Mutants of 7 alpha-hydroxysteroid dehydrogenase St-2-2T15G, T15S and T15A

文档序号：30185 发布日期：2021-09-24 浏览：27次中文

阅读说明：本技术 7α-羟基类固醇脱氢酶St-2-2的突变体T15G、T15S和T15A (Mutants of 7 alpha-hydroxysteroid dehydrogenase St-2-2T15G, T15S and T15A ) 是由祝连彩潘银平唐士金王伯初于 2021-08-16 设计创作，主要内容包括：本发明涉及羟基类固醇脱氢酶,具体涉及7α-羟基类固醇脱氢酶St-2-2的突变体T15G、T15S和T15A。所述突变体的氨基酸序列如SEQ ID NO:2,3或4所示,是氨基酸序列为SEQ ID NO:1的7α-羟基类固醇脱氢酶的第15位氨基酸由Thr定点突变为Gly、Ser或Ala所得。所述突变体在相同底物TCDCA和NADP～(+)的存在下,酶活分别是野生型的3.18、4.27、7.85倍,在生物转化TCDCA获取TUDCA的过程中具有巨大的应用潜力。(The invention relates to hydroxysteroid dehydrogenase, in particular to mutants T15G, T15S and T15A of 7 alpha-hydroxysteroid dehydrogenase St-2-2. The amino acid sequence of the mutant is shown as SEQ ID NO. 2, 3 or 4, and the mutant is obtained by the site-specific mutagenesis of Thr to Gly, Ser or Ala at the 15 th amino acid of 7 alpha-hydroxysteroid dehydrogenase of which the amino acid sequence is SEQ ID NO. 1. The mutant has the same substrates TCDCA and NADP + In the presence of (2), the enzyme activities are respectively 3.18, 4.27 and 3.18 of wild type,7.85 times, has great application potential in the process of obtaining TUDCA by biotransformation of TCDCA.)

1. A mutant 7 α -hydroxysteroid dehydrogenase, characterized by: the amino acid sequence is shown in SEQ ID NO. 2, 3 or 4, and is obtained by changing Thr into Gly, Ser or Ala at the 15 th amino acid of 7 alpha-hydroxysteroid dehydrogenase with the amino acid sequence of SEQ ID NO. 1.

2. A gene encoding the mutant 7 α -hydroxysteroid dehydrogenase according to claim 1.

3. The gene according to claim 2, characterized in that: the nucleotide sequence is shown as SEQ ID NO 6, 7 or 8.

4. An expression cassette, vector or recombinant bacterium comprising the gene of claim 2.

5. The method for producing a 7 α -hydroxysteroid dehydrogenase mutant according to claim 1, wherein: the method comprises the following steps: synthesizing the coding gene of the 7 alpha-hydroxysteroid dehydrogenase mutant, constructing an expression vector, transforming protein expression host bacteria, inducing protein expression and purifying.

6. The method of claim 5, wherein: the nucleotide sequence of the coding gene of the 7 alpha-hydroxysteroid dehydrogenase mutant is shown in SEQ ID NO 6, 7 or 8.

7. A catalyst, characterized by: the 7 α -hydroxysteroid dehydrogenase mutant according to claim 1 as an active ingredient.

8. Use of the mutant of 7 α -hydroxysteroid dehydrogenase according to claim 1 or the catalyst according to claim 7 for asymmetric reduction of carbonyl groups.

9. A method of effecting asymmetric reduction of a carbonyl group of a chemical species, comprising: catalytically reacting a reaction substrate with the mutant 7 α -hydroxysteroid dehydrogenase of claim 1 or the catalyst of claim 7; the reaction substrate is taurochenodeoxycholic acid or glycochenodeoxycholic acid, or a bile acid component containing the taurochenodeoxycholic acid or the glycochenodeoxycholic acid.

10. The method of claim 9, wherein: the catalytic reaction was carried out at room temperature in 50mM Tris-HCl, pH 8.0.

Technical Field

The invention relates to hydroxysteroid dehydrogenase, in particular to mutants T15G, T15S and T15A of 7 alpha-hydroxysteroid dehydrogenase St-2-2.

Background

Asymmetric reduction of carbonyl groups has been one of the hot spots in chemical reaction research. Although chemical methods have achieved certain results at present, the chemical methods often have the disadvantages of limited types and numbers of catalysts, low stereoselectivity, expensive auxiliary reagents, difficult recovery and the like. The enzymatic reaction has high efficiency, chemoselectivity and regioselectivity, and also has high stereoselectivity. The Hydroxysteroid dehydrogenase (HSDH) -mediated enzymatic reaction has relatively stringent stereoselectivity and "not" stringent substrate specificity. For example, scientists have begun to try to synthesize ursodeoxycholic acid (UDCA) by joint epimerization of Chenodeoxycholic acid (CDCA) using 7 α -, 7 β -HSDH produced by microorganisms as early as the eighties of the twentieth century. The free enzyme can also catalyze the conversion of Tauroursodeoxycholic acid (TCDCA), which is a conjugated bile acid, into Tauroursodeoxycholic acid (TUDCA).

The substrate of HSDH is not limited to steroid compounds, and the HSDH can catalyze carbonyl asymmetric reduction of alkyl substituted monocyclic ketone, bicyclic ketone and other substances reported in literature. The excellent catalytic quality of HSDH determines that HSDH has larger application potential in the field of biotransformation. However, the more active HSDH modifications are the basic guarantee for its further application in the field of biotransformation. In recent years, researchers have gradually recognized the great application potential of 7 alpha-, 7 beta-HSDH in the field of biotransformation. Currently, there are 8 functionally-confirmed 7 α -HSDH registered in GenBank, which are respectively from Bacteroides fragilis, Clostridium scindens, Clostridium sordelii, Clostridium absomonum, Stenotrophomonas maltophilia, Eubacterium sp.vpi 12708, Clostridium difficile, and Escherichia coli; the 7 β -HSDH gene from Clostridium absinum and Collinsella aerofaciens has also been successfully cloned. The biological conversion system constructed by the double-enzyme coupling not only overcomes the problem of coenzyme circulation, but also realizes the one-pot type oxidation and reduction of hydroxyl epimerization in a specific chemical region.

The low activity of the enzyme is one of the main factors limiting the industrial application, and almost all natural enzymes need to be modified to meet the requirements of industrial application. The Chinese patent application with the application number of 2019106381160 and the invention name of 7 alpha-hydroxysteroid dehydrogenase and coding gene and application thereof discloses 7 alpha-hydroxysteroid dehydrogenase St-2-2, and the catalytic activity of the enzyme on TCDCA and GCDCA is superior to 57 alpha-HSDHs discovered in the earlier stage of the subject group. At present, no report about the modification of the 15 th amino acid of the enzyme is found.

Disclosure of Invention

In order to further improve the enzyme catalysis efficiency, the invention provides a mutant of 7 alpha-hydroxysteroid dehydrogenase, the amino acid sequence of which is shown as SEQ ID NO. 2, 3 or 4, and the 15 th amino acid of the 7 alpha-hydroxysteroid dehydrogenase with the amino acid sequence of SEQ ID NO. 1 is obtained by changing Thr into Gly, Ser or Ala.

The gene encoding the 7 alpha-hydroxysteroid dehydrogenase mutant also belongs to the protection scope of the invention.

In a preferred embodiment of the invention, the nucleotide sequence of the gene is shown in SEQ ID NO 6, 7 or 8.

Expression cassettes, vectors or recombinant bacteria comprising said genes also belong to the scope of protection of the present invention.

Said vector, which may be a cloning vector, comprises the genes encoding any of said 7 α -HSDH St-2-2 mutants and further elements required for plasmid replication; it may also be an expression vector comprising any of the genes encoding the 7 α -HSDH St-2-2 mutants described and other elements enabling the successful expression of the protein. In some embodiments, the expression vector is a pGEX-6p-2 vector into which the mutant gene is inserted.

The recombinant bacterium can be a recombinant bacterium containing a cloning vector, such as E.coli DH5 alpha, and the 7 alpha-HSDH St-2-2 mutant gene in the cell is replicated by culturing the cell; or a cell comprising an expression vector, e.g., E.coli BL21, cultured under appropriate conditions, e.g., with the addition of an appropriate amount of IPTG to induce expression of the 7 α -HSDH St-2-2 mutant protein at 16 ℃.

The invention also provides a preparation method of the 7 alpha-hydroxysteroid dehydrogenase mutant, which comprises the following steps: synthesizing the coding gene of the 7 alpha-hydroxysteroid dehydrogenase mutant, constructing an expression vector, transforming protein expression host bacteria, inducing protein expression and purifying.

In some embodiments of the invention, in the preparation method, the nucleotide sequence of the gene encoding the 7 α -hydroxysteroid dehydrogenase mutant is shown in SEQ ID NO 6, 7 or 8.

The present invention also provides a catalyst, the active ingredient of which comprises the 7 alpha-hydroxysteroid dehydrogenase mutant. The active ingredient of the catalyst comprises 1, 2 or 3 of 7 alpha-HSDH St-2-2 mutant T15G, T15S and T15A. The catalyst can be used alone or together with other suitable catalysts to improve the catalytic efficiency of the enzyme or to carry out two catalytic reactions in sequence in the same reaction system.

The application of the 7 alpha-hydroxysteroid dehydrogenase mutant or the catalyst in the asymmetric reduction reaction of carbonyl also belongs to the protection scope of the invention.

The invention also provides a method for realizing carbonyl asymmetric reduction of chemical substances, which uses the 7 alpha-hydroxysteroid dehydrogenase mutant or the catalyst to perform catalytic reaction with a reaction substrate; the reaction substrate is taurochenodeoxycholic acid or glycochenodeoxycholic acid, or a bile acid component containing the taurochenodeoxycholic acid or the glycochenodeoxycholic acid.

In some embodiments of the invention, the catalytic reaction is performed at room temperature in 50mM Tris-HCl, pH 8.0.

In the process of enzyme mutant development, we first performed sequence analysis on 7 α -hydroxysteroid dehydrogenase St-2-2 (abbreviated as 7 α -HSDH St-2-2), and found that 7 α -HSDH St-2-2 possesses a conserved region of N-terminal coenzyme binding site (Gly-X-X-X-Gly-X-Gly), and determined that amino acid at position 15 (threonine) is a site affecting the enzymatic properties of 7 α -HSDH St-2-2. Then the threonine at the 15 th position is changed into glycine, serine and alanine respectively through codon replacement, and genes encoding 7 alpha-HSDH St-2-2 mutant T15G, T15S and T15A are obtained by cloning by utilizing a PCR technology. Finally constructing GST fusion expression carrier of mutant geneAnd introducing the mutant into a genetically engineered bacterium E.coli BL21 for induced expression to obtain the mutant zymoprotein. The enzyme activity determination result shows that compared with wild 7 alpha-HSDH St-2-2, the catalytic efficiency of the mutant is obviously improved. In the same substrates TCDCA and NADP⁺In the presence, the enzyme activities of the 7 alpha-HSDH St-2-2 mutant T15G, T15S and T15A are respectively 3.18 times, 4.27 times and 7.85 times of the wild type. In the same substrate GCDCA and NADP⁺In the presence of the mutant T15G, T15S and T15A of the 7 alpha-HSDH St-2-2 mutant, the enzyme activities of the mutant T15G, the mutant T15S and the mutant T15A are respectively 2.27 times, 2.91 and 4.68 times of that of the wild type. Therefore, the 7 alpha-hydroxysteroid dehydrogenase mutant provided by the invention has great application potential in the process of obtaining TUDCA by biotransformation of TCDCA.

Drawings

FIG. 1 is a schematic diagram of the combined transformation of TCDCA with 7. alpha. -HSDH and 7. beta. -HSDH for the preparation of TUDCA.

FIG. 2.7 SDS-PAGE electrophoretograms of the St-2-2 mutant of the alpha-hydroxysteroid dehydrogenase T15G, T15S and T15A; wherein M is a protein molecular weight standard (Marker); lanes 1, 2, 3 are the T15G, T15S and T15A mutant proteins, respectively, with a molecular weight of 28.2 kDa.

FIG. 3 is an SDS-PAGE electrophoresis of wild-type 7. alpha. -hydroxysteroid dehydrogenase St-2-2; wherein M is a protein molecular weight standard (Marker); st-2-2 is wild type 7 alpha-HSDH St-2-2 protein with molecular weight of 28.2 kDa.

FIG. 4. standard curve for NADPH; wherein the abscissa is the concentration (mM) of the NADPH solution, and the ordinate is the light absorption value of the NADPH solution at 340 nm.

FIG. 5 shows relative enzyme activities (TCDCA substrate) of wild-type 7 α -HSDH St-2-2 and mutant, wherein St-2-2 represents wild type, and T15G, T15S and T15A represent mutants.

FIG. 6 shows relative enzyme activities (using GCDCA as a substrate) of wild-type 7 alpha-HSDH St-2-2 and mutants, wherein St-2-2 represents the wild type, and T15G, T15S and T15A are mutants.

Detailed Description

The invention is further described below in connection with specific examples, which are to be construed as merely illustrative and explanatory and not limiting the scope of the invention in any way.

The wild-type 7 alpha-HSDH St-2-2 gene used in the following examples is obtained by cloning by PCR technology using total DNA of black bear feces samples as templates in the early stage of the laboratory, and the amino acid sequence of the gene is shown as SEQ ID NO. 1 and the gene sequence is shown as SEQ ID NO. 5. The isolation of this gene has been disclosed in the patent application No. 2019106381160 entitled "7 α -hydroxysteroid dehydrogenase and its encoding gene and uses," which is incorporated herein by reference in its entirety. The wild-type 7 alpha-HSDH St-2-2 gene can also be obtained by a gene synthesis method.

The pGEX-6p-2 vector used in the following examples is an E.coli protein expression vector purchased from Shanghai Biotech Ltd; the size of the vector is 4985bp, the vector tag is N-GST, and the resistance of the vector is Ampicillin.

Competent cells used in the following examples:

trans5 α competent cells, purchased from holo-gold Biotechnology, Inc., cat #: CD 201-01.

Coli BL21 competent cells, purchased from holo-gold biotechnology limited, cat #: the CD 601.

The main reagents used in the following examples:

prime STAR Max Premix (2 ×), purchased from precious biotechnology limited (da lian), cat #: R045A. BamH I restriction enzyme, purchased from precious biotechnology limited (da lian), cat # k: 1010S. Xho I restriction enzyme, purchased from precious biotechnology limited (da lian), cat #: 1094S. T4 DNA Ligase, purchased from precious biotechnology limited (da lian), cat #: 2011A. PBS buffer dry powder, purchased from beijing solilebao technologies ltd, cat #: and P1010. Glutaminone Sepharose 4B, purchased from GE Healthcare, cat No.: 10223836. PreScission Protease enzyme, purchased from GenScript, Cat. No.: z02799-100. BCA kit, purchased from Beyotime, cat # s: p0006. NADPH, purchased from Sigma-Aldrich, CAS number: 2646-71-1, cat #: 10621692001. NADP⁺Purchased from Sigma-Aldrich, CAS number: 53-59-8, goods number: and N5755. TCDCA (taurochenodeoxycholic acid), purchased from carbofuran technologies, CAS No.: 6009-98-9, cat # 330776. GCDCA (glycochenodeoxycholic acid), purchased from shanghai mclin biochemistry science ltd, CAS no: 16564-43-5, Cat No: G835599.

the formulation of LB medium used in the following examples was: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of sodium chloride and pH 7.4. The preparation method comprises the following steps: at 950mL ddH₂Dissolving 10g tryptone, 5g yeast extract, 10g sodium chloride in O, adjusting pH to 7.4 with NaOH, and adding ddH₂And O is metered to 1L. If a solid medium is prepared, 15g of agar per liter are added. Sterilizing with high pressure steam at 121 deg.C for 20 min.

The reaction buffer (50mM Tris-HCl, pH 8.0) used in the following examples was prepared by the following method: 6.057g of Tris solid powder was dissolved in 1L of deionized water, adjusted to pH 8.0 with hydrochloric acid, and left at room temperature for further use.

Unless otherwise specified, the reagents used in the following examples are conventional in the art, and are either commercially available or formulated according to methods conventional in the art, and may be of laboratory pure grade. Unless otherwise specified, the methods used in the following examples are conventional in the art, and reference may be made to the relevant laboratory manuals or manufacturer's instructions. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Example 1.7 acquisition of alpha-hydroxysteroid dehydrogenase (St-2-2) mutant

1. Mutant design

The amino acid sequence (262aa) of the wild-type 7 α -hydroxysteroid dehydrogenase St-2-2 is as follows:

MKRVENKVALVTSSTRGIGLAIAKTLAKEGARVYLAVRRLDAGQEVANEIIAEGGFAKPVYFDASKVETHMSMIEEVVEAEGRIDILVNNYGSTDVQKDLDLVHGDTEAFFNIVNQNLESVYLPCKVAVPYMIKNGGGSIINISTIGSVNPDLGRIAYVVSKAAINALTQNIAVQYAKKGIRCNAVLPGLIATDAALNNMSEEFLEHFLRHVPLDRTGHPQDIANAVLFFASDESSYITGTLQEVAGGFGMPSPIYGDAVKK(SEQ ID NO:1)。

the gene sequence (789bp) of the wild-type 7 alpha-hydroxysteroid dehydrogenase St-2-2 is as follows:

ATGAAAAGAGTAGAAAATAAAGTAGCATTAGTCACATCTTCTACAAGAGGGATTGGACTTGCTATTGCTAAAACACTTGCTAAAGAAGGTGCACGTGTATACCTTGCAGTAAGAAGATTAGATGCAGGTCAGGAGGTAGCGAATGAAATTATTGCAGAAGGTGGATTTGCTAAGCCTGTTTACTTTGATGCTTCTAAAGTAGAGACACACATGAGTATGATTGAAGAAGTAGTTGAAGCTGAAGGACGTATAGATATTTTAGTCAATAATTATGGTTCAACAGACGTTCAAAAGGACTTAGATCTCGTACATGGAGATACAGAAGCTTTCTTTAATATTGTTAATCAAAATCTTGAAAGTGTTTACTTACCATGTAAGGTGGCGGTACCTTATATGATTAAAAATGGTGGAGGAAGCATTATTAACATTTCTACAATTGGTTCAGTAAACCCTGACCTTGGACGTATTGCTTATGTTGTATCTAAAGCAGCTATCAACGCGCTTACACAAAATATTGCAGTTCAGTATGCAAAAAAAGGGATAAGATGTAATGCTGTTCTTCCAGGTCTTATTGCTACGGATGCAGCCCTTAATAATATGTCAGAGGAGTTCTTAGAACATTTCTTAAGACATGTACCACTTGACCGTACAGGGCATCCTCAAGATATTGCTAATGCAGTACTTTTCTTTGCAAGTGATGAATCTTCTTATATTACAGGTACACTTCAAGAAGTAGCAGGTGGATTTGGTATGCCATCACCTATTTATGGGGATGCTGTTAAGAAATAA(SEQ ID NO:5)。

we found by sequence analysis that 7 α -HSDH St-2-2 possesses a conserved region of the N-terminal coenzyme binding site (Gly-X-X-X-Gly-X-Gly), i.e., sites for Ser13, Ser14, Thr15, Arg16, Gly17, Ile18 and Gly 19; through molecular pairing, Thr15 in 7 alpha-HSDH St-2-2 and coenzyme II adenine ribose 2' -phosphate have interaction, alanine and glycine with smaller steric hindrance have better catalytic activity on a substrate TCDCA, and are probably more favorable for the inlet and outlet of the substrate TCDCA, so that the 15 th amino acid (threonine) is determined to be a site influencing the enzymatic properties of 7 alpha-HSDH St-2-2, and the site corresponds to the 43 th-45 th codon of a 7 alpha-HSDH St-2-2 gene sequence.

The 43 th-45 th codon of the wild 7 alpha-HSDH St-2-2 gene sequence is respectively changed into GGT, TCT and GCT from ACA, namely, the 15 th threonine in the wild 7 alpha-HSDH St-2-2 amino acid sequence is respectively replaced by glycine, serine and alanine to obtain 37 alpha-HSDH St-2-2 mutants which are respectively named as T15G, T15S and T15A mutants, the amino acid sequences of the mutants are shown as SEQ ID NO. 2-4, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 6-8.

2. Construction of expression vectors

2.1 obtaining mutant genes

The experiment uses wild 7 alpha-HSDH St-2-2 gene as template, and adopts site-directed mutagenesis primer to obtain mutant gene by PCR method. The enzyme cutting site BamH I (GGATCC) is introduced at the 5 'end and Xho I (CTCGAG) is introduced at the 3' end of the mutant gene sequences of T15G, T15S and T15A respectively. The nucleotide sequences of the primers are shown in Table 1. Sangon Biotech (China, Shanghai) was entrusted with primer synthesis.

TABLE 17 PCR primers for alpha-HSDH St-2-2 mutant genes

Note: FP denotes the forward primer and RP the reverse primer. The downstream primers for all mutant genes were identical, and X represents G, S or a.

And (3) PCR system:

PCR conditions were as follows:

by means of a recovery kitCycle Pure Kit (OMEGA BIO-TEK, cat # D6493), according to the Kit instructions recorded the operation steps to recover PCR products.

2.2 cleavage and ligation

2.2.1 enzyme digestion

The wild type 7 alpha-HSDH St-2-2 gene, the mutant gene and the pGEX-6p-2 plasmid are subjected to double enzyme digestion by using BamH I and Xho I restriction enzymes respectively.

Enzyme digestion system:

enzyme cutting conditions are as follows: the digestion was carried out at 37 ℃ for 3h (dry bath).

2.2.2 recovery of double digestion products

By means of a recovery kitCycle Pure Kit (OMEGA BIO-TEK, cat # D6493), according to the Kit instructions for the operation steps for enzyme digestion product recovery, as follows:

1) adding equal volume of Binding agent Binding Buffer into each centrifuge tube, mixing with enzyme digestion product, suckingDNA minicolumn in 2mL collection tube, centrifugal (10000 Xg, 1 min).

2) mu.L Binding Buffer was added to the collection tube and centrifuged (14000 Xg, 1 min).

3) The column was washed with 700. mu.L of ethanol diluted SPW Wash Buffer, centrifuged (14000 Xg, 1min) and repeated once.

4) The liquid was discarded and the empty column was centrifuged (14000 Xg, 2 min).

5) Discarding the collection tubeThe DNA mini-column is placed on clean paper, and then the cover is opened and the paper is kept stand for 10min, and the alcohol is fully volatilized. During which a tube of sterile ddH is placed₂O is preheated to 65 ℃ for standby.

6) Will be provided withThe DNA mini-column was placed in a sterilized 1.5mL centrifuge tube, and 50. mu.L of ddH heated to 65 ℃ was added₂O, left at room temperature for 1-2min, DNA eluted, and centrifuged (14000 Xg, 2 min).

7) And (5) measuring the concentration. The DNA concentration was measured by pipetting 2. mu.L of the DNA solution into an ultraspectrophotometer. (concentration unit: ng/. mu.L, 260/280: nucleic acid content).

2.2.3 ligation of linearized vector pGEX-6p-2 with the product of the enzyme digestion of the Gene

Ligation was performed using T4 DNA ligase according to the following system and conditions.

The connection reaction conditions are as follows: ligation was carried out overnight at 16 ℃ to give the following ligation products: pGEX-6p-2/St-2-2, pGEX-6p-2/St-2-2T15G, pGEX-6p-2/St-2-2T15S, pGEX-6p-2/St-2-2T 15A.

2.3 ligation products transformed E.coli DH 5. alpha. competent cells

1) Preparing a culture medium: preparing a proper amount of Luria-Bertani agar culture medium, sterilizing the culture medium for 30min at 121 ℃ by high-pressure steam, cooling the culture medium to 40-50 ℃, and adding ampicillin with the final concentration of 100 mu g/mL into the culture medium. Taking a proper amount of culture medium, uniformly spreading the culture medium in a sterile culture dish, and then placing the culture medium on a super clean bench for solidification. During the period, the-80 ℃ frozen preservation of Trans5 alpha competent cells (full-scale gold, CD201-01) were taken out and rapidly placed on ice, and left for 10min to thaw.

2) Trans5 α competent cells were quickly dispensed as required into sterile 1.5mL centrifuge tubes, 10 μ L of ligation product was added and allowed to stand on ice for 30 min.

3) Heat shock was carried out at 45 ℃ for 45 s.

4) The tube was quickly transferred to ice and left for 2min (do not shake the tube).

5) Adding 500 μ L of sterile Luria-Bertani liquid culture medium, culturing with shaking at 37 deg.C and shaking speed of 180rpm for 45min, and recovering cells.

6) Sucking about 100 μ L of bacterial liquid, and coating on Amp⁺On resistant LB plate Medium (Amp)⁺Final concentration 100. mu.g/mL), incubated overnight at 37 ℃.

2.4 Positive clone screening

1) A single colony was picked and inoculated into a suitable amount of sterile Luria-Bertani liquid medium containing 100. mu.g/mL ampicillin, and cultured by shaking at 37 ℃ and a shaking speed of 220 rpm. Cultured to OD₆₀₀Approximately 0.8-1.

2) Seed preservation: and (3) uniformly mixing the bacterial liquid and 25% sterile glycerol according to the volume ratio of 2:1, quickly freezing by using liquid nitrogen, and storing in a refrigerator at the temperature of-80 ℃.

3) The remaining bacterial liquid is used for plasmid extraction. The Plasmid was extracted using OMEGA Plasmid Mini Kit I (OMEGA BIO-TEK, D6943) according to the protocol described in the Kit instructions, as follows:

1) growth of bacterial liquid to OD₆₀₀The bacteria are obtained by centrifugation at 8000rpm for 5min, about 0.8-1.

2) The supernatant was discarded and the residual liquid immediately blotted with 200. mu.L pipette, and 250. mu.L of Solution I (RNase had been added and stored at 4 ℃) was added immediately and vortexed until the pellet was completely suspended.

3) And adding the uniformly mixed bacterial liquid into a 1.5mL sterile centrifuge tube, adding Solution II with the same volume into the centrifuge tube, and slowly rotating the centrifuge tube to thoroughly mix the sample to obtain a clarified lysate. mu.L of Solution III was immediately added thereto, and the sample was mixed by slowly rotating the centrifuge tube (white flocculent precipitate appeared) and centrifuged (4 ℃, 13000 Xg, 10 min). (Note that this step must be completed within 5min and vigorous mixing cannot be performed, otherwise the chromosomal DNA breaks down and the purity of the resulting plasmid is reduced).

4) The supernatant was carefully aspirated with a 200. mu.L pipette (ensure no aspiration pellet) and transferred to a container2mL collection tubes of DNA mini-columns.

5) Centrifuge (13000 Xg, 1min) and discard the filtrate.

6) mu.L of Buffer HB was added to the collection column, centrifuged (13000 Xg, 1min) and the filtrate discarded.

7) To the collection column, 700. mu.L of DNA Wash Buffer (to which absolute ethanol had been added) was added, centrifuged (13000 Xg, 1min), and the filtrate was discarded to remove impurities. Repeating the steps once, and discarding the liquid.

8) Centrifuging (15000 Xg, 2min) to dryAnd (3) opening the cover of the DNA micro-column, standing for 10min to completely volatilize the absolute ethyl alcohol. During which a tube of sterile ddH is taken₂O was preheated to 65 ℃.

9) Will be provided withThe DNA mini-column was placed in a sterile 1.5mL centrifuge tube and 50. mu.L ddH preheated to 65 ℃ was added₂O, standing at room temperature for 2min, and centrifuging (15000 Xg, 2 min).

10) And (5) measuring the concentration. The DNA concentration was measured by pipetting 2. mu.L of the DNA solution into an ultramicro spectrophotometer. (concentration unit: ng/. mu.L, 260/280: nucleic acid content).

2.5 double restriction enzyme identification of plasmids

Enzyme digestion system:

enzyme cutting conditions are as follows: the enzyme was cleaved at 37 ℃ for 1.5 h. Detecting the enzyme digestion product by agarose gel electrophoresis.

2.6 sequencing confirmation

The recombinant plasmid with correct double enzyme digestion identification is selected and sent to Sangon Biotech (China, Shanghai) company for sequencing, and the recombinant plasmid with correct sequencing result is used as an expression vector of 7 alpha-HSDH St-2-2 wild type, 7 alpha-HSDH St-2-2T15G, 7 alpha-HSDH St-2-2T15S and 7 alpha-HSDH St-2-2T15A mutant.

3. GST fusion heterologous expression of enzyme proteins

3.1 transformation of E.coli BL21 cells with expression vectors

The obtained expression vector was transferred into e.coli BL21 competent cells according to the transformation method described in 2.3 (ligation product transformed e.coli DH5 α competent cells) above to obtain recombinant bacteria for protein expression.

3.2 protein expression and purification

1) Inoculating 100 μ L of the recombinant bacteria into 400mL of sterile LB medium, the final concentration of ampicillin being 100 μ g/mL, shaking-culturing at 37 ℃ and 180 rpm.

2) To be OD₆₀₀When the concentration is about 0.8, IPTG is added to a final concentration of 0.2mM and induction is carried out overnight at 16 ℃ (12 h). Subpackaging the bacterial liquid into high-speed centrifuge bottles, centrifuging at 8000rpm for 5min, and collecting the thallus.

3) Resuspend the cells according to the proportion of adding 30mL of 50mM PBS into 1L of culture system, and break the cells to be clear by ultrasound at 4 ℃. The crushed bacteria liquid is evenly distributed into a sterile 50mL centrifuge tube which is pre-cooled to 4 ℃ and centrifuged at 12000rpm for 20min, the bacteria are centrifugally settled, and after the centrifugation is finished, the supernatant is transferred into the sterile 50mL centrifuge tube by a precision liquid transfer gun.

4) 4mL of Glutathione Sepharose 4B packing was loaded onto an affinity column (GE Healthcare, cat. 10223836), and the column was washed 3 column volumes with sterile 4 ℃ pre-cooled 50mM PBS to remove the absolute ethanol. The supernatant was bound to glutaminone Sepharose 4B for 3h at 4 ℃. The suspension was gently inverted vertically.

5) After the bonding is completed, the filler is precipitated. The supernatant was filtered off, washed with sterile 4 ℃ pre-cooled 50mM PBS (containing 0.25% volume fraction of Tween 20) for 3 to 5 column volumes, then washed with sterile 4 ℃ pre-cooled 50mM PBS for 3 column volumes to remove contaminating proteins, leaving 1mL of 50mM PBS per affinity column at the time of the last elution.

6) 40-60. mu.L of PreScission Protease enzyme (GenScript, cat. No. Z02799-100) was added.

7) The digestion was carried out overnight at 4 ℃. And after enzyme digestion, discharging the supernatant from the chromatographic column to obtain the eluted 7 alpha-HSDH enzyme solution.

8) The obtained enzyme solution was subjected to SDS-PAGE to identify the molecular weight and purity, the molecular weight was about 28.2kDa, and the concentration of the purified protein was determined using BCA kit (Beyotime, cat. No. P0006) according to the kit instructions. Mixing the enzyme solution and 80% of sterile glycerol in a volume ratio of 3:1, packaging the enzyme solution containing glycerol into sterile 1.5mL centrifuge tubes, and storing in a refrigerator at-80 deg.C.

9) The used Glutathione Sepharose 4B filler is soaked in 6mol/L guanidine hydrochloride for 20min, then the filler is largely washed by PBS, and then the filler is soaked in 20% ethanol and stored in a refrigerator at 4 ℃.

SDS-PAGE detection results show that the soluble expression of the mutant enzyme protein and the wild enzyme protein is successful, and the protein bands are single after one-step affinity chromatography (FIG. 2 and FIG. 3). The concentrations of the purified T15G, T15S and T15A mutant enzyme proteins were 1.42mg/mL, 1.31mg/mL and 1.60mg/mL, respectively. The concentration of the purified wild-type enzyme protein was 1.59 mg/mL.

Example 2.7 determination of enzymatic Activity of alpha-hydroxysteroid dehydrogenase (St-2-2) mutant

Preparation of NADPH Standard Curve

A0 mM, 0.1mM, 0.2mM, 0.3mM, 0.4mM NADPH (Sigma-Aldrich, cat. No. 10621692001) solution was prepared using a reaction buffer (50mM Tris-HCl, pH 8.0), respectively. After zeroing with a blank solvent (50mM Tris-HCl, pH 8.0), NADPH solutions of various concentrations were added to 2mL cuvettes, respectively, and the light absorption OD was measured at 340nm at room temperature₃₄₀. And (3) taking the concentration of the NADPH solution as an abscissa and the corresponding light absorption value at 340nm as an ordinate, and drawing a standard curve. The result is shown in fig. 4, and the obtained standard curve equation is that y is 2.7990x-0.001, R²＝0.9999。

2. Enzyme activity assay

1) By ddH₂O configuration 50mM NADP⁺(Sigma-Aldrich, cat # N5755), 50mM TCDCA (Prodweis technology, cat # 330776), 50mM GCDCA (Merlin, cat # G835599).

2) 1955. mu.L of reaction buffer (50mM Tris-HCl, pH 8.0) was added to a 2mL cuvette, followed by 20. mu.L of 50mM NADP⁺And adding 5 μ L of the enzyme protein solution prepared in example 1 into the coenzyme solution, fully mixing, adjusting to zero at a wavelength of 340nm, immediately adding 20 μ L of 50mM TCDCA (taurochenodeoxycholic acid) or 20 μ L of 50mM GCDCA (glycochenodeoxycholic acid) substrate solution into the mixture, fully pumping, fully mixing, recording the change of light absorption at 340nm at room temperature within 30s, and calculating the generation amount of the product according to a standard curve of NADPH. The results were averaged for 3 replicates per enzyme protein sample. The enzyme activity unit is defined as: under the corresponding conditions, the amount of 7 α -HSDH required for the conversion of 1 μmol TCDCA or GCDCA per minute is defined as one enzyme activity unit U. The specific activity of the enzyme is defined as: the number of active units per mg of enzyme protein is given in: u/mg.

3) Calculating enzyme activity: the change in light absorption recorded at 340nm over 30s was taken into the NADPH standard curve y-2.7990 x-0.001, R²The converted substrate concentration mmol/L was calculated at 30s in 0.9999.

Vt: total volume of reaction (mL).

The results show that TCDCA and NADP are present on the same substrate⁺In the presence of (2), the enzyme activities of the 7 alpha-HSDH St-2-2 mutants T15G, T15S and T15A are respectively 3.18 times, 4.27 times and 7.85 times of the wild type of the 7 alpha-HSDH St-2-2 (figure 5). In the same substrate GCDCA and NADP⁺In the presence of (2), the enzyme activities of the 7 alpha-HSDH St-2-2 mutants T15G, T15S and T15A are respectively 2.27 times, 2.91 times and 4.68 times of the wild type of the 7 alpha-HSDH St-2-2 (figure 6).

Sequence listing

<110> university of Chongqing

<120> mutants of the enzyme St-2-hydroxysteroid dehydrogenase T15G, T15S and T15A

<130> P2130663-CQD-CQ-XDW

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 262

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Lys Arg Val Glu Asn Lys Val Ala Leu Val Thr Ser Ser Thr Arg

1 5 10 15

Gly Ile Gly Leu Ala Ile Ala Lys Thr Leu Ala Lys Glu Gly Ala Arg

20 25 30

Val Tyr Leu Ala Val Arg Arg Leu Asp Ala Gly Gln Glu Val Ala Asn

35 40 45

Glu Ile Ile Ala Glu Gly Gly Phe Ala Lys Pro Val Tyr Phe Asp Ala

50 55 60

Ser Lys Val Glu Thr His Met Ser Met Ile Glu Glu Val Val Glu Ala

65 70 75 80

Glu Gly Arg Ile Asp Ile Leu Val Asn Asn Tyr Gly Ser Thr Asp Val

85 90 95

Gln Lys Asp Leu Asp Leu Val His Gly Asp Thr Glu Ala Phe Phe Asn

100 105 110

Ile Val Asn Gln Asn Leu Glu Ser Val Tyr Leu Pro Cys Lys Val Ala

115 120 125

Val Pro Tyr Met Ile Lys Asn Gly Gly Gly Ser Ile Ile Asn Ile Ser

130 135 140

Thr Ile Gly Ser Val Asn Pro Asp Leu Gly Arg Ile Ala Tyr Val Val

145 150 155 160

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

165 170 175

Ala Lys Lys Gly Ile Arg Cys Asn Ala Val Leu Pro Gly Leu Ile Ala

180 185 190

Thr Asp Ala Ala Leu Asn Asn Met Ser Glu Glu Phe Leu Glu His Phe

195 200 205

Leu Arg His Val Pro Leu Asp Arg Thr Gly His Pro Gln Asp Ile Ala

210 215 220

Asn Ala Val Leu Phe Phe Ala Ser Asp Glu Ser Ser Tyr Ile Thr Gly

225 230 235 240

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

245 250 255

Gly Asp Ala Val Lys Lys

260

<210> 2

<211> 262

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

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

1 5 10 15

Gly Ile Gly Leu Ala Ile Ala Lys Thr Leu Ala Lys Glu Gly Ala Arg

20 25 30

Val Tyr Leu Ala Val Arg Arg Leu Asp Ala Gly Gln Glu Val Ala Asn

35 40 45

Glu Ile Ile Ala Glu Gly Gly Phe Ala Lys Pro Val Tyr Phe Asp Ala

50 55 60

Ser Lys Val Glu Thr His Met Ser Met Ile Glu Glu Val Val Glu Ala

65 70 75 80

Glu Gly Arg Ile Asp Ile Leu Val Asn Asn Tyr Gly Ser Thr Asp Val

85 90 95

Gln Lys Asp Leu Asp Leu Val His Gly Asp Thr Glu Ala Phe Phe Asn

100 105 110

Ile Val Asn Gln Asn Leu Glu Ser Val Tyr Leu Pro Cys Lys Val Ala

115 120 125

Val Pro Tyr Met Ile Lys Asn Gly Gly Gly Ser Ile Ile Asn Ile Ser

130 135 140

Thr Ile Gly Ser Val Asn Pro Asp Leu Gly Arg Ile Ala Tyr Val Val

145 150 155 160

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

165 170 175

Ala Lys Lys Gly Ile Arg Cys Asn Ala Val Leu Pro Gly Leu Ile Ala

180 185 190

Thr Asp Ala Ala Leu Asn Asn Met Ser Glu Glu Phe Leu Glu His Phe

195 200 205

Leu Arg His Val Pro Leu Asp Arg Thr Gly His Pro Gln Asp Ile Ala

210 215 220

Asn Ala Val Leu Phe Phe Ala Ser Asp Glu Ser Ser Tyr Ile Thr Gly

225 230 235 240

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

245 250 255

Gly Asp Ala Val Lys Lys

260

<210> 3

<211> 262

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Lys Arg Val Glu Asn Lys Val Ala Leu Val Thr Ser Ser Ser Arg

1 5 10 15

Gly Ile Gly Leu Ala Ile Ala Lys Thr Leu Ala Lys Glu Gly Ala Arg

20 25 30

Val Tyr Leu Ala Val Arg Arg Leu Asp Ala Gly Gln Glu Val Ala Asn

35 40 45

Glu Ile Ile Ala Glu Gly Gly Phe Ala Lys Pro Val Tyr Phe Asp Ala

50 55 60

Ser Lys Val Glu Thr His Met Ser Met Ile Glu Glu Val Val Glu Ala

65 70 75 80

Glu Gly Arg Ile Asp Ile Leu Val Asn Asn Tyr Gly Ser Thr Asp Val

85 90 95

Gln Lys Asp Leu Asp Leu Val His Gly Asp Thr Glu Ala Phe Phe Asn

100 105 110

Ile Val Asn Gln Asn Leu Glu Ser Val Tyr Leu Pro Cys Lys Val Ala

115 120 125

Val Pro Tyr Met Ile Lys Asn Gly Gly Gly Ser Ile Ile Asn Ile Ser

130 135 140

Thr Ile Gly Ser Val Asn Pro Asp Leu Gly Arg Ile Ala Tyr Val Val

145 150 155 160

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

165 170 175

Ala Lys Lys Gly Ile Arg Cys Asn Ala Val Leu Pro Gly Leu Ile Ala

180 185 190

Thr Asp Ala Ala Leu Asn Asn Met Ser Glu Glu Phe Leu Glu His Phe

195 200 205

Leu Arg His Val Pro Leu Asp Arg Thr Gly His Pro Gln Asp Ile Ala

210 215 220

Asn Ala Val Leu Phe Phe Ala Ser Asp Glu Ser Ser Tyr Ile Thr Gly

225 230 235 240

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

245 250 255

Gly Asp Ala Val Lys Lys

260

<210> 4

<211> 262

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Lys Arg Val Glu Asn Lys Val Ala Leu Val Thr Ser Ser Ala Arg

1 5 10 15

Gly Ile Gly Leu Ala Ile Ala Lys Thr Leu Ala Lys Glu Gly Ala Arg

20 25 30

Val Tyr Leu Ala Val Arg Arg Leu Asp Ala Gly Gln Glu Val Ala Asn

35 40 45

Glu Ile Ile Ala Glu Gly Gly Phe Ala Lys Pro Val Tyr Phe Asp Ala

50 55 60

Ser Lys Val Glu Thr His Met Ser Met Ile Glu Glu Val Val Glu Ala

65 70 75 80

Glu Gly Arg Ile Asp Ile Leu Val Asn Asn Tyr Gly Ser Thr Asp Val

85 90 95

Gln Lys Asp Leu Asp Leu Val His Gly Asp Thr Glu Ala Phe Phe Asn

100 105 110

Ile Val Asn Gln Asn Leu Glu Ser Val Tyr Leu Pro Cys Lys Val Ala

115 120 125

Val Pro Tyr Met Ile Lys Asn Gly Gly Gly Ser Ile Ile Asn Ile Ser

130 135 140

Thr Ile Gly Ser Val Asn Pro Asp Leu Gly Arg Ile Ala Tyr Val Val

145 150 155 160

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

165 170 175

Ala Lys Lys Gly Ile Arg Cys Asn Ala Val Leu Pro Gly Leu Ile Ala

180 185 190

Thr Asp Ala Ala Leu Asn Asn Met Ser Glu Glu Phe Leu Glu His Phe

195 200 205

Leu Arg His Val Pro Leu Asp Arg Thr Gly His Pro Gln Asp Ile Ala

210 215 220

Asn Ala Val Leu Phe Phe Ala Ser Asp Glu Ser Ser Tyr Ile Thr Gly

225 230 235 240

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

245 250 255

Gly Asp Ala Val Lys Lys

260

<210> 5

<211> 789

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgaaaagag tagaaaataa agtagcatta gtcacatctt ctacaagagg gattggactt 60

gctattgcta aaacacttgc taaagaaggt gcacgtgtat accttgcagt aagaagatta 120

gatgcaggtc aggaggtagc gaatgaaatt attgcagaag gtggatttgc taagcctgtt 180

tactttgatg cttctaaagt agagacacac atgagtatga ttgaagaagt agttgaagct 240

gaaggacgta tagatatttt agtcaataat tatggttcaa cagacgttca aaaggactta 300

gatctcgtac atggagatac agaagctttc tttaatattg ttaatcaaaa tcttgaaagt 360

gtttacttac catgtaaggt ggcggtacct tatatgatta aaaatggtgg aggaagcatt 420

attaacattt ctacaattgg ttcagtaaac cctgaccttg gacgtattgc ttatgttgta 480

tctaaagcag ctatcaacgc gcttacacaa aatattgcag ttcagtatgc aaaaaaaggg 540

ataagatgta atgctgttct tccaggtctt attgctacgg atgcagccct taataatatg 600

tcagaggagt tcttagaaca tttcttaaga catgtaccac ttgaccgtac agggcatcct 660

caagatattg ctaatgcagt acttttcttt gcaagtgatg aatcttctta tattacaggt 720

acacttcaag aagtagcagg tggatttggt atgccatcac ctatttatgg ggatgctgtt 780

aagaaataa 789

<210> 6

<211> 789

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atgaaaagag tagaaaataa agtagcatta gtcacatctt ctggtagagg gattggactt 60