CN107083412B - Application of medium-short chain acyl coenzyme A synthetase - Google Patents
Application of medium-short chain acyl coenzyme A synthetase Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
Abstract
The invention relates to application of medium-short chain acyl coenzyme A synthetase. The application steps are as follows: (1) preparing medium-short chain acyl coenzyme A synthetase with an amino acid sequence shown as SEQ ID NO. 2; (2) preparing a reaction system by using the medium-short chain acyl coenzyme A synthetase prepared in the step (1); (3) after the reaction, the reaction is stopped, the enzyme protein is removed, and the product is obtained after purification and drying. The invention discloses a technical scheme for preparing 2-heptenoyl coenzyme A and cyclohexanoyl coenzyme A by using medium-short chain acyl coenzyme A synthetase for the first time, overcomes the technical bias that malonyl coenzyme A synthetase only can be catalyzed to form corresponding acyl coenzyme A products by using 1, 3-dicarboxylic acid or salt thereof as a substrate, but cannot catalyze a reaction using monocarboxylic acid as the substrate in the prior art, and widens the application range of the enzyme.
Description
Technical Field
The invention relates to an application of medium-short chain acyl coenzyme A synthetase, in particular to an application of medium-short chain acyl coenzyme A synthetase in preparation of 2-heptenoyl coenzyme A and cyclohexanecarbonyl coenzyme A, belonging to the technical field of enzyme engineering.
Background
Coenzyme a (CoA) is a key cofactor that contributes to acyl activation and transfer during enzymatic reactions, and plays an important role in a wide variety of biological pathways including biosynthesis of fatty acids, macrolides (macrolides), aromatic compounds, catabolic or oxidative processes, modification of sugars, and enzyme regulation. It is estimated that 4% of the known enzymes use CoA as a cofactor.
acyl-CoA (acyl-CoA) is a temporary compound formed by covalently linking a thiol group (-SH) of CoA to a carboxyl group (-COOH) of a free carboxylic acid, and is essential as an acyl donor in primary and secondary metabolism. Macrolides, polyethers, polyenes, flavonoids, stilbenes and other compounds are important sources for discovering natural products with medicinal value, and biosynthesis of the polyketide secondary metabolites relates to short-chain acyl-coas (media) as a structural unit. The diverse acyl-coas are incorporated into the backbone of the polyketide molecule as starter or extender units under catalysis by polyketide synthases (PKSs).
Until now, researchers have had to rely on a variety of acyl-CoA,13C-labelled acyl-CoA, radiolabeled14C-acyl CoA is used for researching the biosynthesis of polyketone natural drugs. Currently, only a few foreign companies such as Sigma-Aldrich can provide commercial acyl-CoA reagents, but the variety is very limited and the price is extremely expensive, and the price per gram is as high as 12.2-53.6 ten thousand yuan, as shown in Table 1 (commercial acyl-CoA reagent information of Sigma-Aldrich company):
TABLE 1
Since the varieties of the existing commercial acyl-CoA derivatives are quite limited, some varieties are economically prohibitive or commercially unavailable, and synthesis is difficult using chemical methods, there is a strong need in China for new acyl-CoA derivatives capable of autonomously synthesizing various forms.
At present, the products of acyl-CoA synthetases with the widest substrate spectrum and the most abundant synthetic acyl-CoA classes are limited to malonyl-CoA, methylmalonyl-CoA, ethylmalonyl-CoA, methoxymalonyl-CoA, hydroxymalonyl-CoA, and the like, but no enzyme capable of synthesizing more acyl-CoA is reported.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides an application of medium-short chain acyl-CoA synthetase.
The technical scheme of the invention is as follows:
an application of short-and-medium-chain acyl-CoA synthetase in preparing 2-heptenoyl-CoA and cyclohexanecarbonyl-CoA is disclosed.
The application comprises the following steps:
(1) preparing medium-short chain acyl coenzyme A synthetase with an amino acid sequence shown as SEQ ID NO. 2;
(2) preparing the following reaction system by using the medium-short chain acyl coenzyme A synthetase prepared in the step (1):
100mM 4-hydroxyethylpiperazine ethanesulfonic acid; 10% glycerol by mass volume, unit g/ml; 100mM magnesium chloride; 15-50 mM 2-heptenoic acid, sodium salt of 2-heptenoic acid, cyclohexanecarboxylic acid or sodium salt of cyclohexanecarboxylic acid; 20mM adenosine triphosphate; 5mM coenzyme A; 10 μ M purified malonyl-CoA synthetase; pH 7.5;
(3) the reaction system reacts for 7.5-10 h at the temperature of 20-24 ℃, the reaction is stopped, the enzyme protein is removed, and the enzyme protein is obtained after purification and drying.
According to the present invention, in the step (1), the medium-short chain acyl-CoA synthetase with the amino acid sequence shown in SEQ ID NO.2 is prepared by the following method:
amplifying malonyl coenzyme A synthetase gene matB of streptomyces coelicolor by using primers matBF and matBR through PCR reaction, then constructing an expression vector and engineering bacteria, performing induced expression, and purifying malonyl coenzyme A synthetase through a Ni-NTA nickel column to obtain the malonyl coenzyme A synthetase;
the nucleotide sequences of the primers matBF and matBR are as follows:
matBF:5’-TCGATTGCACATATGTCCTCTCTCTTCCCGGCCCTCT-3’
matBR:5’-ATCGGATAGCTCGAGTCAGTCACGGTTCAGCGCCCGCTT-3’。
according to the present invention, in the step (3), the enzyme protein is removed by ultrafiltration and centrifugation.
According to the present invention, in the step (3), the purification is performed by HPLC chromatography.
Preferably, in the step (3), the drying is performed by a vacuum freeze-drying method.
The coding gene of the short-chain acyl-CoA synthetase (malonyl-CoA synthetase) is derived from the matB gene of Streptomyces coelicolor ZM12 (purchased from synthetic biology focus laboratory of Chinese academy of sciences), the total length is 1458bp (base pair), as shown in SEQ ID NO.1, the amino acid sequence thereof is 485 residues in total length, as shown in SEQ ID NO.2, and the molecular weight is 50.6 kDa. The enzyme has a larger N-terminal body (residues 1-387) and a smaller C-terminal cap (residue 388-485). The enzyme takes carboxylic acid or salt thereof, ATP and CoA as substrates to catalyze the biosynthesis of various acyl CoA.
The two-step reaction mechanism of the above malonyl-CoA synthetase is shown in FIG. 1:
in the adenylate formation reaction, α -phosphate of ATP is attacked by carboxyl group of carboxylic acid derivative to form acyl-adenylate intermediate and pyrophosphoric acid (PPi); in thioester formation reactions, CoA displaces Adenosine Monophosphate (AMP) to produce acyl CoA and AMP.
The inventors have found, through intensive studies, that the malonyl-CoA synthetase of the present invention has a very broad tolerance to carboxylic acid substrates, which undermines the original knowledge of such malonyl-CoA synthetases, which can use not only 1, 3-dicarboxylic acid and its C-2 substituted derivatives, but also straight-chain and cyclic monocarboxylic acids as substrates, such as 2-heptenoic acid (2-heptenoic acid), cyclohexanecarboxylic acid (cyclohexenecarboxylic acid) to produce 2-heptenoyl-CoA and cyclohexanoyl-CoA. Thus, the malonyl-coa synthetase of the present invention is broadly adaptable to short or medium chain, di-or monocarboxylic acid substrates and catalyzes the formation of the corresponding acyl-coa product.
Advantageous effects
The invention discloses a technical scheme for preparing 2-heptenoyl coenzyme A and cyclohexanoyl coenzyme A by using medium-short chain acyl coenzyme A synthetase for the first time, overcomes the technical bias that only 1, 3-dicarboxylic acid or salt thereof can be used as a substrate by using malonyl coenzyme A synthetase to catalyze the malonyl coenzyme A to form corresponding acyl coenzyme A products, such as malonyl CoA, methylmalonyl CoA, ethylmalonyl CoA, methoxymalonyl CoA and hydroxymalonyl CoA, but the reaction using monocarboxylic acid as the substrate cannot be catalyzed by using malonyl CoA in the prior art, and widens the application range of the enzyme.
Drawings
FIG. 1 is a two-step reaction mechanism for malonyl-CoA synthetase;
FIG. 2 is an N-terminal His6-SDS-PAGE detection map of tag-fused malonyl-coa synthetase;
in the figure: t, S Total protein and supernatant of the expression host strain (control) were obtained in LB medium, under 0.5mM IPTG induction at 15 deg.C, respectively;
ft is Flow-through effluent;
15 is an eluent at an imidazole concentration of 15 mM;
150 is an eluent at an imidazole concentration of 150 mM;
500 is an eluent at an imidazole concentration of 500 mM;
m is a Protein molecular weight standard (PageRuler Broad Range expressed Protein lader);
FIG. 3 shows the HPLC-MS detection results of the test group and the control group;
in the figure: 1) control group, 2) test group.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.
Source of biological sample
Streptomyces coelicolor ZM12 is a known strain, and is purchased from a synthetic biology focus laboratory of Chinese academy of sciences;
coenzyme a was purchased from sigma aldrich (china).
Source of equipment
Ultrafiltration in the examples
Ultra-0.5 Ultra-filtration centrifugal filter device;
detection the retention time (retention time), the ultraviolet absorption peak shape (UV absorbance peak shape) and the molecular weight (molecular weight) of the acyl-CoA product were determined and checked by analyzing a small amount of sample after ultrafiltration protein removal using HPLC coupled MS (electrospray ionization mass spectrometry);
purifying with preparative HPLC column;
drying was performed using a MAXI dry lyo vacuum concentration/lyophilization system (Heto-Holten, Denmark).
Example 1
1. Construction of expression vector and engineering bacterium
And (3) performing PCR amplification on the target gene matB by using primers matBF and matBR and streptomyces coelicolor genome DNA as a template. The purified PCR product was double digested with NdeI and XhoI and purified again, ligated to the same digested and dephosphorylated His containing the N-terminus6-the expression vector of the tag pET-28a (+). The formed plasmid pET-28a (+) -matB is transformed into a host bacterium E.coli BL21(DE3) for protein expression after being verified by DNA sequencing.
matBF:5’-TCGATTGCACATATGTCCTCTCTCTTCCCGGCCCTCT-3’
matBR:5’-ATCGGATAGCTCGAGTCAGTCACGGTTCAGCGCCCGCTT-3’
2. Expression and purification of malonyl-CoA synthetase
The initial culture was grown overnight and used to inoculate LB medium pre-warmed at 37 ℃ with 50mg/l kanamycin. When OD is reached600When the concentration was 0.4, the medium was cooled at 15 ℃ and protein expression was induced by 0.5mM isopropyl-. beta. -D-thiogalactoside (IPTG). After 16h, the cells were harvested by centrifugation, resuspended in lysis buffer (10% by mass volume glycerol, g/ml, 0.5M NaCl, 100mM HEPES pH7.5), sonicated, and centrifuged at 16,000g for 45min to remove cell debris. Cell-free lysates were passed through a nickel-NTA column equilibrated with lysis buffer. The column was washed with lysis buffer containing 15mM imidazole and the proteins were eluted with lysis buffer containing 150mM imidazole.
Polyacrylamide gel electrophoresis (SDS-PAGE) detection, results are as followsFIG. 2 shows that the target protein is N-terminal His6-a tag-fused malonyl-coa synthetase with a molecular weight of 52.8 kDa. The final protein concentration was determined to be 62.7. mu.M using Thermo Scientific Nanodrop 1000.
Example 22 in vitro assay for Heptenoyl-CoA biosynthesis
The reaction system for malonyl-CoA synthetase is shown in Table 2, and the components were added in this order and allowed to stand overnight at a constant temperature under 22 ℃.
TABLE 2
The reaction system of the control group was the same as that of the test group except that malonyl-CoA synthetase was heat-inactivated at 96 ℃ for 10min in advance. Reaction sample through
Ultra-0.5Centrifugal Filter Devices were ultrafiltered to remove protein and analyzed by HPLC-MS (high-resolution positive electrospray ionization mass spectrometry) with HPLC methods as shown in Table 3.
Table 3 HPLC method for mass spectrometric detection [ solvent a: 10mM ammonium acetate (NH)4OAc) aqueous solution, solvent B: acetonitrile (CH)3CN), flow rate 1ml/min, detection of reaction products (adenine, CoA groups) at 260nm]
TABLE 3
Samples were analyzed by HPLC-HR-ESIMS using a calibration Modular HPLC (Shimadzu Japan) and a Dikma reverse phase chromatography column (Agilent 1200, ZORBAX Eclipse XDB-C18, 250X 4.6mm, 5 μm) coupled to an LTQ-Obitrap Velos Pro mass spectrometer (Thermo Scientific), scanning the range 110-;
the results show that the test group 2) showed a new product peak A (2-heptenoyl-CoA) at a retention time of 16.75min, and the observed value of m/z was 878.1981 (theoretical value 878.19565, error: only 2.8 ppm). Meanwhile, substrate peak B (CoA, m/z observed 766.4, theoretical 766.1) was observed in both the test group 2) and the control group 1).
The reaction system for the scale-up preparation of 2-heptenoyl-coa was as previously described with the exception that: the reacted system is subjected to enzyme inactivation and ultrafiltration to remove protein, then a 2-heptenoyl coenzyme A product is separated by preparative HPLC, a MAXI dry lyo vacuum concentration/freeze-drying system (hetero-Holten, Denmark) is used for freeze vacuum drying, and the obtained chromatographically pure 2-heptenoyl coenzyme A freeze-dried powder is packaged in an ampoule bottle and stored at-80 ℃.
Example 3 in vitro assay for the biosynthesis of Cyclohexanecarbonyl-CoA
The reaction system was identical to Table 2 in terms of the components and reaction conditions except that the substrate was replaced with 16mM cyclohexanecarboxylic acid (pH adjusted to 7.2 with NaOH) at the final concentration. The subsequent preparation steps refer to example 2, and 10mg of the purified product of the cyclohexanecarboxyl-CoA is obtained by separation. The detection results are as follows:
1H-NMR (Nuclear magnetic resonance) data (300MHz, D)2O)：δ:0.56(s,H-10'),0.69(s,H-11'),1.55(m,H-5),2.26(t,J＝6.9Hz,H-4'),2.40(t,J＝6.9,H-6),2.55(t,J＝8.1Hz,H-4),2.75(t,J＝6.0Hz,H-1'),3.11(t,J＝6.0,H-3and H-7),3.27(m,H-2'and H-5'),3.35(m,H-5”),3.64(m,H-5”),3.82(s,H-7'),4.04(br s,CH2-9'),4.39(br s, H-4 "), 5.98(d, J ═ 7.5Hz, H-1"), 8.08(s, adenine-CH),8.35(s, adenine-CH). ESI-MS Mass Spectrometry data [ negative ion mode ], m/z]：876.0[M-H]-，437.5[M-2H]2-。
Comparative example
Malonyl-coa synthetase was prepared according to the method disclosed in us Crosby et al 2012 for the preparation of malonyl-coa synthetase from Rhodopseudomonas palustris (Rhodopseudomonas palustris cga009), 2-heptenoyl-coa and cyclohexanoyl-coa were prepared using the methods in example 2 and example 3.
Detection shows that the generation of 2-heptenoyl coenzyme A and cyclohexanoyl coenzyme A is not found in the reaction system. The malonyl-coa synthetase from rhodopseudomonas palustris is unable to catalyze the conversion of short unsaturated linear or cyclic monocarboxylic acids such as 2-heptenoic acid, cyclohexanecarboxylic acid, to the corresponding acyl-coa.
The foregoing is merely an example of the present invention and it should be noted that modifications and embellishments could be made by those skilled in the art without departing from the principle of the present invention and should be considered as within the scope of the present invention.
SEQUENCE LISTING
<110> Liu, super
<120> application of medium-short chain acyl coenzyme A synthetase
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 1458
<212> DNA
<213> Streptomyces coelicolor
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atgtcctctc tcttcccggc cctctccccg gccccgaccg gcgccccggc cgaccggccc 60
gcgctgcggt tcggcgagcg ctccctgacc tacgcggaac tcgccgcggc ggcgggcgcc 120
acggccgggc ggatcggcgg cgccggccgg gtcgcggtct gggccacccc ggcgatggag 180
accggcgtcg ccgtggtggc ggcgctgctg gccggggtcg ccgccgtacc gctcaacccg 240
aagtccggcg acaaggaact cgcgcacatc ctctccgaca gcgcgccctc gctcgtcctg 300
gcgcccccgg acgcggaact cccgcccgcc ctcggggccc tggagcgcgt cgacgtcgac 360
gtgcgggccc gcggggcggt ccccgaggac ggtgccgacg acggcgaccc cgcgctcgtc 420
gtctacacct cgggcaccac gggaccgccg aagggcgccg tcatcccccg gcgggcgctc 480
gccacgaccc tggacgcgct cgccgacgcg tggcagtgga ccggcgagga cgtgctggtg 540
caggggctgc cgctgttcca cgtgcacggg ctggtcctcg gcatcctcgg cccgctgcgc 600
cggggcgggt ccgtgcggca cctgggccgg ttctccaccg agggtgcggc gcgggagctg 660
aacgacggcg cgaccatgct gttcggggtg ccgacgatgt accaccggat cgccgagacg 720
ctccccgccg acccggagct ggcgaaggcg ctcgccgggg cccggctgct ggtgtcgggg 780
tcggccgcgc tgccggtgca cgaccacgag cgcatcgccg ccgccaccgg gcgccgggtg 840
atcgagcggt acggcatgac cgagacgctg atgaacacca gcgtgcgcgc cgacggcgag 900
ccgcgcgccg ggacggtggg cgtgccgctg cccggtgtgg agctgcggct ggtggaggag 960
gacggcacgc cgatcgcggc gctcgacggg gagagcgtcg gcgagatcca ggttcgcggc 1020
ccgaacctgt tcaccgagta cctgaaccgc cccgacgcca ccgccgccgc cttcaccgag 1080
gacggcttct tccgcaccgg cgacatggcg gtgcgcgacc ccgacggcta tgtccgcatc 1140
gtcggccgca aggccaccga cctgatcaag agcggcggtt acaagatcgg ggccggggag 1200
atcgagaacg ccctgctcga acacccggag gtccgggagg ccgccgtcac cggcgaaccc 1260
gaccccgacc tcggggaacg gatcgtggcc tggatcgtcc cggccgaccc cgccgccccg 1320
cccgccctcg gcacgctggc cgaccacgtc gccgcccggc tcgccccgca caagcggccg 1380
cgcgtcgtcc ggtacctcga cgcggtgccc cgcaacgaca tggggaagat catgaagcgg 1440
gcgctgaacc gtgactga 1458
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Met Ser Ser Leu Phe Pro Ala Leu Ser Pro Ala Pro Thr Gly Ala Pro
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Ala Asp Arg Pro Ala Leu Arg Phe Gly Glu Arg Ser Leu Thr Tyr Ala
20 25 30
Glu Leu Ala Ala Ala Ala Gly Ala Thr Ala Gly Arg Ile Gly Gly Ala
35 40 45
Gly Arg Val Ala Val Trp Ala Thr Pro Ala Met Glu Thr Gly Val Ala
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Val Val Ala Ala Leu Leu Ala Gly Val Ala Ala Val Pro Leu Asn Pro
65 70 75 80
Lys Ser Gly Asp Lys Glu Leu Ala His Ile Leu Ser Asp Ser Ala Pro
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Ser Leu Val Leu Ala Pro Pro Asp Ala Glu Leu Pro Pro Ala Leu Gly
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Ala Leu Glu Arg Val Asp Val Asp Val Arg Ala Arg Gly Ala Val Pro
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Glu Asp Gly Ala Asp Asp Gly Asp Pro Ala Leu Val Val Tyr Thr Ser
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Gly Thr Thr Gly Pro Pro Lys Gly Ala Val Ile Pro Arg Arg Ala Leu
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Ala Thr Thr Leu Asp Ala Leu Ala Asp Ala Trp Gln Trp Thr Gly Glu
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Asp Val Leu Val Gln Gly Leu Pro Leu Phe His Val His Gly Leu Val
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Leu Gly Ile Leu Gly Pro Leu Arg Arg Gly Gly Ser Val Arg His Leu
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Gly Arg Phe Ser Thr Glu Gly Ala Ala Arg Glu Leu Asn Asp Gly Ala
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Thr Met Leu Phe Gly Val Pro Thr Met Tyr His Arg Ile Ala Glu Thr
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Leu Pro Ala Asp Pro Glu Leu Ala Lys Ala Leu Ala Gly Ala Arg Leu
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Leu Val Ser Gly Ser Ala Ala Leu Pro Val His Asp His Glu Arg Ile
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Ala Ala Ala Thr Gly Arg Arg Val Ile Glu Arg Tyr Gly Met Thr Glu
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Thr Leu Met Asn Thr Ser Val Arg Ala Asp Gly Glu Pro Arg Ala Gly
290 295 300
Thr Val Gly Val Pro Leu Pro Gly Val Glu Leu Arg Leu Val Glu Glu
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Asp Gly Thr Pro Ile Ala Ala Leu Asp Gly Glu Ser Val Gly Glu Ile
325 330 335
Gln Val Arg Gly Pro Asn Leu Phe Thr Glu Tyr Leu Asn Arg Pro Asp
340 345 350
Ala Thr Ala Ala Ala Phe Thr Glu Asp Gly Phe Phe Arg Thr Gly Asp
355 360 365
Met Ala Val Arg Asp Pro Asp Gly Tyr Val Arg Ile Val Gly Arg Lys
370 375 380
Ala Thr Asp Leu Ile Lys Ser Gly Gly Tyr Lys Ile Gly Ala Gly Glu
385 390 395 400
Ile Glu Asn Ala Leu Leu Glu His Pro Glu Val Arg Glu Ala Ala Val
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Thr Gly Glu Pro Asp Pro Asp Leu Gly Glu Arg Ile Val Ala Trp Ile
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Val Pro Ala Asp Pro Ala Ala Pro Pro Ala Leu Gly Thr Leu Ala Asp
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Claims (10)
1. The application of malonyl-CoA synthetase in the preparation of 2-heptenoyl-CoA is characterized by comprising the following steps:
(1) preparing malonyl-CoA synthetase with an amino acid sequence shown as SEQ ID No. 2;
(2) preparing the following reaction system by using the malonyl coenzyme A synthetase prepared in the step (1):
100mM 4-hydroxyethylpiperazine ethanesulfonic acid; 10% glycerol by mass volume, unit g/ml; 100mM magnesium chloride; 15 to 50mM2 sodium salt of heptenoic acid or 2-heptenoic acid; 20mM adenosine triphosphate; 5mM coenzyme A; 10 μ M purified malonyl-CoA synthetase; pH 7.5;
(3) the reaction system reacts for 7.5-10 h at the temperature of 20-24 ℃, the reaction is stopped, the enzyme protein is removed, and the enzyme protein is obtained after purification and drying.
2. The use according to claim 1, wherein in step (1), malonyl-coa synthetase having an amino acid sequence shown in SEQ ID No.2 is prepared by the following method:
amplifying malonyl coenzyme A synthetase gene matB of streptomyces coelicolor by using primers matBF and matBR through PCR reaction, then constructing an expression vector and engineering bacteria, performing induced expression, and purifying malonyl coenzyme A synthetase through a Ni-NTA nickel column to obtain the malonyl coenzyme A synthetase;
the nucleotide sequences of the primers matBF and matBR are as follows:
matBF:5’-TCGATTGCACATATGTCCTCTCTCTTCCCGGCCCTCT-3’
matBR:5’-ATCGGATAGCTCGAGTCAGTCACGGTTCAGCGCCCGCTT-3’。
3. the use of claim 1, wherein in step (3), the removal of the enzyme protein is carried out by ultrafiltration and centrifugation.
4. The use of claim 1, wherein in step (3), said purification is by HPLC chromatography.
5. The use according to claim 1, wherein in step (3), the drying is performed by a vacuum freeze-drying method.
6. The application of malonyl-CoA synthetase in preparing cyclohexanecarboxyl-CoA is characterized by comprising the following steps:
(1) preparing malonyl-CoA synthetase with an amino acid sequence shown as SEQ ID No. 2;
(2) preparing the following reaction system by using the malonyl coenzyme A synthetase prepared in the step (1):
100mM 4-hydroxyethylpiperazine ethanesulfonic acid; 10% glycerol by mass volume, unit g/ml; 100mM magnesium chloride; 15 to 50mM cyclohexanecarboxylic acid or a sodium salt of cyclohexanecarboxylic acid; 20mM adenosine triphosphate; 5mM coenzyme A; 10 μ M purified malonyl-CoA synthetase; pH 7.5;
(3) the reaction system reacts for 7.5-10 h at the temperature of 20-24 ℃, the reaction is stopped, the enzyme protein is removed, and the enzyme protein is obtained after purification and drying.
7. The use according to claim 6, wherein in step (1), the malonyl-CoA synthetase having an amino acid sequence shown in SEQ ID No.2 is prepared by the following method:
amplifying malonyl coenzyme A synthetase gene matB of streptomyces coelicolor by using primers matBF and matBR through PCR reaction, then constructing an expression vector and engineering bacteria, performing induced expression, and purifying malonyl coenzyme A synthetase through a Ni-NTA nickel column to obtain the malonyl coenzyme A synthetase;
the nucleotide sequences of the primers matBF and matBR are as follows:
matBF:5’-TCGATTGCACATATGTCCTCTCTCTTCCCGGCCCTCT-3’
matBR:5’-ATCGGATAGCTCGAGTCAGTCACGGTTCAGCGCCCGCTT-3’。
8. the use of claim 6, wherein in step (3), the removal of the enzyme protein is carried out by ultrafiltration and centrifugation.
9. The use of claim 6, wherein in step (3), said purification is by HPLC chromatography.
10. The use of claim 6, wherein in step (3), the drying is performed by a vacuum freeze-drying method.
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| CN201710440525.0A CN107083412B (en) | 2017-06-13 | 2017-06-13 | Application of medium-short chain acyl coenzyme A synthetase |
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