CN113462665B - 7 alpha-HSDH enzyme mutant and coding gene and application thereof - Google Patents

Abstract

The invention discloses a 7 alpha-HSDH enzyme mutant and an encoding gene and application thereof. Compared with the wild 7 alpha-HSDH enzyme with the amino acid sequence shown in SEQ ID NO. 2, the amino acid sequence of the 7 alpha-HSDH enzyme mutant carries out pairwise combined mutation, three combined mutation or any mutation of four combined mutations at the 154 th position, the 157 th position, the 194 th position and the 199 th position of the amino acid sequence shown in SEQ ID NO. 2. The 7 alpha-HSDH enzyme mutant can be used for synthesizing and preparing 7-ketolithocholic acid, a substrate CDCA is converted by using the mutant as a biocatalyst to generate 7-KLCA, and the reaction conversion rate of a product after reaction is more than 99 percent through HPLC verification. Compared with wild enzyme, the 7 alpha-HSDH enzyme mutant constructed by the invention has obviously improved catalytic activity, can obviously reduce the use amount of enzyme, and has wide prospect of large-scale industrial application.

Description

7 alpha-HSDH enzyme mutant and coding gene and application thereof

Technical Field

The invention relates to the technical field of biological enzyme engineering, in particular to a 7 alpha-HSDH enzyme mutant and a coding gene and application thereof.

Background

Ursodeoxycholic acid (UDCA), whose chemical name is 3 α,7 β -dihydroxy-5 β -cholestane-24-oic acid, is the 7 β -hydroxy epimer of Chenodeoxycholic acid (CDCA). At present, the bear gall is mainly prepared by two methods of bear gall extraction and artificial synthesis. The extraction source from bear gall is limited, the period is long, the yield is low, and the method is in violation of animal protection, so that the UDCA is mainly prepared by artificial synthesis at present.

7-keto-Lithocholic acid (7-keto-Lithocholic acid, 7-KLCA) is a key intermediate for artificially synthesizing UDCA, and the synthesis mainly comprises two methods, namely a chemical method and an enzymatic method. The chemical method is to oxidize the 7-hydroxyl group by using chenodeoxycholic acid as a raw material and using a chemical oxidant such as chromium trioxide, NBS, PCC and the like, so that the 3-hydroxyl group is oxidized, the product purity is not high, the separation is difficult, the 3-hydroxyl group needs to be protected and deprotected, the synthesis steps are increased, and the used reagent has great pollution to the environment. And the enzyme method (7 alpha-hydroxysteroid dehydrogenase, 7 alpha-HSDH) is adopted to oxidize the 7-hydroxyl, so that the method has high specificity, can not oxidize the 3-hydroxyl, has mild reaction conditions, hardly uses organic solvents and has little environmental pollution.

However, at present, few reports about 7 alpha-HSDH enzymes at home and abroad are available, the enzymes are all original gene sequences, the reactivity is not high, and the industrial application is greatly limited. For example, CN106676079B discloses a 7 α -hydroxysteroid dehydrogenase gene S1-a-2, which encodes a novel 7 α -hydroxysteroid dehydrogenase, and is capable of catalyzing epimerization of 7-hydroxyl group of chenodeoxycholic acid, taurochenodeoxycholic acid (TCDCA), to produce ursodeoxycholic acid, 7-ketolithocholic acid, an intermediate of Tauroursodeoxycholic acid (TUDCA), tauroursodeoxycholic acid (T7K-LCA). However, the catalytic activity of the 7 alpha-hydroxysteroid dehydrogenase coded by the gene on CDCA or TCDCA is only about 2 times of that of 7 alpha-HSDH of clostridium sardinieri, and the enzyme activity is not obviously improved.

A protein three-dimensional structure simulation and protein directed evolution technology is a high-tech technology which is developed in recent years and carries out artificial modification on an original gene sequence so as to meet the requirement of industrial application, wherein the protein directed evolution technology obtains the Nobel chemical prize in 2018. Therefore, combining protein three-dimensional structure simulation and protein directed evolution technology, further searching and developing new hydroxysteroid dehydrogenase suitable for industrial mass production is the hot spot of current research.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to solve the technical problem of providing a 7 alpha-HSDH enzyme mutant, a coding gene and application thereof, so as to solve the problems that the activity of the conventional hydroxysteroid dehydrogenase is not ideal and industrial production is difficult to realize. The invention adopts protein three-dimensional structure simulation and protein directed evolution technology to artificially modify 7 alpha-HSDH enzyme derived from Clostridium cadeveris, and compared with wild enzyme, the modified mutant unit enzyme activity is improved by about 9 times, thereby greatly reducing the use amount of the enzyme in industrial production and having good industrial application prospect.

In order to solve the technical problems, the invention provides the following technical scheme:

the amino acid sequence of the wild type 7 alpha-HSDH enzyme derived from Clostridium cadeveris is shown as SEQ ID NO. 2, and the nucleotide sequence of the coding gene thereof is shown as SEQ ID NO. 1.

The nucleotide sequence of the encoding gene of the 7 alpha-HSDH enzyme is obtained by whole-gene synthesis of Changzhou space biotechnology limited company, and NdeI restriction enzyme sites and HindIII restriction enzyme sites are respectively added at two ends of an encoding region. After the target gene fragment is cut by restriction enzymes NdeI and HindIII, the target gene fragment is connected, transformed and screened with a pET29a (+) vector (Novagen company) cut by the same double enzyme, and the screened positive plasmid 7 alpha-HSDH-pET 29a (+) is transferred into BL21 (DE 3) host bacteria, so that an in-vitro heterologous expression system of the 7 alpha-HSDH enzyme is constructed.

The construction of the mutant of the 7 alpha-HSDH enzyme is obtained by the technical means of directed evolution, namely, the mutant is obtained by utilizing the directed implementation technologies such as error-prone PCR, DNA rearrangement, semi-rational design, three-dimensional structure simulation and the like. Specifically, the invention carries out directed evolution of enzymes by a three-dimensional structure simulation technology. A three-dimensional structure of the 7 alpha-HSDH enzyme is simulated by adopting a homologous modeling method, one or more possible active sites related to catalysis are predicted by utilizing an energy minimum principle and a molecular docking technology, NNK saturation site-specific mutagenesis is carried out on the active sites, and a mutant with remarkably improved activity is screened out.

The more specific process is as follows: the possible active sites predicted by the three-dimensional structure simulation technology are Q154, Y157, A194 and L199. NNK saturation mutation was performed on each of these four sites, and High Pressure Liquid Chromatography (HPLC) was used to screen for mutants. More specifically, the method comprises the following steps: 1. when glutamine (Q) at position 154 is mutated to asparagine (N), the catalytic activity of the mutant is increased relative to the wild-type enzyme; 2. when tyrosine (Y) at the position 157 is mutated into alanine (A), the enzyme activity of the mutant is improved; 3. when alanine (A) at position 194 is mutated into valine (V), the enzyme activity of the mutant is improved compared with that of the wild enzyme; 4. when leucine (L) at position 199 is mutated into phenylalanine (F), the enzyme activity of the mutant is obviously improved. When the mutations at the 4 sites are combined in pairs or three or four, the catalytic activity of the mutant is greatly improved compared with that of a single mutant.

Therefore, in one aspect, the invention claims a 7 alpha-HSDH enzyme mutant, the amino acid sequence of which is compared with the wild-type 7 alpha-HSDH enzyme with the amino acid sequence shown in SEQ ID NO. 2, and any one of two-by-two mutation, three combined mutation or four combined mutation is carried out at the 154 th position, 157 th position, 194 th position and 199 th position of the amino acid sequence shown in SEQ ID NO. 2.

Specifically, the pairwise combined mutation is as follows:

when the 154 th position of the amino acid sequence shown in SEQ ID NO. 2 is mutated from glutamine to asparagine, and the 194 th position is mutated from alanine to valine, the amino acid sequence of the 7 alpha-HSDH enzyme mutant is shown in SEQ ID NO. 3;

or when the 157 th position of the amino acid sequence shown in SEQ ID NO. 2 is mutated from tyrosine to alanine, the 194 th position is mutated from alanine to valine, the amino acid sequence of the 7 alpha-HSDH enzyme mutant is shown in SEQ ID NO. 4;

or when the 194 th site of the amino acid sequence shown in SEQ ID NO. 2 is mutated from alanine to valine and the 199 th site is mutated from leucine to phenylalanine, the amino acid sequence of the 7 alpha-HSDH enzyme mutant is shown in SEQ ID NO. 5.

Specifically, the three combined mutations are:

when the 157 th site of the amino acid sequence shown in SEQ ID NO. 2 is mutated from tyrosine to alanine, the 194 th site is mutated from alanine to valine, the 199 th site is mutated from leucine to phenylalanine, the amino acid sequence of the 7 alpha-HSDH enzyme mutant is shown in SEQ ID NO. 6;

or when the 154 th site of the amino acid sequence shown in SEQ ID NO. 2 is mutated from glutamine to asparagine, the 157 th site is mutated from tyrosine to alanine, the 199 th site is mutated from leucine to phenylalanine, and the amino acid sequence of the 7 alpha-HSDH enzyme mutant is shown in SEQ ID NO. 7.

Specifically, the four combined mutations are:

when the 154 th site of the amino acid sequence shown in SEQ ID NO. 2 is mutated from glutamine to asparagine, the 157 th site is mutated from tyrosine to alanine, the 194 th site is mutated from alanine to valine, the 199 th site is mutated from leucine to phenylalanine, and the amino acid sequence of the 7 alpha-HSDH enzyme mutant is shown in SEQ ID NO. 8.

In another aspect, the invention also claims the coding gene of the 7 alpha-HSDH enzyme mutant.

Specifically, the nucleotide sequence of the coding gene of the 7 alpha-HSDH enzyme mutant with the amino acid sequence shown as SEQ ID NO. 3 is shown as SEQ ID NO. 9;

or, the nucleotide sequence of the coding gene of the 7 alpha-HSDH enzyme mutant with the amino acid sequence shown as SEQ ID NO. 4 is shown as SEQ ID NO. 10;

or, the nucleotide sequence of the coding gene of the 7 alpha-HSDH enzyme mutant with the amino acid sequence shown in SEQ ID NO. 5 is shown in SEQ ID NO. 11;

or, the nucleotide sequence of the coding gene of the 7 alpha-HSDH enzyme mutant with the amino acid sequence shown as SEQ ID NO. 6 is shown as SEQ ID NO. 12;

or, the nucleotide sequence of the coding gene of the 7 alpha-HSDH enzyme mutant with the amino acid sequence shown as SEQ ID NO. 7 is shown as SEQ ID NO. 13;

or, the nucleotide sequence of the coding gene of the 7 alpha-HSDH enzyme mutant with the amino acid sequence shown as SEQ ID NO. 8 is shown as SEQ ID NO. 14.

According to the existing public knowledge, any gene is connected with various expression vectors after being operated or modified, is transformed to a proper host cell, and can excessively express a target protein after being induced under proper conditions.

Therefore, in a further aspect, the invention also claims a vector containing the above-mentioned encoding gene.

Specifically, the vector may be any one of various expression vectors, including but not limited to a pET expression vector, a pCW expression vector, a pUC expression vector, or a pPIC9k expression vector.

In yet another aspect, the invention also claims a host cell containing the above-described encoding gene.

Specifically, the host cell may be any suitable host cell, including but not limited to escherichia coli, pichia pastoris, streptomyces, or bacillus subtilis.

In another aspect, the invention also claims application of the 7 alpha-HSDH enzyme mutant, the coding gene, the vector and the host cell in preparation of 7-ketolithocholic acid.

In still another aspect, the present invention also provides a method for preparing 7-ketolithocholic acid, comprising the steps of:

s1, configuring a reaction system, comprising: 1-10g/L7 alpha-HSDH enzyme mutant, 50-200mM sodium phosphate buffer solution with pH9.0, 0.1-0.5g/L coenzyme NAD +,40-100g/L chenodeoxycholic acid and 1-5g/L NADH oxidase; controlling the temperature of the reaction system to be 30 ℃ and the air flow rate to be 5mL/min, and carrying out stirring reaction;

s2, carrying out HPLC detection after 24h of reaction to obtain the 7-ketolithocholic acid.

The reaction product is detected by HPLC, and the reaction conversion rate is more than 99 percent. Thus, the enzyme mutant can be proved to be used for the biological asymmetric oxidation of chenodeoxycholic acid.

The enzyme capable of performing the above-mentioned biocatalytic reaction may be a pure enzyme, a recombinant cell-resting cell, a crude enzyme solution or a crude enzyme powder, or the like.

Compared with the prior art, the invention has the following beneficial effects:

compared with wild enzyme, the 7 alpha-HSDH enzyme mutant constructed by the invention has obviously improved catalytic activity, can obviously reduce the using amount of enzyme, can completely convert 40-100g/L of substrate into key intermediate within 24 hours at room temperature, and has the conversion rate of more than 99%. The reaction condition is mild, almost no by-product is generated, the coenzyme circulating system is stable, and the method has wide industrial application prospect.

Drawings

FIG. 1 shows the biocatalytic reaction process of 7 alpha-HSDH enzyme mutant.

Detailed Description

The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples, unless otherwise specified, and experimental methods not specified in specific conditions in the examples, are generally commercially available according to conventional conditions, and materials, reagents, and the like used in the following examples, unless otherwise specified.

In the examples, the experimental procedures not specified for the specific conditions were generally carried out according to conventional conditions, for example, as described in molecular cloning, A laboratory Manual (J. SammBruk, D.W. Lassel, huang Peitang, wang Jiaxi, zhu Houchu, et al, third edition, beijing: scientific Press, 2002).

EXAMPLE construction of prokaryotic expression System

The 7 α -HSDH gene fragment was synthesized by Henzhou-based Biotechnology, inc. and recombined onto the PUC57 vector. After double digestion with restriction enzymes NdeI and HindIII (from New England Biolabs, NEB) for 4h at 37 deg.C, the gel was separated by electrophoresis in 1% agarose and recovered by gel cutting (gel recovery kit from Tiangen Biotech (Beijing) Ltd.). Subsequently, the cells were ligated with the expression vector pET29a (+) (Novagen) which had also been subjected to double digestion, overnight at 16 ℃ under the action of T4 DNA ligase (purchased from Takara). The ligation solution was used to transform Top10 competent cells (purchased from Tiangen Biochemical technology, beijing, ltd.), and colony PCR screening and sequencing verification were performed to obtain the positive recombinant plasmid 7 α -HSDH-pET29a (+).

The positive recombinant plasmid 7 alpha-HSDH-pET 29a (+) is transformed into expression host bacteria BL21 (DE 3) (purchased from Tiangen Biochemical technology (Beijing) Co., ltd.), and the prokaryotic expression strain 7 alpha-HSDH-pET 29a (+)/BL 21 (DE 3) is obtained and is used as a primary strain for subsequent directed evolution and fermentation.

An NADH oxidase gene (NOX) for coenzyme regeneration is synthesized by Changzhou Yunyu biotechnology limited, the construction of subsequent recombinant expression plasmid is the same as that of 7 alpha-HSDH-pET 29a (+) plasmid, and the expression strain is obtained after the construction is transferred into BL21 (DE 3).

EXAMPLE two enzyme shake flask fermentation preparation of enzyme lyophilized powder

The expression strains 7 α -HSDH-pET29a (+)/BL 21 (DE 3) and NOx-pET29a (+)/BL 21 (DE 3) constructed above were cultured overnight with shaking at 37 ℃ and 200rpm in 5mL of LB liquid medium [ 10g/L tryptone (OXIOD), 5g/L of yeast powder (OXIOD) and 10g/L of sodium chloride (national reagent) in the presence of 30 μ g/mL of kanamycin sulfate at a final concentration, and then inoculated at a ratio of 1% (V/V) into 500mL of LB liquid medium containing 30 μ g/mL of kanamycin sulfate at 37 ℃ and 200rpm for shaking culture. To be OD ₆₀₀ Between 0.8 and 1.0, the inducer IPTG (isopropyl-. Beta. -D-thiogalactoside, IPTG) was added at a final concentration of 0.1mM and induced overnight at 30 ℃. The cells were collected by centrifugation at 8000rpm at 4 ℃ and suspended in 50mM sodium phosphate buffer pH7.0 and disrupted by sonication (200W, 3s/5)s,20 min), centrifuging at 12000rpm for 20min at 4 deg.C, and freeze drying the supernatant to obtain crude enzyme powder.

EXAMPLE construction and screening of the triple mutants

Construction of mutants: a three-dimensional structure simulation of 7 alpha-HSDH is carried out by adopting a homologous modeling method, and possible catalytic sites are predicted by utilizing molecular docking and an energy minimum principle and are preliminarily determined to be four sites of Q154, Y157, A194 and L199. Next, NNK saturation mutation was performed on each of the four sites using the 7. Alpha. -HSDH-pET29a (+) recombinant plasmid as a template (see Stratagene for concrete mutation procedures)

Site-Directed Mutagenesis Kit instructions).

Wherein:

154 site mutation

Forward primer (SEQ ID NO: 15):

GCACCCATCCGGATATTAGCNNKATTAGCTATGGCACCAGCAA，

reverse primer (SEQ ID NO: 16):

TTGCTGGTGCCATAGCTAATMNNGCTAATATCCGGATGGGTGC；

157 position mutation

Forward primer (SEQ ID NO: 17):

GGATATTAGCCAGATTAGCNNKGGCACCAGCAAAGCGAGC，

reverse primer (SEQ ID NO: 18):

CTCGCTTTGCTGGTGCCMNNGCTAATCTGGCTAATATCC；

194 site mutation

Forward primer (SEQ ID NO: 19):

GGGCATGACCGCGACCGATNNKGTGAAAGATAACCTGACCGAT，

reverse primer (SEQ ID NO: 20):

ATCGGTCAGGTTATCTTTCACMNNATCGGTCGCGGTCATGCCC；

199 site mutation

Forward primer (SEQ ID NO: 21):

CGATGCGGTGAAAGATAACNNKACCGATGATTTTCGCGAT，

reverse primer (SEQ ID NO: 22):

ATCGCGAAAATCATCGGTMNNGTTATCTTTCACCGCATCG。

and (3) mutant culture: the plasmid obtained by the above mutation was transformed into BL21 (DE 3) host cells, plated on LB solid medium containing 30. Mu.g/mL kanamycin, and cultured overnight at 37 ℃ in an inverted state, followed by picking up a single clone from the plate and culturing in a 96-well plate. The overnight cultured bacterial solution was transferred to a 96-well plate containing a fresh LB medium, cultured with shaking at 37 ℃ and 220rpm for 4 hours, induced by the addition of IPTG to a final concentration of 0.1mM, and cultured overnight at 30 ℃. The cells were centrifuged at 4000rpm for 10min at 4 ℃ to collect the cells, and the cells were suspended in 50mM sodium phosphate buffer (pH7.0) to carry out a screening reaction as whole cells.

Screening of mutants: 40g/L substrate concentration, 0.2g/L NAD +,50mM pH9.0 sodium phosphate buffer solution, 2g/L NOX enzyme lyophilized powder, according to the proportion of 10%, adding the whole cell suspension prepared above, placing at 30 ℃, 220rpm oscillation reaction. Samples were taken for HPLC detection at 2h and 24h, respectively.

And (3) performing amplification culture on the clone with the substrate conversion rate remarkably improved in 2h and 24h, and then sequencing to verify the mutation condition. Sequencing results show that the mutant enzyme activity is remarkably improved, and the mutant sites contained in the clone are as follows: when glutamine (Q) at 154 is mutated to asparagine (N), tyrosine (Y) at position 157 is mutated to alanine (a), alanine (a) at position 194 is mutated to valine (V), and leucine (L) at position 199 is mutated to phenylalanine (F).

Two-two combined mutation, three combined mutation and four combined mutation are carried out on the several sites, activity detection finds that the catalytic activity of the combined mutation of some sites is obviously improved compared with single-point mutation, and specific enzyme activity values are shown in the following table 1.

TABLE 1 enzyme Activity of different combinations of mutations

Mutants	Specific enzyme activity	Multiple of improvement
			Wild type 7α-HSDH	55U/mg	--
Q154N	66.2U/mg	1.2
			Y157A	98.2U/mg	1.79
A194V	71.4U/mg	1.3
			L199F	140U/mg	2.5
Q154N/A194V	191.5U/mg	3.49
			Y157A/A194V	302.4U/mg	5.5
A194V/L199F	326.4U/mg	5.93
			Y157A/A194V/L199F	445.5U/mg	8.1
Q154N/Y157A/L199F	434.7U/mg	7.9
			Q154N/Y157A/A194V/L199F	502.4U/mg	9.1

*1U is defined as the amount of enzyme required to produce 1. Mu. Mol of product NMN per unit time (1 min).

Wherein, when the 154 th position of the amino acid sequence shown in SEQ ID NO. 2 is mutated from glutamine to asparagine, and the 194 th position is mutated from alanine to valine, the amino acid sequence of the 7 alpha-HSDH enzyme mutant is shown as SEQ ID NO. 3, and correspondingly, the nucleotide sequence of the coding gene is shown as SEQ ID NO. 9.

When the 157 th position of the amino acid sequence shown in SEQ ID NO. 2 is mutated from tyrosine to alanine, and the 194 th position is mutated from alanine to valine, the amino acid sequence of the 7 alpha-HSDH enzyme mutant is shown in SEQ ID NO. 4, and correspondingly, the nucleotide sequence of the coding gene is shown in SEQ ID NO. 10.

When the 194 th site of the amino acid sequence shown in SEQ ID NO. 2 is mutated from alanine to valine and the 199 th site is mutated from leucine to phenylalanine, the amino acid sequence of the 7 alpha-HSDH enzyme mutant is shown in SEQ ID NO. 5, and correspondingly, the nucleotide sequence of the coding gene is shown in SEQ ID NO. 11.

When the 157 th position of the amino acid sequence shown in SEQ ID NO. 2 is mutated from tyrosine to alanine, the 194 th position is mutated from alanine to valine, the 199 th position is mutated from leucine to phenylalanine, the amino acid sequence of the 7 alpha-HSDH enzyme mutant is shown in SEQ ID NO. 6, and correspondingly, the nucleotide sequence of the coding gene is shown in SEQ ID NO. 12.

When the 154 th site of the amino acid sequence shown in SEQ ID NO. 2 is mutated from glutamine to asparagine, the 157 th site is mutated from tyrosine to alanine, the 199 th site is mutated from leucine to phenylalanine, the amino acid sequence of the 7 alpha-HSDH enzyme mutant is shown in SEQ ID NO. 7, and correspondingly, the nucleotide sequence of the coding gene is shown in SEQ ID NO. 13.

When the 154 th site of the amino acid sequence shown in SEQ ID NO. 2 is mutated from glutamine to asparagine, the 157 th site is mutated from tyrosine to alanine, the 194 th site is mutated from alanine to valine, and the 199 th site is mutated from leucine to phenylalanine, the amino acid sequence of the 7 alpha-HSDH enzyme mutant is shown as SEQ ID NO. 8, and correspondingly, the nucleotide sequence of the coding gene is shown as SEQ ID NO. 14.

EXAMPLE biocatalysis of the four mutants

Dissolving 8g of substrate CDCA in 100mL of 50mM sodium phosphate buffer solution with pH of 9.0, adjusting the pH to 8.0 by using liquid alkali, and adding 0.01g of NAD +, 0.2g of 7 alpha-HSDH mutant freeze-dried powder and 0.2g of NOX enzyme freeze-dried powder after the substrate is completely dissolved. The reaction solution is placed in a constant temperature water bath kettle at 30 ℃, the mechanical stirring reaction is carried out, air is introduced, and the air flow rate is controlled to be 5mL/min. HPLC detection was performed after 24h of reaction, with substrate conversion >99%. The catalytic reaction process is shown in figure 1.

EXAMPLE biocatalysis of the five mutants

Dissolving 10g of substrate CDCA in 100mL of 50mM sodium phosphate buffer solution with pH of 9.0, adjusting the pH to 8.0 by using liquid alkali, and adding 0.01g of NAD +, 0.2g of 7 alpha-HSDH mutant freeze-dried powder and 0.2g of NOX enzyme freeze-dried powder after the substrate is completely dissolved. The reaction solution is placed in a constant temperature water bath kettle at 30 ℃, the mechanical stirring reaction is carried out, air is introduced, and the air flow rate is controlled to be 5mL/min. HPLC detection was performed after 24h of reaction, with substrate conversion >99%.

Finally, it should be noted that the above-mentioned contents are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, and that the simple modifications or equivalent substitutions of the technical solutions of the present invention by those of ordinary skill in the art can be made without departing from the spirit and scope of the technical solutions of the present invention.

Sequence listing

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Lys Ala Ser Ile Asn Tyr Leu Thr Lys Leu Ile Ala Val Gln Cys Ala

165 170 175

Arg Asp Asn Ile Arg Cys Asn Thr Val Leu Pro Gly Met Thr Ala Thr

180 185 190

Asp Val Val Lys Asp Asn Leu Thr Asp Asp Phe Arg Asp Phe Phe Leu

195 200 205

Lys His Thr Pro Ile Lys Arg Met Gly Thr Pro Glu Glu Ile Ala Ala

210 215 220

Ala Ala Leu Tyr Phe Ala Ser Asp Glu Ser Ala Tyr Thr Thr Gly Gln

225 230 235 240

Ile Leu Glu Val Ser Gly Gly Phe Gly Met Pro Thr Pro Val Tyr Gly

245 250 255

Asp Met Ile Glu Met Lys Asn Arg Arg

260 265

<210> 5

<211> 265

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Met Arg Leu Glu Lys Lys Val Val Leu Ile Thr Ala Ser Thr Arg Gly

1 5 10 15

Ile Gly Leu Ser Cys Val Gln Arg Phe Ala Lys Glu Gly Ala Thr Val

20 25 30

Tyr Met Gly Ala Arg Asn Leu Glu Thr Ala Ala Glu Arg Ala Lys Glu

35 40 45

Leu Asn Asp Lys Gly Phe Asn Val Lys Thr Val Tyr Asn Asp Ala Ser

50 55 60

Lys Lys Glu Thr Tyr Ile Ser Met Val Glu Glu Val Ile Lys Asn Glu

65 70 75 80

Gly Lys Ile Asp Val Leu Val Asn Asn Phe Gly Thr Ser Asn Pro Lys

85 90 95

Lys Asp Leu Asp Ile Lys Ser Thr Glu Tyr Ser Glu Phe Ile Ser Thr

100 105 110

Ile Asp Met Asn Leu Ala Ser Val Phe Met Thr Ser Gln Ala Val Ile

115 120 125

Pro His Met Val Ala Asn Gly Gly Gly Ser Ile Ile Asn Ile Ser Ser

130 135 140

Ile Gly Gly Thr His Pro Asp Ile Ser Gln Ile Ser Tyr Gly Thr Ser

145 150 155 160

Lys Ala Ser Ile Asn Tyr Leu Thr Lys Leu Ile Ala Val Gln Cys Ala

165 170 175

Arg Asp Asn Ile Arg Cys Asn Thr Val Leu Pro Gly Met Thr Ala Thr

180 185 190

Asp Val Val Lys Asp Asn Phe Thr Asp Asp Phe Arg Asp Phe Phe Leu

195 200 205

Lys His Thr Pro Ile Lys Arg Met Gly Thr Pro Glu Glu Ile Ala Ala

210 215 220

Ala Ala Leu Tyr Phe Ala Ser Asp Glu Ser Ala Tyr Thr Thr Gly Gln

225 230 235 240

Ile Leu Glu Val Ser Gly Gly Phe Gly Met Pro Thr Pro Val Tyr Gly

245 250 255

Asp Met Ile Glu Met Lys Asn Arg Arg

260 265

<210> 6

<211> 265

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Met Arg Leu Glu Lys Lys Val Val Leu Ile Thr Ala Ser Thr Arg Gly

1 5 10 15

Ile Gly Leu Ser Cys Val Gln Arg Phe Ala Lys Glu Gly Ala Thr Val

20 25 30

Tyr Met Gly Ala Arg Asn Leu Glu Thr Ala Ala Glu Arg Ala Lys Glu

35 40 45

Leu Asn Asp Lys Gly Phe Asn Val Lys Thr Val Tyr Asn Asp Ala Ser

50 55 60

Lys Lys Glu Thr Tyr Ile Ser Met Val Glu Glu Val Ile Lys Asn Glu

65 70 75 80

Gly Lys Ile Asp Val Leu Val Asn Asn Phe Gly Thr Ser Asn Pro Lys

85 90 95

Lys Asp Leu Asp Ile Lys Ser Thr Glu Tyr Ser Glu Phe Ile Ser Thr

100 105 110

Ile Asp Met Asn Leu Ala Ser Val Phe Met Thr Ser Gln Ala Val Ile

115 120 125

Pro His Met Val Ala Asn Gly Gly Gly Ser Ile Ile Asn Ile Ser Ser

130 135 140

Ile Gly Gly Thr His Pro Asp Ile Ser Gln Ile Ser Ala Gly Thr Ser

145 150 155 160

Lys Ala Ser Ile Asn Tyr Leu Thr Lys Leu Ile Ala Val Gln Cys Ala

165 170 175

Arg Asp Asn Ile Arg Cys Asn Thr Val Leu Pro Gly Met Thr Ala Thr

180 185 190

Asp Val Val Lys Asp Asn Phe Thr Asp Asp Phe Arg Asp Phe Phe Leu

195 200 205

Lys His Thr Pro Ile Lys Arg Met Gly Thr Pro Glu Glu Ile Ala Ala

210 215 220

Ala Ala Leu Tyr Phe Ala Ser Asp Glu Ser Ala Tyr Thr Thr Gly Gln

225 230 235 240

Ile Leu Glu Val Ser Gly Gly Phe Gly Met Pro Thr Pro Val Tyr Gly

245 250 255

Asp Met Ile Glu Met Lys Asn Arg Arg

260 265

<210> 7

<211> 265

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Met Arg Leu Glu Lys Lys Val Val Leu Ile Thr Ala Ser Thr Arg Gly

1 5 10 15

Ile Gly Leu Ser Cys Val Gln Arg Phe Ala Lys Glu Gly Ala Thr Val

20 25 30

Tyr Met Gly Ala Arg Asn Leu Glu Thr Ala Ala Glu Arg Ala Lys Glu

35 40 45

Leu Asn Asp Lys Gly Phe Asn Val Lys Thr Val Tyr Asn Asp Ala Ser

50 55 60

Lys Lys Glu Thr Tyr Ile Ser Met Val Glu Glu Val Ile Lys Asn Glu

65 70 75 80

Gly Lys Ile Asp Val Leu Val Asn Asn Phe Gly Thr Ser Asn Pro Lys

85 90 95

Lys Asp Leu Asp Ile Lys Ser Thr Glu Tyr Ser Glu Phe Ile Ser Thr

100 105 110

Ile Asp Met Asn Leu Ala Ser Val Phe Met Thr Ser Gln Ala Val Ile

115 120 125

Pro His Met Val Ala Asn Gly Gly Gly Ser Ile Ile Asn Ile Ser Ser

130 135 140

Ile Gly Gly Thr His Pro Asp Ile Ser Asn Ile Ser Ala Gly Thr Ser

145 150 155 160

Lys Ala Ser Ile Asn Tyr Leu Thr Lys Leu Ile Ala Val Gln Cys Ala

165 170 175

Arg Asp Asn Ile Arg Cys Asn Thr Val Leu Pro Gly Met Thr Ala Thr

180 185 190

Asp Ala Val Lys Asp Asn Phe Thr Asp Asp Phe Arg Asp Phe Phe Leu

195 200 205

Lys His Thr Pro Ile Lys Arg Met Gly Thr Pro Glu Glu Ile Ala Ala

210 215 220

Ala Ala Leu Tyr Phe Ala Ser Asp Glu Ser Ala Tyr Thr Thr Gly Gln

225 230 235 240

Ile Leu Glu Val Ser Gly Gly Phe Gly Met Pro Thr Pro Val Tyr Gly

245 250 255

Asp Met Ile Glu Met Lys Asn Arg Arg

260 265

<210> 8

<211> 265

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Met Arg Leu Glu Lys Lys Val Val Leu Ile Thr Ala Ser Thr Arg Gly

1 5 10 15

Ile Gly Leu Ser Cys Val Gln Arg Phe Ala Lys Glu Gly Ala Thr Val

20 25 30

Tyr Met Gly Ala Arg Asn Leu Glu Thr Ala Ala Glu Arg Ala Lys Glu

35 40 45

Leu Asn Asp Lys Gly Phe Asn Val Lys Thr Val Tyr Asn Asp Ala Ser

50 55 60

Lys Lys Glu Thr Tyr Ile Ser Met Val Glu Glu Val Ile Lys Asn Glu

65 70 75 80

Gly Lys Ile Asp Val Leu Val Asn Asn Phe Gly Thr Ser Asn Pro Lys

85 90 95

Lys Asp Leu Asp Ile Lys Ser Thr Glu Tyr Ser Glu Phe Ile Ser Thr

100 105 110

Ile Asp Met Asn Leu Ala Ser Val Phe Met Thr Ser Gln Ala Val Ile

115 120 125

Pro His Met Val Ala Asn Gly Gly Gly Ser Ile Ile Asn Ile Ser Ser

130 135 140

Ile Gly Gly Thr His Pro Asp Ile Ser Asn Ile Ser Ala Gly Thr Ser

145 150 155 160

Lys Ala Ser Ile Asn Tyr Leu Thr Lys Leu Ile Ala Val Gln Cys Ala

165 170 175

Arg Asp Asn Ile Arg Cys Asn Thr Val Leu Pro Gly Met Thr Ala Thr

180 185 190

Asp Val Val Lys Asp Asn Phe Thr Asp Asp Phe Arg Asp Phe Phe Leu

195 200 205

Lys His Thr Pro Ile Lys Arg Met Gly Thr Pro Glu Glu Ile Ala Ala

210 215 220

Ala Ala Leu Tyr Phe Ala Ser Asp Glu Ser Ala Tyr Thr Thr Gly Gln

225 230 235 240

Ile Leu Glu Val Ser Gly Gly Phe Gly Met Pro Thr Pro Val Tyr Gly

245 250 255

Asp Met Ile Glu Met Lys Asn Arg Arg

260 265

<210> 9

<211> 798

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atgcgcctgg aaaaaaaagt ggtgctgatt accgcgagca cccgcggcat tggcctgagc 60

tgcgtgcagc gctttgcgaa agaaggcgcg accgtgtata tgggcgcgcg caacctggaa 120

accgcggcgg aacgcgcgaa agaactgaac gataaaggct ttaacgtgaa aaccgtgtat 180

aacgatgcga gcaaaaaaga aacctatatt agcatggtgg aagaagtgat taaaaacgaa 240

ggcaaaattg atgtgctggt gaacaacttt ggcaccagca acccgaaaaa agatctggat 300

attaaaagca ccgaatatag cgaatttatt agcaccattg atatgaacct ggcgagcgtg 360

tttatgacca gccaggcggt gattccgcac atggtggcga acggcggcgg cagcattatt 420

aacattagca gcattggcgg cacccatccg gatattagca atattagcta tggcaccagc 480

aaagcgagca ttaactatct gaccaaactg attgcggtgc agtgcgcgcg cgataacatt 540

cgctgcaaca ccgtgctgcc gggcatgacc gcgaccgatg tggtgaaaga taacctgacc 600

gatgattttc gcgatttttt tctgaaacat accccgatta aacgcatggg caccccggaa 660

gaaattgcgg cggcggcgct gtattttgcg agcgatgaaa gcgcgtatac caccggccag 720

attctggaag tgagcggcgg ctttggcatg ccgaccccgg tgtatggcga tatgattgaa 780

atgaaaaacc gccgctaa 798

<210> 10

<211> 798

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atgcgcctgg aaaaaaaagt ggtgctgatt accgcgagca cccgcggcat tggcctgagc 60

tgcgtgcagc gctttgcgaa agaaggcgcg accgtgtata tgggcgcgcg caacctggaa 120

accgcggcgg aacgcgcgaa agaactgaac gataaaggct ttaacgtgaa aaccgtgtat 180

aacgatgcga gcaaaaaaga aacctatatt agcatggtgg aagaagtgat taaaaacgaa 240

ggcaaaattg atgtgctggt gaacaacttt ggcaccagca acccgaaaaa agatctggat 300

attaaaagca ccgaatatag cgaatttatt agcaccattg atatgaacct ggcgagcgtg 360

tttatgacca gccaggcggt gattccgcac atggtggcga acggcggcgg cagcattatt 420

aacattagca gcattggcgg cacccatccg gatattagcc agattagcgc tggcaccagc 480

aaagcgagca ttaactatct gaccaaactg attgcggtgc agtgcgcgcg cgataacatt 540

cgctgcaaca ccgtgctgcc gggcatgacc gcgaccgatg tggtgaaaga taacctgacc 600

gatgattttc gcgatttttt tctgaaacat accccgatta aacgcatggg caccccggaa 660

gaaattgcgg cggcggcgct gtattttgcg agcgatgaaa gcgcgtatac caccggccag 720

attctggaag tgagcggcgg ctttggcatg ccgaccccgg tgtatggcga tatgattgaa 780

atgaaaaacc gccgctaa 798

<210> 11

<211> 798

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

atgcgcctgg aaaaaaaagt ggtgctgatt accgcgagca cccgcggcat tggcctgagc 60

tgcgtgcagc gctttgcgaa agaaggcgcg accgtgtata tgggcgcgcg caacctggaa 120

accgcggcgg aacgcgcgaa agaactgaac gataaaggct ttaacgtgaa aaccgtgtat 180

aacgatgcga gcaaaaaaga aacctatatt agcatggtgg aagaagtgat taaaaacgaa 240

ggcaaaattg atgtgctggt gaacaacttt ggcaccagca acccgaaaaa agatctggat 300

attaaaagca ccgaatatag cgaatttatt agcaccattg atatgaacct ggcgagcgtg 360

tttatgacca gccaggcggt gattccgcac atggtggcga acggcggcgg cagcattatt 420

aacattagca gcattggcgg cacccatccg gatattagcc agattagcta tggcaccagc 480

aaagcgagca ttaactatct gaccaaactg attgcggtgc agtgcgcgcg cgataacatt 540

cgctgcaaca ccgtgctgcc gggcatgacc gcgaccgatg tggtgaaaga taactttacc 600

gatgattttc gcgatttttt tctgaaacat accccgatta aacgcatggg caccccggaa 660

gaaattgcgg cggcggcgct gtattttgcg agcgatgaaa gcgcgtatac caccggccag 720

attctggaag tgagcggcgg ctttggcatg ccgaccccgg tgtatggcga tatgattgaa 780

atgaaaaacc gccgctaa 798

<210> 12

<211> 798

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atgcgcctgg aaaaaaaagt ggtgctgatt accgcgagca cccgcggcat tggcctgagc 60

tgcgtgcagc gctttgcgaa agaaggcgcg accgtgtata tgggcgcgcg caacctggaa 120

accgcggcgg aacgcgcgaa agaactgaac gataaaggct ttaacgtgaa aaccgtgtat 180

aacgatgcga gcaaaaaaga aacctatatt agcatggtgg aagaagtgat taaaaacgaa 240

ggcaaaattg atgtgctggt gaacaacttt ggcaccagca acccgaaaaa agatctggat 300

attaaaagca ccgaatatag cgaatttatt agcaccattg atatgaacct ggcgagcgtg 360

tttatgacca gccaggcggt gattccgcac atggtggcga acggcggcgg cagcattatt 420

aacattagca gcattggcgg cacccatccg gatattagcc agattagcgc tggcaccagc 480

aaagcgagca ttaactatct gaccaaactg attgcggtgc agtgcgcgcg cgataacatt 540

cgctgcaaca ccgtgctgcc gggcatgacc gcgaccgatg tggtgaaaga taactttacc 600

gatgattttc gcgatttttt tctgaaacat accccgatta aacgcatggg caccccggaa 660

gaaattgcgg cggcggcgct gtattttgcg agcgatgaaa gcgcgtatac caccggccag 720

attctggaag tgagcggcgg ctttggcatg ccgaccccgg tgtatggcga tatgattgaa 780

atgaaaaacc gccgctaa 798

<210> 13

<211> 798

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

atgcgcctgg aaaaaaaagt ggtgctgatt accgcgagca cccgcggcat tggcctgagc 60

tgcgtgcagc gctttgcgaa agaaggcgcg accgtgtata tgggcgcgcg caacctggaa 120

accgcggcgg aacgcgcgaa agaactgaac gataaaggct ttaacgtgaa aaccgtgtat 180

aacgatgcga gcaaaaaaga aacctatatt agcatggtgg aagaagtgat taaaaacgaa 240

ggcaaaattg atgtgctggt gaacaacttt ggcaccagca acccgaaaaa agatctggat 300

attaaaagca ccgaatatag cgaatttatt agcaccattg atatgaacct ggcgagcgtg 360

tttatgacca gccaggcggt gattccgcac atggtggcga acggcggcgg cagcattatt 420

aacattagca gcattggcgg cacccatccg gatattagca atattagcgc tggcaccagc 480

aaagcgagca ttaactatct gaccaaactg attgcggtgc agtgcgcgcg cgataacatt 540

cgctgcaaca ccgtgctgcc gggcatgacc gcgaccgatg cggtgaaaga taactttacc 600

gatgattttc gcgatttttt tctgaaacat accccgatta aacgcatggg caccccggaa 660

gaaattgcgg cggcggcgct gtattttgcg agcgatgaaa gcgcgtatac caccggccag 720

attctggaag tgagcggcgg ctttggcatg ccgaccccgg tgtatggcga tatgattgaa 780

atgaaaaacc gccgctaa 798

<210> 14

<211> 798

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

atgcgcctgg aaaaaaaagt ggtgctgatt accgcgagca cccgcggcat tggcctgagc 60

tgcgtgcagc gctttgcgaa agaaggcgcg accgtgtata tgggcgcgcg caacctggaa 120

accgcggcgg aacgcgcgaa agaactgaac gataaaggct ttaacgtgaa aaccgtgtat 180

aacgatgcga gcaaaaaaga aacctatatt agcatggtgg aagaagtgat taaaaacgaa 240

ggcaaaattg atgtgctggt gaacaacttt ggcaccagca acccgaaaaa agatctggat 300

attaaaagca ccgaatatag cgaatttatt agcaccattg atatgaacct ggcgagcgtg 360

tttatgacca gccaggcggt gattccgcac atggtggcga acggcggcgg cagcattatt 420

aacattagca gcattggcgg cacccatccg gatattagca atattagcgc tggcaccagc 480

aaagcgagca ttaactatct gaccaaactg attgcggtgc agtgcgcgcg cgataacatt 540

cgctgcaaca ccgtgctgcc gggcatgacc gcgaccgatg tggtgaaaga taactttacc 600

gatgattttc gcgatttttt tctgaaacat accccgatta aacgcatggg caccccggaa 660

gaaattgcgg cggcggcgct gtattttgcg agcgatgaaa gcgcgtatac caccggccag 720

attctggaag tgagcggcgg ctttggcatg ccgaccccgg tgtatggcga tatgattgaa 780

atgaaaaacc gccgctaa 798

<210> 15

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gcacccatcc ggatattagc nnkattagct atggcaccag caa 43

<210> 16

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ttgctggtgc catagctaat mnngctaata tccggatggg tgc 43

<210> 17

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

ggatattagc cagattagcn nkggcaccag caaagcgagc 40

<210> 18

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

ctcgctttgc tggtgccmnn gctaatctgg ctaatatcc 39

<210> 19

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gggcatgacc gcgaccgatn nkgtgaaaga taacctgacc gat 43

<210> 20

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

atcggtcagg ttatctttca cmnnatcggt cgcggtcatg ccc 43

<210> 21

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

cgatgcggtg aaagataacn nkaccgatga ttttcgcgat 40

<210> 22

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

atcgcgaaaa tcatcggtmn ngttatcttt caccgcatcg 40

Claims (7)

1. A7 alpha-HSDH enzyme mutant is characterized in that compared with a wild type 7 alpha-HSDH enzyme of which the amino acid sequence is shown in SEQ ID NO. 2, the amino acid sequence of the 7 alpha-HSDH enzyme mutant is subjected to any one mutation of pairwise combined mutation, three combined mutation or four combined mutation at the 154 th position, the 157 th position, the 194 th position and the 199 th position of the amino acid sequence shown in SEQ ID NO. 2;

the pairwise combined mutation is as follows:

when the 154 th position of the amino acid sequence shown in SEQ ID NO. 2 is mutated from glutamine to asparagine, and the 194 th position is mutated from alanine to valine, the amino acid sequence of the 7 alpha-HSDH enzyme mutant is shown in SEQ ID NO. 3;

or, when the 157 th position of the amino acid sequence shown in SEQ ID NO. 2 is mutated from tyrosine to alanine, the 194 th position is mutated from alanine to valine, the amino acid sequence of the 7 alpha-HSDH enzyme mutant is shown in SEQ ID NO. 4;

or when the 194 th site of the amino acid sequence shown in SEQ ID NO. 2 is mutated from alanine to valine and the 199 th site is mutated from leucine to phenylalanine, the amino acid sequence of the 7 alpha-HSDH enzyme mutant is shown in SEQ ID NO. 5;

the three combined mutations are:

when the 157 th position of the amino acid sequence shown in SEQ ID NO. 2 is mutated from tyrosine to alanine, the 194 th position is mutated from alanine to valine, the 199 th position is mutated from leucine to phenylalanine, the amino acid sequence of the 7 alpha-HSDH enzyme mutant is shown in SEQ ID NO. 6;

or when the 154 th site of the amino acid sequence shown in SEQ ID NO. 2 is mutated from glutamine to asparagine, the 157 th site is mutated from tyrosine to alanine, the 199 th site is mutated from leucine to phenylalanine, the amino acid sequence of the 7 alpha-HSDH enzyme mutant is shown in SEQ ID NO. 7;

the four combined mutations are:

when the 154 th site of the amino acid sequence shown in SEQ ID NO. 2 is mutated from glutamine to asparagine, the 157 th site is mutated from tyrosine to alanine, the 194 th site is mutated from alanine to valine, the 199 th site is mutated from leucine to phenylalanine, the amino acid sequence of the 7 alpha-HSDH enzyme mutant is shown in SEQ ID NO. 8.

2. The encoding gene of the 7 alpha-HSDH enzyme mutant according to claim 1, wherein the nucleotide sequence of the encoding gene of the 7 alpha-HSDH enzyme mutant, the amino acid sequence of which is shown as SEQ ID NO. 3, is shown as SEQ ID NO. 9;

or, the nucleotide sequence of the coding gene of the 7 alpha-HSDH enzyme mutant with the amino acid sequence shown as SEQ ID NO. 4 is shown as SEQ ID NO. 10;

or, the nucleotide sequence of the coding gene of the 7 alpha-HSDH enzyme mutant with the amino acid sequence shown as SEQ ID NO. 5 is shown as SEQ ID NO. 11;

or, the nucleotide sequence of the coding gene of the 7 alpha-HSDH enzyme mutant with the amino acid sequence shown as SEQ ID NO. 6 is shown as SEQ ID NO. 12;

or, the nucleotide sequence of the coding gene of the 7 alpha-HSDH enzyme mutant with the amino acid sequence shown as SEQ ID NO. 7 is shown as SEQ ID NO. 13;

or, the nucleotide sequence of the coding gene of the 7 alpha-HSDH enzyme mutant with the amino acid sequence shown as SEQ ID NO. 8 is shown as SEQ ID NO. 14.

3. A vector comprising the coding gene according to claim 2.

4. The vector of claim 3, wherein the vector is a pET expression vector, a pCW expression vector, a pUC expression vector or a pPIC9k expression vector.

5. The host cell containing the coding gene of claim 2, wherein the host cell is Escherichia coli, pichia pastoris, streptomyces or Bacillus subtilis.

6. Use of the 7 α -HSDH enzyme mutant according to claim 1, the coding gene according to claim 2, the vector according to claim 3 or 4, or the host cell according to claim 5 for the preparation of 7-ketolithocholic acid.

7. A method for preparing 7-ketolithocholic acid, comprising the steps of:

s1, configuring a reaction system, comprising: 1-10g/L of 7 alpha-HSDH enzyme mutant described in claim 1, 50-200mM pH9.0 sodium phosphate buffer solution, 0.1-0.5g/L coenzyme NAD +,40-100g/L chenodeoxycholic acid, 1-5g/L NADH oxidase; controlling the temperature of the reaction system to be 30 ℃ and the air flow rate to be 5mL/min, and carrying out stirring reaction;

s2, carrying out HPLC detection after 24h of reaction to obtain the 7-ketolithocholic acid.

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