln Val Glu Trp Ile Gly Asn Thr
35 40 45
Asn Glu Leu Asn Ala Ser Tyr Ala Ala Asp Gly Tyr Ala Arg Ile Lys
50 55 60
Gly Leu Gly Ala Leu Val Thr Thr Phe Gly Val Gly Glu Leu Ser Ala
65 70 75 80
Val Asn Gly Ile Ala Gly Ser Tyr Ala Glu Arg Val Pro Val Val Ala
85 90 95
Ile Thr Gly Ala Pro Thr Thr Val Val Glu Gln Ala Lys Arg Tyr Val
100 105 110
His His Ser Leu Gly Glu Gly Val Phe Asp Asp Tyr Gln Arg Met Phe
115 120 125
Glu Pro Ile Thr Cys Gly Gln Ile Tyr Leu Thr Ala Asp Asn Ala Ala
130 135 140
Ile Glu Ile Pro Arg Ile Ile Ala Lys Ala Ile Arg Phe Gln Arg Pro
145 150 155 160
Val His Ile His Leu Pro Ile Asp Val Ala Met Thr Glu Ile Glu Val
165 170 175
Lys Glu Ala Thr Val Gln Pro Met Leu Ser Gln Asp Val Ser Val Phe
180 185 190
Thr Asp Leu Ile Gln Glu Arg Leu Lys Glu Ala Gln Gln Pro Val Leu
195 200 205
Ile Ala Gly His Glu Ile Asn Ser Phe Lys Leu His Glu Glu Leu Glu
210 215 220
Gln Phe Val Asn Gln Thr His Ile Pro Val Ala Gln Leu Ser Leu Gly
225 230 235 240
Lys Gly Ala Phe Asn Glu Glu Asn Pro His Tyr Ile Gly Ile Phe Asp
245 250 255
Gly Lys Ile Ala His Lys Asp Val Arg Asp Tyr Val Asn Ser Ser Asp
260 265 270
Cys Ile Ile Asn Leu Gly Ala Lys Leu Thr Asp Ser Ala Thr Ala Gly
275 280 285
Phe Ser Tyr Glu Phe Lys Thr Glu Asp Val Ile Phe Ile Asn Asp Glu
290 295 300
Asp Phe Arg Val Asp Ser Thr Tyr His Thr Asp Val Ser Leu Arg Asp
305 310 315 320
Met Met Arg Ser Leu Leu Thr Leu Asn Tyr Glu Asn Thr Ala Asn Phe
325 330 335
Thr Pro Phe Asn Arg Glu His Met Thr Asp Phe Ile Leu Asn Asp Glu
340 345 350
Ala Leu Thr Gln Glu Asn Tyr Phe Lys Met Ile Asn His Phe Leu Gln
355 360 365
Gln Asp Asp Val Leu Leu Ala Glu Gln Gly Thr Ser Phe Phe Gly Ala
370 375 380
Tyr Asp Leu Val Leu Tyr Ala Gly Asn Gln Phe Ile Gly Gln Pro Leu
385 390 395 400
Trp Gly Ser Ile Gly Tyr Thr Leu Pro Ala Leu Leu Gly Thr Gln Met
405 410 415
Ala Asp Lys Lys Arg Arg Asn Val Leu Leu Ile Gly Asp Gly Ser Phe
420 425 430
Gln Leu Thr Ala Gln Glu Leu Ser Thr Met Ile Arg His Gln Leu Lys
435 440 445
Pro Ile Ile Phe Leu Ile Asn Asn Asp Gly Tyr Thr Val Glu Arg Leu
450 455 460
Ile His Gly Glu Lys Glu Ile Tyr Asn Asp Ile Gln Met Trp Asp Tyr
465 470 475 480
Thr Ala Leu Pro Ala Val Phe Gly Gly Lys Asp His Val Gln Thr His
485 490 495
Lys Ala Ser Thr Ser Asn Glu Leu Lys Thr Val Met Asp Thr Val Asp
500 505 510
Gln Gln Pro Asp Lys Met His Phe Ile Glu Val Thr Met Gly Met Leu
515 520 525
Asp Ala Pro Glu Lys Leu Val Asn Ile Ser Lys Ala Phe Ala Ala Gln
530 535 540
Asn Lys
545
<210> 2
<211> 368
<212> PRT
<213> Rhodotorula toruloides NP11
<400> 2
Met His Ser Gln Lys Arg Val Val Val Leu Gly Ser Gly Val Ile Gly
1 5 10 15
Leu Ser Ser Ala Leu Ile Leu Ala Arg Lys Gly Tyr Ser Val His Ile
20 25 30
Leu Ala Arg Asp Leu Pro Glu Asp Val Ser Ser Gln Thr Phe Ala Ser
35 40 45
Pro Trp Ala Gly Ala Asn Trp Thr Pro Phe Met Thr Leu Thr Asp Gly
50 55 60
Pro Arg Gln Ala Lys Trp Glu Glu Ser Thr Phe Lys Lys Trp Val Glu
65 70 75 80
Leu Val Pro Thr Gly His Ala Met Trp Leu Lys Gly Thr Arg Arg Phe
85 90 95
Ala Gln Asn Glu Asp Gly Leu Leu Gly His Trp Tyr Lys Asp Ile Thr
100 105 110
Pro Asn Tyr Arg Pro Leu Pro Ser Ser Glu Cys Pro Pro Gly Ala Ile
115 120 125
Gly Val Thr Tyr Asp Thr Leu Ser Val His Ala Pro Lys Tyr Cys Gln
130 135 140
Tyr Leu Ala Arg Glu Leu Gln Lys Leu Gly Ala Thr Phe Glu Arg Arg
145 150 155 160
Thr Val Thr Ser Leu Glu Gln Ala Phe Asp Gly Ala Asp Leu Val Val
165 170 175
Asn Ala Thr Gly Leu Gly Ala Lys Ser Ile Ala Gly Ile Asp Asp Gln
180 185 190
Ala Ala Glu Pro Ile Arg Gly Gln Thr Val Leu Val Lys Ser Pro Cys
195 200 205
Lys Arg Cys Thr Met Asp Ser Ser Asp Pro Ala Ser Pro Ala Tyr Ile
210 215 220
Ile Pro Arg Pro Gly Gly Glu Val Ile Cys Gly Gly Thr Tyr Gly Val
225 230 235 240
Gly Asp Trp Asp Leu Ser Val Asn Pro Glu Thr Val Gln Arg Ile Leu
245 250 255
Lys His Cys Leu Arg Leu Asp Pro Thr Ile Ser Ser Asp Gly Thr Ile
260 265 270
Glu Gly Ile Glu Val Leu Arg His Asn Val Gly Leu Arg Pro Ala Arg
275 280 285
Arg Gly Gly Pro Arg Val Glu Ala Glu Arg Ile Val Leu Pro Leu Asp
290 295 300
Arg Thr Lys Ser Pro Leu Ser Leu Gly Arg Gly Ser Ala Arg Ala Ala
305 310 315 320
Lys Glu Lys Glu Val Thr Leu Val His Ala Tyr Gly Phe Ser Ser Ala
325 330 335
Gly Tyr Gln Gln Ser Trp Gly Ala Ala Glu Asp Val Ala Gln Leu Val
340 345 350
Asp Glu Ala Phe Gln Arg Tyr His Gly Ala Ala Arg Glu Ser Lys Leu
355 360 365
<210> 3
<211> 1638
<212> DNA
<213> Macrococcus hajekii
<400> 3
atgaagcgtc gtgtgggcca gtatctgatt gatcagatta gcgtttatgg cgtggatcag 60
atttttggtg tgccgggcga ttttaatctg gcctttctgg atgatattgt gagccatgaa 120
caggtggaat ggattggcaa taccaatgaa ctgaatgcaa gctatgcagc agatggctat 180
gcccgcatta aaggtctggg cgcactggtt accacctttg gtgtgggtga actgagtgca 240
gtgaatggca ttgccggtag ttatgccgaa cgtgttccgg tggttgcaat taccggtgcc 300
ccgaccaccg ttgtggaaca ggcaaaacgt tatgtgcatc atagtctggg tgaaggtgtt 360
tttgatgatt atcagcgtat gtttgaaccg attacctgcg gtcagattta tctgaccgca 420
gataatgcag ccattgaaat tccgcgtatt attgcaaaag caattcgttt tcagcgcccg 480
gtgcatattc atctgccgat tgatgtggcc atgaccgaaa ttgaagtgaa agaagcaacc 540
gttcagccga tgctgagcca ggatgttagc gtgtttaccg atctgattca ggaacgtctg 600
aaagaagcac agcagccggt tctgattgca ggccatgaaa ttaatagttt taagctgcat 660
gaggaactgg aacagtttgt gaatcagacc catattccgg ttgcccagct gagcctgggc 720
aaaggtgcat ttaatgaaga aaatccgcat tatatcggca tttttgatgg caaaattgcc 780
cataaagatg tgcgtgatta tgttaatagc agtgattgca ttatcaacct gggtgccaaa 840
ctgaccgata gcgcaaccgc aggttttagt tatgaattta aaaccgaaga cgtgattttc 900
attaacgatg aagattttcg tgtggatagt acctatcata ccgatgtgag tctgcgtgat 960
atgatgcgca gtctgctgac cctgaattat gaaaataccg ccaattttac cccgtttaat 1020
cgcgaacaca tgaccgattt tattctgaat gatgaagcac tgacccagga aaattatttt 1080
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gttgaacgtc tgattcatgg cgaaaaagaa atttataacg acattcagat gtgggattat 1440
accgccctgc cggcagtttt tggtggtaaa gatcatgttc agacccataa agccagcacc 1500
agcaatgaac tgaaaaccgt gatggatacc gtggatcagc agccggataa aatgcatttt 1560
attgaagtta ccatgggcat gctggatgca ccggaaaaac tggtgaatat tagtaaagca 1620
ttcgccgccc agaataaa 1638
<210> 4
<211> 1104
<212> DNA
<213> Rhodotorula toruloides NP11
<400> 4
atgcatagtc agaaacgtgt ggttgtgctg ggcagcggcg tgattggtct gagcagcgca 60
ctgattctgg cccgcaaagg ctatagcgtt catattctgg cccgtgatct gccggaagat 120
gttagcagcc agacctttgc cagcccgtgg gcaggtgcaa attggacccc gtttatgacc 180
ctgaccgatg gcccgcgtca ggcaaaatgg gaagaaagta cctttaaaaa atgggtggaa 240
ctggtgccga ccggtcatgc aatgtggctg aaaggtaccc gtcgttttgc ccagaatgaa 300
gatggtctgc tgggtcattg gtataaagat attaccccga attatcgtcc gctgccgagc 360
agcgaatgcc cgcctggtgc cattggcgtt acctatgata ccctgagtgt gcatgccccg 420
aaatattgtc agtatctggc ccgcgaactg cagaaactgg gcgccacctt tgaacgccgt 480
accgtgacca gtctggaaca ggcatttgat ggtgccgatc tggtggtgaa tgcaaccggt 540
ctgggtgcca aaagcattgc cggcattgat gatcaggccg ccgaaccgat tcgtggtcag 600
accgttctgg tgaaaagccc gtgtaaacgt tgtaccatgg atagtagtga tccggccagc 660
ccggcatata ttattccgcg cccgggcggt gaagttattt gcggtggcac ctatggcgtt 720
ggcgattggg atctgagtgt gaatccggaa accgttcagc gcattctgaa acattgtctg 780
cgcctggatc cgaccattag tagcgatggt accattgaag gtattgaagt tctgcgccat 840
aatgtgggtc tgcgtccggc ccgtcgtggc ggtcctagag ttgaagcaga acgcattgtt 900
ctgccgctgg atcgtaccaa aagcccgctg agcctgggcc gtggcagcgc aagagccgct 960
aaagaaaaag aagtgaccct ggtgcatgcc tatggcttta gtagcgcagg ttatcagcag 1020
agttggggcg ccgccgaaga tgtggcacag ctggttgatg aagcctttca gcgctatcat 1080
ggtgcagccc gtgaaagcaa actg 1104
Claims (6)
1. A method for synthesizing chiral 1, 3-dihydroxy-1-substituted aryl acetone by biological catalysis is characterized in that pyruvic acid decarboxylase is used for catalyzing hydroxy pyruvic acid to react with benzaldehyde with different substituents to obtain the chiral 1, 3-dihydroxy-1-substituted aryl acetone, and the method comprises the following steps:
a) carrying out amplification culture on pyruvate decarboxylase genetically engineered bacteria in an LB culture medium, carrying out induced expression to generate target protein, and then centrifuging and collecting bacteria;
b) re-suspending the collected thallus with buffering liquid, adding certain amount of hydroxyl pyruvic acid and benzaldehyde with different substituent groups, and reacting under proper condition for certain time;
c) after reacting for a certain time, centrifuging the reaction system to remove protein, extracting the supernatant for multiple times by using an organic solvent, combining organic phases, drying by using anhydrous sodium sulfate, filtering, and purifying by using a silica gel column to obtain a target product.
2. The method of claim 1, step a), wherein said pyruvate decarboxylase is a gene sequence obtained by gene mining in the NCBI database, and the nucleic acid sequence of which is SEQ ID NO: 3, the amino acid sequence of pyruvate decarboxylase is SEQ ID NO: 1, carrying out gene synthesis to obtain a corresponding gene, constructing a plasmid vector, and transferring the plasmid into engineering bacteria to obtain the pyruvate decarboxylase gene engineering bacteria.
3. The method according to claim 1, wherein the benzaldehyde of different substituents is selected from the group consisting of benzaldehyde, 4-fluorobenzaldehyde, 4-methylthiobenzaldehyde, 4-bromobenzaldehyde, 4-chlorobenzaldehyde, 4-methylbenzaldehyde, 3-chlorobenzaldehyde, 3-fluorobenzaldehyde and 3-methylbenzaldehyde.
4. The method according to claim 1, step b), characterized in that said hydroxypyruvate is added directly or by converting D-serine by adding a D-amino acid oxidase.
5. Step b) according to claim 4, wherein said D-amino acid oxidase converts D-serine to hydroxypyruvate, which is reacted as a substrate without isolation.
6. Step b) according to claim 4, wherein the D-amino acid oxidase has the nucleic acid sequence EQ ID NO: 4, the amino acid sequence of which is SEQ ID NO: 2.
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