Acyl-coenzyme A synthetase mutant ACS01-Des4 as well as coding gene, expression strain and application of acyl-coenzyme A synthetase mutant ACS01-Des4

Abstract

The invention discloses an acyl coenzyme A synthetase mutant ACS01-Des4 as well as a coding gene, an expression strain and application thereof. According to the invention, acyl-coenzyme A synthetase from marine uncultured microorganisms is taken as a basis, mutation sites are designed through PROSS, and then whole-gene synthesis is carried out, so that mutant genes are obtained. After the engineering bacteria containing the mutant plasmids are subjected to induced expression, the acyl-coenzyme A 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 in-vitro diagnostic kit for detecting free fatty acid by an enzyme method.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an acyl-CoA synthetase mutant ACS01-Des4, a coding gene, 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 cause 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 beneficial for 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 suitable for 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-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 a mutant gene by full gene synthesis after designing mutation sites through PROSS. After induction expression of the engineered bacteria containing the mutant plasmid, an acyl-CoA synthetase with improved stability is obtained. Under 37°C conditions, the stability of the mutant is increased by 3 times. In the enzymatic detection of free fatty acids in vitro diagnostic kit, the linear fitting curve R of the same unit mutant enzyme for serum sample detection value 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, the histidineamide at position 26 in the amino acid sequence of acyl-CoA synthetase ACS01 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 The 260th serine mutated to alanine, the 269th serine mutated to alanine, the 274th phenylalanine mutated to leucine, the 275th cysteine ​​mutated to valine, the 283rd glycine mutated to methionine, the 287th alanine mutated to proline, the 293rd serine mutated to aspartic acid, the 295th valine mutated to phenylalanine, the 305th serine mutated to threonine, the 308th serine mutated to cysteine, the 316th glutamine mutated to alanine, the 333rd alanine mutated to valine, and the 316th glutamine mutated to alanine. The 343rd alanine mutated to glutamine, the 344th glycine mutated to alanine, the 350th methionine mutated to lysine, the 355th alanine mutated to cysteine, the 372th isoleucine mutated to phenylalanine, the 386th alanine mutated to proline, the 418th methionine mutated to lysine, the 456th methionine mutated to lysine, the 478th glycine mutated to threonine, the 528th methionine mutated to valine, the 530th arginine mutated to lysine, and the 550th serine 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 Escherichia coli BL21(DE3) / pET-28a(+)-ACS01-Des4 has been sent to the China Center for Type Culture Collection (CCTCC) for preservation, with the deposit number being CCTCC NO: M 20242459, the deposit date being November 7, 2024, and the deposit address being 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: 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 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 the mutation design predicted to be soluble is selected to determine the target amino acid for mutation. The whole gene is synthesized and connected to the expression vector pET-28a(+), and the expression host is Escherichia coli BL21(DE3), to obtain an engineered strain containing the mutant gene of the present invention.

[0013] The expression plasmid vector described in the above construction method includes pCold, pET15, pET22 or pET28, etc.

[0014] 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.

[0015] The acyl-CoA synthetase mutant of the present invention can be obtained by fermenting the engineered strain.

[0016] The invention discloses an application of the acyl-CoA synthetase mutant in preparing a free fatty acid detection reagent.

[0017] When palmitic acid was used as substrate, at 37°C and pH 7.0, the stability of the mutant increased by 3 times, and the specific enzyme activity of the mutant increased by 1.14 times. In the in vitro diagnostic kit for free fatty acid detection by enzymatic method, 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.

[0018] 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 under the condition of 37°C, the stability of the mutant is increased by 3 times. When palmitic acid is used as a substrate, the specific enzyme activity of the mutant is increased by 1.14 times. Compared with the starting enzyme, the optimum pH of the mutant is changed from 8.0 to 7.0; the optimum temperature is changed from 30°C to 45°C. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 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 the purified flow-through, 5 is ACS01 - Des4 is the supernatant, 6 is ACS01 - Des4 pure enzyme, M is a protein marker.

[0020] Figure 2 Where a is the optimum pH and b is the optimum temperature.

[0021] Figure 3 is the half-life of the starting enzyme ACS01 and the mutant ACS01-Des4 at 37°C. DETAILED DESCRIPTION

[0022] The implementation methods in the following embodiments are all conventional methods unless otherwise specified. (I) Construction of an expression strain containing the acyl-CoA synthetase mutant gene of the present invention

[0023] 1. Selection of mutation sites of acyl-CoA synthetase gene 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 acyl-CoA synthetase ACS01 was simulated using Swiss-Model using the structure of ttLC-FACS as a template.

[0024] 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. The E. coli solubility was predicted, and the mutation designs with predicted soluble results 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 The isoleucine at position 30 mutated to leucine, the serine at position 231 mutated to threonine, the isoleucine at position 239 mutated to methionine, the serine at position 242 mutated to leucine, the asparagine at position 244 mutated to alanine, the serine at position 260 mutated to alanine, the serine at position 269 mutated to alanine, the phenylalanine at position 274 mutated to leucine, the cysteine ​​at position 275 mutated to valine, the glycine at position 283 mutated to methionine, the alanine at position 287 mutated to proline, the serine at position 293 mutated to aspartic acid, the valine at position 295 mutated to phenylalanine, the serine at position 305 mutated to threonine, the serine at position 308 mutated to The 316th glutamine mutated to alanine, the 333rd alanine mutated to valine, the 343rd alanine mutated to glutamine, the 344th glycine mutated to alanine, the 350th methionine mutated to lysine, the 355th alanine mutated to cysteine, the 372th isoleucine mutated to phenylalanine, the 386th alanine mutated to proline, the 418th methionine mutated to lysine, the 456th methionine mutated to lysine, the 478th glycine mutated to threonine, the 528th methionine mutated to valine, the 530th arginine mutated to lysine, and the 550th serine mutated to glutamic acid.

[0025] 2. Construction of genetically engineered strains producing acyl-CoA synthetase mutations The acyl-CoA synthetase ACS01 mutant gene in step 1 was fully synthesized and ligated into the expression vector pET-28a(+). The cloning site was N I and XOt I, the 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.

[0026] 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 sent to the China Center for Type Culture Collection (CCTCC) for preservation, with the deposit number being CCTCC NO: M 20242459, the deposit date being November 7, 2024, and the deposit address being Wuhan University, Wuhan, China. (II) Expression and protein purification of genetically engineered bacteria containing the acyl-CoA synthetase mutation of the present invention

[0027] The engineered strain obtained in (a) Escherichia coli BL21(DE3) / pET-28a(+)-ACS01-Des4 was inoculated into 400 mL LB liquid medium containing ampicillin and cultured at 37℃ 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 culture was continued at 16℃ and 120 rpm for 16 hours; the cells were collected by centrifugation at 4℃ and 8000 g, 3 times the volume of the bacterial liquid K2HPO4-KH2PO4 (pH7.0) buffer was added, and the cells were broken by ultrasonication at 350 W in an ice bath for 30 min, and the supernatant was collected by centrifugation at 12000 g to obtain the crude enzyme solution. SDS-PAGE of the whole bacterial protein 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 elution solution was 200 mM and the elution was performed for 3 column volumes. The obtained protein was tested to reach the purity of SDS-PAGE. (III) Detection of the optimal pH and temperature of the acyl-CoA synthetase mutant of the present invention

[0028] The principle of the acyl-CoA synthetase assay reaction is as follows:

[0029] The reaction system includes two steps: The first step reaction system was 1 mL, including 0.2M K2HPO4-KH2PO4 (pH7.0), 10mM ATP, 10mM MgCl2, 1mM Palmitic acid, 5% (w / v) Triton X-100, and 10 mM CoA.

[0030] 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.

[0031] 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. 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 550 nm was measured. Enzyme activity (U) is defined as the amount of enzyme required to convert 1 μmol of Palmiticacid into acyl-CoA per minute at 37°C.

[0032] 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 7.0, and the enzyme can show more than 85% enzyme activity in the pH range of 6.5-7.5. The optimum temperature of the mutant is 45°C, and the enzyme can show more than 80% enzyme activity in the range of 40°C-50°C. (IV) Application of the acyl-CoA synthetase mutant of the present invention in a NEFA detection kit

[0033] The R1 reaction solution in the free fatty acid detection kit includes (final concentration): pH 7.0 phosphate buffer, 0.2-0.5mmol coenzyme A (CoA), 2-5mmol adenosine-5'-hydrated triphosphate disodium (ATP), 2-5mmol 4-aminoantipyrine, 2-5KU / L ascorbic acid oxidase; the R2 reaction solution includes: 2-5KU / L acyl-CoA oxidase (ACOD), 2-5KU / L peroxidase (POD), 1-5mmol 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 500 mg / L-800 mg / L. In the entire detection system, the sample volume ratio is: R1:R2=4:200:50.

[0034] 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.

[0035] The test results show that when the same unit enzyme amount is added, the mutant protein obtained by the present invention has a comparable reactivity to the free fatty acid standard compared to 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, abbreviated as ACS01-Des4, whose amino acid sequence is shown in SEQ ID NO:

1.

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

2.

3. A mutant plasmid comprising the coding gene according to claim 2.

4. A strain expressing the acyl-CoA synthetase mutant according to claim 1, comprising the mutant plasmid according to claim 3.

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

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

7. The use according to claim 6, 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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