CN115808398A - Method for preparing conjugate - Google Patents

Abstract

The present application relates to methods of making conjugates. Specifically, provided is a preparation method of the conjugate, which comprises the following steps: 1) Providing a glucose-6-phosphate dehydrogenase mutant; 2) Providing a hapten; 3) The glucose-6-phosphate dehydrogenase mutant and the hapten are mixed according to a molar ratio of 1:1 coupling. The glucose-6-phosphate dehydrogenase mutant of the present application comprises one or a combination of mutations selected from the group consisting of: D306C, D375C, G C. The detection kit prepared by using the conjugate has the advantages of strong specificity, high sensitivity, convenient operation, short detection time, accurate quantification and suitability for high-throughput detection.

Description

Preparation method of the conjugate

This application claims priority to patent application 201910017764.4 filed on January 9, 2019 and patent application 201910423122.4 filed on May 21, 2019. This application is a divisional application of the patent application 2019113721472 "Glucose-6-phosphate dehydrogenase mutant and its use in the preparation of detection reagents" submitted on December 27, 2019.

technical field

This application relates to the field of biological detection, in particular to an enzyme 6-phosphate glucose dehydrogenase (G6PDH for short) with multi-site mutation and its application in detection kits.

Background technique

Haptens, certain small molecular substances (molecular weight less than 4000Da), which alone cannot induce an immune response, that is, they are not immunogenic, but when they are cross-linked or combined with macromolecular proteins or non-antigenic polylysine and other carriers Afterwards, immunogenicity can be obtained and an immune response can be induced. These small molecular substances can be combined with the response effect products and have antigenicity. They are only immunoreactive, not immunogenic, and are also called incomplete antigens.

A hapten can combine with the corresponding antibody to produce an antigen-antibody reaction, but it cannot stimulate the human or animal body to produce antibodies alone. It is only immunoreactive, not immunogenic, also known as incomplete antigen. Most polysaccharides, lipids, hormones, and small molecule drugs are haptens. If the hapten is chemically combined with a certain protein molecule (carrier), new immunogenicity will be obtained and animals will be stimulated to produce corresponding antibodies. Once a hapten is bound to a protein, it constitutes an antigenic cluster of that protein. Some chemically active group substances (such as penicillin, sulfonamide, etc.) that have a smaller molecular weight than general haptens but have specific structures are called simple haptens.

Due to the lack of two or more sites that can be used as a sandwich method for small molecule antigens or haptens, the double-antibody sandwich method cannot be used for determination, and the competition mode is mostly used. The principle is that the antigen in the specimen competes with a certain amount of enzyme-labeled antigen to bind to the solid-phase antibody. The more antigen content in the sample, the less enzyme-labeled antigen combined on the solid phase, and the lighter the color. This method is often used in the determination of small molecule hormones and drugs by ELISA.

Cholyglycin (CG), as a specific example of a hapten, is a conjugated cholic acid formed by combining cholic acid and glycine, and is one of the main components of bile acids. Cholesterol undergoes a series of complex enzyme-catalyzed reactions in liver cells to form primary bile acids, including cholic acid (CA) and chenodeoxycholic acid (CDCA). There are three hydroxyl groups (C3, C7, C12) on the steroid nucleus of cholic acid ), the hydroxyl group at the end of the side chain is combined with glycine through a peptide bond to form glycocholic acid (Figure 1).

Glycocholic acid is synthesized by liver cells, discharged into the gallbladder through capillaries and bile ducts, and enters the duodenum with bile to help digestion and absorption of fat in food. 95% of bile acids are reabsorbed in the ileum and colon, and then return to the liver through the portal vein, where they are taken up and reused by liver cells, and the reabsorbed glycocholic acid enters the liver-enteric circulation. Through this mechanism, the body can make full use of glycocholic acid .

Under normal circumstances, the content of cholic acid in the peripheral blood is extremely small, and the content of glycocholic acid in the peripheral blood of normal people is at a very low level no matter on an empty stomach or after a meal. When human liver cells are damaged or cholestasis occurs, it will cause glycocholic acid metabolism and circulation disorders, which will reduce the ability of liver cells to absorb glycocholic acid, resulting in increased glycocholic acid content in the blood, and the level of glycocholic acid is related to Correlates with the severity of liver cell damage and bile acid metabolism disorder.

Determination of glycocholic acid content in serum is one of the sensitive indicators for evaluating liver cell function and circulation of hepatobiliary substances. Compared with routine liver function tests such as ALT, AST, total bilirubin (TBIL), alkaline phosphatase (ALP), glutamyl transpeptidase (GGT), serum albumin (ALB), the determination of glycocholic acid is more accurate sensitive. Therefore, glycocholic acid can be used as a better detection index in the detection of liver function such as chronic hepatitis, acute hepatitis, liver cirrhosis, liver cancer, obstructive liver disease, enterohepatic circulation disorder, bile duct and gallbladder excretory dysfunction.

Currently known glycocholic acid detection methods mainly include: radioimmunoassay, enzyme-linked immunoassay, chemiluminescence immunoassay, high performance liquid chromatography, gas-liquid chromatography, gas chromatography and mass spectrometry. However, there are many defects in these detection methods, such as radioimmunoassay isotopes have radioactive contamination, short validity period, inconvenient operation and many other disadvantages, and enzyme-linked immunosorbent assay is cumbersome to operate and takes a long time, so it is not suitable for clinical application. use on. Although chemiluminescence has good sensitivity, it needs supporting special equipment, and the high cost of putting it into use is not conducive to popularization. In the process of clinical detection and diagnosis, homogeneous enzyme immunoassay (EMIT) and latex-enhanced immunoturbidimetric assay are the main methods.

The principle of homogeneous enzyme immunoassay: In the liquid homogeneous reaction system, the enzyme-labeled antigen (such as G6PDH-CG) and the non-labeled antigen (CG) compete with the quantitative antibody (CG antibody) for binding, when the antibody and the non-labeled The more the antigen is bound, the more the activity of the enzyme-labeled antigen will be released, and the more the enzyme will catalyze the substrate NAD+ to generate NADH. The change of the absorbance of NADH at 340nm wavelength can be used to calculate the content of CG in the liquid.

Contents of the invention

In view of the needs in the art, the present application provides a novel glucose-6-phosphate dehydrogenase mutant and its use in the preparation of a glycocholic acid detection kit.

According to some embodiments, a glucose-6-phosphate dehydrogenase mutant is provided. Different from the mutant of glucose-6-phosphate dehydrogenase of the published patent US006090567A (Homogeneous immunoassays using mutant glucose-6-phosphatedehydrogenases), the mutant of glucose-6-phosphate dehydrogenase of the present application comprises a mutation selected from the following : D306C, G426C, D375C.

According to some embodiments, a 6-phosphate glucose dehydrogenase mutant is provided, and the 6-phosphate glucose dehydrogenase mutant is shown in a sequence selected from the following: SEQ ID No.2, SEQ ID No.3 , SEQ ID No.4.

According to some embodiments, there is provided a polynucleotide encoding the glucose-6-phosphate dehydrogenase mutant of the present application.

According to some embodiments, there is provided an expression vector comprising the polynucleotide of the present application.

According to some embodiments, there is provided a host cell comprising the expression vector of the present application. Host cells can be prokaryotic (such as bacteria) or eukaryotic (such as yeast).

According to some embodiments, a conjugate is provided, which is formed by coupling the glucose-6-phosphate dehydrogenase mutant of the present application with a hapten at a molar ratio of 1:n.

In some embodiments, n is 1 to 50, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50.

In some specific embodiments, the glucose-6-phosphate dehydrogenase mutant of the present application is coupled to the hapten in a molar ratio of preferably 1:1.

In some specific embodiments, the hapten has a molecular weight of 100 Da to 4000 Da, for example: 100, 150, 200, 250, 300, 350, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 . ,2200,2300,2400,2500,2600,2700,2800,2900,3000,3100,3200,3300,3400,3500,3600,3700,3800,3900,4000.

From the present application, the skilled person will understand that "hapten" also includes derivative forms thereof. In order to facilitate coupling with 6-phosphate glucose dehydrogenase, those haptens (such as CG) that do not have a coupling group (such as a group that reacts with a sulfhydryl group) themselves can be modified to have a linker, in order to covalently bond with sulfhydryl groups. Therefore, in this application, a hapten derivative refers to a hapten modified to have a sulfhydryl reactive group.

Haptens are selected from: small molecule drugs (such as antibiotics, psychotropic drugs), hormones, metabolites, sugars, lipids, amino acids, and short peptides (when the molecular weight is less than 4000kDa, or the amino acid length does not exceed 50 residues).

Haptens such as but not limited to: Vitamin D, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, folic acid, cardiac glycosides, enzyme phenolic acid, rapamine, cyclosporine A, amiodarone, formazan Amhotrexate, tacrolimus, serum amino acids, bile acid, glycochlic acid, phenylalanine, ethanol, urinary nicotine metabolite cotinine, urinary morphine, urinary monohydric phenol derivatives, neuropeptide tyrosine Acid, plasma galanin, polyamine, histamine, thyroid-stimulating hormone, prolactin, placental lactogen, growth hormone, follicle-stimulating hormone, luteinizing hormone, adrenocorticotropic hormone, vasopressin, calcitonin, calcitonin Protoxin, parathyroid hormone, thyroxine, triiodothyronine, trans-triiodothyronine, free thyroxine, free triiodothyronine, cortisol, urinary 17-hydroxycorticosteroid, urinary 17 - Ketosteroids, dehydroepiandrosterone and sulfate esters, aldosterone, urinary vanillyl mandelic acid, plasma renin, angiotensin, erythropoietin, testosterone, dihydrotestosterone, androstenedione, 17α-hydroxyprogesterone, Estrone, estriol, estradiol, progesterone, human chorionic gonadotropin, insulin, proinsulin, C-peptide, gastrin, plasma prostaglandin, plasma 6-keto prostaglandin F1α, prostacyclin, adrenal Catecholamine, norepinephrine, cholecystokinin, nasulin, cyclic adenosine monophosphate, cyclic guanosine monophosphate, vasoactive peptide, somatostatin, secretin, P-substance, neurotensin, thromboxane A2 , Thromboxane B2, 5 serotonin, neuropeptide Y, osteocalcin.

In a specific embodiment, the hapten is glycocholic acid or a derivative thereof. Although glycocholic acid is used as a specific example, the skilled person can understand that the technical effect of the present application does not depend on the specific type of hapten, and it is applicable to any hapten that can be immunologically detected by means of a competition method.

In a specific embodiment, the hapten is a glycocholic acid derivative bearing a sulfhydryl reactive group, such as leimide, bromoacetyl, vinylsulfone or aziridine. In a specific embodiment, the hapten is a glycocholic acid derivative, as shown in Formula I:

According to some embodiments, there is provided a reagent comprising a conjugate of the present application.

According to some embodiments, a use of the glucose-6-phosphate dehydrogenase mutant of the present application in the preparation of a detection reagent is provided.

According to some embodiments, the use of the conjugate of the present application in the preparation of a detection reagent is provided.

In a specific embodiment, the detection reagent is selected from: enzyme-linked immunoassay detection reagents, chemiluminescent immunoassay detection reagents, homogeneous enzyme immunoassay detection reagents, latex-enhanced immunoturbidimetric assay reagents.

In a specific embodiment, the detection reagent is preferably a detection reagent based on a competition method.

According to some embodiments, there is provided a glycocholic acid detection kit comprising:

- a first reagent comprising a substrate and an antibody to glycocholic acid; the substrate being a substrate of glucose-6-phosphate dehydrogenase;

- a second reagent comprising a conjugate of the present application;

- optionally, a calibrator comprising 10 mM to 500 mM buffer, 0 mg/L to 40 mg/L glycocholic acid; and

- Optionally, a quality control product comprising 10 mM to 500 mM buffer, 0 mg/L to 40 mg/L glycocholic acid.

According to one embodiment, a glycocholic acid detection kit is provided, comprising:

a first reagent comprising:

10mM to 500mM buffer,

5mM to 25mM substrate,

0.1mg/L to 1mg/L glycocholic acid antibody,

10mM to 300mM NaCl,

0.1g/L to 5g/L stabilizer,

0.1g/L to 5g/L surfactant,

0.1g/L to 5g/L preservatives;

a second reagent comprising:

10mM to 500mM buffer,

The conjugate described in claim 5,

0.1g/L to 5g/L stabilizer,

0.1g/L to 5g/L surfactant,

0.1g/L to 5g/L preservatives.

In some embodiments, the buffer is selected from one or a combination of: tromethamine buffer, phosphate buffer, Tris-HCl buffer, citric acid-sodium citrate buffer, barbiturate buffer solution, glycine buffer, borate buffer, trimethylolmethane buffer; preferably, phosphate buffer; the concentration of the buffer is 10mmol/L to 500mmol/L, preferably 100mM; the buffer The pH is 6.5 to 7.5, preferably 7.2 or 7.0.

In some embodiments, the stabilizer is selected from one or a combination of: bovine serum albumin, trehalose, glycerin, sucrose, mannitol, glycine, arginine, polyethylene glycol 6000, polyethylene glycol 8000; preferably bovine serum albumin.

In some embodiments, the surfactant is selected from one or a combination of: Brij35, Triton X-100, Triton X-405, Tween20, Tween30, Tween80, coconut oil fatty acid diethanolamide, AEO7, preferably Tween20.

In some embodiments, the preservative is selected from one or a combination of: azide, MIT, PC-300, thimerosal; and the azide is selected from: sodium azide, lithium azide.

In some embodiments, the substrate comprises: glucose-6-phosphate, β-nicotinamide adenine dinucleotide.

In some embodiments, the concentration of the buffer is 100 mM.

In some embodiments, the substrate concentration of the reaction catalyzed by the G6PDH enzyme is 5 mM.

In some embodiments, the concentration of the glycocholic acid antibody is 0.1 mg/L.

In some embodiments, the concentration of NaCl is 300 mM.

In some embodiments, the concentration of stabilizer is 0.5 g/L.

In some embodiments, the concentration of surfactant is 0.1 g/L.

In some embodiments, the concentration of preservative is 1 g/L.

According to some embodiments, a method for preparing a conjugate is provided, comprising the steps of:

1) Provide a hapten or derivative thereof according to the present application, especially provide a hapten or a derivative thereof according to the present application in an aprotic solvent (such as but not limited to acetonitrile, dimethylformamide, dimethyl sulfoxide) derivative;

2) Provide the 6-phosphate glucose dehydrogenase mutant of the present application, preferably in a buffer (which provides a reaction environment, such as but not limited to PBS, Tris, TAPS, TAPSO, the pH of the buffer is 6.0 to 8.0) Glucose 6-phosphate dehydrogenase mutant;

3) At 18°C to 28°C, contact the 6-phosphate glucose dehydrogenase mutant with the hapten or its derivatives at a molar ratio of 1:n for 1 hour to 4 hours (preferably 2 hours to 3 hours) Coupling the hapten or its derivative with the glucose-6-phosphate dehydrogenase mutant to obtain the conjugate;

4) Optionally purify the conjugate, such as desalting treatment, etc., as required.

In some embodiments, n is 1 to 50, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50.

In some specific embodiments, steps 1) and 2) can be interchanged or run in parallel.

In some specific embodiments, prior to conjugation, the glucose-6-phosphate dehydrogenase contains a free sulfhydryl group, thereby allowing a 1:1 directional reaction with a hapten or a derivative thereof.

Description of drawings

Figure 1. Structural diagram of glycocholic acid.

Figure 2. Structural diagram of glycocholic acid derivatives.

Figure 3A. G6PDH (wild type) amino acid sequence (SEQ ID No. 1); derived from Leuconostoc pseudomesenteroides.

Figure 3B. G6PDH(D306C) amino acid sequence (SEQ ID No. 2).

Figure 3C. G6PDH (D375C) amino acid sequence (SEQ ID No. 3).

Figure 3D. G6PDH (G426C) amino acid sequence (SEQ ID No. 4).

Detailed ways

Example

Embodiment 1. Synthesis of Glycocholic Acid Derivatives

Add glycocholic acid (1.0eq), maleimidoethylamine (1.0g, 1.0eq) and triethylamine (3.0eq) into a dry and clean 25mL two-necked bottle;

Then add dimethylformamide (5mL) and stir until completely dissolved, add dichloroethane (1.25eq), and stir at 25°C for 2h;

HPLC monitoring, until the completion of the reaction;

Add the above reaction mixture into water (25mL), add ethyl acetate 20mL×3 for extraction;

The organic phases were combined, dried over anhydrous Na ₂ SO ₄ , concentrated under reduced pressure, and the resulting oil was purified by column chromatography to obtain 1.04 g of milky white powdery solid, yield 45%, M+: 602.72.

The function of this embodiment is to make CG have a group that can bind to enzymes, and the technical effect of this application does not depend on specific hapten derivatives.

Example 2. Coupling of Glycocholic Acid Derivatives to G6PDH Molecules

According to the G6PDH-glycocholic acid conjugate of the present application, the coupling is carried out in the following manner: the sulfhydryl reactive group (such as maleimide group) on the glycocholic acid derivative molecule co-operates with the thiol group on the G6PDH molecule. price combination.

1. Solution preparation:

Glycocholic acid derivative solution: 10 mg/ml of the glycocholic acid derivative prepared in Example 1 was dissolved in DMF;

G6PDH solution: 6.7mg/mL G6PDH (mutant of the present application or control mutant), PB 100mmol, NaCl 100mmol, pH=8.0;

Coupling solution: 100mM PB/K, 100mM EDTA, 150mM NaCl, pH=7.2;

Desalting solution: 100 mM PB/K, 100 mM EDTA, 150 mM NaCl, pH=7.2.

2. Coupling operation: 1.6ml G6PDH solution, 6ml coupling solution and 0.40ml glycocholic acid derivative solution were reacted at room temperature (20 to 25° C.) for 4 hours.

3. After shaking the above reaction system at room temperature for 4 hours, use the above desalting solution to elute with a desalting column to collect the protein peak, and the obtained product is G6PDH-glycocholic acid conjugate.

Embodiment 3. Preparation of kit

Prepare the following kit for detecting glycocholic acid, which comprises:

Reagent R1, comprising:

100mM PB buffer, pH 7.2

15mM Glucose 6-phosphate

15mM β-nicotinamide adenine dinucleotide

0.1mg/L Glycocholic acid antibody

200mM NaCl

0.5g/L bovine serum albumin

0.1g/L Tween20

1g/L sodium azide;

Reagent R2, including:

100mM PB buffer, pH 7.2

0.1mg/L G6PDH-CG conjugate

0.5g/L bovine serum albumin

0.1g/L Tween 20

1g/L sodium azide;

Calibrator: 100mM PB buffer, pH 7.2, and 0, 2.5, 5.0, 10, 20, 40mg/L glycocholic acid (or add as needed);

Quality control: 100mM PB buffer, pH 7.2, and 1.5, 8.0, 25, 35mg/L glycocholic acid (or add as needed).

Test case

Reaction time: 10 minutes, of which the incubation time is 4.7 minutes. After adding reagent R2 and incubating for 1 minute, measure and read the absorbance A1, and after incubation for 1 minute, measure and read the absorbance A2, and calculate ΔA=(A2-A1)/min. The content of glycocholic acid in the sample is calculated by the calibration curve: CG=absorbance of sample tube*concentration of calibrator/absorbance of calibrator.

The performance of the glycocholic acid detection kit prepared in Example 3 was tested, and the main detection performances were total imprecision, repeatability, recovery, linearity, specificity, etc.

Table 1. Parameters of automatic biochemical analyzer

Detection model Hitachi 7180 analysis/time/point 2 points speed/10min/20-24 points R1/R2/S 120:40:9 Wavelength (sub / main) 405/340 response type increment Calibration type Spine type Calibration point 6 Calibrator Concentration 0/2.50/5.00/10.00/20.00/40.00

Test example 1. Glycocholic acid detection kit calibration absorbance

Table 2. Glycocholic acid detection kit calibration absorbance

Test Example 2. Total Imprecision of Glycocholic Acid Detection Kit

Table 3. Total Imprecision

Test Example 3. Reproducibility of Glycocholic Acid Detection Kit

Table 4. Repeatability

Test Example 4. Recovery of Glycocholic Acid Detection Kit

Table 5. Recovery

Test Example 5. Glycocholic Acid Detection Kit Linearity

Table 6. Linearity

Detection Example 6. Anti-specificity of Glycocholic Acid Detection Kit

Table 7. Specificity

Interfering substances (40μg/ml) Reagent for this application (D306C) Control reagent (A45C mutant) glycodeoxycholic acid 18.61% 30.20% glycochenodeoxycholic acid 1.63% 37.91% chenodeoxycholic acid 0.61% 16.28% ursodeoxycholic acid -0.42% 6.55% sodium cholate 61.21% 61.90% Sodium deoxycholate 4.22% 11.74%

The reagent of the present application has little or no cross-reaction with structural analogues of glycocholic acid.

Test Example 7. Antibody Inhibition Rate

1. Detection principle of antibody inhibition rate

When the antibody is combined with the G6PDH-CG conjugate, the enzymatic activity of G6PDH is affected due to steric hindrance, which reduces the efficiency of catalyzing the conversion of NAD to NADH. By detecting the change in the amount of NADH, it is possible to compare the addition of the antibody with the non-addition of the antibody The difference between the experimental groups, this difference is reflected in the ability of the antibody to inhibit G6PDH.

2. Reaction system:

Table 8. Preparation of detection reagents for antibody inhibition rate

Table 9. Parameters for the detection of antibody inhibition rate

Detection model Hitachi 7180 analysis/time/point 2 points speed/10min/20-24 points R1/S 120:20 Wavelength (sub / main) 405/340 response type increment

3. Results:

The absorbance of the G6PDH-CG conjugate is measured by comparing the time when the antibody is added and when the antibody is not added, and the inhibition of the antibody on G6PDH can be obtained.

Antibody inhibition rate = change value of absorbance of G6PDH-CG when containing antibody/change value of absorbance of G6PDH-CG when not containing antibody.

Compared with the published mutation sites, the mutants of the present application have significantly improved antibody inhibition rate, which can reach more than 30%, up to 50%. However, the inhibition rate of commonly used mutation sites (such as A45C, K55C) is only about 20% or even lower.

Table 10. Antibody Inhibition Rates of Different G6PDH Mutants

Although not limited to a specific theory, it can be partially explained as: compared with the G6PDH mutant in the prior art, the mutation site (ie, the site where a free sulfhydryl group is introduced) in the enzyme mutant (D306C, D375C, G426C) of the present application It is the position of coupling with hapten (such as hormone, small molecule drug, etc.). When the hapten is combined with the hapten-specific antibody at this position, the steric hindrance formed has the greatest impact on the activity of the G6PDH enzyme, and at the same time, the spatial folding of the molecule cannot be substantially affected after the mutation is introduced. Therefore, the location of this mutation site is very important, and it is necessary to take into account the activity of the G6PDH enzyme, the spatial folding of the coupling molecule, and the full exposure of the hapten epitope.

Since the enzyme mutant has a significant increase in the antibody inhibition rate, it can have a clear advantage in calibrating the absorbance. After the enzyme mutant and the hapten-coupled conjugate are formulated into a kit, due to the improvement of the calibration curve, the reagent has obvious performance in terms of repeatability, total imprecision, linearity, specificity, etc. promote.

Claims (3)

1. A method of preparing a conjugate comprising the steps of:

1) Providing a glucose-6-phosphate dehydrogenase mutant;

2) Providing a hapten;

3) The glucose-6-phosphate dehydrogenase mutant and the hapten are mixed according to a molar ratio of 1:1, coupling;

the hapten is glycocholic acid or a glycocholic acid derivative;

the steps 1) and 2) can be performed in parallel or in an interchangeable order;

preferably, the glucose-6-phosphate dehydrogenase mutant comprises a mutation selected from any one of the following mutations compared to the wild-type glucose-6-phosphate dehydrogenase of leuconostoc pseudomesenteroides: D306C, D375C, G C.

2. The method of claim 1, wherein the glucose-6-phosphate dehydrogenase mutant is represented by any one of the sequences selected from the group consisting of: SEQ ID No.2, SEQ ID No.3, SEQ ID No.4.

3. The method of claim 1, wherein the glycocholic acid derivative is of formula I:

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