CN104480077A - Recombination acetyl coenzyme A synthetase - Google Patents
Recombination acetyl coenzyme A synthetase Download PDFInfo
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Abstract
The invention discloses recombination acetyl coenzyme A synthetase. The amino acid sequence of therecombination acetyl coenzyme A synthetase is shown in SEQ ID No.2 and the nucleotide sequence of the recombination acetyl coenzyme A synthetase is shown in SEQ ID NO.1.The recombination acetyl coenzyme A synthetase has the characteristics of high thermal stability and high activity; the retaining rate of enzyme activity is 59.6% after the recombination acetyl coenzyme A synthetase is treated for 30 min at the temperature of 60 DEG C and is much improved compared with constitutive enzyme; an apparent Km value of a catalyzed oleic acid acetylation reaction is 1.2*10<-5>M; the thermal stability of enzyme in a kit can be improved, and temperature influences can be avoided or reduced in the transportation and utilization processes of the kit.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of recombinate acetyl-CoA-synthetase and the application in test kit thereof.
Background technology
Free fatty acids (NEFA) refers to that in blood fat, white protein carries the non-esterified fatty acid of transhipment, and the NEFA content in human normal plasma is few, and be 50 ~ 1200 μm of ol/L, only account for 5% ~ 10% of total fatty acids in blood plasma, fluctuation range is large.Free fatty acids not only participates in formation, the energy metabolism of body, also participates in the adjustment of the numerous physiological function of body, the such as synthesis of many physiological regulation materials.
The abnormal of NEFA all has important diagnostic value in numerous disease detection, it raises the diagnosis being usually used in the aspects such as diabetes, acromegaly, hyperthyroidism, hepatitis, its reduction is usually used in Hypothyroidism, and antidiabetic drug or Regular Insulin use the checkout and diagnosis of the aspect such as excessive.
At present, the main method detecting NEFA clinically has vapor-phase chromatography and enzyme process, and wherein enzyme process detects due to simple to operate, quick, particularly can be adapted to the batch quantity analysis of full automatic biochemical apparatus, so the application widely obtained.
The reaction principle that enzyme process detects NEFA is: NEFA to be detected is under the effect of acetyl-CoA-synthetase (Acyl-CoA Synthetase, ACS); acetyl-CoA (Acyl-CoA) is generated with coenzyme A in test kit; acetyl-CoA is oxidized by oxygen under the effect of ACOD (Acyl-CoA Oxidase, ACO), and reaction generates product hydrogen peroxide (H
2o
2), the latter utilizes Trinder reaction and peroxidase (Peroxidase, POD), there is lower and chromogen substance and generate coloured quinone-imine compound, by the content of absorption peak change detection NEFA under visible light in developer 4-AA (4-AAP).
Concrete reactions steps is divided into following three parts:
ACS is in enzymatic assays NEFA method, there is important effect, the first step reaction of its catalysis determines sensitivity and the precision of enzymatic assays to a great extent, but existing ACS poor heat stability in the market, be difficult to the impact meeting detection kit temperature in transport and process of clinical application, thus the decline of the sensitivity causing test kit to detect and precision.
Summary of the invention
Technical problem to be solved by this invention is, overcomes the shortcoming of above prior art: provide a kind of high thermal stability, highly active restructuring acetyl-CoA-synthetase.
Technical solution of the present invention is as follows: a kind of restructuring acetyl-CoA-synthetase, and its aminoacid sequence is as shown in SEQ ID No.2; Encode the gene nucleotide series of this enzyme as shown in SEQ ID No.1.
High thermal stability of the present invention, highly active restructuring acetyl-CoA-synthetase obtain by the following method:
1) with the ACS gene order of Pseudomonas fluorescens bacterial strain for template, carry out fallibility pcr amplification, set up ACS libraries of random mutants.
2), under the ACS mutated library of acquisition being connected to plasmid pET28a T7 promotor, importing in E.coli expression strain BL21 (DE32), mutated library is carried out recombinant expressed.
3) sieved again by dull and stereotyped development process high flux screening and shake-flask culture, compare the retention rate alive of the enzyme after thermal treatment, screening obtains high thermal stability, highly active ACS.
Gene order containing acetyl-CoA-synthetase in Pseudomonas fluorescens bacterial strain, the present invention with this gene order for template design primer, be respectively 5 '-ATCCTTAAAGAATTCACGGC-3 ', 3 '-CGTTAAATAGGATCCTAAAG-5 ', through fallibility pcr amplification, gained fragment is the ACS gene order containing random mutation, reclaim mutant fragments, with EcoR 1 and BamH 1 respectively enzyme cut GAATTC and GGATCC site, be connected in the pET28a containing T7 promotor with T4 ligase enzyme, be transformed in E.coli expression strain BL21 (DE32), the LB coated containing 20 ~ 500mg/L penbritin (AMP) is dull and stereotyped, 37 DEG C of incubated overnight, form mono-clonal bacterium colony, on the LB flat board of utilize seal method to copy to transfer to another block to contain isopropylthiogalactoside (IPTG) that final concentration is 0.05 ~ 1mM and 20 ~ 500mg/L AMP, 25 DEG C of inducing culture 5 ~ 15h, express acetyl-CoA-synthetase, then cell pyrolysis liquid is added, in 60 DEG C of process 30min, after being disposed, flat board evenly sprays reaction solution, and 37 DEG C of temperature bath process 10-30min, observe the change of mono-clonal colony colour, select the obvious mono-clonal of change, next step utilizes shake-flask culture to sieve bacterial strain again, wherein cell pyrolysis liquid consists of 10 ~ 500mM tri-(methylol) aminomethane hydrochloride buffer (Tris-HCl), 0.01 ~ 1% sodium laurylsulfonate (SDS), 20 ~ 2000U/ml N,O-Diacetylmuramidase, 0.05 ~ 2% Triton (TritonX-100), metachromasia liquid consists of 10 ~ 500mM Tris-HCl, 0.01 ~ 2mM oleic acid, 0.1 ~ 10mM Triphosaden (ATP), 0.1 ~ 10mM coenzyme A (CoA), 1 ~ 200U/ml ACO, 5 ~ 500U/ml peroxidase (POD), 0.01 ~ 10mM N-ethyl-N-TOOS (TOOS), 0.02 ~ 20mM 4-AAP.
Select the strain of above-mentioned colour-change obvious mono-clonal bacterium colony 8 ~ 10, be inoculated in 5 ~ 50ml 2YT liquid nutrient medium, 37 DEG C are cultured to OD when being 0.6 ~ 1.0, inoculate in 100 ~ 500ml 2YT liquid nutrient medium, 37 DEG C are cultured to OD when being 0.6 ~ 1.0, add the IPTG that final concentration is 0.05 ~ 1mM, be cooled to 25 DEG C of inducing culture 5 ~ 15h, collected by centrifugation thalline.
Get the thalline of collected by centrifugation, add the cell pyrolysis liquid of 2 ~ 10 times of volumes, add appropriate Ni filler matrix, vertical mixing is spent the night, centrifugal 5 ~ the 10min of 5000 ~ 8000rpm, abandoning supernatant, in precipitation, add 2 ~ 10 times of volume elutriants 1, vertical mixing is spent the night, the centrifugal 5 ~ 10min of 5000 ~ 8000rpm, abandoning supernatant, in precipitation, add 2 ~ 10 times of volume elutriants 2, vertical mixing is spent the night, the centrifugal 5 ~ 10min of 5000 ~ 8000rpm, get supernatant liquor, namely obtain the higher ACS enzyme solution of purity; The cell pyrolysis liquid of this step consists of 10 ~ 500mM Tris-HCl, 0.01 ~ 1%SDS, 20 ~ 2000U/ml N,O-Diacetylmuramidase, 0.05 ~ 2%TritonX-100; Elutriant 1 consists of 20 ~ 200mM Tris-HCl, 0.2 ~ 2M sodium-chlor (NaCl), 20 ~ 50mM imidazoles; Elutriant 2 consists of 20 ~ 200mM Tris-HCl, 0.2 ~ 2M NaCl, 200 ~ 500mM imidazoles.
Above-mentioned obtained often group ACS enzyme solution is carried out thermostability experiment: by enzyme solution respectively through 60 DEG C of water bath processing 30min, then the enzyme solution after untreated and thermal treatment gets 0.1 ~ 1ml, add the enzyme reaction solution alive of 10 times of volumes, 37 DEG C of temperature bath insulation 10min, absorbancy change is measured under 546nm, absorbancy is reacted in thermal treatment and untreated enzyme solution compare, compared by thermal stability data, obtain ACS and the expression strain thereof of high thermal stability; Enzyme reaction solution alive is 10 ~ 500mM Tris-HCl, 0.01 ~ 2mM oleic acid, 0.001 ~ 2mM ATP, 0.1 ~ 10mM CoA, 1 ~ 200U/ml ACO, 5 ~ 500U/ml POD, 0.01 ~ 10mM TOOS, 0.02 ~ 20mM 4-AAP.
Measure the heat treatment stability effect of the acetyl-CoA-synthetase of the high thermal stability of above-mentioned acquisition:
By enzyme solution after 60 DEG C of temperature bath process 30min, get 100 ~ 1000 μ l enzyme solution, add metachromasia liquid, 37 DEG C of reaction 10min, add 50 ~ 100 μ l 100% trichoroacetic acid(TCA) (TCA) termination reactions immediately.Get 1ml reaction solution, measure absorbancy changing value, react compare with untreated enzyme solution under spectrophotometer with 546nm wavelength, after obtaining this enzyme 60 DEG C process 30min, enzyme retention rate alive is 59.6%; Wherein metachromasia liquid is 10 ~ 500mM Tris-HCl, 0.01 ~ 2mM oleic acid, 0.001 ~ 2mM ATP, 0.1 ~ 10mMCoA, 1 ~ 200U/ml ACO, 5 ~ 500U/ml POD, 0.01 ~ 10mM TOOS, 0.02 ~ 20mM 4-AAP.
Measure the apparent Km value that above-mentioned shaking flask sieves the acetyl-CoA-synthetase catalysis oleic acid acetylization reaction of the high thermal stability of acquisition again:
Configuration enzyme reaction system: 10 ~ 500mM Tris-HCl, 0.001 ~ 2mM ATP, 0.1 ~ 10mM CoA, 1 ~ 200U/ml ACO, 5 ~ 500U/ml POD, 0.01 ~ 10mM TOOS, 0.02 ~ 20mM 4-AAP; Be divided into 15 parts, every part of 1ml, add the oleic acid that final concentration is 0.001mM, 0.002mM, 0.003mM, 0.005mM, 0.008mM, 0.01mM, 0.02mM, 0.03mM, 0.04mM, 0.05mM, 0.08mM, 0.1mM, 0.2mM, 0.3mM, 0.5mM respectively, hatch to 37 DEG C, add a certain amount of enzyme solution, reaction 10min, adds 50 ~ 100 μ l 100%TCA termination reactions immediately.Get 1ml reaction solution, measure absorbancy change with 546nm wavelength under spectrophotometer, calculate speed of response, utilize origin8.0 software connecting curve.Measuring and obtaining the acetylizad apparent Km value of catalysis oleic acid is 1.2*10
-5m.
High thermal stability of the present invention, highly active restructuring acetyl-CoA-synthetase, pass through DNA sequencing reaction, this enzyme nucleotide coding sequence is as shown in SEQ ID No.1, compared with original gene order, having the change that 5 place's nucleotide sequences occur, is 189 T → A respectively, 506 T → A, 1132 G → C, 1470 C → T, 1510 G → C.
By nucleotide sequence analysis, the protein amino acid sequence of this acetyl-CoA-synthetase mutation expression, as shown in SEQ ID No.2, finds compared with original aminoacid sequence, 3 place's aminoacid sequences are had to there occurs change, 169 F → Y respectively, 378 G → R, 504 V → L.
Above-mentioned restructuring acetyl-CoA-synthetase is applied in NEFA enzyme process detection kit.NEFA test kit is made up of R1 and R2 two portions.R1 consists of: 1 ~ 500mM Tris-HCl damping fluid or MOPS damping fluid, 0.01 ~ 1mM CoA, 0.01 ~ 5mM ATP, 0.001 ~ 2mM MgCl
2, 0.001 ~ 2%Triton X-100,0.5mM ~ 10mM TOOS, 0.05 ~ 100U/mL recombinate acetyl-CoA-synthetase, pH7.2 ~ 8.2; R2 consists of: 1 ~ 800mM Tris-HCl damping fluid or MOPS damping fluid, 0.2 ~ 5mM 4-AAP, 0.01 ~ 5mM NEM, 1 ~ 200U/mL POD, 0.1 ~ 100U/mL ACO, pH7.2 ~ 9.2.
The invention has the beneficial effects as follows:
(1) the restructuring acetyl-CoA-synthetase that the present invention obtains has high thermal stability, highly active characteristic, and after 60 DEG C of process 30min, enzyme retention rate alive is 59.6%, and comparatively constitutive enzyme is significantly improved; The apparent Km value of catalysis oleic acid acetylization reaction is 1.2*10
-5m.
(2) the restructuring acetyl-CoA-synthetase of the present invention's acquisition, thermostability is high, can improve the thermostability of enzyme in test kit, is conducive to avoiding or reduce the transport of test kit and the temperature impact of use procedure.
(3) the restructuring acetyl-CoA-synthetase activity that obtains of the present invention is high, can reduce its consumption in free-fat acid detection kit, reduce test kit production cost.
(4) the restructuring acetyl-CoA-synthetase that the present invention obtains can obtain high expression in Recombinant organism, and the mode of production is simple, cost is low, can scale operation, effectively reduction free-fat acid-enzyme hydrolysis method detection kit production cost.
Embodiment
With specific embodiment, the present invention is described in further details below, but the present invention is not only confined to following specific embodiment.
Embodiment 1:
According to the acetyl coenzyme A synthetase gene sequence in Pseudomonas fluorescens bacterial strain, design primer sequence:
Forward primer: 5 '-ATCCTTAAAGAATTCACGGC-3 '
Reverse primer: 3 '-CGTTAAATAGGATCCTAAAG-5 '
Apply above-mentioned primer, utilize fallibility PCR to increase, every 100 μ l fallibility PCR amplification system consist of:
10* amplification buffer 10 μ l
DNTP mixture 20 μm of ol
Primer 50pmol
Template 0.5ug
Taq DNA polymerase 0.25mU
Mg
2+0.15mmol
Mn
2+5mmol
All the other are distilled water.
PCR reaction conditions is: 94 DEG C of 5min; 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 2min, 30 circulations; 72 DEG C of 10min, 4 DEG C.
Get above-mentioned 3 μ l pcr amplification products and carry out agarose gel electrophoresis, observe object product band at 2.1 ~ 2.2kb place.Reclaim electrophoresis product band, with EcoR 1 and BamH 1 respectively enzyme cut GAATTC and GGATCC site, be connected in the pET28a containing T7 promotor with T4 ligase enzyme, be transformed in E.coli expression strain BL21 (DE32), coat on the LB flat board containing 100mg/L AMP, 37 DEG C of overnight incubation, obtain ACS random mutation expression strain.
Embodiment 2:
Mono-clonal seal method on the LB flat board of mutation expression bacterial strain embodiment 1 obtained copies on the LB flat board transferred to containing final concentration 0.4mM IPTG, 100mg/L AMP, cultivates 10h for 25 DEG C, the expression of induction acetyl-CoA-synthetase.
Even application cell pyrolysis liquid on expression flat board, is then placed on thermal treatment 30min in 60 DEG C of baking ovens by flat board.Cell pyrolysis liquid consist of 100mM Tris-HCl, 0.5%SDS, 500U/ml N,O-Diacetylmuramidase, 1%TritonX-100.The percentage concentration adopted in the present invention is quality concentration of volume percent (g/ml) conventional in this area.
On heat treated LB flat board, even application metachromasia liquid, abundant reaction 10min, metachromasia liquid consist of 50mM Tris-HCl, 1mM oleic acid, 2mM ATP, 2mM CoA, 20U/ml ACO, 40U/ml POD, 0.5mM TOOS, 0.5mM 4-AAP, pH7.2.
After having reacted, observe color reaction, select significant reaction, present single bacterium colony of intense violet color and amount to 10 strains, carry out next step shaking flask and sieve experiment again.
Embodiment 3:
Be inoculated in the 2YT liquid nutrient medium of 200ml by the 10 strain mono-clonals that dull and stereotyped for embodiment 2 preliminary screening obtains, 37 DEG C are cultured to OD when being 0.6, add the IPTG that final concentration is 0.4mM, cultivate 10h for 25 DEG C.
By centrifugal for fermented liquid 10000rpm 10min, collect thalline, add 5 times of volume cells lysates, add 5ml Ni filler matrix, vertical mixing is spent the night, the centrifugal 5min of 8000rpm, abandoning supernatant, adds 5 times of volume elutriants 1 in precipitation, and vertical mixing is spent the night, the centrifugal 5min of 8000rpm, abandoning supernatant, adds 5 times of volume elutriants 2 in precipitation, vertical mixing is spent the night, the centrifugal 10min of 8000rpm, gets supernatant liquor, and the supernatant liquor of acquisition is highly purified acetyl-CoA-synthetase enzyme solution; Wherein cell pyrolysis liquid consist of 100mM Tris-HCl, 0.5%SDS, 200U/ml N,O-Diacetylmuramidase, 1%TritonX-100; Elutriant 1 consist of 100mM Tris-HCl, 1M NaCl, 50mM imidazoles; Elutriant 2 consist of 100mM Tris-HCl, 0.5M NaCl, 400mM imidazoles.
Embodiment 4:
The acetyl-CoA-synthetase enzyme solution of 10 groups of fallibility PCR random mutations embodiment 3 obtained carries out thermostability experiment.
By enzyme solution respectively through 60 DEG C of water bath processing 30min, then the enzyme solution after untreated and thermal treatment gets 0.1ml, and the enzyme adding 2ml is lived reaction solution, 37 DEG C of temperature bath insulation 10min, live reaction solution in contrast with the enzyme adding pure water, under 546nm, measures absorbancy change.Absorbancy change is in table 1.Relatively thermal stability data, the thermostability of known No. 5 bacterial strains is higher.Namely the ACS expression strain of high thermal stability is obtained.What described enzyme lived reaction solution consists of 200mM Tris-HCl, 0.5mM oleic acid, 1mM ATP, 1mM CoA, 20U/mlACO, 50U/ml POD, 1mM TOOS, 1mM 4-AAP, pH7.5.
When table 1 different strains shaking flask is sieved again, the change of 60 DEG C of process 30min absorbancys
Embodiment 5:
Embodiment 4 shaking flask is sieved again No. 5 bacterial strains obtained, purification acetyl-CoA-synthetase solution.
By No. 5 inoculation in 20ml 2YT liquid nutrient medium, 37 DEG C are cultured to OD when being about 1.0, transfer in 1L 2YT liquid nutrient medium, and 37 DEG C are cultured to OD when being about 0.8, add the IPTG that final concentration is 0.2mM, 28 DEG C of inducing culture 8h.
By centrifugal for fermented liquid 10000rpm 10min, collect thalline, add 3 times of volume cells lysates, add 20ml Ni filler matrix, vertical mixing is spent the night, the centrifugal 10min of 5000rpm, abandoning supernatant, adds 3 times of volume elutriants 1 in precipitation, and vertical mixing is spent the night, the centrifugal 8min of 6000rpm, abandoning supernatant, adds 3 times of volume elutriants 2 in precipitation, vertical mixing is spent the night, the centrifugal 6min of 7000rpm, gets supernatant liquor, and the supernatant liquor of acquisition is highly purified acetyl-CoA-synthetase enzyme solution; Wherein cell pyrolysis liquid consist of 50mM Tris-HCl, 0.8%SDS, 400U/ml N,O-Diacetylmuramidase, 0.5%Triton X-100; Elutriant 1 consist of 50mM Tris-HCl, 0.8M NaCl, 30mM imidazoles; Elutriant 2 consist of 50mMTris-HCl, 0.5M NaCl, 500mM imidazoles.
Embodiment 6:
Acetyl-CoA-synthetase embodiment 5 prepared measures thermal treatment rear stability data.
By ACS enzyme solution, 60 DEG C of water-bath thermal treatment 30min, then get 0.5ml, add 5ml metachromasia liquid, 37 DEG C of temperature bath 10min, absorbancy changing value under mensuration 546nm, to add the metachromasia liquid of pure water as blank, experiment is in triplicate, thermal treatment and untreated ACS enzyme solution absorbancy changing value are in table 3, calculating mean value, obtains this enzyme after 60 DEG C of process 30min, and enzyme retention rate alive is 59.6%; Wherein metachromasia liquid consist of 100mM Tris-HCl, 0.3mM oleic acid, 1.2mM ATP, 1.2mM CoA, 40U/mlACO, 70U/ml POD, 1.2mM TOOS, 1.4mM 4-AAP, pH7.8.
Thermal stability data after table 2ACS 60 DEG C of thermal treatment 30min
Embodiment 7:
Acetyl-CoA-synthetase embodiment 5 prepared measures the apparent Km value of catalysis oleic acid acetylization reaction.
Configuration enzyme reaction system 100mM Tris-HCl, 2mM ATP, 2mM CoA, 100U/ml ACO, 200U/mlPOD, 1mM TOOS, 1mM 4-AAP, be divided into 15 parts, every part of 1ml, add respectively final concentration be 0.001,0.002,0.003,0.005,0.008,0.01,0.02,0.03,0.04,0.05,0.08,0.1,0.2,0.3, the oleic acid of 0.5mM, hatch to 37 DEG C, add 10 μ l enzyme solution, reaction 10min, adds 100 μ l 100%TCA termination reactions immediately.Get 1ml reaction solution, measure absorbancy change with 546nm wavelength under spectrophotometer, calculate speed of response, in table 2.Utilize origin8.0 software coupling Michaelis-Menton equation curve, obtain the apparent Km value of this enzyme catalysis oleic acid acetylization reaction for 1.2*10
-2mM, i.e. 1.2*10
-5m.
The acetylizad speed of response of table 3ACS catalysis different concns oleic acid
Embodiment 8:
Restructuring acetyl-CoA-synthetase embodiment 5 prepared is applied in NEFA enzyme process detection kit.NEFA test kit is made up of R1 and R2 two portions.R1 consists of: 100mM Tris-HCl damping fluid, 0.5mMCoA, 1mM ATP, 2mM MgCl
2, 0.1%Triton X-100,5mM TOOS, 10U/mL recombinate acetyl-CoA-synthetase, pH7.5; R2 consists of: 200mM MOPS damping fluid, 0.5mM 4-AAP, 1mM NEM, 60U/mL POD, 20U/mL ACO, pH8.3.
Below be only that feature of the present invention implements example, scope is not constituted any limitation.The technical scheme that all employings exchange on an equal basis or equivalence is replaced and formed, all drops within rights protection scope of the present invention.
SEQUENCE LISTING
<110> Ningbo Meikang Biotechnology Co., Ltd.
<120> recombinates acetyl-CoA-synthetase
<130> 001
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 2217
<212> DNA
<213> Pseudomonas fluorescens
<400> 1
atggctgctg tgatttcaaa ttcttctggg acagagctgt atgtggatcc caccaaagca 60
ataccagatt tatgtataaa tgatgtagtc ctatccactc caccatgcta tgctgaagaa 120
actcttgtgc agtcttctgc tgacatcaac acaagtaaca gcagcgagaa gcccatagtt 180
cctgaaatat cctcttcaga tgtcatggtc cagtcagctg aaaacctgcc aacatctgac 240
atgaagctgt ggacagcaca gagggacagt gcagtgaaat tgcgactgga agattcggat 300
gttgccagct tgccccctgt taccattcac cagttgttcc aagagactgt aaataaatac 360
ggagattatg tggcccttgc gtctaagcag ggagaccagt ggcacaaaat gtcttacaag 420
caatattatg agcagtgcag aatagcagcc aaaggtttca ttaagttggg tttggaaaga 480
taccatggtg tgggcatcct ggggtataac tctgcagaat ggtttattgc ggatgttgga 540
gctatttttg ctggtggctt tgctgtgggc atctatacca caaactctgc tgaggcttgt 600
cattatgttg cacagaactg tgaagccaac attattgtgg tggagaatca gaaacaactt 660
caaaaaatcc ttcaggttca ggatcagctg cctcacctta aagcaatcat ccagtacaag 720
gacgagctga aggagaagag gcctaatctg tatacatgga aagagtttat gcagctggga 780
aaagacattc cagactctca attagaccag atcatttcat ctcagaagcc caaccaatgt 840
tgcaccctca tctatacttc tgggaccact gggcaaccta aaggtgtcat gcttagccat 900
gataatatca cttggaccgc agcagcagca ggaaagactg tgcgactaag agaggctact 960
gatctgcagg aaattgtggt tagctacctg cctcttagcc acattgcagc acagatgatc 1020
gatatttggt tgaccatgaa gtatggagga gccacttact ttgctcagcc tgatgcacta 1080
aagggctctc tagccatcac cttgcgtgaa gtgaggccaa cagcctttat gcgtgttcca 1140
agagtgtggg agaaaatgca ggaaaaaatg aaagctgttg gagccaagtc atctacaatc 1200
aaacgcaaaa tggcaacttg ggccaaagga gtgggcttag agacaaatct gaagaaaatg 1260
aatggatcaa caccccatcc aatgaagtat cacgtggcaa ataaattggt tttcaaaaaa 1320
gtccgcaagg ctcttggact ggaccgatgc acaaagtgtt acacaggggc agcccccatc 1380
acgaaggaca ccttggagtt ttttctaagc ctcaatattc ctgtttatga gctttatggg 1440
atgagtgaaa gttctggccc acataccatt tctttgcctg atgctttcag aatcaccagt 1500
tgtggaaaac taatttcagg gtgcaagaca aagatccatc agccagacag tgatggcagt 1560
ggggagatcc ttttctgggg acgtcatgtc tttatgggat atttgaacat ggaagacaaa 1620
acacacgagt ccttggatga ggaaggctgg ttgcattctg gtgatattgg gaaacacgat 1680
gagaatggat ttctttatat tactggaaga atcaaagagc tgataataac ggcaggtgga 1740
gagaacatcc ctcctgttcc aactgaagat gctgtaaaag agcaagttcc aataatcagt 1800
aatgccatgt tgattggaga caagaagaag ttcctttcca tgcttctaac tcttaagtgc 1860
aatgtgaatg cagacacggg ggagccagag gatgaactca cgcctgaagc cattcagttt 1920
tgtcggcaga tcggcagcaa agccacactg gtatctgaca tagttggggg caaagacaca 1980
gctgtatatg ctgcaatcca ggaaggggta aattcagtaa atgagaagtc aacctcaaat 2040
gctcagaagg tccaaaagtg gctcattctt gaaaaagatt tttctatcac tggaggagaa 2100
ttgggcccca ccatgaagtt aaaacgtcca gtagtggcaa agatgtacaa agaccaaatt 2160
gatagcttct atcaggatgc aggaacccct acggaaaact tcacccctcc taagtag 2217
<210> 2
<211> 738
<212> PRT
<213> Pseudomonas fluorescens
<400> 2
Met Ala Ala Val Ile Ser Asn Ser Ser Gly Thr Glu Leu Tyr Val Asp
1 5 10 15
Pro Thr Lys Ala Ile Pro Asp Leu Cys Ile Asn Asp Val Val Leu Ser
20 25 30
Thr Pro Pro Cys Tyr Ala Glu Glu Thr Leu Val Gln Ser Ser Ala Asp
35 40 45
Ile Asn Thr Ser Asn Ser Ser Glu Lys Pro Ile Val Pro Glu Ile Ser
50 55 60
Ser Ser Asp Val Met Val Gln Ser Ala Glu Asn Leu Pro Thr Ser Asp
65 70 75 80
Met Lys Leu Trp Thr Ala Gln Arg Asp Ser Ala Val Lys Leu Arg Leu
85 90 95
Glu Asp Ser Asp Val Ala Ser Leu Pro Pro Val Thr Ile His Gln Leu
100 105 110
Phe Gln Glu Thr Val Asn Lys Tyr Gly Asp Tyr Val Ala Leu Ala Ser
115 120 125
Lys Gln Gly Asp Gln Trp His Lys Met Ser Tyr Lys Gln Tyr Tyr Glu
130 135 140
Gln Cys Arg Ile Ala Ala Lys Gly Phe Ile Lys Leu Gly Leu Glu Arg
145 150 155 160
Tyr His Gly Val Gly Ile Leu Gly Tyr Asn Ser Ala Glu Trp Phe Ile
165 170 175
Ala Asp Val Gly Ala Ile Phe Ala Gly Gly Phe Ala Val Gly Ile Tyr
180 185 190
Thr Thr Asn Ser Ala Glu Ala Cys His Tyr Val Ala Gln Asn Cys Glu
195 200 205
Ala Asn Ile Ile Val Val Glu Asn Gln Lys Gln Leu Gln Lys Ile Leu
210 215 220
Gln Val Gln Asp Gln Leu Pro His Leu Lys Ala Ile Ile Gln Tyr Lys
225 230 235 240
Asp Glu Leu Lys Glu Lys Arg Pro Asn Leu Tyr Thr Trp Lys Glu Phe
245 250 255
Met Gln Leu Gly Lys Asp Ile Pro Asp Ser Gln Leu Asp Gln Ile Ile
260 265 270
Ser Ser Gln Lys Pro Asn Gln Cys Cys Thr Leu Ile Tyr Thr Ser Gly
275 280 285
Thr Thr Gly Gln Pro Lys Gly Val Met Leu Ser His Asp Asn Ile Thr
290 295 300
Trp Thr Ala Ala Ala Ala Gly Lys Thr Val Arg Leu Arg Glu Ala Thr
305 310 315 320
Asp Leu Gln Glu Ile Val Val Ser Tyr Leu Pro Leu Ser His Ile Ala
325 330 335
Ala Gln Met Ile Asp Ile Trp Leu Thr Met Lys Tyr Gly Gly Ala Thr
340 345 350
Tyr Phe Ala Gln Pro Asp Ala Leu Lys Gly Ser Leu Ala Ile Thr Leu
355 360 365
Arg Glu Val Arg Pro Thr Ala Phe Met Arg Val Pro Arg Val Trp Glu
370 375 380
Lys Met Gln Glu Lys Met Lys Ala Val Gly Ala Lys Ser Ser Thr Ile
385 390 395 400
Lys Arg Lys Met Ala Thr Trp Ala Lys Gly Val Gly Leu Glu Thr Asn
405 410 415
Leu Lys Lys Met Asn Gly Ser Thr Pro His Pro Met Lys Tyr His Val
420 425 430
Ala Asn Lys Leu Val Phe Lys Lys Val Arg Lys Ala Leu Gly Leu Asp
435 440 445
Arg Cys Thr Lys Cys Tyr Thr Gly Ala Ala Pro Ile Thr Lys Asp Thr
450 455 460
Leu Glu Phe Phe Leu Ser Leu Asn Ile Pro Val Tyr Glu Leu Tyr Gly
465 470 475 480
Met Ser Glu Ser Ser Gly Pro His Thr Ile Ser Leu Pro Asp Ala Phe
485 490 495
Arg Ile Thr Ser Cys Gly Lys Leu Ile Ser Gly Cys Lys Thr Lys Ile
500 505 510
His Gln Pro Asp Ser Asp Gly Ser Gly Glu Ile Leu Phe Trp Gly Arg
515 520 525
His Val Phe Met Gly Tyr Leu Asn Met Glu Asp Lys Thr His Glu Ser
530 535 540
Leu Asp Glu Glu Gly Trp Leu His Ser Gly Asp Ile Gly Lys His Asp
545 550 555 560
Glu Asn Gly Phe Leu Tyr Ile Thr Gly Arg Ile Lys Glu Leu Ile Ile
565 570 575
Thr Ala Gly Gly Glu Asn Ile Pro Pro Val Pro Thr Glu Asp Ala Val
580 585 590
Lys Glu Gln Val Pro Ile Ile Ser Asn Ala Met Leu Ile Gly Asp Lys
595 600 605
Lys Lys Phe Leu Ser Met Leu Leu Thr Leu Lys Cys Asn Val Asn Ala
610 615 620
Asp Thr Gly Glu Pro Glu Asp Glu Leu Thr Pro Glu Ala Ile Gln Phe
625 630 635 640
Cys Arg Gln Ile Gly Ser Lys Ala Thr Leu Val Ser Asp Ile Val Gly
645 650 655
Gly Lys Asp Thr Ala Val Tyr Ala Ala Ile Gln Glu Gly Val Asn Ser
660 665 670
Val Asn Glu Lys Ser Thr Ser Asn Ala Gln Lys Val Gln Lys Trp Leu
675 680 685
Ile Leu Glu Lys Asp Phe Ser Ile Thr Gly Gly Glu Leu Gly Pro Thr
690 695 700
Met Lys Leu Lys Arg Pro Val Val Ala Lys Met Tyr Lys Asp Gln Ile
705 710 715 720
Asp Ser Phe Tyr Gln Asp Ala Gly Thr Pro Thr Glu Asn Phe Thr Pro
725 730 735
Pro Lys
Claims (3)
1. a restructuring acetyl-CoA-synthetase, is characterized in that: its aminoacid sequence is as shown in SEQ ID No.2.
2. a gene for coding restructuring acetyl-CoA-synthetase according to claim 1, is characterized in that: its nucleotide sequence is as shown in SEQ ID No.1.
3. press the application of restructuring acetyl-CoA-synthetase according to claim 1 in NEFA enzyme process detection kit for one kind; Described NEFA test kit is made up of R1 and R2 two portions; R1 consists of: 1 ~ 500mM Tris-HCl damping fluid or MOPS damping fluid, 0.01 ~ 1mM CoA, 0.01 ~ 5mM ATP, 0.001 ~ 2mM MgCl
2, 0.001 ~ 2% Triton X-100,0.5mM ~ 10mM TOOS, 0.05 ~ 100U/mL recombinate acetyl-CoA-synthetase, pH7.2 ~ 8.2; R2 consists of: 1 ~ 800mM Tris-HCl damping fluid or MOPS damping fluid, 0.2 ~ 5mM 4-AAP, 0.01 ~ 5mM NEM, 1 ~ 200U/mL POD, 0.1 ~ 100U/mL ACO, pH7.2 ~ 9.2.
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| CN201410804903.5A CN104480077B (en) | 2014-12-22 | 2014-12-22 | Restructuring acetyl-CoA-synthetase |
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| CN104480077B CN104480077B (en) | 2017-03-01 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106442496A (en) * | 2016-09-20 | 2017-02-22 | 上海科华生物工程股份有限公司 | Free fatty acid detection kit |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6058095A (en) * | 1983-09-08 | 1985-04-04 | Eiken Kagaku Kk | Determination of free fatty acid in serum |
| CN1418957A (en) * | 2001-11-12 | 2003-05-21 | 杭州华大基因研发中心 | High temp. resistant acetyl-coA carboxylase gene and coded polypeptide and preparation process thereof |
| CN103207175A (en) * | 2013-03-15 | 2013-07-17 | 绍兴圣康生物科技有限公司 | Free fatty acid determination reagent kit |
-
2014
- 2014-12-22 CN CN201410804903.5A patent/CN104480077B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6058095A (en) * | 1983-09-08 | 1985-04-04 | Eiken Kagaku Kk | Determination of free fatty acid in serum |
| CN1418957A (en) * | 2001-11-12 | 2003-05-21 | 杭州华大基因研发中心 | High temp. resistant acetyl-coA carboxylase gene and coded polypeptide and preparation process thereof |
| CN103207175A (en) * | 2013-03-15 | 2013-07-17 | 绍兴圣康生物科技有限公司 | Free fatty acid determination reagent kit |
Non-Patent Citations (1)
| Title |
|---|
| KENSUKE KAMEDA ET AL.: "isolation and characterization of the multiple charge isoforms of acyl-coa synthetase from escherichia coli.", 《BIOCHIMICA ET BIOPHYSICA ACTA(BBA)-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106442496A (en) * | 2016-09-20 | 2017-02-22 | 上海科华生物工程股份有限公司 | Free fatty acid detection kit |
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| CN104480077B (en) | 2017-03-01 |
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Address after: 315104 Zhejiang city of Ningbo province Yinzhou District Qiming Road No. 299 Patentee after: Meikang biological Polytron Technologies Inc Address before: 315104 Zhejiang city of Ningbo province Yinzhou District Qiming Road No. 299 Patentee before: Ningbo Meikang Biotechnology Co., Ltd. |