An acyl-coa synthetase mutant, its coding gene, expression strain and application thereof

Abstract

The application discloses an acyl-CoA synthetase mutant, a coding gene thereof, an expression strain and application thereof. The application is based on an acyl-CoA synthetase from a marine uncultured microbial source, a mutation site is designed by using Funclib, and then a whole gene is synthesized to obtain a mutant gene. After expression of an engineering bacterium containing the mutant plasmid, an acyl-CoA synthetase with improved specific enzyme activity and stability is obtained. When palmitic acid is used as a substrate, the specific enzyme activity of the mutant is improved by 7.0 times, and the mutant has potential application value in an enzyme method for detecting free fatty acids in vitro.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an acyl-CoA synthase mutant, its encoding gene, expression strain, and its applications. Background Technology

[0002] Free fatty acids (NEFAs) are a class of organic acids, also known as non-esterified fatty acids. NEFAs are one of the substances derived from the breakdown of neutral fats, mainly composed of oleic acid, palmitic acid, and linoleic acid. Most NEFAs are bound to albumin and exist in the blood. NEFAs are directly related to oxidative stress, which can lead to diabetes by damaging pancreatic islet cells and reducing the sensitivity of peripheral tissues to insulin. NEFA concentrations can increase due to diseases such as diabetes, severe liver damage, and hyperthyroidism. Clinical detection of NEFAs is helpful in the diagnosis of these diseases. Currently, the main methods for detecting NEFAs clinically are gas chromatography and enzymatic methods. Enzymatic methods are particularly suitable for batch analysis in fully automated biochemical analyzers due to their simplicity, speed, and suitability for such procedures.

[0003] The main principle of the free fatty acid assay kit is that, in the presence of coenzyme A (CoA) and ATP, NEFA in the sample is acted upon by acyl-CoA synthase (ACS). The generated acyl-CoA is then oxidized by acyl-CoA oxidase (ACOD), simultaneously generating hydrogen peroxide. 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 resulting compound exhibits maximum absorption at a specific wavelength. By measuring the absorbance value, the NEFA content can be determined.

[0004] Acyl-CoA synthase (EC6.2.1.3, long-chain-fatty-acid-CoAligase) is a key rate-limiting enzyme in enzymatic methods for the detection of NEFA, determining the sensitivity and precision of the enzyme assay. Acyl-CoA synthase 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] This invention provides an acyl-CoA synthase mutant, its encoding gene, expression strain, and its applications. Based on acyl-CoA synthase derived from uncultured marine microorganisms, this invention uses Funclib to design mutation sites and then synthesizes the entire gene to obtain the mutant gene. After induction expression in engineered bacteria containing the mutant plasmid, an acyl-CoA synthase with increased specific activity and improved stability is obtained. When palmitic acid is used as a substrate, the mutant's specific activity is increased by 7.0 times. In an in vitro diagnostic kit for the enzymatic detection of free fatty acids, the linear fitting curve R between the same unit of the mutant enzyme and the commercial enzyme for serum sample detection is shown. 2 With a value of 0.995, this mutant has potential application value in in vitro diagnostic kits for the enzymatic detection of free fatty acids.

[0006] The present invention relates to a mutant acyl-CoA synthase, the amino acid sequence of which is as shown in SEQ ID No: 1, wherein asparagine at position 314 is mutated to serine, arginine at position 315 is mutated to proline, arginine at position 438 is mutated to lysine, and glutamine at position 479 is mutated to glutamic acid.

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

[0008] The coding gene for the acyl-CoA synthase mutant of the present invention has the nucleotide sequence shown in SEQ ID No: 2.

[0009] The mutant plasmid of this invention is the encoding gene of the acyl-CoA synthase mutant as described in SEQ ID No: 2.

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

[0011] The engineered strain expressing the acyl-CoA synthase mutant of this invention, classified as Escherichiacoli BL21(DE3) / pET28a(+)-ACS-K1, has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCCNO: M20221867, on December 5, 2022, at Wuhan University, Wuhan, China.

[0012] The present invention discloses a method for constructing an engineered strain expressing an acyl-CoA synthase mutant, comprising the following steps:

[0013] First, using the structure of acyl-CoA synthase from *Thermus thermophilus* as a template, homology modeling of the acyl-CoA synthase structure was performed using the Swiss-Model. A semi-rational design strategy was employed, selecting non-completely conserved amino acid residues near the catalytic active site as candidate sites. Funclib site-specificity analysis and free energy calculations yielded the top 50 free energy mutation designs, all of which underwent molecular docking using AutoDockVina. Mutations with high affinity for the substrate adenosine palmitate (AMP-PAL) were selected to determine the target amino acid. The entire gene was synthesized and ligated into the expression vector pET-28a(+), with the expression host being *Escherichiacoli* BL21(DE3), resulting in an engineered strain containing the mutant gene of this invention.

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

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

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

[0017] The application of the acyl-CoA synthase mutant of the present invention is to prepare a detection reagent for the enzymatic detection of free fatty acids using the acyl-CoA synthase mutant.

[0018] When palmitic acid was used as a substrate, the specific enzyme activity of the mutant was increased by 7.0 times at 37℃ and pH 7.0. In an in vitro diagnostic kit for the enzymatic detection of free fatty acids, the linear fitting curve R between the detection values ​​of the same unit of mutant enzyme protein on serum samples and those of the commercial enzyme was [not specified]. 2 The value was 0.995. This mutant has potential application value in enzymatic detection kits for free fatty acids in vitro.

[0019] This invention measured and compared the specific enzyme activity, optimal pH, optimal temperature, and stability of the mutant protein and the original wild-type protein. The results showed that, using palmitic acid as a substrate, the specific enzyme activity of the mutant obtained in this invention was 7.0 times higher than that of the starting enzyme ACS-K. Compared to the starting enzyme, the optimal pH of the mutant remained at 7.5, and its adaptability to alkaline environments was increased; the optimal temperature changed from 45℃ to 40℃. Attached Figure Description

[0020] Figure 1SDS-PAGE spectra of the purified mutant protein and the starting enzyme ACS-K: Figure 1 In the diagram, 1 is the ACS-K lysis supernatant, 2 is the purified permeate, 3 is the purified ACS-K enzyme, 4 is the ACS-K1 lysis supernatant, 5 is the purified permeate, 6 is the purified ACS-K1 enzyme, and M is the protein marker.

[0021] Figure 2 In this context, a represents the optimal pH, and b represents the optimal temperature. Detailed Implementation

[0022] Unless otherwise specified, the implementation methods in the following embodiments are all conventional methods.

[0023] (I) Construction of expression strains containing the acyl-CoA synthase mutant gene of the present invention

[0024] 1. Selection of mutation sites in acyl-CoA synthase genes

[0025] Based on sequence alignment, ACS-K is most similar to ttLC-FACS (PDB code: 1V26), an acyl-CoA synthase from *Thermus thermophilus*, with an amino acid sequence identity of 40%. Using the structure of ttLC-FACS as a template, the structure of acyl-CoA synthase ACS-K was simulated using the Swiss-Model method.

[0026] Based on simulated structures and multiple sequence alignment information, non-completely conserved amino acid residues near the catalytic active site were selected as candidate sites. Free energy rankings were obtained through site-specific analysis and free energy calculations using Funclib (https: / / funclib.weizmann.ac.il / bin / steps). AutoDockVina was used to simulate molecular docking energy between candidate enzymes with higher free energy rankings and the intermediate substrate molecule adenosine palmitate (AMP-PAL). Mutation designs with high affinity for AMP-PAL were selected to determine the target amino acids for mutation, namely asparagine N at position 314, arginine R at position 315, arginine R at position 438, and glutamine Q at position 479. The mutation directions were: asparagine N at position 314 replaced by serine S; arginine R at position 315 replaced by proline P; arginine R at position 438 replaced by lysine K; and glutamine Q at position 479 replaced by glutamate E.

[0027] 2. Construction of acyl-CoA synthase mutant genetically engineered strains

[0028] The acyl-CoA synthase ACS-K mutant gene from step 1 was synthesized in its entirety and ligated into the expression vector pET-28a(+). The cloning sites were NdeI and XhoI, and the expression host was EscherichiacoliBL21(DE3), resulting in the engineered strain EscherichiacoliBL21(DE3) / pET28a(+)-ACS-K1 containing the mutant gene of this invention.

[0029] The strain Escherichiacoli BL21(DE3) / pET28a(+)-ACS-K1 of this invention has been deposited at the China Center for Type Culture Collection (CCTCC), with accession number M20221867, on December 5, 2022. The depositary address is China Center for Type Culture Collection, Wuhan University, China.

[0030] (II) Expression and protein purification of genetically engineered bacteria containing the acyl-CoA synthase mutant of the present invention

[0031] The engineered strain Escherichiacoli BL21(DE3) / pET-28a(+)-ACS-K1 obtained in step (I) was inoculated into 400 mL of LB liquid medium containing ampicillin and cultured at 37°C and 200 rpm until the OD600 reached 0.6 (using a UNICOUV2102 UV-Vis spectrophotometer, with LB medium as a blank). Induction was initiated with 0.25 mM IPTG, and the cells were cultured for another 17 hours at 16°C and 120 rpm. Cells were collected by centrifugation at 8000 g at 4°C, and three volumes of K2HPO4-KH2PO4 (pH 7.0) buffer were added. The cells were lysed by sonication at 350 W on ice for 30 min, and the supernatant was collected by centrifugation at 12000 g to obtain the crude enzyme solution. SDS-PAGE analysis of the whole-cell protein showed that the protein expression level was over 90% of the total bacterial protein. The crude enzyme solution was purified by Ni-NTA column chromatography. The imidazole concentration in the elution buffer was 200 mM, and elution was performed for 3 column volumes. The obtained protein was tested and found to be of SDS-PAGE purity.

[0032] (III) Detection of the optimal pH and optimal temperature of the acyl-CoA synthase mutant containing the present invention

[0033] The reaction principle for the detection of acyl-CoA synthase is as follows:

[0034]

[0035] The reaction system consists of two steps:

[0036] The first step reaction system was 1 mL, including 0.2 M K2HPO4-KH2PO4 (pH 7.0), 10 mM ATP, 10 mM MgCl2, 1 mM Palmicacid, 5% (w / v) Triton X-100, and 10 mM CoA.

[0037] The second step reaction system is 2 mL, including 0.2 M K2HPO4-KH2PO4 (pH 7.0), 20 mM MN-ethylmaleimide (NEM), 15 mM 4-AA, 15 mM MEHA, 85 U / mL LPOD, and 125 U / mL ACOD.

[0038] First, preheat 1 ml of the first-step reaction mixture at 37°C for 5 min, then add the enzyme solution and react for 10 min. Next, add the second-step reaction mixture, mix well, and react for 5 min. Three parallel experiments were performed in the experimental group, while the control group used buffer solution instead of enzyme solution. Zero the instrument using the control group and measure the absorbance at 550 nm. Enzyme activity (U) is defined as the amount of enzyme required to convert 1 μmol of Palmiticacid to acyl-CoA per minute at 37°C.

[0039] The test results showed that, using Palmiticacid as a substrate, the optimal pH for the mutant obtained in this invention was 7.5, and the enzyme exhibited over 90% activity within the pH range of 7.0-7.5. The optimal temperature for the mutant was 40℃, and the enzyme exhibited over 60% activity within the temperature range of 35℃-45℃.

[0040] (iv) Application of the acyl-CoA synthase mutant of the present invention in the NEFA detection kit

[0041] The free fatty acid assay kit includes the following in reaction solution R1: phosphate buffer, coenzyme A (CoA), adenosine-5'-hydrated disodium triphosphate (ATP), 4-aminoantipyrine, and ascorbic acid oxidase; and reaction solution R2: acyl-CoA oxidase (ACOD), peroxidase (POD), and 3-methyl-N-ethyl-N-aniline (MEHA). The concentration of ACS in reaction solution R1 is between 2 KU / L and 4 KU / L, and the concentration of ACOD in reaction solution R2 is between 500 mg / L and 800 mg / L. The overall assay system has a sample:R1:R2 ratio of 4:200:50.

[0042] The detection process of the free fatty acid assay kit includes: first, incubating the free fatty acid standard with reaction solution R1 at 37°C for 5 minutes, and then measuring the absorbance value OD1 at the dominant wavelength of 546 nm. Next, adding reaction solution R2 and mixing well, incubating at 37°C for 5 minutes, and then measuring the absorbance OD2 at the secondary wavelength of 700 nm. Finally, calculating the difference between the absorbance value at the secondary wavelength and the absorbance value at the dominant wavelength. Two parallel experiments were performed in the experimental group, while the control group used pure water instead of the free fatty acid standard.

[0043] The test results showed that, when the same unit amount of enzyme was added, the mutant protein obtained by this invention had comparable reactivity to the free fatty acid standard compared to the commercial enzyme. When the mutant obtained by this invention was used in combination with the commercial acyl-CoA oxidase, it met the third-party quality control testing standards (deviation <15%). The R-squared value of the linear fitting curve between the mutant obtained by this invention and the commercial enzyme for serum sample detection results was [not specified in the original text]. 2 It is 0.995.

Claims

1. An acyl-CoA synthase mutant, characterized in that: The amino acid sequence of the acyl-CoA synthase mutant is as shown in SEQ ID No: 1, where asparagine at position 314 is mutated to serine, arginine at position 315 is mutated to proline, arginine at position 438 is mutated to lysine, and glutamine at position 479 is mutated to glutamic acid.

2. The gene encoding the acyl-CoA synthase mutant of claim 1, characterized in that: The nucleotide sequence of the encoding gene is shown in SEQ ID No:

2.

3. An engineered strain expressing the acyl-CoA synthase mutant of claim 1, characterized in that: The engineered strain is classified as Escherichia coli (Escherichia coli). Escherichia coli BL21(DE3) / pET28a(+)-ACS-K1 has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20221867, on December 5, 2022, at Wuhan University, Wuhan, China.

4. The application of the acyl-CoA synthase mutant according to claim 1, characterized in that: A detection reagent for the enzymatic detection of free fatty acids was prepared using the acyl-CoA synthase mutant.

5. The application according to claim 4, characterized in that: When palmitic acid is used as a substrate, the optimal pH is 7.0-7.5 and the optimal temperature is 35℃-45℃.

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