Acyl-coenzyme A oxidase mutant as well as expression strain and application thereof

Abstract

The invention discloses an acyl-coenzyme A oxidase mutant as well as an expression strain and application thereof. On the basis of acyl coenzyme A oxidase derived from marine microorganisms, mutation sites are designed through Saprot and scored, and mutation is introduced through overlapping extension PCR (polymerase chain reaction), so that a mutant gene is obtained. After the engineering bacteria containing the mutant plasmids are subjected to induced expression, the acyl-coenzyme A oxidase with improved stability and specific enzyme activity is obtained. Under the condition of 37 DEG C, the stability of the mutant is improved by 1.3 times. When palmitoyl coenzyme A is used as a substrate, the specific enzyme activity of the mutant is improved to 1.5 times. The mutant has application value in an in-vitro diagnostic kit for detecting free fatty acid by an enzyme method.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an acyl-CoA oxidase mutant ACOD-ID-S106A, its 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. Under normal circumstances, NEFAs account for only 5% to 10% of the total fatty acid content in the human body, with a typical physiological concentration range of 0.1-1.8 mmol / L. 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. Plasma NEFA concentration is used as a diagnostic marker; monitoring changes in glucose and NEFA during metabolism will provide more accurate diagnostic methods and lead to more effective disease prevention and management. The half-life of NEFAs in plasma is 2–3 minutes. High concentrations of NEFAs are highly toxic to healthy cells. NEFA concentrations can increase due to diseases such as diabetes, severe liver damage, and hyperthyroidism, damaging intracellular organelles and leading to enhanced cytokine toxicity. Therefore, NEFA concentration is an important marker for monitoring human glucose and lipid metabolism, assisting in the diagnosis of diabetes, and monitoring obesity, metabolic syndrome, cardiovascular disease, and other malignant diseases. Accurate detection of serum NEFA concentration plays a crucial role in assessing human health. Clinical NEFA testing is beneficial for assisting in the diagnosis of diseases. Currently, the main methods for clinical NEFA detection are gas chromatography and enzymatic methods. Among these, enzymatic methods are simple and rapid, and are particularly suitable for batch analysis in fully automated biochemical analyzers.

[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 then undergoes oxidative condensation with phenol and 4-aminoantipyrine (4-AA) under the action of peroxidase (POD). The resulting compound has maximum absorption at a specific wavelength (500 nm). The NEFA content is determined by measuring the absorbance value.

[0004] Acyl coenzyme A oxidase (EC 1.3.3.6) is one of the key enzymes in the enzymatic detection of NEFA, determining the sensitivity and precision of the detection. ACOD 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 oxidase mutant, ACOD-ID-S106A, its expression strain, and its applications. Based on the marine microbial acyl-CoA oxidase ACOD-ID, this invention designs and scores mutation sites using Saprot, introduces mutations via overlap extension PCR, and obtains the mutant gene. Engineered bacteria containing the mutant gene plasmid, after induction expression, yield an acyl-CoA oxidase with increased stability and specific activity. At 37°C, the specific activity of the mutant is increased by 1.5 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 The value is 0.99913, indicating that this mutant has application value in in vitro diagnostic kits for the enzymatic detection of free fatty acids.

[0006] The acyl-CoA oxidase mutant of the present invention, abbreviated as ACOD-ID-S106A, has the amino acid sequence shown in SEQ ID NO: 1. Specifically, the serine at position 106 of the amino acid sequence of the acyl-CoA oxidase ACOD-ID is mutated to alanine. The amino acid sequence of the acyl-CoA oxidase mutant of the present invention may also include a combination of nonsense mutations or synonymous mutations in the sequence.

[0007] The encoding gene of the acyl-CoA oxidase mutant of the present invention has the nucleotide sequence shown in SEQ ID NO: 2.

[0008] The present invention also provides a plasmid containing the encoding gene of the acyl-CoA oxidase mutant as described in SEQ ID NO: 2.

[0009] The strain expressing the acyl-CoA oxidase mutant of the present invention contains the above-mentioned plasmid.

[0010] The engineered strain expressing the acyl-CoA oxidase mutant of this invention, classified as Escherichiacoli BL21(DE3) / pET-28a(+)-ACOD-ID-S106A, has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026018, deposit date January 6, 2026, and deposit address: Wuhan University, Wuhan, China.

[0011] The method for constructing an engineered strain expressing an acyl-CoA oxidase mutant according to the present invention includes the following steps:

[0012] First, the structure of acyl-CoA oxidase ACOD-ID was modeled using AlphaFold. Then, Saprot, a sequence- and structure-based prediction scoring method, was used to predict and score full-sequence saturation mutations. The mutation with the highest score and located within the active pocket was selected. After determining the target amino acid of the mutation, a single-point mutation was introduced via PCR amplification and ligated into the expression vector pET-28a(+). The expression host was Escherichia coli BL21(DE3), resulting in an engineered strain containing the mutant gene of this invention.

[0013] The expression plasmid vectors described in the above construction methods include pCold, pET15, and the pET series.

[0014] 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.

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

[0016] The application of the acyl-CoA oxidase mutant of the present invention in the preparation of free fatty acid detection reagents.

[0017] The detection system has a pH range of 6.0-9.5 and a temperature range of 15℃-60℃.

[0018] Further optimization is achieved by using a pH of 6.0-8.5 and a temperature of 15℃-50℃.

[0019] When palmitoyl-CoA was used as a substrate, the stability of the mutant increased by 1.3 times and the specific activity increased by 1.5 times under the conditions of 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 on commercial enzymes was [not specified]. 2 The value was 0.99913. This mutant has application value in in vitro diagnostic kits for the enzymatic detection of free fatty acids.

[0020] 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 the stability of the mutant increased by 1.3 times at 37°C. With palmitoyl-CoA as a substrate, the specific enzyme activity of the mutant increased by 1.5 times. Compared to the starting enzyme, the optimal pH of the mutant changed from 7.5 to 7.0; the optimal temperature remained unchanged. Attached Figure Description

[0021] Figure 1 This is an SDS-PAGE image of the purified mutant protein and the starting enzyme ACOD-ID. In the image, 1 is the ACOD-ID lysis supernatant, 2 is the ACOD-ID purification breakthrough buffer, 3 is the ACOD-ID washing buffer, 4 is the ACOD-ID purified enzyme, 5 is the ACOD-ID-S106A lysis supernatant, 6 is the ACOD-ID-S106A purification breakthrough buffer, 7 is the ACOD-ID-S106A washing buffer, 8 is the ACOD-ID-S106A purified enzyme, and M is the protein marker.

[0022] Figure 2 Figure a shows the investigation of the optimal pH, and Figure b shows the investigation of the optimal temperature.

[0023] Figure 3 The half-life at 37°C is the starting enzyme ACOD-ID and the mutant ACOD-ID-S106A. Detailed Implementation

[0024] Unless otherwise specified, the implementation methods in the following embodiments are all conventional methods.

[0025] (i) Construction of expression strains containing the acyl-CoA oxidase mutant gene of the present invention

[0026] 1. Selection of mutation sites in acyl-CoA oxidase gene

[0027] First, the structure of acyl-CoA oxidase was modeled using AlphaFold. Then, Saprot, a sequence- and structure-based prediction scoring method, was used to predict full-sequence saturation mutations. Higher scores generally indicate a closer approximation of a specific characteristic or function, demonstrating higher accuracy and reliability. The mutation design with the highest score and located near the active pocket was selected, and the target amino acid, serine at position 106, was mutated to alanine.

[0028] 2. Construction of acyl-CoA synthase mutant genetically engineered strains

[0029] The mutation site of acyl-CoA oxidase ACOD-ID in step 1 was amplified by PCR and introduced into the gene, which was then ligated into the expression vector pET-28a(+). The restriction sites were NdeI and XhoI, and the expression host was Escherichia coli BL21(DE3), resulting in the engineered strain Escherichia coli BL21(DE3) / pET-28a(+)-ACOD-ID-S106A containing the mutant gene of this invention.

[0030] The engineered strain expressing the acyl-CoA oxidase mutant of this invention, classified as Escherichiacoli BL21(DE3) / pET-28a(+)-ACOD-ID-S106A, has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026018, deposit date January 6, 2026, and deposit address: Wuhan University, Wuhan, China.

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

[0032] The engineered strain *Escherichia coli* BL21(DE3) / pET-28a(+)-ACOD-ID-S106A obtained in step (I) was inoculated into 400 mL LB liquid medium containing kanamycin and cultured at 37°C and 200 rpm until the OD600 reached 0.6 (UNICO UV2102 UV-Vis spectrophotometer, with uncultured LB medium as a blank). IPTG was added to a final concentration of 0.25 mM for induction, and the culture was continued at 16°C and 120 rpm for 16 hours. The cells were collected by centrifugation at 8000 g at 4°C, resuspended in 1 / 10 volume of K2HPO4-KH2PO4 (pH 7.0) buffer, and sonicated at 350 W on ice for 30 min to lyse the cells. The supernatant was collected by centrifugation at 12000 g to obtain the crude enzyme solution. The crude enzyme solution was purified by Ni-NTA column chromatography. The imidazole concentration in the elution buffer was 120 mM, and elution was performed for 3 column volumes. The obtained protein was tested and found to be of SDS-PAGE purity.

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

[0034] The reaction principle of acyl-CoA synthase detection is as follows:

[0035]

[0036] reaction system

[0037] The reaction system consisted of 1 mL of 0.2 M K2HPO4-KH2PO4 (pH 7.0), 15 mM 4-AA, 85 U / mL POD, 1 mM Palmitic acid, 0.2 % (w / v) Phenol Solution, 1 % (w / v) Triton X-100, and 5 mM Palmitoyl-CoA.

[0038] During the oxidation of fatty acyl-CoA by acyl-CoA oxidase, hydrogen peroxide (H₂O₂) is produced. Upon the addition of peroxidase, phenol undergoes a quantitative oxidative condensation reaction with hydrogen peroxide, ultimately producing a red substance. This can be quantitatively determined using ultraviolet spectroscopy at a specific wavelength (500 nm). The enzyme activity of acyl-CoA oxidase is defined as the amount of ACOD enzyme required to generate 1 μmol of H₂O₂ per minute at 37 °C; one enzyme activity unit is defined as this amount.

[0039] The test results showed that, using palmitoyl-CoA as a substrate, the optimal pH for the mutant obtained in this invention was 7.0, and the enzyme exhibited more than 50% activity within the pH range of 6.0-8.5. The optimal temperature for the mutant was 35℃, and the enzyme exhibited more than 50% activity within the temperature range of 15℃-50℃.

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

[0041] The free fatty acid assay kit includes the following solutions: R1 (final concentration): pH 7.0 phosphate buffer, 0.2-0.5 mmol CoA, 2-5 mmol adenosine-5'-hydrated disodium triphosphate (ATP), 2-5 mmol 4-aminoantipyrine, 2-5 KU / L ascorbic acid oxidase, and 2-4 KU / L acyl-CoA synthase (ACS); R2 includes: 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 concentration of ACS in R1 is 2-4 KU / L, and the concentration of ACOD in R2 is 2-5 KU / L. The sample volume ratio in the entire assay system is: sample:R1:R2 = 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 min, and measuring the absorbance value OD1 at the main wavelength of 546 nm; then adding reaction solution R2, mixing well, incubating at 37°C for 5 min, and measuring the absorbance OD2 at the secondary wavelength of 700 nm. Finally, the difference between the absorbance value at the secondary wavelength and the absorbance value at the main wavelength 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. The results showed that, with the same amount of enzyme, the mutant protein obtained in this invention showed comparable reactivity to the free fatty acid standard compared to the commercial enzyme. When the mutant obtained in this invention is used in combination with the commercial acyl-CoA oxidase, it meets the third-party quality control testing standards (deviation <15%). The R-value of the linear fitting curve between the mutant obtained in this invention and the commercial enzyme for serum sample detection results is [not specified in the original text]. 2It is 0.99913.

Claims

1. An acyl-CoA oxidase mutant, abbreviated as ACOD-ID-S106A, characterized in that... Its amino acid sequence is shown in SEQ ID NO:

1.

2. The encoding gene of the acyl-CoA oxidase 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 oxidase mutant of claim 1, characterized in that: The engineered strain is classified and named Escherichia coli BL21(DE3) / pET-28a(+)-ACOD-ID-S106A has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026018, deposit date January 6, 2026, and address: Wuhan University, Wuhan, China.

4. The application of the acyl-CoA oxidase mutant of claim 1 in the preparation of a free fatty acid detection reagent.

5. The application according to claim 4, characterized in that: The detection system has a pH range of 6.0-9.5 and a temperature range of 15℃-60℃.

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