CN116200352A

CN116200352A - 7 alpha-hydroxysteroid dehydrogenase mutant and application thereof in 7-oxo-lithocholic acid synthesis

Info

Publication number: CN116200352A
Application number: CN202211637423.5A
Authority: CN
Inventors: 张荣珍; 柳志永
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2022-12-16
Filing date: 2022-12-16
Publication date: 2023-06-02

Abstract

The invention discloses a 7 alpha-hydroxysteroid dehydrogenase mutant and application thereof in 7-oxo-lithocholic acid synthesis, belonging to the technical fields of genetic engineering and biocatalysis. According to the invention, the Brucella melitensis 7 alpha-hydroxysteroid dehydrogenase (Bm7alpha-HSDH) pocket specific amino acid is subjected to site-directed mutagenesis, and partial dominant mutagenesis is combined, so that the mutant with obviously improved thermal stability and catalytic efficiency is obtained. In the catalytic substrate goose deoxidizationIn the reaction of synthesizing 7-oxo-lithocholic acid from cholic acid, the enzyme activity of the mutant is improved by about 45 times at most, the catalytic efficiency is improved by about 122 times, and T is improved _m The maximum value can be raised by about 12 ℃, which provides high-quality biological enzyme and precursor compound for synthesizing ursodeoxycholic acid by biological method catalysis and lays a solid research foundation for realizing industrial production.

Description

7 alpha-hydroxysteroid dehydrogenase mutant and application thereof in 7-oxo-lithocholic acid synthesis

Technical Field

The invention relates to a 7 alpha-hydroxysteroid dehydrogenase mutant and application thereof in 7-oxo-lithocholic acid synthesis, belonging to the technical fields of genetic engineering and biocatalysis.

Background

Chenodeoxycholic acid (3α,7α -dihydroxy-5- β -cholanic acid) is a steroid containing 2 hydroxyl groups, and has the main effect of reducing the saturation of bile cholesterol. 7-oxo-lithocholic acid (3α -hydroxy-7-oxo-5β -cholanic acid) is a reduction product of chenodeoxycholic acid, acting as an intermediate for the synthesis of ursodeoxycholic acid (3α,7β -dihydroxy-5β -cholan-24-oic acid). Ursodeoxycholic acid and chenodeoxycholic acid are stereoisomers, and are mainly used for treating cholelithiasis, fatty liver and biliary dyspepsia, preventing new coronavirus, etc.

7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH, EC 1.1.1.159) is one of the main enzymes for the production of 7-oxo-lithocholic acid. 7 alpha-hydroxysteroid dehydrogenase belongs to the short-chain dehydrogenase/reductase, comprising Rossmann-folded NAD (P) H/NAD (P) ⁺ A binding domain and an active catalytic triplet consisting of Ser-Tyr-Lys. 10 different sources of 7α -HSDH genes have been cloned and functionally validated. These 7α -HSDH have amino acid sequence homologies ranging from 17.57% to 73.93% and exhibit different catalytic efficiencies and thermostabilities in 7-oxo-lithocholic acid production. However, the natural 7. Alpha. -HSDH enzyme has poor thermostability and low efficiency in the production of 7-oxo-lithocholic acid. It was reported that 7 a-HSDH from Xanthomonas maltophilia gave only 80% yield of 7-oxo-lithocholic acid after 24h of reaction. After incubation of 7α -HSDH from Clostridium absonum for 2h at 37 ℃, its residual activity was less than 50%.

The inventor screens out a 7alpha-HSDH from Brucella melitensis, which can catalyze and synthesize 7-oxo-lithocholic acid by taking chenodeoxycholic acid as a substrate, but the stability and the catalytic efficiency of wild type enzyme are lower, so that higher time cost and economic cost are consumed in the biological preparation process. Therefore, the thermal stability and the catalytic efficiency of the 7 alpha-HSDH are improved, and a solid research foundation can be laid for the industrial production of the intermediate 7-oxo-lithocholic acid of ursodeoxycholic acid synthesized by the enzyme.

Disclosure of Invention

The present inventors have found that 7α -HSDH from b.melitensis is capable of catalytic synthesis of 7-oxo-lithocholic acid with chenodeoxycholic acid as substrate, but the wild-type enzyme has lower stability and catalytic efficiency, resulting in higher time and economic costs in the biological preparation process.

The invention optimizes the codon of the 7alpha-HSDH gene from B.melitensis according to the preference of the escherichia coli codon and artificially synthesizes the gene, which is named Bm7alpha-HSDH; then, through mutating specific amino acid, the 7 alpha-HSDH mutant with improved catalytic efficiency and thermal stability is obtained, and can be used for realizing the efficient synthesis of 7-oxo-lithocholic acid.

The invention provides a 7 alpha-HSDH gene derived from B.melitensis, which contains 915bp base and is obtained by optimizing the codon preference of escherichia coli, and the nucleotide sequence of the gene 7 alpha-HSDH is shown as SEQ ID NO. 1. The 7 alpha-HSDH encoded by the gene shown in SEQ ID NO.1 comprises 305 amino acids, and the amino acid sequence is shown in SEQ ID NO. 2.

The invention provides a 7 alpha-hydroxysteroid dehydrogenase mutant, which is obtained by mutating one or more amino acids at 262 th, 301 th and 258 th of 7 alpha-hydroxysteroid dehydrogenase with an amino acid sequence shown as SEQ ID NO. 2.

The invention provides a 7 alpha-hydroxysteroid dehydrogenase mutant, which is obtained by mutating lysine at position 262 of the amino acid sequence of 7 alpha-hydroxysteroid dehydrogenase with an amino acid sequence shown as SEQ ID NO.2 into threonine, and is named as: K262T;

or, the 7 alpha-hydroxysteroid dehydrogenase mutant is obtained by mutating the 301 th glutamine of the amino acid sequence of the 7 alpha-hydroxysteroid dehydrogenase with the amino acid sequence shown as SEQ ID NO.2 into isoleucine, and is named as: Q301I;

or, the 7 alpha-hydroxysteroid dehydrogenase mutant is obtained by mutating the 258 th isoleucine of the amino acid sequence of the 7 alpha-hydroxysteroid dehydrogenase with the amino acid sequence shown in SEQ ID NO.2 into methionine and mutating the 262 th lysine of the amino acid sequence of the 7 alpha-hydroxysteroid dehydrogenase with the amino acid sequence shown in SEQ ID NO.2 into threonine, and is named as threonine: I258M/K262T.

The invention also provides a gene for encoding the 7 alpha-hydroxysteroid dehydrogenase mutant, which can be obtained by cloning and artificial total sequence synthesis.

The invention also provides a recombinant vector carrying the gene.

In one embodiment of the present invention, the recombinant expression vector may be constructed by ligating the coding gene to various commercially available empty vectors by a method conventional in the art.

In one embodiment of the present invention, the recombinant vector is an expression vector of pET-21a, pET-21a or pRSFDuet-1.

The invention also provides a recombinant cell for expressing the mutant, carrying the gene or carrying the recombinant vector.

In one embodiment of the invention, the recombinant cell is an expression host which is bacterial or fungal.

In one embodiment of the present invention, the recombinant cell is an E.coli cell as an expression host.

The invention also provides a recombinant expression transformant containing the coding gene of the 7 alpha-HSDH mutant or the recombinant expression vector thereof.

In one embodiment of the present invention, the recombinant expression transformant can be prepared by transforming the above recombinant expression vector into a corresponding host cell by a conventional technique in the art.

In one embodiment of the present invention, the host cell is a conventional host cell in the art, so long as it is capable of stably self-replicating the recombinant expression vector and the 7α -HSDH gene encoded thereby is capable of being efficiently expressed.

The invention also provides a catalyst for catalyzing chenodeoxycholic acid to synthesize 7-oxo-lithocholic acid, wherein the catalyst is the 7 alpha-hydroxysteroid dehydrogenase mutant or enzyme solution containing the 7 alpha-hydroxysteroid dehydrogenase mutant prepared by adopting the recombinant cells or lyophilized powder thereof.

In one embodiment of the invention, the catalyst is in any one of the following forms: culturing the recombinant expression transformant of the present invention, and isolating the transformant cell containing the 7α -hydroxysteroid dehydrogenase mutant; or, culturing the recombinant expression transformant of the present invention, and separating the crude enzyme solution containing the 7α -hydroxysteroid dehydrogenase mutant; or, culturing the recombinant expression transformant of the present invention, separating to obtain a crude enzyme solution containing the 7α -hydroxysteroid dehydrogenase mutant, and drying the crude enzyme solution to obtain a crude enzyme powder. The culture method and conditions of the recombinant expression transformant are conventional methods and conditions, and different optimization conditions are adopted for different host systems, so that efficient expression of the protein is realized.

The invention also provides a method for synthesizing 7-oxo-lithocholic acid, which comprises the steps of adding the mutant or the mutant prepared by adopting the recombinant cells into a recombinant cell containing chenodeoxycholic acid and coenzyme NAD ⁺ In the reaction system of (2), catalyzing and preparing to obtain 7-oxo-lithocholic acid;

in one embodiment of the present invention, the reaction system further comprises a buffer salt solution having a pH of 8.0 to 9.5.

In one embodiment of the present invention, the buffer salt solution may be any buffer conventionally used in the art, as long as its pH ranges from 8.0 to 9.5, such as sodium phosphate, potassium phosphate, tris-HCl buffer, sodium carbonate buffer, preferably sodium carbonate-sodium bicarbonate buffer having a pH of 9.5.

In one embodiment of the present invention, the concentration of the buffer is 0.05 to 0.2M.

In one embodiment of the invention, the concentration of the substrate chenodeoxycholic acid is 2 to 50mM.

In one embodiment of the present invention, the reaction temperature in the reaction system is 20 to 40 ℃.

In one embodiment of the invention, the reaction temperature is 30 ℃.

The reaction conditions are as follows: the preparation is carried out in a buffer salt solution with the pH value of 8.0-9.5, the concentration of the substrate chenodeoxycholate is 2-50 mM, and the temperature is 20-40 ℃.

The invention also provides application of the mutant, the gene, the recombinant vector or the recombinant cell in preparing 7-oxo-lithocholic acid or a product containing 7-oxo-lithocholic acid.

Advantageous effects

(1) The invention uses 7 alpha-HSDH of the amino acid sequence shown in a sequence table SEQ ID NO.2 as a female parent, finds key amino acids near a substrate binding site through homologous sequence and structure comparison, adopts an alanine scanning strategy to determine candidate sites for pseudo mutation, and adopts a site-directed mutagenesis method to successfully realize remarkable changes of the catalytic efficiency and the thermal stability of the 7 alpha-HSDH. Based on the above, the 7 alpha-HSDH mutant with improved catalytic efficiency is obtained through enzyme activity measurement, CD detection and liquid phase detection. Compared with the wild type, the preferred mutant can efficiently catalyze chenodeoxycholic acid and has improved thermal stability.

(2) The invention constructs 7 alpha-HSDH gene in plasmid pET-21a, expresses Escherichia coli BL (DE 3), and obtains pure enzyme after purifying crude enzyme liquid by His-Trap affinity chromatography column. Na at pH 9.5 by optimizing the enzyme activity assay conditions ₂ CO ₃ -NaHCO ₃ Specific enzyme activity was determined in buffer at 30 ℃. The enzyme activity of the combined mutant I258M/K262T is improved by about 45 times by enzyme activity measurement, T _m The value was increased by about 12 ℃.

(3) The 7 alpha-HSDH provides a new way for synthesizing ursodeoxycholic acid by combining the asymmetric transformation reaction with coenzyme circulation for the hydroxysteroid dehydrogenase, solves the problem of insufficient raw material for synthesizing ursodeoxycholic acid, provides excellent strains and enzymes for synthesizing ursodeoxycholic acid intermediates in industry, and is beneficial to greatly reducing the production cost.

Drawings

Fig. 1: SDS-PAGE analysis of wild type, single point dominant mutants and combinations of mutations. M: protein molecular weight standard, CK: e.coli cell disruption supernatant; lane 1 is the expression of WT in cells; lanes 2-5: purified products of WT, K262T, Q301I, I M/K262T protein, respectively.

Detailed Description

Chenodeoxycholic acid referred to in the following examples was purchased from: the largehead, inc.

The following examples relate to the following media:

LB medium: peptone 1%, yeast extract 0.5%, naCl 1%, pH7.0. Ampicillin (100. Mu.g.mL) was added before use if necessary ^-1 ) 1.5% of agar powder is added to the solid medium.

The detection method involved in the following examples is as follows:

determination of 7 alpha-HSDH enzyme Activity:

the enzyme activity of 7α -HSDH to oxidize CDCA was measured at 30℃by measuring the rate of change of absorbance of NADH at 340 nm. Enzyme activity measurement standard: 200. Mu.L of reaction volume was added with Na at a final concentration of 100mM ₂ CO ₃ -NaHCO ₃ (pH 9.5), 5mM NAD ⁺ And 5mM CDCA, incubated in a metal bath at 30deg.C for 3min, and appropriate amount of pure enzyme (10-200 μM) was added, and the absorbance change at 340nm was scanned in a Bio-Tek Cystation 5 cell imaging multifunctional detection system.

The enzyme activity is defined as: under the above conditions, the amount of enzyme catalyzing the production of 1. Mu. Mol NADH per minute is defined as one unit U.

The calculation formula of the enzyme activity is as follows: enzyme activity (U) =ew×v×10 ³ /(6220×0.5)

The calculation formula of specific activity: specific activity (U.mg) ^-1 ) =enzyme activity (U)/protein amount (mg)

Wherein, EW: a change in absorbance at 340nm within 1 min; v: volume of reaction solution (mL); 6220: molar extinction coefficient L.mol ^-1 ·cm ^-1 ) The method comprises the steps of carrying out a first treatment on the surface of the 0.5: optical path distance (cm).

Determination of kinetic parameters:

according to the milbey equation v=v _max ×[S]/K _m +[S]Calculating K of enzyme on different substrates _m Values.

Wherein v: reaction Rate (U.mg) ^-1 )；V _max : maximum reaction Rate (U.mg) ^-1 )；[S]: substrate concentration (mM); k (K) _m : the reaction speed V reaches half of 1/2V _max Substrate concentration at that time.

When the substrate concentration is saturated, k _cat ＝V _max Et, calculate K _cat Values.

Wherein V is _max : maximum reaction rate; et: concentration of enzyme sites.

Kinetic parameter determination:

the total reaction volume was 200. Mu.L, 0.1M Na was added ₂ CO ₃ -NaHCO ₃ Buffer (pH 9.5), 5.0mM NAD ⁺ The temperature is kept at 30 ℃ for 2min, and a proper amount of pure enzyme solution is added to measure the specific enzyme activities under different substrate concentrations (0.1 mM-5.0 mM). Fitting of enzyme dynamics curves is realized through GraphPad software, substrate concentration and specific enzyme activity are taken as horizontal and vertical coordinates respectively, and a Mie equation model is selected for nonlinear fitting to obtain V _max Value of K _m Value, k is obtained by data conversion _cat Value and k _cat /K _m Values (table 3).

CD determination

Circular dichromatic measurements were performed using a Jasco J720 spectropolarimeter. Wavelength scan data were collected from 190 to 250nm in phosphate buffer (pH 8.0) using the following instrument setup (30 scans on average): response, 1s; the sensitivity is 100 mm; at a speed of 50nm min ^-1 . The scan is repeated every 3℃between 20℃and 80 ℃. The T of the different proteins was calculated in 50mM Na/K phosphate buffer (pH 8.0) at a protein concentration of about 0.01M, with the decrease in the circular dichroism signal with increasing temperature recorded at 220nm _m Values.

Example 1:7 alpha-HSDH gene codon optimization and synthesis thereof

The method comprises the following specific steps:

(1) The melitensis-derived 7α -HSDH gene contains 915bp bases (NCBI accession number: MW 202238). And (3) carrying out codon optimization on the 7 alpha-HSDH gene by referring to the codon preference of the escherichia coli, wherein the optimized gene sequence is shown as SEQ ID NO. 1.

(2) 6 histidine tag genes are introduced into the C end of the gene, bamH I and Xho I restriction enzyme cutting site sequences are respectively added into the two ends of the gene, and finally the gene is connected with pET-21a to obtain recombinant plasmid pET-21a-7α -HSDH.

Example 2: construction of 7 alpha-HSDH mutant gene and recombinant E.coli expression System

(1) Construction of 7α -HSDH mutants

Design of 7alpha-HSDH by full plasmid PCR technique

Site-directed mutagenesis, designing a primer sequence (Table 1) by using a recombinant plasmid pET-21a-7α -HSDH as a template, and carrying out the following mutations respectively: K262C, K262F, K262G, K T, K262V, K262Y, Q301F, Q301I, Q301L, Q301M, Q301N, I M/K262T.

Table 1: construction of mutant primer design Table

pmol·μl ^-1 ) 1.0 μl, template 1.0 μl, ddH ₂ O 22.0μl；

PCR amplification conditions: pre-deformation: 98 ℃ for 30s; denaturation: 98 ℃ for 10s; annealing: 15s at 55 ℃; extension: 72 DEG C

60s; rear extension: 72 ℃ for 10min; and (3) preserving: 4 ℃.

After the PCR amplification is finished, the amplified product is digested with Dpn I for 2 hours at 37 ℃ to remove the template plasmid, the digested product is transformed into E.coli BL21 (DE 3) competent cells, a single colony test tube is selected for culture, and the sequence of the mutant is verified by sequencing.

(2) E.coli BL21 (DE 3) transformed with the recombinant plasmid:

add 10. Mu.L of the PCR product from step (1) to 100. Mu.L of E.coli BL21 (DE 3) competent cell suspension per tube, mix gently and stand in ice bath for 30min. Transferring into a water bath at 42 ℃ and thermally striking for 90s. Transfer to ice bath rapidly and cool for 3min. mu.L of LB liquid medium was added to each tube, and the culture was performed at 37℃for 1 hour with shaking at 100 rpm. Centrifuging the cultured bacterial liquid at 3,000Xg for 2min, discarding 700 μL of supernatant, mixing the rest bacterial liquid uniformly, and coating to obtain a liquid containing 50 μg/mL ^-1 Ampicillin was cultured on LB plates at 37℃overnight in an inverted manner.

(3) Selection of positive clones:

4 clones were picked and transferred to a 5 mL-containing 100. Mu.g.mL cell ^-1 Ampicillin was cultured in LB medium at 37℃for 8 hours, and plasmid extraction kit Mini-Plasmid Rapid Isolation Kit (Nanjino)Praise biotechnology limited) to extract plasmids.

Enzyme digestion verification is carried out by using the following reaction system: 10 XBuffer 2. Mu.L, plasmid DNA 5. Mu.L, bamH I0.5. Mu.L, xho I0.5. Mu.L, ddH ₂ O made up the system to 20. Mu.L.

Positive clones were obtained separately: E.coli/pET-21a-K262C, E.coli/pET-21a-K262F, E.coli/pET-21a-K262G, E.coli/pET-21a-K262T, E.coli/pET-21a-K262V, E.coli/pET-21a-K262Y, E.coli/pET-21a-Q301F, E.coli/pET-21a-Q301I, E.coli/pET-21a-Q301L, E.coli/pET-21a-Q301M, E.coli/pET-21a-Q301N, E.coli/pET-21a-I258M/K262T.

Meanwhile, recombinant bacteria containing wild enzymes are prepared according to the method: E.coli/pET-21a-7α -HSDH.

Example 3: induction expression culture of recombinant bacteria

The method comprises the following specific steps:

(1) Positive clones containing mutants prepared in example 2 were picked up and inoculated with 10mL of single colony containing wild-type enzyme containing 100. Mu.g.mL ^-1 Ampicillin was cultured overnight in LB liquid medium at 37℃with shaking at 200 rpm.

(2) Transferring 10mL of the culture solution obtained in the step (1) to 1L of culture solution containing 100 mug.mL ^-1 In LB liquid medium of ampicillin, shake culture was carried out at 37℃and 200rpm until OD was reached ₆₀₀ About 0.6 to about 0.8, and preparing a culture; isopropyl-BETA-D-thiogalactoside was added to the culture at a final concentration of 0.1mM, and induction culture was performed at 25℃for 12 hours.

(3) After the induction culture was completed, the culture solution was centrifuged at 6,000Xg for 10min to collect recombinant E.coli cells, and washed three times with physiological saline to collect the cells.

Example 4: purification of recombinant proteins

Purification of recombinant protein using His-Trap HP affinity column:

(1) 2g of the wet cells collected in example 3 were weighed, and cells were suspended in an appropriate amount of 20mM Tris-HCl (pH 8.0) buffer, and sonicated in an ice bath (working for 2s at 3s intervals for 10 min). The supernatant was collected as a crude enzyme solution by centrifugation at 12,000Xg for 30min at 4 ℃.

(2) The crude enzyme solutions were purified by His-Trap affinity chromatography produced by Cytiva, respectively, and purified products of wild type and its dominant mutant proteins were analyzed by SDS-PAGE (FIG. 1), and the bands expressed in cells of the wild type and after purification were all shown as non-single bands, probably the flexible ends of the whole-length protein affected the expression of the protein. The pure enzyme solution is used for enzyme activity determination after ultrafiltration and desalination.

The pure enzyme solutions containing the wild-type enzyme WT, the pure enzyme solution containing the K262C, the pure enzyme solution containing the K262F, the pure enzyme solution containing the K262G, the pure enzyme solution containing the K262T, the pure enzyme solution containing the K262V, the pure enzyme solution containing the K262Y, the pure enzyme solution containing the Q301F, the pure enzyme solution containing the Q301I, the pure enzyme solution containing the Q301L, the pure enzyme solution containing the Q301M, the pure enzyme solution containing the Q301N and the pure enzyme solution containing the I258M/K262T are prepared respectively.

Example 5: specific enzyme activity determination

The specific enzyme activities of the wild-type enzyme and the mutant enzyme obtained in example 4 to chenodeoxycholate were measured respectively, and the results are shown in the following table.

Table 2: comparison of specific enzyme Activity of wild-type and mutant

Annotation: and n.d. ^a Indicating no activity was detected; the naming mode of the mutant is as follows: "amino acid substituted at the original amino acid position" is used to denote a mutant. As in K262C, the amino acid at position 262 is replaced by cysteine Cys from lysine Lys of the parent 7α -HSDH, and the numbering of the position corresponds to the corresponding position of the amino acid sequence of the parent 7α -HSDH.

As shown in Table 2, the specific enzyme activities of the wild type and the mutant catalytic chenodeoxycholic acid are respectively measured, and the result shows that the specific enzyme activity of the single mutant K262C, K262G, K262T, Q I is obviously improved relative to the wild type, and the specific activity of other single mutants is reduced to different degrees.

The dominant single mutant is subjected to combined mutation, and finally, I258M/K262T with greatly improved specific enzyme activity is obtained through screening. The results show that the steric hindrance and the polarity change of the side chains of the different amino acids at the 258, 262 and 301 sites play an important role in catalyzing the catalytic activity of chenodeoxycholic acid.

Example 6: kinetic parameter determination

Kinetic parameter determination: the total reaction volume was 200. Mu.L, 0.1M Na was added ₂ CO ₃ -NaHCO ₃ Buffer (pH 9.5), 5.0mM NAD ⁺ The temperature is kept at 30 ℃ for 2min, and a proper amount of pure enzyme solution is added to measure the specific enzyme activities of different substrates (0.1 mM-5.0 mM). Calculation of k of 7 alpha-HSDH wild type and mutant _cat 、K _m 、k _cat /K _m . (Table 3).

Table 3: kinetic parameters of wild type and mutant

As shown in Table 3, K262G, K262T, Q I and I258M/K262T mutant catalyzes the K of the substrate chenodeoxycholic acid _cat /K _m The value is obviously improved, which is beneficial to improving the catalytic efficiency of synthesizing 7-oxo-lithocholic acid by catalyzing substrate chenodeoxycholic acid by enzyme.

Example 7: CD determination

By characterizing the thermal stability of the mutant obtained in example 4, a mutant having both improved thermal stability and catalytic efficiency was obtained as an excellent enzyme for industrial conversion synthesis of 7-oxo-lithocholic acid. Round two-chromatographic measurements using a Jasco J720 spectropolarimeter to calculate T for different proteins _m Values (table 4).

Table 4: thermal stability analysis of 7 alpha-HSDH wild type and dominant mutants

The results are shown in Table 4, T for K262T, Q I and I258M/K262T relative to the wild type _m The values all showed significant improvement, in combination with example 6K262T and I258M/K262T show both improvements in thermal stability and catalytic efficiency.

Example 8: catalytic synthesis of 7-oxo-lithocholic acid by 7 alpha-HSDH

The method comprises the following specific steps:

the pure enzyme catalytic reaction system was carried out in a 50mL centrifuge tube, and 10mL of the reaction mixture was purified by 0.1M Na ₂ CO ₃ -NaHCO ₃ Buffer (pH 9.5), 2mM NAD ⁺ 2mM chenodeoxycholic acid and 20 μg pure enzyme at 30deg.C, 200r.min ^-1 The reaction was allowed to react for 12 hours with shaking, and samples were taken to check the product yield. The reaction was stopped by adjusting the pH to 10.0-11.0 with 2M NaOH, extracting three times with an equal volume of acetonitrile, mixing the extracts, concentrating by rotary evaporation until crystals are separated out, removing the solvent after suction filtration, drying to constant weight, and detecting by HPLC. The reaction conversion was analyzed by liquid chromatography using a C-18 column (250 mm. Times.4.6 mm) with acetonitrile as mobile phase: water (0.1% phosphoric acid) =60:40, column temperature 30 ℃, flow rate 1.0mL min ^-1 The detection wavelength is 195nm. The results are shown in Table 5.

Table 5: yield of 7α -HSDH wild type and mutant

As shown in Table 5, the K262T, I M/K262T mutant exhibited a significant improvement in yield, and the remaining mutants had no significant improvement in yield over the wild type due to changes in enzyme activity or thermostability. Indicating that the improvement of the mutant in the aspects of catalytic efficiency and thermal stability can play a decisive role in efficiently preparing 7-oxo-lithocholic acid from chenodeoxycholic acid.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A 7 alpha-hydroxysteroid dehydrogenase mutant, which is characterized in that the 7 alpha-hydroxysteroid dehydrogenase mutant is obtained by mutating lysine at position 262 of the amino acid sequence of 7 alpha-hydroxysteroid dehydrogenase with the amino acid sequence shown as SEQ ID No.2 into threonine;

or, the 7 alpha-hydroxysteroid dehydrogenase mutant is obtained by mutating the 301 th glutamine of the amino acid sequence of 7 alpha-hydroxysteroid dehydrogenase with the amino acid sequence shown as SEQ ID NO.2 into isoleucine;

or, the 7 alpha-hydroxysteroid dehydrogenase mutant is obtained by mutating the 258 th isoleucine of the amino acid sequence of the 7 alpha-hydroxysteroid dehydrogenase with the amino acid sequence shown as SEQ ID NO.2 into methionine and mutating the 262 th lysine of the amino acid sequence of the 7 alpha-hydroxysteroid dehydrogenase with the amino acid sequence shown as SEQ ID NO.2 into threonine.

2. A gene encoding the mutant of claim 1.

3. A recombinant vector carrying the gene of claim 2.

4. The recombinant vector according to claim 3, wherein the recombinant vector is an expression vector of pET-21a, pET-21a or pRSFDuet-1.

5. A recombinant cell expressing the mutant of claim 1, or carrying the gene of claim 2, or carrying the recombinant vector of claim 3 or 4.

6. The recombinant cell of claim 5, wherein the recombinant cell is an expression host that is bacterial or fungal.

7. A catalyst for catalyzing chenodeoxycholic acid to synthesize 7-oxo-lithocholic acid, which is characterized in that the catalyst is the 7 alpha-hydroxysteroid dehydrogenase mutant according to claim 1 or an enzyme solution containing the 7 alpha-hydroxysteroid dehydrogenase mutant or a lyophilized powder thereof prepared by using the recombinant cell according to claim 5 or 6.

8. A method for synthesizing 7-oxo-lithocholic acid, which comprises adding the mutant of claim 1 or the mutant obtained by using the recombinant cell of claim 5 or 6 to a recombinant cell containing chenodeoxycholic acid as a substrate and NAD as a coenzyme ⁺ In the reaction system of (2), 7-oxo-lithocholic acid is prepared by catalysis.

9. The method according to claim 8, wherein the reaction system further comprises a buffer salt solution having a pH of 8.0 to 9.5; the concentration of the substrate chenodeoxychol is 2-50 mM, and the reaction temperature is 20-40 ℃.

10. Use of the mutant according to claim 1, or the gene according to claim 2, or the recombinant vector according to claim 3 or 4, or the recombinant cell according to claim 5 or 6 for the preparation of 7-oxo-lithocholic acid or a product containing 7-oxo-lithocholic acid.