CN111826358A - 12-hydroxycholate dehydrogenase and use thereof - Google Patents
12-hydroxycholate dehydrogenase and use thereof Download PDFInfo
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Abstract
The invention discloses a novel 12-hydroxycholate dehydrogenase derived from Eggerthella lenta and a coding gene thereof, and the enzyme is used as a biocatalyst to catalyze 12-hydroxy oxidation of cholic acid and derivatives thereof to carbonyl substrates. The gene for coding the enzyme has low homology with the currently known 12-hydroxystearic acid and derivatives thereof, and has high enzyme activity on the 12-hydroxycholate and the derivatives thereof, and in the process of culturing and expressing escherichia coli of the gene, antibiotics do not need to be added, and the high-efficiency expression can be realized without using the induction of the traditional isopropyl-beta-D-thiogalactopyranoside (IPTG). In the transformation process, after the thalli are concentrated, the concentration of a substrate can reach 200g/L, the transformation rate is more than 95%, no organic solvent is used in the reaction process, and the transformation method is green and environment-friendly.
Description
Technical Field
The present invention relates to 12-hydroxycholate dehydrogenase from Eggerthella lenta, sequences encoding this enzyme, methods of obtaining this enzyme, and methods of oxidizing the 12-hydroxyl group in cholic acid and derivatives thereof.
Background
Ursodeoxycholic acid can be used for treating fatty liver complicated with hyperlipidemia (combined with ursodeoxycholic acid has effects of treating fatty liver complicated with hyperlipidemia, Weihong, 2017, 30,133), and ursodeoxycholic acid can be used for treating primary biliary cirrhosis hepatitis. The current methods for synthesizing ursodeoxycholic acid are mostly obtained by chemical oxidation-reduction methods using cholic acid as a substrate, and only recently, The development of biocatalysis has led to The wide application of enzyme catalysis in ursodeoxycholic acid (The enzyme catalysis and chemical synthesis of ursodeoxycholic and chemical consumption from cholic acid, Sutherland, J.D. Macdonald, I.A. Forrest, T.P. Prep.biochem.,1982,12, 307; (321)).
In the synthesis of ursodeoxycholic acid (formula 1), which is currently involved in biological methods, the main research point is how to oxidize 12 α -OH and 7 α -OH regioselectively using cholic acid as a substrate (One-step synthesis 12-ketoursodeoxycholic acid from hydrolytic acid using a multienzyme system, Liu, L.Braun, M.Gebhardt, G.Wester-Botz, D.Gross, R.Schmid, R.D.Appl.Microbiol.Biotechnol.2013, 97, 633. 639; Multi-enzymic One-pot reaction of hydrolytic acid, 12-ketodeoxychol acid-cells, B.B.68, B.Biotechnol.20177, Biotechnol.D.Biotechnol.2013, C.20176, B.68, B.12. Biotechnol.12. Biotechnol.2013, C.20111, C.D.12. Biotechnol.Biotechnol.D.Biotechnol.Biotechnol.2013, Chalcohol.2013, C.68, C.12. Biotechnol.D.D.. In the current industrial production, the ursodeoxycholic acid is synthesized by using cheap and easily-obtained cholic acid as a substrate. Ursodeoxycholic acid is prepared by the process of reducing a 12-carbonyl group with Huang Minlon by regioselective oxidation of the hydroxyl groups at the 7, 12 positions followed by reduction of the carbonyl group at the 7-position to a beta hydroxyl group.
At present, when cholic acid is used as a substrate for oxidation by a chemical method, 3-carbonyl byproducts are often generated due to the lack of regioselectivity of an oxidizing agent. Enzymatic implementation of 12-oxidation of cholic acid enables regioselective specific oxidation of 12-hydroxy group, e.g. 12-hydroxysteroid dehydrogenase from Clostridium spThe oxidation of the hydroxyl group, the co-enzyme specificity of this enzyme is NADPH (12 α -Hydroxysteroid dehydrogenase from Clostridium group P, strain C48-50 Production, purification and charaterization, Manfred Braun, Heinrich Lunsdorf, Andrea F. Buckmann, Eur. J. biochem,1991,196, 439-450). Up to now, no natural NAD (H) -dependent 12-hydroxy steroid dehydrogenase sequence has been reported, because the cofactor NAD (P) is added in the reaction process+Increasing the reaction rate and thus the substrate concentration, but NADP+The price of (A) is NAD+About five times, therefore, the discovery and the use of NAD (H) -dependent 12-hydroxysteroid dehydrogenase can obviously reduce the cost of the cofactor in the reaction, and the understanding of people on the environmental protection problem is improved in consideration of the close attention of China to the environmental problem at present, so that the establishment of a green catalytic process with high substrate concentration and high conversion rate is very necessary.
Disclosure of Invention
In order to solve the problems, the invention utilizes the combined action of 12-hydroxycholate dehydrogenase and flavin reductase to realize green oxidation of 12-hydroxyl substrate of cholic acid and derivatives thereof, the byproduct is only water, and the excellent 12-hydroxyl cholic acid dehydrogenase is selected in the process of culturing thalli without using antibiotics for culture and induction by isopropyl-beta-D-thiogalactopyranoside (IPTG), so that the preparation of high-efficiency and low-cost biocatalyst and the high-concentration transformation of the substrate can be realized. The invention comprises a novel method and a post-treatment process for synthesizing 12-carbonyl cholic acid and derivatives thereof by using whole cells, a bacteria-breaking liquid, a crude enzyme, a pure enzyme and an immobilized enzyme.
The optimized 12-hydroxycholate dehydrogenase derived from Eggerthella lenta has a nucleotide sequence shown as SEQ ID No.1 and an amino acid sequence shown as SEQ ID No. 2.
The present invention provides a method for preparing 12-hydroxycholate dehydrogenase.
The preparation method comprises the following steps: (1) synthesizing a new 12-hydroxycholate dehydrogenase gene and constructing the gene on a pET21a expression vector to obtain a recombinant plasmid with a target enzyme gene. (2) The recombinant plasmid is transferred into host bacterial cells (preferably Escherichia coli BL21(DE3)) to obtain corresponding engineering strains. (3) Construction of 12-Hydroxycholic acid dehydrogenase Gene and flavin reductase Gene onto the Dual expression vector pRSFDuet-1 (4) the engineered strain was inoculated into an industrial medium and cultured at 32 ℃ for 20 hours. (5) And (4) centrifugally collecting thalli, and breaking the thalli to obtain a supernatant.
The invention also provides a method for converting substrate cholic acid and derivatives thereof by using the 12-hydroxycholate dehydrogenase recombinant bacteria as a biocatalyst. Specifically, cholic acid or derivatives thereof are directly added into the fermentation liquor until the final concentration is 10-60 g/L, and the auxiliary substrate riboflavin is 0.1-5g/L (flavin reductase) or the auxiliary substrate pyruvic acid is 5-50g/L (lactate dehydrogenase), and the reaction can be completed at 30-35 ℃ after conversion for 8-12 h.
The invention has the beneficial effects that: the invention utilizes a gene mining method to obtain a new 12-hydroxycholate dehydrogenase sequence in a genome database, and utilizes escherichia coli as a host after gene synthesis to successfully and efficiently express the 12-hydroxycholate dehydrogenase. The novel 12-hydroxycholate dehydrogenase has a broad substrate spectrum and has high activity on cholic acid and derivatives thereof. The recombinant 12-hydroxycholate dehydrogenase bacteria is used as a biocatalyst, cholic acid or derivatives thereof are used as a substrate, a target product 12-carbonyl cholic acid or 12-carbonyl cholic acid derivatives with high yield can be obtained with high substrate feeding concentration, the yield is not lower than 95%, the conversion rate is higher than 98%, the conversion time is short, the dosage of the biocatalyst is small, the preparation method is simple and convenient, the conditions are mild, and the method is environment-friendly.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention.
Example 1: synthesis of 12-hydroxy cholic acid dehydrogenase gene and construction of gene engineering bacteria
1.112 Synthesis of Hydroxycholic acid dehydrogenase Gene
A possible 12-hydroxycholate dehydrogenase sequence derived from Eggerthella lenta is excavated by a gene mining method, the gene is synthesized by codon optimization according to a protein sequence, and the gene is constructed into a pET21a expression vector, wherein the insertion sites of the gene are NdeI and XhoI.
1.2 transformation of recombinant plasmids
Preparing competent Escherichia coli cells by calcium chloride method.
(1) mu.L of the recombinant plasmid was placed in 50. mu.L of E.coli BL21(DE3) competent cells and ice-cooled for 30 min.
(2) And (3) carrying out heat shock on the mixture in a water bath at 42 ℃ for 45s, and quickly placing the mixture on ice for 1-2 min.
(3) Adding 600 mu L of fresh LB liquid culture medium, and carrying out shake culture at 37 ℃ for 45-60 min.
(4) And (3) coating 200 mu L of bacterial liquid on the surface of LB solid medium containing ampicillin, and culturing at 37 ℃ for 12-16 h until single colonies appear.
Example 2: construction of Co-expressing bacteria
The pRSFDuet-flavin reductase vector which is available in a laboratory is subjected to double enzyme digestion by NdeI/XhoI and then is recovered, pET-12-steroid dehydrogenase is subjected to double enzyme digestion by NdeI/XhoI and then is recovered to obtain a gene fragment, the gene fragment and the linear vector are connected by T4 DNA ligase and then are transformed into BL21(DE3) competence, and a single clone is selected for verification and activity detection, so that the pRSFDuet-flavin reductase-12-hydroxysteroid dehydrogenase co-expression strain is obtained.
The pRSFDuet-lactate dehydrogenase carrier which is available in a laboratory is subjected to double enzyme digestion by NdeI/XhoI and then is recovered, pET-12-steroid dehydrogenase is subjected to double enzyme digestion by NdeI/XhoI and then is recovered to obtain a gene fragment, the gene fragment and the linear carrier are connected by T4 DNA ligase and then are transformed into BL21(DE3) competence, and a single clone is selected for verification and activity detection, so that the pRSFDuet-lactate dehydrogenase-12-hydroxysteroid dehydrogenase co-expression strain is obtained.
Example 3: induced expression of single expression bacterium and co-expression bacterium
Preparing 50mL of seed liquid, wherein the culture medium is LB liquid culture medium (peptone 10g/L, yeast powder 5g/L, NaCl 10g/L), picking single colony of the genetically engineered bacteria by using an inoculating loop, inoculating into the culture medium, and culturing at 37 ℃ and 200rpm overnight. The seed solution cultured overnight was inoculated to a fermentation medium (industrial medium) at an inoculum size of 1%, and cultured at 32 ℃ and 200rpm for 20 hours. And (3) taking 1mL of fermentation liquor, and performing ultrasonic bacteria breaking to detect the activities of 12-hydroxycholate dehydrogenase and flavin reductase. 12-Hydroxycholic acid dehydrogenase enzyme Activity definition: the enzyme amount required for generating 1. mu. mol NADH per minute is 1 enzyme activity unit (U); definition of enzymatic Activity of flavin: the amount of enzyme required to consume 1. mu. mol NADH per minute was 1 enzyme activity unit (U). The enzyme-labeling instrument detects that the enzyme activity of 12-hydroxycholate dehydrogenase of the fermentation liquor reaches 23.7U/ml, and the enzyme activity of flavin reductase reaches 26.6U/ml.
Example 4: method for converting cholic acid by 12-hydroxycholate dehydrogenase recombinant bacterium fermentation broth
The 12-hydroxycholate dehydrogenase is induced and expressed according to the method of the embodiment 3, 1L of fermentation liquor is obtained, 0.1g/L of riboflavin is added, a substrate is dissolved in an aqueous solution with the pH of 9.0, the aqueous solution is dripped into the fermentation liquor, the final concentration is 40g/L, the reaction pH is controlled to be about 8.0, and the reaction process is detected by thin layer chromatography. Detecting after 14 hours, completely reacting the substrate, adjusting the pH value of the reaction to 9.0, centrifuging to remove thalli, readjusting the pH value of the reaction to 6.0 to obtain a large amount of 12-carbonyl cholic acid precipitate, and filtering to obtain 38.6g of crude product.
Example 5: method for converting methyl cholate by 12-hydroxy cholic acid dehydrogenase recombinant bacterium fermentation broth
The 12-hydroxycholate dehydrogenase and the flavin reductase were expressed by induction according to the method of example 3 to obtain 1L fermentation broth, 0.3g/L riboflavin was added, the substrate was dissolved in methanol solution and added dropwise to the fermentation broth to a final concentration of 20g/L, the reaction pH was controlled at about 8.0, and the progress of the reaction was checked by thin layer chromatography. After 12 hours, the substrate reaction was complete, and the solvent was removed under reduced pressure after ethyl acetate extraction to give 18.4g of crude product.
Example 6: method for converting cholic acid ethyl ester by 12-hydroxyl cholic acid dehydrogenase recombinant bacterium fermentation broth
The 12-hydroxycholate dehydrogenase and the flavin reductase were expressed by induction according to the method of example 3 to obtain 1L fermentation broth, 0.5g/L riboflavin was added, the substrate was dissolved in ethanol solution and added dropwise to the fermentation broth to a final concentration of 60g/L, the reaction pH was controlled at about 8.0, and the progress of the reaction was checked by thin layer chromatography. After 6 hours, the substrate reaction was complete, and the solvent was removed under reduced pressure after ethyl acetate extraction to give 58.3g of crude product.
Example 7: method for converting 12-hydroxycholate dehydrogenase recombinant bacterium fermentation broth into taurocholic acid
The 12-hydroxycholate dehydrogenase and the flavin reductase were expressed by induction according to the method of example 3 to obtain 1L fermentation broth, 0.2g/L riboflavin was added, the substrate was dissolved in an alkaline aqueous solution, the final concentration was 40g/L riboflavin was added dropwise to the fermentation broth, the reaction pH was controlled at about 8.0, and the progress of the reaction was checked by thin layer chromatography. And detecting after 16 hours, completely reacting the substrate, adjusting the pH value of the reaction to 9.0, centrifuging to remove thalli, readjusting the pH value of the reaction to 6.0, and filtering to obtain 38.3g of crude product.
Example 8: method for converting glycocholic acid by using 12-hydroxycholate dehydrogenase recombinant bacterium fermentation broth
The 12-hydroxycholate dehydrogenase and the flavin reductase were expressed by induction according to the method of example 3 to obtain 1L fermentation broth, 0.4g/L riboflavin was added, the substrate was dissolved in an alkaline aqueous solution, the final concentration was 60g/L in the fermentation broth by dropwise addition, the reaction pH was controlled at about 8.0, and the progress of the reaction was checked by thin layer chromatography. After 10 hours, detection is carried out, the substrate completely reacts, the pH value of the reaction is adjusted to 9.0, thalli are removed by centrifugation, the pH value of the reaction is adjusted to 6.0 again, and crude products of 56.3g are obtained by filtration.
Example 9: method for converting 7-Keto cholic acid by 12-hydroxycholate dehydrogenase recombinant strain fermentation broth
The 12-hydroxycholate dehydrogenase and the flavin reductase were expressed by induction according to the method of example 3 to obtain 1L fermentation broth, 0.05g/L riboflavin was added, the substrate was dissolved in an alkaline aqueous solution, the final concentration was 50g/L riboflavin was added dropwise to the fermentation broth, the reaction pH was controlled at about 8.0, and the progress of the reaction was checked by thin layer chromatography. And detecting after 22 hours, completely reacting the substrate, adjusting the pH value of the reaction to 9.0, centrifuging to remove thalli, readjusting the pH value of the reaction to 6.0, and filtering to obtain 48.3g of crude product.
Example 10: method for converting cholic acid by 12-hydroxycholate dehydrogenase recombinant bacterium fermentation broth
Inducing and expressing 12-hydroxycholate dehydrogenase and flavin reductase according to the method of example 3 to obtain 3L of fermentation liquid, concentrating the volume of the fermentation liquid to 1L, adding 0.5g/L riboflavin, dissolving the substrate in a pH9.0 aqueous solution, dropwise adding the substrate into the fermentation liquid to obtain the final concentration of 60g/L, controlling the reaction pH to be about 8.0, and detecting the reaction process by thin layer chromatography. After 20 hours, detection shows that the substrate completely reacts, the pH value of the reaction is adjusted to 9.0, the thalli are removed by centrifugation, the pH value of the reaction is adjusted to 6.0 again, and crude product 56.9g is obtained by filtration.
Example 11: method for converting methyl cholate by 12-hydroxy cholic acid dehydrogenase recombinant bacterium fermentation broth
Performing inducible expression on 12-hydroxycholate dehydrogenase and flavin reductase according to the method of example 3 to obtain 3L of fermentation broth, concentrating the volume of the fermentation broth to 1L, adding 1g/L of riboflavin, dissolving the substrate in methanol solution, and dropwise adding NAD (nicotinamide adenine dinucleotide) to the fermentation broth with the final concentration of 100g/L+The final concentration is 0.01g/L, the reaction pH is controlled to be about 8.0, and the reaction process is detected by thin layer chromatography. After 20 hours, the substrate reaction was complete, and after ethyl acetate extraction and drying, ethyl acetate was removed under reduced pressure to obtain 96.9g of crude product.
Example 12: method for converting cholic acid ethyl ester by 12-hydroxyl cholic acid dehydrogenase recombinant bacterium fermentation broth
Performing inducible expression on 12-hydroxycholate dehydrogenase and flavin reductase according to the method in example 3 to obtain 3L of fermentation liquid, concentrating the volume of the fermentation liquid to 1L, adding 1g/L of riboflavin, dissolving the substrate in ethanol solution, and dropwise adding NAD (nicotinamide adenine dinucleotide) with the final concentration of 150g/L into the fermentation liquid+The final concentration is 0.01g/L, the reaction pH is controlled to be about 8.0, and the reaction process is detected by thin layer chromatography. After 20 hours, the substrate reaction was complete, and after ethyl acetate extraction and drying, ethyl acetate was removed under reduced pressure to obtain 142.9g of crude product.
Example 13: method for converting 12-hydroxycholate dehydrogenase recombinant bacterium fermentation broth into taurocholic acid
The 12-hydroxycholate dehydrogenase and the flavin reductase were expressed by induction according to the method of example 3 to obtain 3L of fermentation broth, the volume of the fermentation broth was concentrated to 1L, and 0.5g/L riboflavin, NAD, was added+Dissolving the substrate in water solution with pH of 9.0 at final concentration of 0.1g/L, dropwise adding into fermentation liquor at final concentration of 200g/L, controlling reaction pH at about 8.0, and detecting reaction process by thin layer chromatography. After 20 hours, detection shows that the substrate completely reacts, the pH value of the reaction is adjusted to 9.0, the thalli are removed by centrifugation, the pH value of the reaction is adjusted to 6.0 again, and crude products of 196.9g are obtained by filtration.
Example 14: method for converting glycocholic acid by using 12-hydroxycholate dehydrogenase recombinant bacterium fermentation broth
According toExample 3 the method of inducible expression of 12-hydroxy cholic acid dehydrogenase and flavin reductase to obtain 3L fermentation broth, concentrating the volume of fermentation broth to 1L, adding 0.5g/L riboflavin, NAD+Dissolving the substrate in water solution with pH of 9.0 at final concentration of 0.1g/L, dropwise adding into fermentation liquor at final concentration of 150g/L, controlling reaction pH at about 8.0, and detecting reaction process by thin layer chromatography. After 20 hours, detection shows that the substrate completely reacts, the pH value of the reaction is adjusted to 9.0, the thalli are removed by centrifugation, the pH value of the reaction is adjusted to 6.0 again, and crude products of 146.9g are obtained by filtration.
Example 15: method for converting 7-Keto cholic acid by 12-hydroxycholate dehydrogenase recombinant strain fermentation broth
The 12-hydroxycholate dehydrogenase and the flavin reductase were expressed by induction according to the method of example 3 to obtain 3L of fermentation broth, the volume of the fermentation broth was concentrated to 1L, and 1.0g/L of riboflavin, NAD, was added+Dissolving the substrate in water solution with pH of 9.0 at final concentration of 0.05g/L, dropwise adding into fermentation liquor at final concentration of 150g/L, controlling reaction pH at about 8.0, and detecting reaction process by thin layer chromatography. Detecting after 20 hours, completely reacting the substrate, adjusting the pH value of the reaction to 9.0, centrifuging to remove thalli, readjusting the pH value of the reaction to 6.0, and filtering to obtain 139.9g of crude product.
Example 16: method for converting cholic acid by using 12-hydroxycholate dehydrogenase bacteria breaking liquid
Inducing and expressing 12-hydroxycholate dehydrogenase and flavin reductase according to the method of example 3 to obtain 3L of fermentation liquid, centrifuging the fermentation liquid, collecting thallus, breaking the thallus in a high-pressure homogenizer, concentrating to 1L, adding 0.5g/L of riboflavin, NAD (nicotinamide adenine dinucleotide), and adding riboflavin and NAD (nicotinamide adenine dinucleotide) in+Dissolving a substrate in a water solution with the pH of 9.0 to obtain a final concentration of 0.05g/L, dropwise adding the substrate into a fermentation liquid with the final concentration of 150g/L, controlling the reaction pH to be about 8.0, detecting the reaction process by thin layer chromatography, detecting after 10 hours until the substrate completely reacts, adjusting the reaction pH to 9.0, centrifuging to remove thalli, readjusting the reaction pH to 6.0, and filtering to obtain a crude product of 142.9 g.
Example 17: method for converting cholic acid by using crude enzyme solution of 12-hydroxycholate dehydrogenase
Inducing and expressing 12-hydroxycholate dehydrogenase and flavin reductase according to the method of example 3 to obtain 1L fermentation liquid, centrifuging the fermentation liquid, collecting thallus, and breaking the thallus in a high-pressure homogenizerAdding ammonium sulfate for precipitation to obtain 12-hydroxycholate dehydrogenase and flavin reductase, diluting to 300mL, adding 0.5g/L riboflavin and NAD+Dissolving a substrate in a water solution with the pH of 9.0 to the final concentration of 0.05g/L, dropwise adding the substrate into a fermentation liquid with the final concentration of 100g/L, controlling the reaction pH to be about 8.0, detecting the reaction process by thin layer chromatography, detecting after 8 hours until the substrate completely reacts, adjusting the reaction pH to 9.0, centrifuging to remove thalli, readjusting the reaction pH to 6.0, and filtering to obtain a crude product of 92.3 g.
Example 18: method for converting cholic acid by using crude enzyme solution of 12-hydroxycholate dehydrogenase
The 12-hydroxycholate dehydrogenase and the flavin reductase were expressed by induction according to the method of example 3 to obtain 1L fermentation broth, the fermentation broth was centrifuged to collect the cells, the cells were immobilized with calcium alginate for 12-hydroxycholate dehydrogenase and flavin reductase, 0.5g/L riboflavin was added, phosphate was added to buffer the volume to 200mL, NAD+Dissolving a substrate in a water solution with the pH of 9.0 to the final concentration of 0.04g/L, dropwise adding the substrate into a fermentation liquid with the final concentration of 120g/L, controlling the reaction pH to be about 8.0, detecting the reaction process by thin layer chromatography, detecting after 8 hours until the substrate completely reacts, adjusting the reaction pH to 9.0, centrifuging to remove thalli, readjusting the reaction pH to 6.0, and filtering to obtain 112.7g of a crude product.
Example 19: method for converting cholic acid by 12-hydroxycholate dehydrogenase recombinant bacterium fermentation broth
The 12-hydroxycholate dehydrogenase and lactate dehydrogenase are induced and expressed according to the method of the embodiment 3, 1L of fermentation liquor is obtained, 20g/L of pyruvic acid is added, the substrate is dissolved in aqueous solution with the pH of 9.0, the final concentration is 40g/L by dropping the substrate into the fermentation liquor, the reaction pH is controlled to be about 8.0, and the reaction process is detected by thin layer chromatography. And detecting after 14 hours, completely reacting the substrate, adjusting the pH value of the reaction to 9.0, centrifuging to remove thalli, readjusting the pH value of the reaction to 6.0 to obtain a large amount of 12-carbonyl cholic acid precipitate, and filtering to obtain a crude product 37.6 g.
Example 20: method for converting cholic acid by 12-hydroxycholate dehydrogenase recombinant bacterium fermentation broth
The 12-hydroxycholate dehydrogenase and lactate dehydrogenase are induced and expressed according to the method of the embodiment 3, 1L of fermentation liquor is obtained, 40g/L of pyruvic acid is added, a substrate is dissolved in an aqueous solution with the pH of 9.0, the aqueous solution is dripped into the fermentation liquor, the final concentration is 80g/L, the reaction pH is controlled to be about 8.0, and the reaction process is detected by thin layer chromatography. Detecting after 14 hours, completely reacting the substrate, adjusting the pH value of the reaction to 9.0, centrifuging to remove thalli, readjusting the pH value of the reaction to 6.0 to obtain a large amount of 12-carbonyl cholic acid precipitate, and filtering to obtain a crude product of 77.4 g.
Example 21: method for converting cholic acid by 12-hydroxycholate dehydrogenase recombinant bacterium fermentation broth
The 12-hydroxycholate dehydrogenase and lactate dehydrogenase are induced and expressed according to the method of the embodiment 3, 1L of fermentation liquor is obtained, 10g/L of pyruvic acid is added, the substrate cholic acid is dissolved in aqueous solution with the pH of 9.0, the final concentration of the cholic acid is dropped into the fermentation liquor to be 20g/L, the reaction pH is controlled to be about 8.0, and the reaction process is detected by thin layer chromatography. Detecting after 14 hours, completely reacting the substrate, adjusting the pH value of the reaction to 9.0, centrifuging to remove thalli, readjusting the pH value of the reaction to 6.0 to obtain a large amount of 12-carbonyl cholic acid precipitate, and filtering to obtain 18.6g of crude product.
Sequence listing
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Met Asp Met Gly Leu Lys Asp Lys Val Val Leu Ile Thr Gly Gly Gly
1 5 10 15
Gly Gly Ile Ala Arg Gly Ile Glu Arg Ala Phe Ala Thr Glu Gly Ala
20 25 30
Lys Phe Ile Leu Thr Asp Leu Phe Ser Gly Gly Leu Glu Ala Ala Lys
35 40 45
Glu Glu Leu Glu Arg Asp Phe Gly Ser Glu Val Phe Thr Ile Leu Ala
50 55 60
Asn Gly Ser Val Glu Glu Glu Val Arg Ala Ala Val Glu Ala Gly Ala
65 70 75 80
Glu His Phe Gly Gly Arg Ile Asp Val Leu Ile Asn Asn Ala Gln Ala
85 90 95
Ser Ala Ser Gly Leu Thr Leu Val Gln His Ser Glu Glu Asp Phe Asp
100 105 110
Leu Ala Val Arg Ser Gly Leu Tyr Ala Thr Phe Phe Tyr Met Lys His
115 120 125
Ala Tyr Pro Tyr Leu Lys Glu Thr Ala Gly Ser Val Ile Asn Phe Ala
130 135 140
Ser Gly Ala Gly Ile Gly Gly Asn Pro Gly Gln Ser Ser Tyr Ala Ala
145 150 155 160
Ala Lys Glu Gly Ile Arg Gly Met Ser Arg Val Ala Ala Ser Glu Trp
165 170 175
Gly Pro Asp Asn Ile Asn Val Asn Ile Val Cys Pro Ile Val Met Thr
180 185 190
Lys Ala Leu Glu Glu Trp Arg Glu Arg Glu Pro Glu Met Tyr Glu Lys
195 200 205
Asn Val Lys Ala Ile Pro Leu Gly Arg Phe Gly Asp Ala Glu Lys Asp
210 215 220
Val Gly Arg Val Cys Val Phe Leu Ala Ser Pro Asp Ala Ser Phe Val
225 230 235 240
Thr Gly Asp Thr Ile Met Val Gln Gly Gly Ser Gly Met Lys Pro
245 250 255
Claims (8)
1. 12-hydroxycholate dehydrogenase, the amino acid sequence of which is shown in SEQ ID No. 2.
2. The use of 12-hydroxycholate dehydrogenase as claimed in claim 1 as a biocatalyst for the oxidation of the 12-hydroxyl group in cholic acid and its derivatives, including cholic acid, cholic acid methyl ester, cholic acid ethyl ester, taurocholic acid, glycocholic acid, 7-Keto-cholic acid.
3. Use according to claim 2, wherein the biocatalyst is derived from Eggerthella lenta and has the amino acid sequence shown in SEQ ID No. 2.
4. The use of claim 2, wherein the biocatalyst is a protein having 12-hydroxycholate dehydrogenase activity derived from 12-hydroxycholate dehydrogenase represented by SEQ ID No.2 by substitution, deletion or addition of one or several amino acid residues in its amino acid sequence.
5. The use as claimed in claim 2, wherein 12-hydroxycholate dehydrogenase is expressed heterologously with flavin reductase in E.coli as an engineered bacterium.
6. The use as claimed in claim 2, wherein 12-hydroxycholate dehydrogenase is expressed heterologously with lactate dehydrogenase in E.coli as an engineered bacterium.
7. The use of claim 2, wherein the conversion reaction of cholic acid or its derivatives is directly carried out by using engineering bacteria fermentation broth without adding cofactor NAD+After addition of the cosubstrate, conversion of the cholic acid and cholic acid derivative concentration10-60 g/l, conversion rate>95％。
8. Use according to claim 2, characterized in that the conversion is carried out directly with the fermentation broth, the cofactor NAD being added+The concentration is 0.01-0.5 g/L, after adding co-substrate, the concentration of cholic acid and cholic acid derivative is 20-200 g/L, and the conversion rate>95％。
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| CN115786292A (en) * | 2022-08-25 | 2023-03-14 | 福州大学 | 3 alpha-hydroxy steroid dehydrogenase and application thereof in preparation of dehydroepiandrosterone |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108546691A (en) * | 2018-05-09 | 2018-09-18 | 华东理工大学 | 7 beta-hydroxy sterol dehydrogenase mutants and its application in preparing ursodesoxycholic acid |
| CN109306342A (en) * | 2017-07-26 | 2019-02-05 | 中国科学院天津工业生物技术研究所 | A kind of new 17 beta-hydroxy steroid dehydrogenase types, its gene and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109306342A (en) * | 2017-07-26 | 2019-02-05 | 中国科学院天津工业生物技术研究所 | A kind of new 17 beta-hydroxy steroid dehydrogenase types, its gene and application |
| CN108546691A (en) * | 2018-05-09 | 2018-09-18 | 华东理工大学 | 7 beta-hydroxy sterol dehydrogenase mutants and its application in preparing ursodesoxycholic acid |
Non-Patent Citations (1)
| Title |
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| GENBANK DATABASE: "Accession NO.: WP_114518444.1，SDR family oxidoreductase [Eggerthella lenta]", 《GENBANK DATABASE》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115786292A (en) * | 2022-08-25 | 2023-03-14 | 福州大学 | 3 alpha-hydroxy steroid dehydrogenase and application thereof in preparation of dehydroepiandrosterone |
| CN115786292B (en) * | 2022-08-25 | 2023-09-29 | 福州大学 | 3 beta-hydroxy steroid dehydrogenase and application thereof in preparation of dehydroepiandrosterone |
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