Glucose-6-phosphate dehydrogenase mutant as well as expression strain and application thereof

Abstract

The invention discloses a glucose-6-phosphate dehydrogenase mutant as well as an expression strain and application thereof. According to the invention, on the basis of glucose-6-phosphate dehydrogenase from marine microorganisms, after a mutation site is selected by combining Evcouplings design with Fold X scoring, a recombinant plasmid is constructed, and a mutant gene is obtained. After the engineering bacteria containing the mutant plasmids are subjected to induced expression, the glucose-6-phosphate dehydrogenase G6PDH202-Des3 with improved specific enzyme activity is obtained. Under the condition of 37 DEG C, the specific enzyme activity of the mutant is improved to 1.6 times, and the stability is improved to 1.2 times. The mutant has potential application value in an in-vitro diagnostic kit for detecting creatine kinase activity by an enzyme method.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a glucose-6-phosphate dehydrogenase mutant G6PDH202-Des3, its expression strain, and its applications. Background Technology

[0002] Myocardial infarction is a life-threatening emergency, referring to a sudden and sustained reduction or interruption of blood flow to the coronary arteries—the main blood vessels supplying the heart—due to blockage. This leads to ischemia, hypoxia, and necrosis of a portion of the myocardium. During a myocardial infarction, irreversible damage occurs to myocardial cells, and creatine kinase (CK) stored within these cells enters the bloodstream. With the continuous release of CK from numerous necrotic myocardial cells, the concentration of CK in the blood significantly increases, exceeding the upper limit of the normal range. CK catalyzes the production of ATP and creatine from ADP and creatine phosphate. ATP, under the catalysis of hexokinase (HK), transfers a γ-phosphate group to the hydroxyl group at the 6-position of glucose, generating glucose-6-phosphate (G6P). Subsequently, G6P loses a hydrogen atom under the catalysis of glucose-6-phosphate dehydrogenase (G6PDH) to generate glucose-6-phosphate delta-lactone and NADH. Therefore, the coupling of glucose-6-phosphate dehydrogenase with hexokinase can be used to monitor CK activity in the blood, thereby assessing the size of the myocardial infarction and the likelihood of recurrence. Currently, the main methods for detecting CK activity in clinical practice are immunosuppression and enzymatic methods. Among them, enzymatic methods are simple and fast, making them suitable for batch analysis in fully automated biochemical analyzers.

[0003] The main principle of the CK activity assay kit is as follows: creatine phosphate and ADP in the sample are catalyzed by CK to generate ATP. ATP and glucose are then reacted with hexokinase (HK) to generate glucose-6-phosphate. Subsequently, glucose-6-phosphate is acted upon by glucose-6-phosphate dehydrogenase (G6PDH), which transfers an H+ to NAD+ to generate glucose-6-phosphate delta-lactone. NAD+ is then reduced to NADH. NADH exhibits maximum absorption at a specific wavelength. By measuring the absorbance value, the activity of CK can be determined.

[0004] Glucose-6-phosphate dehydrogenase (EC1.1.1.49) is a key enzyme in enzymatic methods for detecting CK activity, determining the sensitivity and precision of the assay. G6PDH, with its good enzyme activity and stability, plays an important role in improving the accuracy and stability of in vitro CK activity detection kits. Summary of the Invention

[0005] This invention provides a glucose-6-phosphate dehydrogenase mutant G6PDH202-Des3, its expression strain, and its applications. Based on the glucose-6-phosphate dehydrogenase G6PDH202 from marine microorganisms, this invention utilizes Evcouplings design combined with Fold X scoring to select mutation sites, constructs a recombinant plasmid, and obtains the mutant gene. After induction expression in engineered bacteria containing the mutant plasmid, a glucose-6-phosphate dehydrogenase with increased specific enzyme activity is obtained. At 37℃, the specific enzyme activity of the mutant is increased by 1.6 times. In an in vitro diagnostic kit for detecting CK activity using an enzymatic method, the linear fitting curve R between the detection value of the mutant enzyme on serum samples and the detection value of the commercial enzyme for the same unit is shown. 2 The value was 0.9975, indicating that this mutant has potential application value in in vitro diagnostic kits for the enzymatic detection of CK activity.

[0006] The present invention relates to a glucose-6-phosphate dehydrogenase mutant, abbreviated as G6PDH202-Des3, the amino acid sequence of which is shown in SEQ ID NO: 1. Specifically, the leucine at position 232 of the amino acid sequence of glucose-6-phosphate dehydrogenase G6PDH202 is mutated to methionine.

[0007] The glucose-6-phosphate dehydrogenase mutant of the present invention may further include a combination of nonsense mutations or synonymous mutations in its amino acid sequence.

[0008] The encoding gene of the glucose-6-phosphate dehydrogenase mutant of the present invention has the nucleotide sequence shown in SEQ ID NO: 2.

[0009] The mutant plasmid of this invention contains the encoding gene of the glucose-6-phosphate dehydrogenase mutant as described in SEQ ID NO: 2.

[0010] The strain expressing the glucose-6-phosphate dehydrogenase mutant of the present invention contains the mutant plasmid.

[0011] The engineered strain expressing the glucose-6-phosphate dehydrogenase mutant of this invention, classified as Escherichiacoli BL21(DE3) / pET-22b(+)-G6PDH202-Des3, has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026017, deposited on January 6, 2026, at Wuhan University, Wuhan, China.

[0012] The present invention discloses a method for constructing an engineered strain expressing a glucose-6-phosphate dehydrogenase mutant, comprising the following steps:

[0013] First, the structure of glucose-6-phosphate dehydrogenase was modeled using Alpha fold. Evcouplings (https: / / evcouplings.org) was used to score the epistatic effects of specific amino acid interactions or functional synergistic mutations into different amino acids. Fold X was then used to score these sites. After determining the target amino acid for the mutation based on multiple design, a single-point mutation was introduced using overlap extension PCR with designed primers and ligated into the expression vector pET-22b(+). The expression host was Escherichia coli 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, pET series expression vectors, 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 glucose-6-phosphate dehydrogenase mutant of the present invention can be obtained by fermentation of the engineered strain.

[0017] The application of the glucose-6-phosphate dehydrogenase mutant of the present invention in the preparation of creatine kinase detection reagent.

[0018] The detection system has a pH of 7.0-9.0 and a temperature of 25℃-50℃.

[0019] Using glucose-6-phosphate as a substrate, at 37℃ and pH 7.0, the mutant's specific enzyme activity increased by 1.6 times and its stability increased by 1.2 times. In an in vitro diagnostic kit for the enzymatic detection of CK activity, the linear fitting curve R between the same unit of mutant enzyme protein and the detection value of the commercial enzyme in serum samples was [not specified]. 2 The value was 0.9975. This mutant has potential application value in enzymatic diagnostic kits for detecting CK activity.

[0020] This invention measured and compared the specific enzyme activity, optimal pH, optimal temperature, and stability of the mutant protein and the starting enzyme. The results showed that the optimal pH for the mutant was 8.5, and the optimal temperature was 40℃. At 37℃, using glucose-6-phosphate as a substrate, the specific enzyme activity of the mutant increased by 1.6 times, and its stability increased by 1.2 times. Attached Figure Description

[0021] Figure 1 The SDS-PAGE pattern of the purified mutant protein. Figure 1Lane 1 contains the supernatant from the G6PDH202-Des3 lysis, Lane 2 contains the G6PDH202-Des3 purification and breakthrough buffer, Lane 3 contains the G6PDH202-Des3 30 mM imidazole elution buffer, and Lane 4 contains the G6PDH202-Des3 200 mM imidazole elution buffer. M stands for protein marker.

[0022] Figure 2 In this context, a represents the optimal pH, and b represents the optimal temperature.

[0023] Figure 3 The stability of the starting enzyme G6PDH202 and the mutant G6PDH202-Des3 at 37 °C was determined.

[0024] Figure 4 The linear fitting curve between the detection results of serum samples of mutant G6PDH202-Des3 and the detection results of commercial enzymes. Detailed Implementation

[0025] Unless otherwise specified, the implementation methods in the following embodiments are all conventional methods.

[0026] (i) Construction of expression strains containing the glucose-6-phosphate dehydrogenase mutant gene of the present invention

[0027] 1. Selection of mutation sites in glucose-6-phosphate dehydrogenase gene

[0028] First, the structure of glucose-6-phosphate dehydrogenase was modeled using Alphafold. Evcouplings (https: / / evcouplings.org) was used to score the epistatic effects of specific amino acid interactions or functional synergistic mutations into different amino acids. These sites were then scored using Fold X. Based on multiple design, the target amino acid for the mutation was determined. Primers were designed to introduce a single-point mutation using overlap extension PCR and ligated into the expression vector pET-22b(+). The expression host was Escherichia coli BL21(DE3), resulting in an engineered strain containing the mutant gene of this invention. Mutation sites whose prediction results matched the scores from both software programs were selected to determine the target amino acid, leucine at position 232, as a mutation to methionine.

[0029] 2. Construction of mutant strains of glucose-6-phosphate dehydrogenase

[0030] The mutation site of glucose-6-phosphate dehydrogenase G6PDH202 in step 1 was amplified by PCR and introduced into the gene, which was then ligated into the expression vector pET-22b(+). The cloning sites were NdeI and XhoI, and the expression host was Escherichia coli BL21(DE3), resulting in the engineered strain Escherichia coli BL21(DE3) / pET-22b(+)-G6PDH202-Des3 containing the mutant gene of this invention.

[0031] The engineered strain expressing the glucose-6-phosphate dehydrogenase mutant of this invention, classified as Escherichiacoli BL21(DE3) / pET-22b(+)-G6PDH202-Des3, has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026017, deposited on January 6, 2026, at Wuhan University, Wuhan, China.

[0032] (II) Expression and protein purification of engineered bacteria containing the glucose-6-phosphate dehydrogenase mutant gene of the present invention

[0033] The engineered strain Escherichia coli BL21(DE3) / pET-22b(+)-G6PDH202-Des3 obtained in (I) was inoculated into 400 mL LB liquid medium containing ampicillin and cultured at 37℃ and 200 rpm until OD. 600 The concentration was 0.6 (UNICO UV2102 UV-Vis spectrophotometer, with LB medium as blank); IPTG was added to a final concentration of 0.25 mM for induction, and the cells were cultured at 16℃ and 120 rpm for 16 hours; the cells were collected by centrifugation at 8000 g at 4℃, and 3 times the volume of K2HPO4-KH2PO4 (pH 7.0) buffer was added. The cells were lysed by sonication at 350 W on ice for 15 min, and the supernatant was collected by centrifugation at 12000 g to obtain crude enzyme solution, which was then purified by Ni-NTA column chromatography.

[0034] (III) Detection of the optimal pH and optimal temperature of the glucose-6-phosphate dehydrogenase mutant of the present invention

[0035] The reaction principle of glucose-6-phosphate dehydrogenase detection is as follows:

[0036]

[0037] Reaction system:

[0038] The reaction system consisted of 0.75 mL of 50 mM Tris-HCl (pH 8.0), 2.0 mM NAD+, 3.3 mM D-Glucose-6-phosphate, and 3.0 mM MgCl2.

[0039] In the first step, the reaction mixture of Tris-HCl, NAD+, D-Glucose-6-phosphate, and MgCl2 was preheated at 37°C for 5 min, and then glucose-6-phosphate dehydrogenase enzyme solution was added, and the reaction was allowed to proceed for 5 min. Three parallel experiments were performed in the experimental group, while the control group used buffer solution instead of the enzyme solution. The absorbance at 340 nm was measured using the control group as the zeroing point. Enzyme activity (U) was defined as the amount of enzyme required to convert 1 NAD+ to NADH per minute at 37°C.

[0040] The test results showed that, using glucose-6-phosphate as a substrate, the optimal pH for the mutant obtained in this invention was 8.5, and the enzyme exhibited more than 50% activity in the pH range of 6.0-9.5. The optimal temperature for the mutant was 45℃, and the enzyme exhibited more than 50% activity in the temperature range of 25℃-48℃.

[0041] (iv) Application of the glucose-6-phosphate dehydrogenase mutant of the present invention in an in vitro CK activity detection kit

[0042] The reaction solution in the CK activity in vitro detection kit includes:

[0043] R1 reaction solution: 50-200 mM Tris-HCl buffer (pH 7.0), 1-100 mM glucose, 1-5 mM adenosine diphosphate (ADP), 1-5 mM nicotinamide adenine dinucleotide.

[0044] R2 reaction solution: 100-500 mmol / L creatine phosphate (CP), 1-10 KU / L hexokinase, 1-10 KU / L glucose-6-phosphate dehydrogenase, 0.1-5 g / L preservative.

[0045] The CK activity assay kit detection process includes: first, incubating the CK standard and R1 reaction solution in a 37°C water bath for 5 min, and measuring the absorbance OD1 at the dominant wavelength of 340 nm; then adding the R2 reaction solution, mixing well, incubating in a 37°C water bath for 5 min, and measuring the absorbance OD2 at the secondary wavelength of 405 nm. Finally, the difference between the secondary wavelength absorbance and the dominant wavelength absorbance is calculated. Two parallel experiments were performed in the experimental group, while the control group used pure water instead of the CK standard. The results showed that, with the same unit amount of enzyme added, the mutant protein obtained in this invention showed comparable reactivity to the CK standard compared to the commercial enzyme. When the mutant obtained in this invention is used in combination with the commercial hexokinase, 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]. 2 It is 0.9975.

Claims

1. A glucose-6-phosphate dehydrogenase mutant, abbreviated as G6PDH202-Des3, characterized in that... Its amino acid sequence is shown in SEQ ID NO:

1.

2. The encoding gene of the glucose-6-phosphate dehydrogenase mutant according to claim 1, characterized in that... Its nucleotide sequence is shown in SEQ ID NO:

2.

3. The expression strain of the glucose-6-phosphate dehydrogenase mutant according to claim 1, characterized in that: The expressed strain is classified as Escherichia coli BL21(DE3) / pET-22b(+)-G6PDH202-Des3 and has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026017, deposit date January 6, 2026, and deposit address: Wuhan University, Wuhan, China.

4. The use of the glucose-6-phosphate dehydrogenase mutant of claim 1 in the preparation of creatine kinase detection reagent.

5. The application according to claim 4, characterized in that: The detection system has a pH of 7.0-9.0 and a temperature of 25℃-50℃.

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