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Recombination acetyl coenzyme A synthetase

An acetyl coenzyme and synthetase technology, applied in the biological field, can solve the problems of decreased sensitivity and precision of the kit, difficult to meet the influence of the temperature of the detection kit, poor thermal stability, etc., and achieves low cost, reduced temperature influence, and thermal stability. high sex effect

Active Publication Date: 2015-04-01
NINGBO MEDICAL SYSTEM BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] ACS plays an important role in the enzymatic determination of NEFA. The first step reaction it catalyzes determines the sensitivity and precision of the enzymatic determination to a large extent. However, the ACS currently on the market has poor thermal stability and is difficult to Meet the temperature impact of the detection kit during transportation and clinical application, resulting in a decrease in the sensitivity and precision of the detection kit

Method used

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  • Recombination acetyl coenzyme A synthetase
  • Recombination acetyl coenzyme A synthetase
  • Recombination acetyl coenzyme A synthetase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] According to the acetyl-CoA synthetase gene sequence in Pseudomonas fluorescens bacterial strain, design primer sequence:

[0036] Forward primer: 5'-ATCCTTAAAAGAATTCACGGC-3'

[0037] Reverse primer: 3'-CGTTAAATAGGATCCTAAAG-5'

[0038] Apply the above primers and use error-prone PCR to amplify, and the composition of each 100 μl error-prone PCR amplification system is:

[0039] 10*amplification buffer 10μl

[0040] dNTP mix 20 μmol

[0041] Primer 50pmol

[0042] Template 0.5ug

[0043] TaqDNA polymerase 0.25mU

[0044] Mg 2+ 0.15mmol

[0045] mn 2+ 5mmol

[0046] The rest is double distilled water.

[0047] The PCR reaction conditions were: 94°C for 5 min; 30 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 2 min; 72°C for 10 min, 4°C.

[0048] The above 3 μl PCR amplification product was taken for agarose gel electrophoresis, and the target product band was observed at 2.1-2.2 kb. Recover the electrophoresis product bands, use EcoR 1 and BamH 1 to digest t...

Embodiment 2

[0050] Copy and transfer the single clone on the LB plate of the mutant expression strain obtained in Example 1 to an LB plate containing a final concentration of 0.4mM IPTG and 100mg / L AMP, and culture it at 25°C for 10h to induce the activity of acetyl-CoA synthetase Express.

[0051] The cell lysate was evenly sprayed on the expression plate, and then the plate was placed in a 60°C oven for heat treatment for 30min. The composition of the cell lysate is 100mM Tris-HCl, 0.5% SDS, 500U / ml lysozyme, 1% TritonX-100. The percentage concentration used in the present invention is the mass volume percentage concentration (g / ml) commonly used in the art.

[0052] On the heat-treated LB plate, evenly spray the color-changing reaction solution and fully react for 10 minutes. mM TOOS, 0.5 mM 4-AAP, pH 7.2.

[0053] After the reaction was completed, the color reaction was observed, and a total of 10 single colonies with obvious reaction and dark purple color were selected, and then t...

Embodiment 3

[0055] Inoculate 10 single clones obtained from preliminary screening on the plate in Example 2 into 200ml of 2YT liquid medium, culture at 37°C until the OD is 0.6, add IPTG with a final concentration of 0.4mM, and culture at 25°C for 10h.

[0056] Centrifuge the fermentation broth at 10,000rpm for 10min, collect the bacteria, add 5 times the volume of cell lysate, add 5ml Ni filler matrix, mix vertically overnight, centrifuge at 8000rpm for 5min, discard the supernatant, add 5 times the volume of eluent 1 to the precipitate , mixed vertically overnight, centrifuged at 8000rpm for 5min, discarded the supernatant, added 5 times the volume of eluent 2 to the precipitate, mixed vertically overnight, centrifuged at 8000rpm for 10min, took the supernatant, and the obtained supernatant was high-purity Acetyl-CoA synthetase enzyme solution; the composition of the cell lysate is 100mM Tris-HCl, 0.5% SDS, 200U / ml lysozyme, 1% TritonX-100; the composition of the eluent 1 is 100mM Tris-H...

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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 [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant acetyl-CoA synthetase and its application in a kit. Background technique [0002] Free fatty acids (NEFA) refer to non-esterified fatty acids carried and transported by albumin in blood lipids. The content of NEFA in normal human plasma is very small, ranging from 50 to 1200 μmol / L, accounting for only 5% to 10% of the total fatty acids in plasma. The range is large. Free fatty acids not only participate in the composition and energy metabolism of the body, but also participate in the adjustment of many physiological functions of the body, such as the synthesis of many physiological regulatory substances. [0003] The abnormality of NEFA has important diagnostic value in the detection of many diseases. Its increase is often used in the diagnosis of diabetes, acromegaly, hyperthyroidism, hepatitis, etc., and its decrease is often used in the diagnosis of hyp...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N15/52C12Q1/28C12Q1/26C12Q1/25
CPCC12N9/93C12Q1/25C12Q1/26C12Q1/28
Inventor 邹炳德邹继华章玉胜贾江花
Owner NINGBO MEDICAL SYSTEM BIOTECHNOLOGY CO LTD
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