Acyl-CoA synthetase mutant, its encoding gene, expression strain and application thereof

Abstract

The present invention discloses an acyl-CoA synthetase mutant, its encoding gene, expression strain and application thereof. The present invention is based on the acyl-CoA synthetase from an uncultured marine microorganism source, and the mutant gene is obtained by full gene synthesis after the mutation site is designed by Funclib. After the engineered bacteria containing the mutant plasmid are induced to express, the acyl-CoA synthetase with improved specific enzyme activity and improved stability is obtained. When palmitic acid is used as a substrate, the specific enzyme activity of the mutant is increased by 4.4 times. While the specific activity is improved, the stability of the mutant is also improved, and the mutant has potential application value in an in vitro diagnostic kit for enzymatic detection of free fatty acids.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an acyl-CoA synthetase mutant, a coding gene thereof, an expression strain and an application thereof. Background Art

[0002] Free fatty acids (NEFA) are a class of organic acids, also known as non-esterified fatty acids. NEFA is one of the substances decomposed from neutral fat, mainly composed of oleic acid, palmitic acid, linoleic acid, etc. Most NEFA is bound to albumin and exists in the blood. NEFA is directly related to oxidative stress, which can lead to the occurrence of diabetes by damaging pancreatic cells and reducing the sensitivity of peripheral tissues to insulin. The concentration of NEFA will increase due to diseases such as diabetes, severe liver disorders, and hyperthyroidism. Clinical detection of NEFA is helpful in assisting the diagnosis of the occurrence of diseases. At present, the main methods for clinical detection of NEFA are gas chromatography and enzymatic method. Among them, enzymatic detection is simple and fast to operate, especially it can be adapted to batch analysis of fully automatic biochemical analyzers.

[0003] The main principle of the free fatty acid detection kit is that NEFA in the sample is acted upon by acyl-CoA synthetase (ACS) in the presence of coenzyme A (CoA) and ATP, and the generated acyl-CoA is oxidized by acyl-CoA oxidase (ACOD), and hydrogen peroxide is generated at the same time. The generated hydrogen peroxide undergoes quantitative oxidative condensation with 3-methyl-ethyl-hydroxyaniline (MEHA) and 4-aminoantipyrine (4-AA) under the action of peroxidase (POD). The generated compound has maximum absorption at a specific wavelength, and the NEFA content is obtained by measuring the absorbance value.

[0004] Acyl-CoA synthetase (EC 6.2.1.3, long-chain-fatty-acid-CoAligase) is the key rate-limiting enzyme in the enzymatic detection of NEFA, which determines the sensitivity and precision of the enzymatic detection. ACS with good enzyme activity and stability plays an important role in improving the accuracy and stability of free fatty acid detection kits. Summary of the invention

[0005] The present invention provides an acyl-CoA synthetase mutant, its encoding gene, expression strain and application thereof. The present invention is based on the acyl-CoA synthetase from uncultured marine microorganisms, and obtains the mutant gene by full gene synthesis after designing the mutation site through Funclib. After the engineered bacteria containing the mutant plasmid are induced to express, the acyl-CoA synthetase with improved specific enzyme activity and improved stability is obtained. When palmitic acid is used as a substrate, the specific enzyme activity of the mutant is increased by 4.4 times. While the specific activity is improved, the stability of the mutant is also improved. Under the conditions of 4°C and pH 7.0, the stability of the mutant is improved by 22.2% compared with the starting enzyme and 27.3% compared with the commercial enzyme. In the enzymatic detection of free fatty acids in vitro diagnostic kit, the linear fitting curve R of the same unit mutant enzyme detection value for serum samples and the commercial enzyme detection value 2 The mutant has potential application value in the in vitro diagnostic kit for enzymatic detection of free fatty acids.

[0006] The acyl-CoA synthetase mutant of the present invention has an amino acid sequence of the acyl-CoA synthetase shown in SEQ ID No: 1, wherein the serine at position 311 is mutated to alanine, the asparagine at position 314 is mutated to serine, the glutamic acid at position 436 is mutated to methionine, the arginine at position 438 is mutated to histidine, and the glutamine at position 479 is mutated to glutamic acid.

[0007] The amino acid sequence of the acyl-CoA synthetase mutant of the present invention may also include a combination of nonsense mutations or synonymous mutations in the sequence.

[0008] The nucleotide sequence of the gene encoding the acyl-CoA synthetase mutant of the present invention is shown in SEQ ID No: 2.

[0009] The mutant plasmid of the present invention contains the coding gene of the acyl-CoA synthetase mutant as described in SEQ ID No: 2.

[0010] The strain expressing the acyl-CoA synthetase mutant of the present invention contains the mutant plasmid.

[0011] The engineered strain expressing the acyl-CoA synthetase mutant of the present invention is classified and named Escherichiacoli BL21(DE3) / pET28a(+)-ACS-K33, and has been sent to the China Center for Type Culture Collection (CCTCC) for preservation. The preservation number is CCTCC NO: M 20221558, the preservation time is October 11, 2022, and the preservation address is: Wuhan University, Wuhan, China.

[0012] The method for constructing an engineered strain expressing an acyl-CoA synthetase mutant of the present invention comprises the following steps:

[0013] First, the structure of acyl-CoA synthetase from Thermus thermophilus was used as a template, and Swiss-Model was used to perform homology modeling on the structure of acyl-CoA synthetase. A semi-rational design strategy was adopted, and non-completely conserved amino acid residues near the catalytic active center were selected as candidate sites. The top 50 mutation designs with free energy were obtained through Funclib site-specific analysis and free energy calculation. All of them were molecularly docked using AutoDockVina, and mutation designs with higher affinity to the substrate adenosine palmitate (AMP-PAL) were selected to determine the target amino acid for mutation. The whole gene was synthesized and connected to the expression vector pET-28a(+), and the expression host was Escherichia coliBL21(DE3), thereby obtaining an engineered strain containing the mutant gene of the present invention.

[0014] The expression plasmid vector described in the above construction method includes pCold, pET15, pET22 or pET28, etc.

[0015] The host bacteria in the above construction method include E. coli BL21 (DE3), E. coli DH5α, E. coli JM109 or E. coli Rosetta and the like.

[0016] The acyl-CoA synthetase mutant of the present invention can be obtained by fermenting the engineered strain.

[0017] The application of the acyl-CoA synthetase mutant of the present invention is to apply the acyl-CoA synthetase mutant to an in vitro diagnostic kit for detecting free fatty acids by enzymatic method.

[0018] When palmitic acid was used as the substrate, the specific enzyme activity of the mutant increased by 4.4 times at 37°C and pH 7.0. While the specific activity was increased, the stability of the mutant was also improved. At 4°C and pH 7.0, the stability of the mutant was increased by 22.2% compared with the original enzyme and 27.3% compared with the commercial enzyme. In the enzymatic free fatty acid in vitro diagnostic kit, the linear fitting curve R of the same unit mutant enzyme protein for serum sample detection value and commercial enzyme detection value 2 The mutant has potential application value in the in vitro diagnostic kit for enzymatic detection of free fatty acids.

[0019] The present invention measures and compares the specific enzyme activity, optimum pH, optimum temperature, stability, etc. of the mutant protein and the original wild-type protein. The measurement results show that when palmitic acid is used as a substrate, the specific enzyme activity of the mutant obtained by the present invention is increased by 4.4 times relative to the starting enzyme ACS-K. While the specific activity is improved, the stability of the mutant is also improved. Under the conditions of 37°C and pH 7.0, the half-life of the mutant is extended by 22.2% relative to the starting enzyme ACS-K. Compared with the starting enzyme, the optimum pH of the mutant is changed from 7.0 to 7.5, and the adaptability to alkaline environment is increased; the optimum temperature is changed from the original 45°C to 40°C, and the adaptability to low temperature environment (25-35°C) is increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The SDS-PAGE profile of the purified mutant protein and the starting enzyme ACS-K is shown below: Figure 1 In the figure, 1 is the supernatant of ACS_K, 2 is the purified permeate, 3 is the pure enzyme of ACS-K, 4 is the supernatant of ACS-K33, 5 is the purified permeate, 6 is the pure enzyme of ACS-K33, and M is a protein marker.

[0021] Figure 2 Where a is the optimum pH and b is the optimum temperature.

[0022] Figure 3 Temperature stability at 4°C and pH 7.0. DETAILED DESCRIPTION

[0023] The implementation methods in the following embodiments are all conventional methods unless otherwise specified.

[0024] (I) Construction of an expression strain containing the acyl-CoA synthetase mutant gene of the present invention

[0025] 1. Selection of mutation sites of acyl-CoA synthetase gene

[0026] Based on sequence alignment, ACS-K is most similar to ttLC-FACS (PDB code: 1V26) from Thermus thermophilus, with an amino acid sequence identity of 40%. The structure of ttLC-FACS was used as a template to simulate the structure of acyl-CoA synthetase ACS-K using Swiss-Model.

[0027] According to the simulated structure and multiple sequence alignment information, non-completely conserved amino acid residues near the catalytic active center were selected as candidate sites. The free energy ranking was obtained by site-specific analysis and free energy calculation using Funclib (https: / / funclib.weizmann.ac.il / bin / steps). AutoDockVina was used to perform molecular docking energy simulation between the candidate enzymes with higher free energy rankings and the intermediate substrate molecule adenosine palmitate (AMP-PAL) in the catalytic reaction. Mutation designs with higher affinity for AMP-PAL were selected to determine the target amino acids for mutation, which were serine S at site 311, asparagine N at site 314, glutamate E at site 436, arginine R at site 438, and glutamine Q at site 479. The mutation directions were as follows: serine S at site 311 was replaced by alanine A, asparagine N at site 314 was replaced by serine S, glutamate E at site 436 was replaced by methionine M, arginine R at site 438 was replaced by histidine H, and glutamine Q at site 479 was replaced by glutamate E.

[0028] 2. Construction of genetically engineered strains producing acyl-CoA synthetase mutations

[0029] The acyl-CoA synthetase ACS-K mutant gene in step 1 is fully synthesized and connected to the expression vector pET-28a(+), the cloning sites are NdeI and XhoI, and the expression host is Escherichia coliBL21(DE3), to obtain the engineered strain Escherichia coliBL21(DE3) / pET-28a(+)-ACS-K33 containing the mutant gene of the present invention.

[0030] The strain Escherichia coli BL21 (DE3) / pET28a (+) -ACS-K33 of the present invention has been sent to the China Center for Type Culture Collection (CCTCC) for preservation, with the preservation number being CCTCC NO: M 20221558, the preservation date being October 11, 2022, and the preservation address being: Wuhan University, Wuhan, China.

[0031] (II) Expression and protein purification of genetically engineered bacteria containing the acyl-CoA synthetase mutation of the present invention

[0032] The engineered strain Escherichia coli BL21 (DE3) / pET-28a (+) -ACS-K33 obtained in (a) was inoculated into 400 mL LB liquid culture medium containing ampicillin, and cultured at 37°C and 200 rpm until OD600 was 0.6 (UNICO UV2102 UV-visible spectrophotometer, with LB culture medium as blank); IPTG with a final concentration of 0.25 mM was added for induction, and cultured for 17 hours at 16°C and 120 rpm; the cells were collected by centrifugation at 4°C and 8000 g, 3 times the volume of the bacterial solution was added, and the cells were broken by ultrasonication at 350 W ice bath for 30 min, and the supernatant was collected by centrifugation at 12000 g to obtain a crude enzyme solution. The whole bacterial protein SDS-PAGE showed that the protein expression amount accounted for more than 90% of the whole bacterial protein. The crude enzyme solution was purified by Ni-NTA column chromatography, the imidazole concentration in the eluent was 200 mM, and the elution was 3 column volumes. The obtained protein reached the SDS-PAGE purity after detection.

[0033] (III) Detection of the optimal pH and temperature of the acyl-CoA synthetase mutant of the present invention

[0034] The principle of the acyl-CoA synthetase assay reaction is as follows:

[0035]

[0036]

[0037]

[0038] The reaction system includes two steps:

[0039] The first step reaction system is 1 mL, including 0.2M K2HPO4-KH2PO4 (pH7.0), 10mM ATP, 10mM MgCl2, 1mM Palmitic acid, 5% (w / v) Triton X-100, and 10mM CoA.

[0040] The reaction system of the second step was 2 mL, including 0.2 M K2HPO4-KH2PO4 (pH 7.0), 20 mM N-ethylmaleimide (NEM), 15 mM 4-AA, 15 mM MEHA, 85 U / mL POD, and 125 U / mL ACOD.

[0041] First, preheat 1 ml of the first step reaction mixture at 37°C for 5 minutes, then add the enzyme solution. After reacting for 10 minutes, add the second step reaction solution and mix well, and react for 5 minutes. The experimental group performed 3 parallel experiments, and the control group used buffer instead of enzyme solution. The control group was set to zero and the light absorption value at 550nm was measured. Enzyme activity (U) is defined as: the amount of enzyme required to convert 1 μmol Palmitic acid into acyl-CoA per minute at 37°C.

[0042] The test results show that when palmitic acid is used as a substrate, the optimum pH of the mutant obtained by the present invention is 7.5, and the enzyme can show more than 70% enzyme activity in the pH range of 6.0-9.0. The optimum temperature of the mutant is 40°C, and the enzyme can show more than 60% enzyme activity in the range of 25°C-50°C.

[0043] (IV) Detection of the stability of the acyl-CoA synthetase mutants of the present invention at 4°C

[0044] The mutant enzyme was stored at 4°C and pH 7.0, and samples were taken every 2 or 3 days. The initial enzyme activity was taken as 100%, and the residual enzyme activity after a certain period of time was calculated according to the following formula: residual enzyme activity = (initial enzyme activity - loss enzyme activity) / initial enzyme activity × 100%. The measurement results showed that the half-life of the mutant obtained by the present invention under this condition was 22 days, which was 22.2% higher than that of the original enzyme.

[0045] (V) Application of the Acyl-CoA Synthetase Mutant of the Present Invention in a NEFA Detection Kit

[0046] The R1 reaction solution in the free fatty acid detection kit includes: phosphate buffer, coenzyme A (CoA), adenosine-5'-dibasic triphosphate (ATP), 4-aminoantipyrine, ascorbic acid oxidase; the R2 reaction solution includes: acyl-CoA oxidase (ACOD), peroxidase (POD), 3-methyl-N-ethyl-N-aniline (MEHA). Among them, the amount of ACS in the R1 reaction solution is 2KU / L-4KU / L, and the amount of ACOD in the R2 reaction solution is 500mg / L-800mg / L. In the entire detection system, sample: R1: R2 = 4:200:50.

[0047] The detection process of the free fatty acid detection kit includes: first, incubate the free fatty acid standard and R1 reaction solution in a 37℃ water bath for 5 minutes, detect the absorbance value OD1 at the main wavelength of 546nm, then add R2 reaction solution and mix, incubate in a 37℃ water bath for 5 minutes, and detect the absorbance OD2 at the secondary wavelength of 700nm. Finally, calculate the difference between the absorbance value of the secondary wavelength and the absorbance value of the main wavelength. The experimental group performed 2 parallel experiments, and the control group replaced the free fatty acid standard with pure water.

[0048] The test results show that when the same unit amount of enzyme is added, the mutant protein obtained by the present invention has a comparable reactivity to the free fatty acid standard compared with the commercial enzyme. When the mutant obtained by the present invention is used in combination with the commercial acyl-CoA oxidase, it meets the third-party quality control test standard (deviation <15%). The R value of the linear fitting curve of the test results of serum samples and the test results of the commercial enzyme obtained by the mutant obtained by the present invention is 2 is 0.998.

Claims

1. An acyl-CoA synthetase mutant, characterized in that: The amino acid sequence of the acyl-CoA synthetase mutant is the amino acid sequence of the acyl-CoA synthetase shown in SEQ ID No: 1, in which the serine at position 311 is mutated to alanine, the asparagine at position 314 is mutated to serine, the glutamic acid at position 436 is mutated to methionine, the arginine at position 438 is mutated to histidine, and the glutamine at position 479 is mutated to glutamic acid.

2. A gene encoding the acyl-CoA synthetase mutant according to claim 1, characterized in that: Its nucleotide sequence is shown in SEQ ID No:

2.

3. An engineered strain expressing the acyl-CoA synthetase mutant according to claim 1, characterized in that: The engineering strain is classified and named Escherichia coli BL21(DE3) / pET28a(+)-ACS-K33, and has been sent to the China Center for Type Culture Collection (CCTCC) for preservation. The preservation number is CCTCC NO: M 20221558. The preservation time is October 11, 2022. The preservation address is: Wuhan University, Wuhan, China.

4. A use of the acyl-CoA synthetase mutant according to claim 1, characterized in that: The acyl-CoA synthetase mutant is used to prepare an in vitro diagnostic kit for detecting free fatty acids by enzymatic method.

5. The use according to claim 4, characterized in that: Taking palmitic acid as substrate, the optimum pH of the acyl-CoA synthetase mutant is 7.5 and the optimum temperature is 40°C.

6. The use according to claim 4, characterized in that: The acyl-CoA synthetase mutant can show more than 70% enzyme activity in the pH range of 6.0-9.0; the acyl-CoA synthetase mutant can show more than 60% enzyme activity in the pH range of 25°C-50°C.

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