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

Method for preparing conjugate

This application claims priority from patent application 201910017764.4 filed on 2019, 1, 9 and patent application 201910423122.4 filed on 2019, 5, 21. The application is a divisional application of patent application 2019113721472 'glucose-6-phosphate dehydrogenase mutant and application thereof in preparation of detection reagents', which is filed in 2019, 12 months and 27 days.

Technical Field

The application relates to the field of biological detection, in particular to a multi-site mutant enzyme, namely 6-phosphoglucose dehydrogenase (G6 PDH for short) and application thereof in a detection kit.

Background

Haptens, certain small molecular substances (molecular weight less than 4000 Da), alone are not capable of inducing an immune response, i.e. are not immunogenic, but can gain immunogenicity when cross-linked or conjugated with carriers such as macromolecular proteins or non-antigenic polylysines, inducing an immune response. These small molecule substances can bind to response effector products, have antigenicity, are immunoreactive only and are not immunogenic, and are also called incomplete antigens.

The hapten can be combined with a corresponding antibody to generate an antigen-antibody reaction, and can not singly stimulate the human or animal body to generate the antigen of the antibody. It is immunoreactive only, has no immunogenicity, and is also called incomplete antigen. Most polysaccharides, lipids, hormones, and small molecule drugs are haptens. If a hapten is chemically bound to a protein molecule (carrier), it will acquire new immunogenicity and will stimulate the production of the corresponding antibody in the animal. Haptens, once bound to a protein, constitute an antigenic cluster of the protein. Some chemical active group substances (such as penicillin, sulfanilamide, etc.) with molecular weight smaller than that of general hapten and with specific structures are called simple haptens.

Small molecule antigens or haptens lack two or more sites that can be used in sandwich assays, and therefore cannot be measured using the double antibody sandwich assay, and often use a competition mode. The principle is that the antigen in the specimen and a certain amount of enzyme-labeled antigen compete to bind with the solid-phase antibody. The more the amount of antigen in the specimen is, the less the enzyme-labeled antigen bound to the solid phase is, and the lighter the color development is. ELISA measurement of small molecule hormone, medicine, etc. is used in different methods.

Glycocholic acid (CG), which is a specific example of a hapten, is a conjugated cholic acid formed by binding cholic acid and glycine, and is one of main components of bile acid. Cholesterol undergoes a series of complex enzyme catalytic reactions in liver cells to form primary bile acid, which comprises Cholic Acid (CA) and chenodeoxycholic acid (CDCA), wherein the steroid nucleus of the cholic acid has three hydroxyl groups (C3, C7 and C12), and the hydroxyl group at the tail end of a side chain is combined with glycine by a peptide bond to form glycocholic acid (figure 1).

Glycocholic acid is synthesized by liver cells, is discharged into a gall bladder through a bile capillary and a bile duct, enters duodenum along with bile, and helps digestion and absorption of fat in food. 95% bile acid is reabsorbed in ileum and colon, returns to liver through portal vein, is absorbed and reused by liver cells, and the reabsorbed glycocholic acid enters liver-intestine circulation again, and through the mechanism, the organism can fully utilize glycocholic acid.

Normally, the content of cholic acid in peripheral blood is extremely low, and the content of glycocholic acid in peripheral blood is very low in normal people regardless of fasting or postprandial conditions. When human liver cells are damaged or bile is deposited, metabolism and circulation disorder of glycocholic acid can be caused, so that the capacity of the liver cells for absorbing glycocholic acid is reduced, the content of glycocholic acid in blood is increased, and the glycocholic acid value is related to the severity of liver cell damage and bile acid metabolic disturbance.

The determination of the content of glycocholic acid in serum is one of the sensitive indexes for evaluating the function of liver cells and the circulation function of liver and gall series substances. The glycocholic acid is more sensitive to liver function tests than conventional liver function tests such as ALT, AST, total Bilirubin (TBIL), alkaline phosphatase (ALP), glutamyltranspeptidase (GGT), serum Albumin (ALB) and the like. Therefore, glycocholic acid can be used as a better detection index in liver function detection such as chronic hepatitis, acute hepatitis, liver cirrhosis, liver cancer, obstructive liver disease, liver and intestine circulatory disturbance, bile duct and gallbladder excretion dysfunction.

The currently known glycocholic acid detection methods mainly include: radioimmunoassay, enzyme-linked immunosorbent assay, chemiluminescence immunoassay, high performance liquid chromatography, gas-liquid chromatography, gas chromatography, mass spectrometry, etc. However, these detection methods all have many defects, such as radioactive contamination of radioimmunoassay isotope, short validity period, inconvenient operation and the like, and the enzyme-linked immunosorbent assay is relatively complicated in operation, takes a long time and is not suitable for clinical use. Although the chemiluminescence has good sensitivity, the chemiluminescence needs special equipment, and the cost of the chemiluminescence is high, so that the chemiluminescence is not beneficial to popularization. In the clinical detection and diagnosis process, homogeneous enzyme immunoassay (EMIT) and latex enhanced immunoturbidimetry are mainly used for detection.

Principle of homogeneous enzyme immunoassay: in a liquid homogeneous reaction system, an enzyme-labeled antigen (such as G6 PDH-CG) and a non-labeled antigen (CG) compete to be combined with a quantitative antibody (CG antibody), when the antibody is more combined with the non-labeled antigen, the more activity released by the enzyme-labeled antigen is, the more NADH is generated by catalyzing a substrate NAD +, and the change of absorbance of NADH is detected at a wavelength of 340nm, so that the content of CG in the liquid can be calculated.

Disclosure of Invention

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

According to some embodiments, a glucose-6-phosphate dehydrogenase mutant is provided. In contrast to the already published mutant of glucose-6-phosphate dehydrogenase of patent US006090567a (homogenomic immunological systems using mutant glucose-6-phosphate dehydrogenes), the glucose-6-phosphate dehydrogenase mutant of the present application comprises a mutation selected from the group consisting of: D306C, G426C, D C.

According to some embodiments, there is provided a glucose-6-phosphate dehydrogenase mutant, the glucose-6-phosphate dehydrogenase mutant being represented by a sequence selected from the group consisting of: SEQ ID No.2, SEQ ID No.3, SEQ ID No.4.

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

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

According to some embodiments, there is provided a host cell comprising an expression vector of the present application. The host cell may be prokaryotic (e.g., bacteria) or eukaryotic (e.g., yeast).

According to some embodiments, there is provided a conjugate of a glucose-6-phosphate dehydrogenase mutant of the present application and a hapten in a molar ratio of 1: n is coupled.

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 preferably present in a molar ratio of 1: 1.

In some specific embodiments, the hapten has a molecular weight of from 100Da to 4000Da, for example: 100. 150, 200, 250, 300, 350, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 520, 550, 570, 600, 620, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000.

According to the present application, the skilled person will understand that "hapten" also comprises forms of its derivatives. To facilitate conjugation to glucose-6-phosphate dehydrogenase, haptens (e.g., CG) that do not themselves carry a coupling group (e.g., a group that reacts with a thiol group) can be engineered to carry a linker for covalent binding to the thiol group. Thus, in this application, a hapten derivative refers to a hapten that has been engineered to carry a thiol-reactive group.

The hapten is selected from: small molecule drugs (e.g. antibiotics, psychotropic drugs), hormones, metabolites, sugars, lipids, amino acids, short peptides (molecular weight less than 4000kDa, or amino acids no longer than 50 residues in length).

Haptens are exemplified by, but not limited to: vitamin D, 25 hydroxyvitamin D, 1, 25 dihydroxyvitamin D, folic acid, cardiac glycoside, mycophenolic acid, lei Paming, cyclosporin A, amiodarone, methotrexate, tacrolimus, serum amino acids, bile acids, glycocholic acid, phenylalanine, ethanol, cotinine metabolite cotinine, uromorphine, urinary monohydroxyphenol derivatives, neuropeptide tyrosine, plasma galanin, polyamines, histamine, thyroid stimulating hormone, prolactin, placental prolactin, growth hormone, follicle stimulating hormone, luteinizing hormone, adrenocorticotropic hormone, antidiuretic hormone, calcitonin, procalcitonin, parathyroid hormone, thyroxine, triiodothyronine, anti-triiodothyronine, free thyroxine, free triiodothyronine, cortisol urinary 17-hydroxycorticosteroids, urinary 17-ketosteroids, dehydroepiandrosterone and sulfates, aldosterone, urinary vanillylmandelic acid, plasma renin, angiotensin, erythropoietin, testosterone, dihydrotestosterone, androstenedione, 17 α hydroxyprogesterone, estrone, estriol, estradiol, progesterone, human chorionic gonadotropin, insulin, proinsulin, C-peptide, gastrin, plasma prostaglandin, plasma 6-one prostaglandin F1 α, prostacyclin, epinephrine, catecholamine, norepinephrine, cholecystokinin, nalin, cyclic adenosine monophosphate, cyclic guanosine monophosphate, vasoactive peptides, somatostatin, secretin, P-substance, neurotensin, thromboxane A2, thromboxane B2, 5 hydroxytryptamine, neuropeptide Y, osteocalcin.

In a particular embodiment, the hapten is glycocholic acid or a derivative thereof. Although glycocholic acid is taken as a specific example, it will be understood by the skilled person that the technical effect of the present application is independent of the particular type of hapten, which is applicable to any hapten which can be immunologically detected by means of competition methods.

In particular embodiments, the hapten is a glycocholic acid derivative, which carries a thiol-reactive group, such as, for example, a maleimide, bromoacetyl, vinyl sulfone, or aziridine. In a particular embodiment, the hapten is a glycocholic acid derivative, represented by formula I:

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

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

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

In specific embodiments, the detection reagent is selected from the group consisting of: enzyme-linked immunosorbent assay reagent, chemiluminescence immunoassay reagent, homogeneous enzyme immunoassay reagent and latex enhanced immunoturbidimetry reagent.

In a specific embodiment, the detection reagent is preferably a reagent for detection 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 a glycocholic acid antibody; the substrate is a substrate for glucose-6-phosphate dehydrogenase;

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

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

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

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

a first reagent comprising:

10mM to 500mM buffer solution,

5mM to 25mM substrate,

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

10mM to 300mM NaCl,

0.1g/L to 5g/L stabilizer,

0.1g/L to 5g/L of surfactant,

0.1g/L to 5g/L preservative;

a second reagent comprising:

10mM to 500mM buffer solution,

The conjugate of claim 5,

0.1g/L to 5g/L stabilizer,

0.1g/L to 5g/L of surfactant,

0.1g/L to 5g/L preservative.

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

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

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; the azide is selected from: sodium azide and lithium azide.

In some embodiments, the substrate comprises: 6-phosphoglucose, beta-nicotinamide adenine dinucleotide.

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

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

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

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

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

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

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

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

1) Providing a hapten or a derivative thereof according to the present application, in particular in an aprotic solvent (such as but not limited to acetonitrile, dimethylformamide, dimethylsulfoxide);

2) Providing a glucose-6-phosphate 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 buffer pH being 6.0 to 8.0);

3) (ii) contacting the glucose-6-phosphate dehydrogenase mutant and the hapten or derivative thereof in a molar ratio of 1: n for 1 to 4 hours (preferably 2 to 3 hours) to allow the hapten or derivative thereof and the glucose-6-phosphate dehydrogenase mutant to be conjugated to obtain the seed conjugate;

4) The conjugate is optionally purified, for example, desalted or the like, 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) are interchangeable or concurrent.

In some specific embodiments, the glucose-6-phosphate dehydrogenase, prior to conjugation, comprises a free sulfhydryl group, thereby allowing for 1: 1.

Drawings

FIG. 1 shows the structure of glycocholic acid.

FIG. 2 shows the structure of glycocholic acid derivatives.

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

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

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

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

Detailed Description

Examples

EXAMPLE 1 Synthesis of Glycocholic acid derivatives

Dry, clean 25mL two-necked bottles were charged with glycocholic acid (1.0 eq), maleimidoethylamine (1.0 g,1.0 eq), and triethylamine (3.0 eq);

then dimethylformamide (5 mL) is added and stirred to be completely dissolved, dichloroethane (1.25 eq) is added and stirred for 2h at 25 ℃;

monitoring by HPLC until the reaction is finished;

the reaction mixture was added to water (25 mL), and ethyl acetate (20 mL. Times.3) was added to conduct extraction;

the organic phases were combined and anhydrous Na ₂ SO ₄ Drying, concentrating under reduced pressure, and purifying the resulting oil by column chromatography to give 1.04g of a milky white powdery solid in a yield of 45%, M +:602.72.

the effect of this example is to make CG with a group that can bind to the enzyme, and the technical effect of the present application is independent of the particular hapten derivative.

Example 2 coupling of Glycocholic acid derivatives to G6PDH molecules

The coupling was performed according to the G6 PDH-glycocholic acid conjugate of the present application in the following manner: the thiol-reactive group (e.g., maleimide group) on the glycocholic acid derivative molecule is covalently bound to a thiol group on the G6PDH molecule.

1. Solution preparation:

glycocholic acid derivative solution: the glycocholic acid derivative prepared in example 1 at 10mg/ml was dissolved in DMF;

g6PDH solution: 6.7mg/mL G6PDH (mutant or control mutant of the present application), PB 100mmol, naCl 100mmol, pH =8.0;

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

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

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

3. And (3) oscillating the reaction system at room temperature for 4h, eluting with the desalting solution by using a desalting column, and collecting protein peaks to obtain a product, namely the G6 PDH-glycocholic acid conjugate.

Example 3 preparation of the kit

A kit for detecting glycocholic acid was prepared, which comprises:

a reagent R1 comprising:

100mM PB buffer, pH 7.2

15mM glucose 6-phosphate

15mM beta-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;

a reagent R2 comprising:

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;

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

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

Example of detection

Reaction time: 10min, wherein the incubation time is 4.7min, after 1min of incubation after adding the reagent R2, measuring the read absorbance A1, after 1min of incubation, measuring the read absorbance A2, calculating Δ a = (A2-A1)/min. The glycocholic acid content of the sample was calculated by a calibration curve: CG = sample tube absorbance calibrator concentration/calibrator absorbance.

The glycocholic acid detection kit prepared in example 3 is subjected to performance detection, and the main detection performances are total inaccuracy, repeatability, recovery, linearity, specificity and the like.

TABLE 1 parameters of fully automatic biochemical analyzer

Detecting machine type	Hitachi 7180
		analysis/time/Point	2 point speed/10 min/20-24 points
R1/R2/S	120:40:9
		Wavelength (auxiliary/main)	405/340
Type of reaction	Incremental increase
		Calibration type	Spine type
Calibration point	6
		Concentration of calibrator	0/2.50/5.00/10.00/20.00/40.00

Detection example 1 Glycocholic acid detection kit calibration Absorbance

TABLE 2 Glycocholic acid detection kit calibration absorbance

Detection example 2 Total inaccuracy degree of Glycocholic acid detection kit

TABLE 3 Total inaccuracy

Detection example 3 Glycocholic acid detection kit repeatability

TABLE 4 repeatability

Detection example 4 Glycocholic acid detection kit recovery

TABLE 5 recovery

Detection example 5 Glycocholic acid detection kit Linearity

TABLE 6 linearity

Detection example 6 Glycocholic acid detection kit anti-specificity

TABLE 7 specificity

Interferent (40. Mu.g/ml)	Reagent of the present 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％
			Cholesterol acid sodium salt	61.21％	61.90％
Deoxycholic acid sodium salt	4.22％	11.74％

The reagent of the present application has little to no cross-reaction with the structural analog 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 activity of G6PDH enzyme is influenced due to steric hindrance, so that the efficiency of catalyzing NAD to be converted into NADH is reduced, and the difference between an experimental group in which the antibody is added and an experimental group in which the antibody is not added is compared by detecting the change of NADH amount, wherein the difference is represented by the inhibition capacity of the antibody on G6 PDH.

2. Reaction system:

TABLE 8 preparation of reagents for measuring antibody inhibition

TABLE 9 antibody inhibition Rate testing of on-machine parameters

Detecting machine type	Hitachi 7180
		analysis/time/Point	2 point speed/10 min/20-24 points
R1/S	120:20
		Wavelength (auxiliary/main)	405/340
Type of reaction	Incremental increase

3. As a result:

and comparing the added antibody with the unadditized antibody, and respectively detecting the absorbance values of the G6PDH-CG conjugate to obtain the inhibition situation of the antibody on G6 PDH.

Antibody inhibition = absorbance change of G6PDH-CG with antibody/absorbance change of G6PDH-CG without antibody.

Compared with published mutation sites, the mutant of the application has obviously improved antibody inhibition rate which can reach more than 30 percent and can reach 50 percent. Whereas the inhibition rate of the mutation site (such as A45C, K C) which is commonly used before is only about 20 percent or even lower.

TABLE 10 antibody inhibition of different G6PDH mutants

While not being bound to a particular theory, it may be explained in part as: compared with the G6PDH mutant in the prior art, the mutant (D306C, D375C, G C) of the enzyme of the present application has a mutation site (i.e., a site for introducing a free thiol) at a position where coupling with a hapten (such as hormone, small molecule drug and the like) occurs. When the hapten binds to a hapten-specific antibody at this position, the steric hindrance formed has the greatest effect on the activity of the G6PDH enzyme, and after the introduction of the mutation, it cannot substantially affect the steric folding of the molecule. Therefore, the position of this mutation site is very important, and needs to be compatible with the activity of G6PDH enzyme, the spatial folding of the coupling molecule, and the sufficient exposure of the hapten epitope.

The enzyme mutant has obvious advantage in calibration absorbance due to obvious improvement of the antibody inhibition rate. After the conjugate obtained by coupling the enzyme mutant and the hapten is prepared into the kit, the kit has obvious performance improvement in the aspects of repeatability, total inaccuracy, linearity, specificity and the like due to the improvement of a calibration curve.

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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