An acyl-coa synthetase mutant acs01-des4, its coding gene, expression strain and application thereof

Abstract

The application discloses an acyl-CoA synthetase mutant ACS01-Des4, a coding gene thereof, an expression strain thereof and application of the mutant, and is based on acyl-CoA synthetase from a marine uncultured microbial source. After a mutation site is designed by PROSS, a mutant gene is obtained through whole gene synthesis. After the expression of an engineering bacterium containing the mutant plasmid is induced, the acyl-CoA synthetase with improved stability and specific enzyme activity is obtained. Under the condition of 37 DEG C, the stability of the mutant is improved by 3 times. When palmitic acid is used as a substrate, the specific enzyme activity of the mutant is improved by 1.14 times. The mutant has potential application value in an enzyme method for detecting free fatty acid in vitro diagnostic kits.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to an acyl-CoA synthetase mutant ACS01-Des4, its encoding gene, an expression strain and applications thereof. Background Art

[0002] Free fatty acids (NEFAs) are a class of organic acids, also known as non-esterified fatty acids. NEFAs are one of the substances broken down from neutral fats, primarily composed of oleic acid, palmitic acid, and linoleic acid. Most NEFAs are bound to albumin and present in the blood. NEFAs are directly linked to oxidative stress, which can contribute to the development of diabetes by damaging pancreatic cells and reducing peripheral tissue sensitivity to insulin. NEFA concentrations can be elevated in conditions such as diabetes, severe liver disease, and hyperthyroidism. Clinical NEFA testing can be helpful in assisting in the diagnosis of these conditions. Currently, the main methods for clinically detecting NEFAs are gas chromatography and enzymatic methods. Enzymatic methods are simple and rapid, and are particularly amenable to batch analysis using fully automated 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. The resulting 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 resulting compound has maximum absorption at a specific wavelength, and the NEFA content is determined by measuring the absorbance value.

[0004] Acyl-CoA synthetase (EC 6.2.1.3, long-chain-fatty-acid-CoA ligase) 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 ACS01-Des4, its encoding gene, expression strain and application thereof. The present invention is based on the acyl-CoA synthetase ACS01 from uncultured marine microorganisms, and obtains the mutant gene by whole-gene synthesis after designing the mutation site through PROSS. After the engineered bacteria containing the mutant plasmid are induced to express, an acyl-CoA synthetase with improved stability is obtained. At 37°C, the stability of the mutant is increased by 3 times. In the in vitro diagnostic kit for the enzymatic detection of free fatty acids, the linear fitting curve R of the same unit mutant enzyme detection value for serum samples and the commercial enzyme detection value is 2 The mutant has potential application value in in vitro diagnostic kits for enzymatic detection of free fatty acids.

[0006] The acyl-CoA synthetase mutant of the present invention is abbreviated as ACS01-Des4, and its amino acid sequence is shown in SEQ ID NO: 1. Specifically, in the amino acid sequence of acyl-CoA synthetase ACS01, the histidine at position 26 is mutated to tyrosine, the glycine at position 30 is mutated to valine, the leucine at position 43 is mutated to lysine, the methionine at position 46 is mutated to phenylalanine, the glutamic acid at position 51 is mutated to histidine, the aspartic acid at position 59 is mutated to tyrosine, the tyrosine at position 68 is mutated to histidine, the asparagine at position 72 is mutated to histidine, the valine at position 77 is mutated to proline, the leucine at position 121 is mutated to phenylalanine, the alanine at position 126 is mutated to valine, and the methionine at position 137 is mutated to phenylalanine. The glutamic acid at position 1 was mutated to histidine, the alanine at position 138 was mutated to threonine, the valine at position 143 was mutated to leucine, the phenylalanine at position 150 was mutated to histidine, the glycine at position 157 was mutated to alanine, the lysine at position 185 was mutated to proline, the serine at position 192 was mutated to glutamine, the serine at position 225 was mutated to alanine, the isoleucine at position 230 was mutated to leucine, the serine at position 231 was mutated to threonine, the isoleucine at position 239 was mutated to methionine, the serine at position 242 was mutated to leucine, and the asparagine at position 244 was mutated to glutamine. Serine at position 260 mutated to alanine, serine at position 269 mutated to alanine, phenylalanine at position 274 mutated to leucine, cysteine ​​at position 275 mutated to valine, glycine at position 283 mutated to methionine, alanine at position 287 mutated to proline, serine at position 293 mutated to aspartic acid, valine at position 295 mutated to phenylalanine, serine at position 305 mutated to threonine, serine at position 308 mutated to cysteine, glutamine at position 316 mutated to alanine, and alanine at position 333 mutated to valine. The amino acids at position 343 were mutated to glutamine, the glycine at position 344 was mutated to alanine, the methionine at position 350 was mutated to lysine, the alanine at position 355 was mutated to cysteine, the isoleucine at position 372 was mutated to phenylalanine, the alanine at position 386 was mutated to proline, the methionine at position 418 was mutated to lysine, the methionine at position 456 was mutated to lysine, the glycine at position 478 was mutated to threonine, the methionine at position 528 was mutated to valine, the arginine at position 530 was mutated to lysine, and the serine at position 550 was 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 gene encoding 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 Escherichia coli BL21(DE3) / pET-28a(+)-ACS01-Des4 has been deposited in the China Center for Type Culture Collection (CCTCC) with the deposit number M 20242459 on November 7, 2024. The deposit 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 ACS01 from Thermus thermophilus, which has the highest amino acid sequence consistency with ACS01, was used as a template to perform homology modeling on the structure of acyl-CoA synthetase using Swiss-Model. Using PROSS, an automated design method based on sequence and structure, small molecule ligands and interactive chains are specified in PROSS. The amino acid positions around these ligands and those in contact with the chains will not mutate, and their conformations will remain fixed. Completely conserved amino acids are specified, and they also remain fixed during the mutation process. An automated algorithm based on atomic Rosetta modeling and phylogenetic sequence information obtains 9 mutation designs with increasing mutation rates. They are predicted to be soluble in Escherichia coli, and mutation designs with predicted results of solubleness are selected to determine the target amino acids for mutation. The entire gene is synthesized and connected into the expression vector pET-28a(+). The expression host is Escherichia coli BL21(DE3) to obtain an engineered strain containing the mutant gene of the present invention.

[0014] The expression plasmid vectors described in the above construction method include 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 et al.

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

[0017] Application of the acyl-CoA synthetase mutant of the present invention in preparing a free fatty acid detection reagent.

[0018] When palmitic acid was used as the substrate, the stability of the mutant was increased by 3 times at 37°C and pH 7.0, and the specific enzyme activity of the mutant was increased by 1.14 times. In the in vitro diagnostic kit for the enzymatic detection of free fatty acids, the linear fitting curve R of the same unit mutant enzyme protein for serum sample detection value and commercial enzyme detection value was 2 The mutant has potential application value in in vitro diagnostic kits for enzymatic detection of free fatty acids.

[0019] The present invention measured and compared the specific enzymatic activity, optimal pH, optimal temperature, and stability of the mutant protein with those of the original wild-type protein. The results showed that at 37°C, the mutant's stability was three times greater. Using palmitic acid as a substrate, the mutant's specific enzymatic activity increased 1.14 times. Compared to the original enzyme, the mutant's optimal pH increased from 8.0 to 7.0, and its optimal temperature increased from 30°C to 45°C. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The following is the SDS-PAGE profile of the purified mutant protein and the starting enzyme ACS01: Figure 1 1 is the purified permeate of ACS01, 2 is the supernatant of ACS01, 3 is the pure enzyme of ACS01, 4 is the - Des4 is purified flow-through, 5 is ACS01 - Des4 is the supernatant of the crushing, 6 is ACS01 - Des4 pure enzyme, M is a protein marker.

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

[0022] Figure 3 is the half-life of the starting enzyme ACS01 and the mutant ACS01-Des4 at 37°C. DETAILED DESCRIPTION

[0023] The implementation methods in the following examples 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 in acyl-CoA synthetase genes

[0026] Based on sequence alignment, ACS01 is most similar to the acyl-CoA synthetase 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 ACS01 using Swiss-Model.

[0027] PROSS (https: / / pross.weizmann.ac.il / ), an automated design method based on sequence and structure, was used. Small molecule ligands, interactive chains, and some completely conserved amino acids were specified in PROSS. An automated algorithm based on atomic Rosetta modeling and phylogenetic sequence information obtained 9 mutation designs with increasing mutation rates. These designs were used for E. coli solubility prediction. The mutation designs predicted to be soluble were selected to determine the target amino acids for mutation, including histidylamide at position 26 to tyrosine, glycine at position 30 to valine, leucine at position 43 to lysine, methionine at position 46 to phenylalanine, and methionine at position 50 to phenylalanine. The glutamic acid at position 1 mutated to histidine, the aspartic acid at position 59 mutated to tyrosine, the tyrosine at position 68 mutated to histidine, the asparagine at position 72 mutated to histidine, the valine at position 77 mutated to proline, the leucine at position 121 mutated to phenylalanine, the alanine at position 126 mutated to valine, the glutamic acid at position 137 mutated to histidine, the alanine at position 138 mutated to threonine, the valine at position 143 mutated to leucine, the phenylalanine at position 150 mutated to histidine, the glycine at position 157 mutated to alanine, the lysine at position 185 mutated to proline, the serine at position 192 mutated to glutamine, the serine at position 225 mutated to alanine, and the Isoleucine at position 30 mutated to leucine, serine at position 231 mutated to threonine, isoleucine at position 239 mutated to methionine, serine at position 242 mutated to leucine, asparagine at position 244 mutated to alanine, serine at position 260 mutated to alanine, serine at position 269 mutated to alanine, phenylalanine at position 274 mutated to leucine, cysteine ​​at position 275 mutated to valine, glycine at position 283 mutated to methionine, alanine at position 287 mutated to proline, serine at position 293 mutated to aspartic acid, valine at position 295 mutated to phenylalanine, serine at position 305 mutated to threonine, and serine at position 308 mutated to threonine. The 316th amino acid sequence of the glutamine residue was mutated to cysteine, the 316th amino acid sequence of the glutamine residue was mutated to alanine, the 333th amino acid sequence of the alanine residue was mutated to valine, the 343th amino acid sequence of the alanine residue was mutated to glutamine, the 344th amino acid sequence of the glycine residue was mutated to alanine, the 350th amino acid sequence of the methionine residue was mutated to lysine, the 355th amino acid sequence of the alanine residue was mutated to cysteine, the 372th amino acid sequence of the isoleucine residue was mutated to phenylalanine, the 386th amino acid sequence of the alanine residue was mutated to proline, the 418th amino acid sequence of the methionine residue was mutated to lysine, the 456th amino acid sequence of the methionine residue was mutated to lysine, the 478th amino acid sequence of the threonine residue was mutated to threonine, the 528th amino acid sequence of the methionine residue was mutated to valine, the 530th amino acid sequence of the arginine residue was mutated to lysine, and the 550th amino acid sequence of the serine residue was mutated to glutamic acid.

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

[0029] The acyl-CoA synthetase ACS01 mutant gene in step 1 was synthesized and ligated into the expression vector pET-28a(+). The cloning site was Nde I and Xho I, expression host is Escherichia coli BL21 (DE3), to obtain an engineered strain containing the mutant gene of the present invention Escherichia coli BL21(DE3) / pET-28a(+)-ACS01-Des4.

[0030] The engineered strain expressing the acyl-CoA synthetase mutant of the present invention is classified and named Escherichia coli BL21(DE3) / pET-28a(+)-ACS01-Des4 has been deposited in the China Center for Type Culture Collection (CCTCC) with the deposit number M 20242459 on November 7, 2024. The deposit address is Wuhan University, Wuhan, China.

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

[0032] The engineered strain obtained in (1) Escherichia coli BL21(DE3) / pET-28a(+)-ACS01-Des4 was inoculated into 400 mL of LB liquid medium containing ampicillin and cultured at 37°C and 200 rpm to an OD600 of 0.6 (using a UNICO UV2102 UV-visible spectrophotometer, with the LB medium as a blank). IPTG was added for induction at a final concentration of 0.25 mM and cultured for 16 hours at 16°C and 120 rpm. The cells were harvested by centrifugation at 8000 g at 4°C, and three volumes of K2HPO4-KH2PO4 (pH 7.0) buffer were added. Cells were disrupted by sonication at 350 W for 30 minutes in an ice bath, and the supernatant was collected by centrifugation at 12000 g to obtain a crude enzyme solution. SDS-PAGE analysis of the whole-cell protein showed that the protein expressed accounted for over 90% of the total protein. The crude enzyme solution was purified by Ni-NTA column chromatography. The imidazole concentration in the elution buffer was 200 mM, and the elution was performed over 3 column volumes. The obtained protein was tested for purity by SDS-PAGE.

[0033] (III) Detection of the Optimal pH and Optimal Temperature of the Acyl-CoA Synthetase Mutant Containing the Present Invention

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

[0035]

[0036] The reaction system includes two steps:

[0037] 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 Palmitic acid, 5% (w / v) Triton X-100, and 10 mM CoA.

[0038] 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.

[0039] First, preheat 1 mL of the first-step reaction mixture at 37°C for 5 minutes before adding the enzyme solution. After reacting for 10 minutes, add the second-step reaction solution, mix thoroughly, and react for 5 minutes. Three replicates were performed for the experimental group, while the control group used buffer instead of enzyme solution. Zero the control group and measure absorbance at 550 nm. Enzyme activity (U) was defined as the amount of enzyme required to convert 1 μmol of palmitic acid to acyl-CoA per minute at 37°C.

[0040] Test results showed that when palmitic acid was used as a substrate, the mutant obtained in the present invention had an optimal pH of 7.0, and the enzyme exhibited over 85% of its activity within the pH range of 6.5-7.5. The mutant also had an optimal temperature of 45°C, and the enzyme exhibited over 80% of its activity within the 40°C-50°C range.

[0041] (IV) Application of the Acyl-CoA Synthetase Mutant of the Present Invention in a NEFA Detection Kit

[0042] The R1 reaction solution in the free fatty acid assay kit contains (final concentrations): pH 7.0 phosphate buffer, 0.2-0.5 mmol coenzyme A (CoA), 2-5 mmol adenosine-5'-triphosphate disodium hydrate (ATP), 2-5 mmol 4-aminoantipyrine, and 2-5 kU / L ascorbate oxidase. The R2 reaction solution contains 2-5 kU / L acyl-CoA oxidase (ACOD), 2-5 kU / L peroxidase (POD), and 1-5 mmol 3-methyl-N-ethyl-N-aniline (MEHA). The amount of ACS in the R1 reaction solution ranges from 2 kU / L to 4 kU / L, and the amount of ACOD in the R2 reaction solution ranges from 500 mg / L to 800 mg / L. The sample volume ratio of the entire assay system is R1:R2 = 4:200:50.

[0043] The free fatty acid assay kit involves incubating the free fatty acid standard with the R1 reaction solution in a 37°C water bath for 5 minutes. The absorbance (OD1) is measured at a primary wavelength of 546 nm. The R2 reaction solution is then added and mixed thoroughly. The mixture is then incubated in a 37°C water bath for 5 minutes. The absorbance (OD2) is measured at a secondary wavelength of 700 nm. Finally, the difference between the secondary and primary wavelength absorbances is calculated. Two parallel experiments were performed in the experimental group, while the control group used pure water instead of the free fatty acid standard.

[0044] The test results showed that when the same unit amount of enzyme was added, the mutant protein obtained by the present invention had a comparable reactivity to the free fatty acid standard compared to the commercial enzyme. When the mutant obtained by the present invention was used in combination with the commercial acyl-CoA oxidase, it met the third-party quality control test standards (deviation <15%). The R value of the linear fitting curve of the test results of the mutant obtained by the present invention on serum samples and the test results of the commercial enzyme was 2 is 0.998.

Claims

1. An acyl-CoA synthetase mutant, characterized in that: The acyl-CoA synthetase mutant is abbreviated as ACS01-Des4, and its amino acid sequence is shown in SEQ ID NO:

1.

2. The gene encoding the acyl-CoA synthetase mutant according to claim 1, characterized in that: The nucleotide sequence of the coding gene is shown in SEQ ID NO:

2.

3. A plasmid containing the coding gene according to claim 2.

4. A strain expressing the acyl-CoA synthetase mutant according to claim 1, characterized in that: The strain is classified and named Escherichia coli BL21(DE3) / pET-28a(+)-ACS01-Des4, and has been sent to the China Center for Type Culture Collection (CCTCC) for preservation. The preservation number is CCTCC NO: M 20242459, the preservation date is November 7, 2024, and the preservation address is Wuhan University, Wuhan, China.

5. Use of the acyl-CoA synthetase mutant according to claim 1 in preparing a free fatty acid detection reagent.

6. The use according to claim 5, characterized in that: When palmitic acid is used as the substrate, the reaction pH is 5.0-8.5 and the reaction temperature is 25°C-55°C.

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