CN114921427B - Artificial enzyme with wide substrate spectrum for converting carboxylic acid into chiral beta-hydroxy carboxylic acid - Google Patents
Artificial enzyme with wide substrate spectrum for converting carboxylic acid into chiral beta-hydroxy carboxylic acid Download PDFInfo
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Abstract
The invention discloses an artificial enzyme for converting carboxylic acid into beta-hydroxycarboxylic acid, and belongs to the field of protein engineering. The carboxylic acid beta-hydroxylase P450 of the invention BSβ The mutant is prepared from wild type carboxylic acid hydroxylase P450 BSβ Is mutated at positions 78, 85 and 290. Carboxylic acid beta-hydroxylase P450 BSβ L78I/Q85H/G290I is beta-hydroxylase P450 in fatty acids BSβ The artificial enzyme with higher yield and wider substrate range is obtained after the improvement on the basis of the L78A/Q85A/V170I/G290I. The substrate range catalyzed by the artificial enzyme constructed by the invention is expanded into natural straight-chain saturated fatty acids, natural unsaturated fatty acids and non-natural carboxylic acids with the chain length of C6-C20. The present invention allows one-step conversion of these substrates to the corresponding (R) - β -hydroxycarboxylic acids.
Description
Technical Field
The invention relates to an artificial enzyme for converting carboxylic acid into beta-hydroxycarboxylic acid by using hydrogen peroxide as an oxidant, and belongs to the field of protein engineering.
Background
Beta-hydroxycarboxylic acids are an important class of compounds. Beta-hydroxycarboxylic acids (C10-C14) containing 10-14 carbon atoms have natural herbicidal and antifungal properties. When combined with amino acids or carbohydrates, they can create more natural products of structural complexity and biological significance, such as antibiotics, organic compounds with insecticidal and immunomodulatory activity. Beta-hydroxycarboxylic acids are also useful as synthetic precursors for the synthesis of diols, beta-amino acids, beta-lactones, beta-lactams, bicycloheptanones and polyhydroxyalkanoates, and have many more applications in natural product synthesis, drug discovery and biomaterials. Thus, the preparation of β -hydroxycarboxylic acids has received a great deal of attention.
Common chemical methods for synthesizing β -hydroxycarboxylic acids include asymmetric aldol reactions of linear aldehydes, reformatsky reactions, and asymmetric hydrogenation reactions of β -ketocarboxylic acids, typically requiring transition metals and complex catalytic ligands or stoichiometric amounts of chiral auxiliary reagents. Ketoreductase enzymes can also asymmetrically reduce β -ketocarboxylic acids, providing an enzymatic route to chiral β -hydroxycarboxylic acids.
In earlier studies, by engineering P450 BSβ Variants (P450) BSβ -L78A/Q85A/V170I/G290I) beta-C-H oxidative functionalization of bioavailable fatty acids. Although this is a direct, stepwise economical and eco-friendly reaction with excellent regioselectivity and enantioselectivity, acceptable substrates are limited to fatty acids with chain lengths exceeding 10 carbon atoms, hampering their wide application in β -carboxylic acid synthesis.
Disclosure of Invention
The invention aims to solve the technical problems of overcoming the prior art defect and providing an artificial enzyme for converting carboxylic acid into beta-hydroxycarboxylic acid by using hydrogen peroxide as an oxidant, and the prepared carboxylic acid beta-hydroxylase P450 BSβ Mutant catalysis to produce beta-hydroxy carboxylic acid and expand substrate range to C 6 -C 20 Natural straight-chain saturated fatty acids of chain length, oleic acid, linoleic acid and non-natural carboxylic acids with different functional groups. The invention has the advantages of high reaction activity, high regioselectivity, enantioselectivity, wide substrate range, simple system, high synthesis efficiency, low cost and the like.
The invention is implemented by P450 BSβ Directed evolution of hydroxylase effects a highly regio-and enantioselective oxidation of the unactivated β -C-H bond of carboxylic acids. Obtained P450 BSβ The hydroxylase mutant can directly use cheap hydrogen peroxide as an oxidant, and takes natural linear saturated fatty acid, natural unsaturated fatty acid (oleic acid or linoleic acid) with the chain length of C6-C20 as a substrate to generate R-type chiral beta-hydroxycarboxylic acid by taking non-natural carboxylic acid containing different functional groups such as alkene, alkyne, bromine, amine, nitrile, ester, dihydroxyl, cyclopentyl, furan, pyrrole, benzyloxy (ortho, meta, para), fluorophenyl, chlorophenyl, benzoyl methoxy and the like as a substrate.
In order to solve the technical problems, the invention provides a carboxylic acid beta-hydroxylase P450 BSβ Mutants containing the sequence shown in SEQ ID NO. 1The amino acid sequences shown.
The invention also provides a method for encoding the carboxylic acid beta-hydroxylase P450 BSβ The nucleotide sequence of the mutant is shown as SEQ ID NO. 2.
The present invention also provides a beta-hydroxylase P450 comprising the carboxylic acid as described above BSβ A vector of the nucleotide sequence of the mutant.
The invention also provides a method for expressing the carboxylic acid beta-hydroxylase P450 BSβ Genetically engineered bacteria of the mutants.
The invention also provides a method for obtaining carboxylic acid beta-hydroxylase P450 BSβ Mutant methods, wild-type carboxylic acid hydroxylase P450 BSβ The wild-type P450 is obtained by mutating leucine at position 78 to isoleucine, glutamine at position 85 to histidine, and glycine at position 290 to isoleucine BSβ The sequence is shown as SEQ ID NO. 3.
The invention also provides a carboxylic acid beta-hydroxylase P450 BSβ A method for preparing a mutant comprising the steps of:
construction and expression of wild-type carboxylic acid hydroxylase P450 BSβ And transforming the plasmid into E.coli;
through three rounds of site-directed saturation mutation, a saturation mutation library is constructed, and P450 which expresses a C-H group at beta position of carboxylic acid with high regioselectivity and enantioselectivity is screened BSβ Mutant plasmid of the mutant;
carboxylic acid beta-hydroxylase P450 BSβ Expression and purification of the mutant.
Carboxylic acid beta-hydroxylase P450 BSβ The method for obtaining and preparing the mutant comprises the following steps:
(1) Construction of expression wild-type carboxylic acid beta-hydroxylase P450 BSβ Is a plasmid of (2): the nucleotide sequence of SEQ ID NO. 4 is recombined on an expression vector through enzyme digestion, and the plasmid is transformed into escherichia coli;
(2) Through three rounds of site-directed saturation mutation, a saturation mutation library is constructed, and beta-C-H radical of carboxylic acid which can be highly regioselectively and enantioselectively oxidized (hydroxylated) is screened and expressedP450 of a bolus BSβ Mutant plasmid of the mutant;
(3) Carboxylic acid beta-hydroxylase P450 BSβ Expression of the mutant: culturing the strain obtained in the step (2) in an LB liquid medium (Kan/Cam) overnight, adding a TB liquid medium (Kan/Cam) for expansion culture, adding heme precursor delta-aminolevulinic acid and IPTG for induction expression in a logarithmic growth phase, and centrifuging to collect the strain;
(4) Containing carboxylic acid beta-hydroxylase P450 BSβ Preparation of cell lysates of mutants: re-suspending the strain obtained in the step (3) by using a buffer solution, performing ultrasonic disruption and cell lysis, and centrifuging to obtain a supernatant;
(5) Carboxylic acid beta-hydroxylase P450 BSβ Purification of the mutants: adding the supernatant obtained in the step (4) into a nickel ion affinity column which is pretreated in advance to enable target protein with His tag to be combined with nickel ions, removing impurity protein by using imidazole with lower concentration, eluting the target protein by using buffer solution of high concentration imidazole, dialyzing the eluted target protein to remove the imidazole, and storing at-80 ℃ after measuring the concentration of the target protein.
The invention also provides a preparation method of beta-hydroxy carboxylic acid, which uses natural straight-chain saturated fatty acid, natural unsaturated fatty acid and non-natural carboxylic acid with chain length of C6-C20 as substrates, hydrogen peroxide as oxidant is added, then reaction buffer solution is added, and the carboxylic acid beta-hydroxylase P450 is used BSβ The mutant is used as a catalyst for catalytic reaction.
Further, the natural unsaturated fatty acid is oleic acid or linoleic acid; the non-natural carboxylic acids contain one or more functional groups of alkene, alkyne, bromine, amine, nitrile, ester, dihydroxy, cyclopentyl, furan, pyrrole, benzyloxy (ortho, meta, para), fluorophenyl, chlorophenyl, benzoylmethoxy.
Further, the beta-hydroxycarboxylic acid is an R-type beta-hydroxycarboxylic acid.
Further, the catalytic reaction uses 5% ethanol as a cosolvent.
Further, the reaction is carried out at room temperature, the reaction system is an aqueous phase system, and the reaction conditions are mild.
The invention also provides the carboxylic acid beta-hydroxylase P450 BSβ The application of the mutant in preparing and synthesizing beta-hydroxy carboxylic acid.
Beta-hydroxylase P450 BSβ The method for preparing beta-hydroxycarboxylic acid by mutant catalysis comprises the following specific steps:
the carboxylic acid beta-hydroxylation reaction system comprises bufferA (0.1M KPi,0.3M KCl,pH 7.0), and purified beta-hydroxylase P450 BSβ Mutant (5. Mu.M/10. Mu.M), substrate (5 mM), triton X-100 (0.625%), 5% ethanol as co-solvent and hydrogen peroxide (7.5 mM+2.5mM, 2.5min apart) were added a second time. The reaction was reacted at room temperature for 45min in a final volume of 100 mL. After completion of the reaction, the reaction was quenched with HCl, 1mL of the reaction mixture was removed, extracted, and TMSCHN was used 2 Processed and analyzed by GC. The remaining reaction solution was extracted with ethyl acetate, and dried over anhydrous MgSO 4 Drying and filtering. The filtrate was concentrated in vacuo and purified by silica gel column chromatography (n-hexane: ethyl acetate: acetic acid=85:15:1-30:70:1) to give (R) - β -hydroxycarboxylic acid. All experiments were performed in triplicate to calculate the yield.
The carboxylic acid beta-hydroxylase P450 of the invention BSβ The mutant is prepared from wild type carboxylic acid hydroxylase P450 BSβ Is mutated at positions 78, 85 and 290. P450 s BSβ The hydroxylase mutant has high reaction activity, and the substrate such as natural straight-chain saturated fatty acid, natural unsaturated fatty acid (oleic acid or linoleic acid) with the chain length of C6-C20 and the non-natural carboxylic acid containing different functional groups such as alkene, alkyne, bromine, amine, nitrile, ester, dihydroxyl, cyclopentyl, furan, pyrrole, benzyloxy (ortho, meta, para), fluorophenyl, chlorophenyl, benzoyl methoxy and the like is directly converted into the corresponding beta-hydroxycarboxylic acid by directly using cheap hydrogen peroxide as an oxidant. P450 s BSβ Directed evolution of hydroxylase enables oxidation of the unactivated beta C-H bond altitude region and enantioselectivity of carboxylic acids. Carboxylic acid beta-hydroxylase P450 because of high reactivity, high selectivity and low cost BSβ The mutant has good industrial production prospect in the aspect of synthesizing beta-hydroxycarboxylic acid.
Advantageous effects
(1) The artificial enzyme constructed by the invention directly uses cheap hydrogen peroxide as an oxidant, natural straight-chain saturated fatty acid with the chain length of C6-C20, natural unsaturated fatty acid (oleic acid or linoleic acid) and non-natural carboxylic acid containing different functional groups such as alkene, alkyne, bromine, amine, nitrile, ester, dihydroxyl, cyclopentyl, furan, pyrrole, benzyloxy (ortho, meta, para), fluorophenyl, chlorophenyl, benzoylmethoxy and the like as substrates, and synthesizes (R) -beta-hydroxycarboxylic acid in one step, so that the catalytic substrate range is wider.
(2) Most P450 hydroxylases require expensive biological coenzymes NAD (P) H and additionally prepared redox partners to mediate electron transfer for hydroxylation reactions, thus the reaction system is complex and costly. And the carboxylic acid beta-hydroxylase P450 BSβ Mutant only needs H 2 O 2 As a direct redox donor and without the mediation of a redox partner, the carboxylic acid is catalyzed to synthesize the (R) -beta-hydroxycarboxylic acid, and NAD (P) H redox partner and cofactor are not needed, so that the reaction system has the advantages of simplicity and low cost.
(3) The carboxylic acid beta-hydroxylase P450 BSβ The mutant has the characteristics of high reaction activity, high regioselectivity, enantioselectivity, mild reaction conditions and the like.
Detailed Description
The invention is further described below. The following examples are only for more clearly illustrating the technical aspects of the present invention, and are not intended to limit the scope of the present invention.
Unless specifically stated, all of the following compounds and reagents were purchased from Sigma-Aldrich, tokyo Chemical Industry, EMD Millipore, macklin Biochemical, bidepharm Biochemical, new England Biolabs.
Example 1
The preparation method of the carboxylic acid beta-hydroxylase P450BS beta mutant comprises the following specific steps:
(1) Construction of carboxylic acid beta-hydroxylase P450BS beta gene expression vector: the P450BS beta fragment synthesized by the whole gene (the sequence is shown as SEQ ID NO:4 and synthesized by the biological Co., ltd. Of Suzhou Jin Weizhi) is connected to an expression vector pET28a (+) cut by the same enzyme by T4 ligase after the restriction enzymes NcoI and XhoI are subjected to enzyme cutting operation according to the specification; transforming the ligation product into E.coli DE3 competent cells; a successfully transformed monoclonal colony was picked from a solid LB medium plate containing 50. Mu.g/ml kanamycin and 34. Mu.g/ml chloramphenicol, and cultured overnight at 37℃and a shaker rotation of 220rpm in LB liquid medium containing the same concentrations of kanamycin and chloramphenicol. Extracting recombinant plasmid from the bacterial liquid cultured overnight by using a plasmid small extraction kit, and sending the extracted plasmid to sequencing and identification;
(2) Screening of carboxylic acid beta-hydroxylase P450BS beta mutant plasmids: constructing a site-specific saturated mutant plasmid library by using the sequenced plasmid as a PCR template through a Quickchange method, screening the site-specific saturated mutant plasmid library through three rounds of saturated mutant library to obtain mutant plasmids expressing P450BS beta mutant capable of oxidizing (hydroxylating) carboxylic acid beta-site C-H groups with high regioselectivity and enantioselectivity, and carrying out sequencing identification;
(3) Preparation of carboxylic acid beta-hydroxylase P450BS beta mutant: the glycerinum preserved in a refrigerator at the ultralow temperature of minus 80 ℃ is selected and inoculated into 40ml of LB liquid medium (Kan/Cam) for overnight culture at 37 ℃. 5ml of the overnight cultured bacterial liquid is inoculated into 500ml of TB medium (Kan/Cam) and is subjected to expansion culture at 37 ℃ and a shaking table rotating speed of 220 rpm; waiting for OD 600 At a value of about 0.6, 250ul of precursor ALA (delta-aminolevulinic acid) was added at a final concentration of 0.5mM and 500ul of IPTG at a final concentration of 1mM was added to induce expression. Culturing at 25deg.C and shaking table rotation speed of 220rpm for 16 hr. The cells were harvested by centrifugation at 5000rpm for 0min and resuspended in 75ml buffer A (0.1M KPi,0.3M KCl,pH 7.0) containing 10mM imidazole and disrupted by sonication. Centrifugal for 45min at 4 ℃ and 10000rpm to obtain supernatant. Loading the supernatant containing the target protein into a nickel ion affinity column which is arranged in advance, and combining the target protein carrying the His tag with nickel ions. The protein of interest was removed with buffer A containing 35mM imidazole, followed by elution with buffer B (0.1M KPi,0.3M KCl,250mM imidazole, pH 7.0). The purity of the target protein was identified using the P450-CO spectroscopic method. Buffer for eluting target proteinA dialysis, 4 ℃ overnight, remove imidazole. Subpackaging the protein and preserving at-80 ℃.
Example 2P450 BSβ Purification of mutant proteins by large amounts of expression
The glycerinum preserved in a refrigerator at the ultralow temperature of minus 80 ℃ is selected and inoculated into 40ml TB liquid medium (Kan/Cam) for overnight culture at 37 ℃. Inoculating the overnight cultured bacterial liquid into 500ml of TB culture medium (Kan/Cam), and performing expansion culture at 37 ℃ and a shaking table rotation speed of 220 rpm; waiting for OD 600 About 0.6, 250. Mu.l of precursor ALA (. Delta. -aminolevulinic acid) was added at a final concentration of 0.5mM and 500. Mu.l of IPTG at a final concentration of 1mM were added to induce expression, and the mixture was incubated at 25℃and at a shaking table rotation speed of 220rpm for 16 hours. The cells were harvested by centrifugation at 5000rpm for 0min and resuspended in 75ml buffer A (0.1M KPi,0.3M KCl,pH7.0) before disruption by sonication. The supernatant was obtained by centrifugation at 10000rpm at 4℃for 45min. Loading the supernatant containing the target protein into a nickel ion affinity column which is arranged in advance, so that the target protein carrying the His tag is combined with nickel ions. The target protein was eluted with buffer A containing 35mM imidazole and then buffer B (0.1M KPi,0.3M KCl,250mM imidazole, pH 7.0). The eluted target protein was dialyzed against buffer A to remove imidazole, and overnight at 4 ℃. The concentration of the protein was determined by the P450-CO method. Subpackaging the protein and preserving at-80 ℃.
EXAMPLE 3 beta-hydroxylase P450 BSβ Catalytic production of beta-hydroxycarboxylic acid by mutants
The carboxylic acid beta-hydroxylation reaction system comprises bufferA (0.1M KPi,0.3M KCl,pH 7.0), and purified beta-hydroxylase P450 BSβ Mutant (5. Mu.M/10. Mu.M), substrate (5 mM), triton X-100 (0.625%), 5% ethanol as co-solvent and hydrogen peroxide (7.5 mM+2.5mM, 2.5min apart) were added a second time. The reaction was reacted at room temperature for 45min in a final volume of 100 mL. After completion of the reaction, the reaction was quenched with HCl, 1mL of the reaction mixture was removed, extracted, and TMSCHN was used 2 Processed and analyzed by GC. The remaining reaction solution was extracted with ethyl acetate, and dried over anhydrous MgSO 4 Drying and filtering. The filtrate was concentrated in vacuo and purified by silica gel column chromatography (n-hexane: ethyl acetate: acetic acid=85:15:1-3)0:70:1) to give (R) - β -hydroxycarboxylic acids. All experiments were performed in triplicate to calculate the yield.
TABLE 1 beta-hydroxylase P450 BSβ Mutants catalyze different saturated fatty acid substrates
Reaction conditions: beta-hydroxylase P450 BSβ Mutant (5. Mu.M), substrate (5 mM), hydrogen peroxide (7.5 mM+2.5mM, interval 2.5 min), 0.1M KPi (pH 7.0), 0.3M KCl,0.625%TritonX-100,5% ethanol (co-solvent), 100ml system, room temperature, 1h. ee: enantioselectivity. [a] By crude product 1 H-NMR measurement.
TABLE 2 beta-hydroxylase P450 BSβ Mutants catalyze carboxylic acid substrates of different functional groups
Reaction conditions: beta-hydroxylase P450 BSβ Mutant (5. Mu.M), substrate (5 mM), hydrogen peroxide (7.5 mM+2.5mM, interval 2.5 min), 0.1M KPi (pH 7.0), 0.3M KCl,0.625%TritonX-100,5% ethanol (co-solvent), 100ml system, room temperature, 1h. ee: enantioselectivity. [a] By crude product 1 H-NMR measurement.
EXAMPLE 4 beta-hydroxylation P450 BSβ Mutant product identification assay
GC, taking 500. Mu.l of the extracted ethyl acetate extract, adding 300. Mu.l of methanol, mixing well, adding 75. Mu.l of 10% trimethylsilyl diazomethaneThe n-hexane solution was esterified. Samples were taken, and detected and quantified by gas chromatography. Chromatographic column: DB-WAX. The procedure is as follows: maintaining at 40deg.C for 1min and 10deg.C for min -1 Keeping at 250deg.C for 15min for 40min.
GC-MS after the reaction of the substrate with natural linear saturated fatty acids of chain length C6-C20, natural unsaturated fatty acids (oleic acid or linoleic acid) and with various functional groups such as alkene, alkyne, bromine, amine, nitrile, ester, dihydroxy, cyclopentyl, furan, pyrrole, benzyloxy (ortho, meta, para), fluorophenyl, chlorophenyl, benzoylmethoxy, etc., the reaction is terminated with 5N HCl and extracted with an equal volume of ethyl acetate. The samples were taken and the corresponding β -hydroxycarboxylic acid was quantified by gas chromatography-mass spectrometry. Chromatographic column: TG-5MS. The procedure is as follows: maintaining at 60deg.C for 1min and 20deg.C for min -1 The total time was 14min at 320 ℃.
HPLC using a CHIRALPAK IA column (Daicel, 4.6X105 mm,5 μm column) on a high performance liquid chromatograph (Agilent 1260 Infinicity) using hexane and isopropanol (isopropanol: hexane: =1:99-8:92) as mobile phases (flow rate: 1.0mL min) -1 Detection wavelength: 210 nm). For UV detection, the purified β -hydroxycarboxylic acid and its racemic standard prepared by the enzymatic reaction are converted to the corresponding benzyl ester according to the disclosed procedure.
NMR purification of beta-hydroxycarboxylic acid by silica gel column with CDCl 3 Dissolving and transferring into nuclear magnetic tube, and recording on Agilent DD2 400MHz nuclear magnetic resonance spectrometer 1 H-sum 13 C-NMR spectrum.
The artificial enzyme constructed by the invention can utilize natural straight-chain saturated fatty acid with the chain length of C6-C20, natural unsaturated fatty acid (oleic acid or linoleic acid) and non-natural carboxylic acid containing different functional groups such as alkene, alkyne, bromine, amine, nitrile, ester, dihydroxyl, cyclopentyl, furan, pyrrole, benzyloxy (ortho, meta, para), fluorophenyl, chlorophenyl, benzoyl methoxy and the like as substrates, and hydrogen peroxide as an oxidant to synthesize the beta-hydroxycarboxylic acid in one step. The method has the advantages that: 1) The novel carboxylic acid hydroxylase P450 BSβ Mutant catalytic substrate range is greaterThe catalyst is wide in range, and can catalyze not only natural straight-chain saturated fatty acids, but also non-natural carboxylic acids. 2) The novel carboxylic acid hydroxylase P450 BSβ The mutant synthesizes beta-hydroxycarboxylic acid by catalyzing carboxylic acid, NAD (P) H redox chaperonin and auxiliary factors are not needed, and the reaction system is simple and low in cost. 3) The novel carboxylic acid hydroxylase P450 BSβ The mutant has the characteristics of high reaction activity, high regioselectivity, enantioselectivity, mild reaction conditions and the like.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and variations could be made by those skilled in the art without departing from the technical principles of the present invention, and such modifications and variations should also be regarded as being within the scope of the invention.
Sequence listing
<110> university of Yangzhou
<120> an artificial enzyme with broad substrate spectrum for the specific conversion of carboxylic acid to chiral beta-hydroxycarboxylic acid
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 425
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 1
Met Asp Glu Gln Ile Pro His Asp Lys Ser Leu Asp Asn Ser Leu Thr
1 5 10 15
Leu Leu Lys Glu Gly Tyr Leu Phe Ile Lys Asn Arg Thr Glu Arg Tyr
20 25 30
Asn Ser Asp Leu Phe Gln Ala Arg Leu Leu Gly Lys Asn Phe Ile Cys
35 40 45
Met Thr Gly Ala Glu Ala Ala Lys Val Phe Tyr Asp Thr Asp Arg Phe
50 55 60
Gln Arg Gln Asn Ala Leu Pro Lys Arg Val Gln Lys Ser Ile Phe Gly
65 70 75 80
Val Asn Ala Ile His Gly Met Asp Gly Ser Ala His Ile His Arg Lys
85 90 95
Met Leu Phe Leu Ser Leu Met Thr Pro Pro His Gln Lys Arg Leu Ala
100 105 110
Glu Leu Met Thr Glu Glu Trp Lys Ala Ala Val Thr Arg Trp Glu Lys
115 120 125
Ala Asp Glu Val Val Leu Phe Glu Glu Ala Lys Glu Ile Leu Cys Arg
130 135 140
Val Ala Cys Tyr Trp Ala Gly Val Pro Leu Lys Glu Thr Glu Val Lys
145 150 155 160
Glu Arg Ala Asp Asp Phe Ile Asp Met Val Asp Ala Phe Gly Ala Val
165 170 175
Gly Pro Arg His Trp Lys Gly Arg Arg Ala Arg Pro Arg Ala Glu Glu
180 185 190
Trp Ile Glu Val Met Ile Glu Asp Ala Arg Ala Gly Leu Leu Lys Thr
195 200 205
Thr Ser Gly Thr Ala Leu His Glu Met Ala Phe His Thr Gln Glu Asp
210 215 220
Gly Ser Gln Leu Asp Ser Arg Met Ala Ala Ile Glu Leu Ile Asn Val
225 230 235 240
Leu Arg Pro Ile Val Ala Ile Ser Tyr Phe Leu Val Phe Ser Ala Leu
245 250 255
Ala Leu His Glu His Pro Lys Tyr Lys Glu Trp Leu Arg Ser Gly Asn
260 265 270
Ser Arg Glu Arg Glu Met Phe Val Gln Glu Val Arg Arg Tyr Tyr Pro
275 280 285
Phe Ile Pro Phe Leu Gly Ala Leu Val Lys Lys Asp Phe Val Trp Asn
290 295 300
Asn Cys Glu Phe Lys Lys Gly Thr Ser Val Leu Leu Asp Leu Tyr Gly
305 310 315 320
Thr Asn His Asp Pro Arg Leu Trp Asp His Pro Asp Glu Phe Arg Pro
325 330 335
Glu Arg Phe Ala Glu Arg Glu Glu Asn Leu Phe Asp Met Ile Pro Gln
340 345 350
Gly Gly Gly His Ala Glu Lys Gly His Arg Cys Pro Gly Glu Gly Ile
355 360 365
Thr Ile Glu Val Met Lys Ala Ser Leu Asp Phe Leu Val His Gln Ile
370 375 380
Glu Tyr Asp Val Pro Glu Gln Ser Leu His Tyr Ser Leu Ala Arg Met
385 390 395 400
Pro Ser Leu Pro Glu Ser Gly Phe Val Met Ser Gly Ile Arg Arg Lys
405 410 415
Ser Leu Glu His His His His His His
420 425
<210> 2
<211> 1278
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
atggatgagc agattccaca tgacaaaagt ctcgataaca gtctgacact gctgaaggaa 60
gggtatttat ttattaaaaa cagaacagag cgctacaatt cagatctgtt tcaggcccgt 120
ttgttgggaa aaaactttat ttgcatgact ggcgctgagg cggcgaaggt gttttatgat 180
acggatcgat tccagcggca gaacgctttg cctaagcggg tgcagaaatc gatttttggt 240
gttaatgcga ttcacggaat ggatggcagc gcgcatatcc atcggaagat gctttttctg 300
tcattgatga caccgccgca tcaaaaacgt ttggctgagt tgatgacaga ggagtggaaa 360
gcagcagtca caagatggga gaaggcagat gaggttgtgt tatttgaaga agcaaaagaa 420
atcctgtgcc gggtagcgtg ctattgggca ggtgttccgt tgaaggaaac ggaagtcaaa 480
gagagagcgg atgacttcat tgacatggtc gacgcgttcg gtgctgtggg accgcggcat 540
tggaaaggaa gaagagcaag gccgcgtgcg gaagagtgga ttgaagtcat gattgaagat 600
gctcgtgccg gcttgctgaa aacgacttcc ggaacagcgc tgcatgaaat ggcttttcac 660
acacaagaag atggaagcca gctggattcc cgcatggcag ccattgagct gattaatgta 720
ctgcggccta ttgtcgccat ttcttacttt ctggtgtttt cagctttggc gcttcatgag 780
catccgaagt ataaggaatg gctgcggtct ggaaacagcc gggaaagaga aatgtttgtg 840
caggaggtcc gcagatatta tccgttcatt ccgtttttag gggcgcttgt caaaaaagat 900
tttgtatgga ataactgtga gtttaagaag ggcacatcgg tgctgcttga tttatatgga 960
acgaaccacg accctcgtct atgggatcat cccgatgaat tccggccgga acgatttgcg 1020
gagcgggaag aaaatctgtt tgatatgatt cctcaaggcg gggggcacgc cgagaaaggc 1080
caccgctgtc caggggaagg cattacaatt gaagtcatga aagcgagcct ggatttcctc 1140
gtccatcaga ttgaatacga tgttccggaa caatcactgc attacagtct cgccagaatg 1200
ccatcattgc ctgaaagcgg cttcgtaatg agcggaatca gacgaaaaag tctcgagcac 1260
caccaccacc accactga 1278
<210> 3
<211> 425
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 3
Met Asp Glu Gln Ile Pro His Asp Lys Ser Leu Asp Asn Ser Leu Thr
1 5 10 15
Leu Leu Lys Glu Gly Tyr Leu Phe Ile Lys Asn Arg Thr Glu Arg Tyr
20 25 30
Asn Ser Asp Leu Phe Gln Ala Arg Leu Leu Gly Lys Asn Phe Ile Cys
35 40 45
Met Thr Gly Ala Glu Ala Ala Lys Val Phe Tyr Asp Thr Asp Arg Phe
50 55 60
Gln Arg Gln Asn Ala Leu Pro Lys Arg Val Gln Lys Ser Leu Phe Gly
65 70 75 80
Val Asn Ala Ile Gln Gly Met Asp Gly Ser Ala His Ile His Arg Lys
85 90 95
Met Leu Phe Leu Ser Leu Met Thr Pro Pro His Gln Lys Arg Leu Ala
100 105 110
Glu Leu Met Thr Glu Glu Trp Lys Ala Ala Val Thr Arg Trp Glu Lys
115 120 125
Ala Asp Glu Val Val Leu Phe Glu Glu Ala Lys Glu Ile Leu Cys Arg
130 135 140
Val Ala Cys Tyr Trp Ala Gly Val Pro Leu Lys Glu Thr Glu Val Lys
145 150 155 160
Glu Arg Ala Asp Asp Phe Ile Asp Met Val Asp Ala Phe Gly Ala Val
165 170 175
Gly Pro Arg His Trp Lys Gly Arg Arg Ala Arg Pro Arg Ala Glu Glu
180 185 190
Trp Ile Glu Val Met Ile Glu Asp Ala Arg Ala Gly Leu Leu Lys Thr
195 200 205
Thr Ser Gly Thr Ala Leu His Glu Met Ala Phe His Thr Gln Glu Asp
210 215 220
Gly Ser Gln Leu Asp Ser Arg Met Ala Ala Ile Glu Leu Ile Asn Val
225 230 235 240
Leu Arg Pro Ile Val Ala Ile Ser Tyr Phe Leu Val Phe Ser Ala Leu
245 250 255
Ala Leu His Glu His Pro Lys Tyr Lys Glu Trp Leu Arg Ser Gly Asn
260 265 270
Ser Arg Glu Arg Glu Met Phe Val Gln Glu Val Arg Arg Tyr Tyr Pro
275 280 285
Phe Gly Pro Phe Leu Gly Ala Leu Val Lys Lys Asp Phe Val Trp Asn
290 295 300
Asn Cys Glu Phe Lys Lys Gly Thr Ser Val Leu Leu Asp Leu Tyr Gly
305 310 315 320
Thr Asn His Asp Pro Arg Leu Trp Asp His Pro Asp Glu Phe Arg Pro
325 330 335
Glu Arg Phe Ala Glu Arg Glu Glu Asn Leu Phe Asp Met Ile Pro Gln
340 345 350
Gly Gly Gly His Ala Glu Lys Gly His Arg Cys Pro Gly Glu Gly Ile
355 360 365
Thr Ile Glu Val Met Lys Ala Ser Leu Asp Phe Leu Val His Gln Ile
370 375 380
Glu Tyr Asp Val Pro Glu Gln Ser Leu His Tyr Ser Leu Ala Arg Met
385 390 395 400
Pro Ser Leu Pro Glu Ser Gly Phe Val Met Ser Gly Ile Arg Arg Lys
405 410 415
Ser Leu Glu His His His His His His
420 425
<210> 4
<211> 1278
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
atggatgagc agattccaca tgacaaaagt ctcgataaca gtctgacact gctgaaggaa 60
gggtatttat ttattaaaaa cagaacagag cgctacaatt cagatctgtt tcaggcccgt 120
ttgttgggaa aaaactttat ttgcatgact ggcgctgagg cggcgaaggt gttttatgat 180
acggatcgat tccagcggca gaacgctttg cctaagcggg tgcagaaatc gctgtttggt 240
gttaatgcga ttcagggaat ggatggcagc gcgcatatcc atcggaagat gctttttctg 300
tcattgatga caccgccgca tcaaaaacgt ttggctgagt tgatgacaga ggagtggaaa 360
gcagcagtca caagatggga gaaggcagat gaggttgtgt tatttgaaga agcaaaagaa 420
atcctgtgcc gggtagcgtg ctattgggca ggtgttccgt tgaaggaaac ggaagtcaaa 480
gagagagcgg atgacttcat tgacatggtc gacgcgttcg gtgctgtggg accgcggcat 540
tggaaaggaa gaagagcaag gccgcgtgcg gaagagtgga ttgaagtcat gattgaagat 600
gctcgtgccg gcttgctgaa aacgacttcc ggaacagcgc tgcatgaaat ggcttttcac 660
acacaagaag atggaagcca gctggattcc cgcatggcag ccattgagct gattaatgta 720
ctgcggccta ttgtcgccat ttcttacttt ctggtgtttt cagctttggc gcttcatgag 780
catccgaagt ataaggaatg gctgcggtct ggaaacagcc gggaaagaga aatgtttgtg 840
caggaggtcc gcagatatta tccgttcggc ccgtttttag gggcgcttgt caaaaaagat 900
tttgtatgga ataactgtga gtttaagaag ggcacatcgg tgctgcttga tttatatgga 960
acgaaccacg accctcgtct atgggatcat cccgatgaat tccggccgga acgatttgcg 1020
gagcgggaag aaaatctgtt tgatatgatt cctcaaggcg gggggcacgc cgagaaaggc 1080
caccgctgtc caggggaagg cattacaatt gaagtcatga aagcgagcct ggatttcctc 1140
gtccatcaga ttgaatacga tgttccggaa caatcactgc attacagtct cgccagaatg 1200
ccatcattgc ctgaaagcgg cttcgtaatg agcggaatca gacgaaaaag tctcgagcac 1260
caccaccacc accactga 1278
Claims (10)
1. The carboxylic acid beta-hydroxylase P450BS beta mutant is characterized in that the amino acid sequence of the carboxylic acid beta-hydroxylase P450BS beta mutant is shown as SEQ ID NO. 1.
2. A nucleotide sequence encoding the carboxylic acid beta-hydroxylase P450BS beta mutant of claim 1, said nucleotide sequence being set forth in SEQ ID No. 2.
3. The method for producing a carboxylic acid beta-hydroxylase P450BS beta mutant according to claim 1, wherein the wild-type P450BS beta sequence is shown in SEQ ID NO. 3, wherein leucine at position 78 is mutated to isoleucine, glutamine at position 85 is mutated to histidine, and glycine at position 290 is mutated to isoleucine.
4. The method for producing a carboxylic acid beta-hydroxylase P450BS beta mutant according to claim 1, comprising the steps of:
constructing a plasmid expressing wild-type carboxylic acid hydroxylase P450BS beta, and transforming the plasmid into escherichia coli;
constructing a saturated mutation library through three rounds of site-directed saturation mutation, and screening mutant plasmids expressing P450BS beta mutants capable of oxidizing carboxylic acid beta-site C-H groups with high regioselectivity and enantioselectivity;
expression and purification of carboxylic acid beta-hydroxylase P450BS beta mutant.
5. The method for producing a carboxylic acid beta-hydroxylase P450BS beta mutant according to claim 4, wherein the expression and purification of the carboxylic acid beta-hydroxylase P450BS beta mutant comprises the steps of:
culturing the selected mutant plasmid in LB liquid medium overnight, adding TB liquid medium for expansion culture, adding heme precursor delta-aminolevulinic acid and IPTG for induction expression in logarithmic growth phase, centrifuging and collecting bacteria;
suspending the obtained strain with buffer solution, performing ultrasonic disruption to lyse cells, and centrifuging to obtain supernatant;
adding the supernatant into a nickel ion affinity column, combining target protein with His tag with nickel ion, removing impurity protein, eluting target protein, and obtaining carboxylic acid beta-hydroxylase P450BS beta mutant.
6. A process for the preparation of β -hydroxycarboxylic acids, characterized in that a catalytic reaction is carried out using natural linear saturated fatty acids, natural unsaturated fatty acids and unnatural carboxylic acids of chain length C6-C20 as substrates, hydrogen peroxide as oxidizing agent and the carboxylic acid β -hydroxylase P450BS β mutant of claim 1 as catalyst.
7. The method for producing β -hydroxycarboxylic acid as claimed in claim 6, wherein the natural unsaturated fatty acid is oleic acid or linoleic acid; the non-natural carboxylic acids contain one or more functional groups of alkene, alkyne, bromine, amine, nitrile, ester, dihydroxy, cyclopentyl, furan, pyrrole, benzyloxy, fluorophenyl, chlorophenyl, benzoylmethoxy.
8. The method for producing β -hydroxycarboxylic acid as claimed in claim 6, wherein the β -hydroxycarboxylic acid is R-type β -hydroxycarboxylic acid.
9. The method for producing β -hydroxycarboxylic acid as claimed in claim 6, wherein the reaction is carried out at room temperature, and the reaction system is an aqueous phase system.
10. Use of a carboxylic acid β -hydroxylase P450BS β mutant according to claim 1 for the preparation of β -hydroxycarboxylic acids.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108779476A (en) * | 2015-06-30 | 2018-11-09 | 道达尔销售服务公司 | The production of alpha-olefin |
| CN112111477A (en) * | 2020-09-30 | 2020-12-22 | 扬州大学 | Artificial biocatalyst for converting fatty acid into olefin by using hydrogen peroxide |
| CN113774033A (en) * | 2021-06-11 | 2021-12-10 | 合肥生融生物科技有限公司 | Artificial biocatalyst for converting fatty acid into beta-hydroxy fatty acid |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108779476A (en) * | 2015-06-30 | 2018-11-09 | 道达尔销售服务公司 | The production of alpha-olefin |
| CN112111477A (en) * | 2020-09-30 | 2020-12-22 | 扬州大学 | Artificial biocatalyst for converting fatty acid into olefin by using hydrogen peroxide |
| CN113774033A (en) * | 2021-06-11 | 2021-12-10 | 合肥生融生物科技有限公司 | Artificial biocatalyst for converting fatty acid into beta-hydroxy fatty acid |
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