NAD (H) dependent 3alpha-hydroxysteroid dehydrogenase mutant E139S

Abstract

The invention belongs to the technical field of biology, and particularly relates to an NAD (H) dependent type 3 alpha-hydroxysteroid dehydrogenase mutant E139S, the amino acid sequence of the mutant E139S is shown as SEQ ID NO: 1, and the mutant E139S is obtained by changing the 139th amino acid of wild type 3 alpha-hydroxysteroid dehydrogenase of which the amino acid sequence is SEQ ID NO: 2 into Ser from Glu. The DNA coding sequence of the gene is as shown in SEQ ID NO: 3. The mutant E139S can catalyze oxidation of alpha hydroxyl at C3 site of taurochenodeoxycholic acid (TCDCA) and tauroursodeoxycholic acid (TUDCA), and the activity of the mutant E139S is 1.26 times and 1.73 times that of wild type 3 alpha-hydroxysteroid dehydrogenase. The application prospect in clinical examination of the total bile acid content is huge.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a NAD(H)-dependent 3alpha-hydroxysteroid dehydrogenase mutant E139S. BACKGROUND

[0002] Bile acid is an important component of bile and plays an important role in fat metabolism. The determination of the content of total bile acid in blood, tissue fluid or other systems has been one of the research hotspots. At present, 3alpha-hydroxysteroid dehydrogenase is often used in clinical detection to catalyze the oxidation reaction of C3 hydroxyl of bile acid in the sample to be detected, and the oxidation type coenzyme I or II is reduced to reduced coenzyme I or II at the same time, and the content of total bile acid in the sample to be detected is calculated by measuring the light absorption change value at 340 nm. In the detection process, 3alpha-hydroxysteroid dehydrogenase has high chemical selectivity and stereoselectivity, and the activity of the enzyme is one of the decisive factors for whether the enzyme has wide application in clinical detection. The existing 3alpha-hydroxysteroid dehydrogenase has low catalytic activity, so that the detection efficiency of bile acid is low. At present, the mining and discovery of new enzymes with high catalytic activity and stability is one of the research hotspots in the field of hydroxysteroid dehydrogenase. SUMMARY

[0003] The present application solves the problem that the existing 3alpha-hydroxysteroid dehydrogenase has low catalytic activity, so that the detection efficiency of bile acid is low, and provides a NAD(H)-dependent 3alpha-hydroxysteroid dehydrogenase mutant E139S. The 3alpha-hydroxysteroid dehydrogenase mutant E139S catalyzes the oxidation of C3 alpha-hydroxyl of taurocholic deoxycholic acid (TCDCA) and taurocholic deoxycholic acid (TUDCA) to generate Tauro-3-dehydro-CDCA and Tauro-3-dehydro-UDCA, respectively, and the activity is 1.26 times and 1.73 times that of 3alpha-hydroxysteroid dehydrogenase (Accession NO. UAW21665), which has great application value in clinical detection.

[0004] The technical scheme claimed in the present application is as follows:

[0005] A 3alpha-hydroxysteroid dehydrogenase mutant E139S, the amino acid sequence of which is shown in SEQ ID NO: 1, and the 139th amino acid of the wild-type 3alpha-hydroxysteroid dehydrogenase with the amino acid sequence of SEQ ID NO: 2 is changed from Glu to Ser.

[0006] The present application also provides a gene encoding the 3alpha-hydroxysteroid dehydrogenase mutant E139S, the nucleotide sequence of which is shown in SEQ ID NO: 3.

[0007] The application also provides an expression cassette comprising the gene.

[0008] The application also provides a vector comprising the gene or the expression cassette.

[0009] The application also provides a recombinant cell comprising the gene, the expression cassette or the vector.

[0010] The application also provides a method for preparing the 3α-hydroxysteroid dehydrogenase mutant E139S, which comprises culturing the recombinant cell under conditions that can successfully induce protein expression and isolating the 3α-hydroxysteroid dehydrogenase mutant E139S.

[0011] The application also provides a catalyst, which comprises the 3α-hydroxysteroid dehydrogenase mutant E139S as an effective component.

[0012] Preferably, the catalyst further comprises other reagents that can improve the catalytic efficiency of the enzyme or increase the stability of the enzyme when used simultaneously with the 3α-hydroxysteroid dehydrogenase mutant E139S.

[0013] The application also provides a method for realizing asymmetric reduction of carbonyl of a chemical substance, which comprises using the 3α-hydroxysteroid dehydrogenase mutant E139S or the catalyst to catalyze the oxidation of the C3 α-hydroxyl of TCDCA and TUDCA to generate Tauro-3-dehydro-CDCA and Tauro-3-dehydro-UDCA, respectively.

[0014] Advantages

[0015] The application provides an NAD(H)-dependent 3α-hydroxysteroid dehydrogenase mutant E139S, which is obtained by comparing the similarities and differences between the wild-type 3α-HSDH and homologous enzyme proteins from the primary structure to the higher structure in multiple angles and multiple layers, determining that the site affecting the enzymatic properties of 3α-HSDH is the 139th residue, i.e., glutamic acid. Then, the glutamic acid (Glu) is changed into serine (Ser) by codon substitution, and the gene of the 3α-HSDH mutant E139S is obtained by using the whole gene synthesis technology. Finally, the GST fusion expression vector of the mutant gene is constructed and introduced into the genetically engineered bacteria E. coli BL21 to induce expression, and the 3α-HSDH mutant E139S enzyme protein is obtained. The enzymatic reaction rate is determined, and the results show that the catalytic efficiency of the mutant E139S on the substrates NAD + and TCDCA is 1.26 times that of the wild type. +The catalytic efficiency of the application and TUDCA is 1.73 times of the wild type. The application has great application value in clinical test of total bile acid content, and can solve the problem that the catalytic activity of the existing 3alpha-hydroxysteroid dehydrogenase is low, so that the detection efficiency of bile acid is low.

[0016] SEQUENCE LISTING

[0017] SEQ ID NO: 1, Amino acid sequence of 3a-hydroxysteroid dehydrogenase mutant E139S MGTYVVTGAASGIGKAVAEQISEEGHRVVAVDLRNTELTADLSDRESCKKVIEQILERAPEGLDGLVPCAGVGPDVARRELIPLVNYFAVVDLVEGLLAALQQRKGSIVLISSNSSQMMEYNASFMDAMLDDDRERALSVAADIGGQDAYGGSKQALARWMRRNNQNISRSGVRMNAIAPGHTETGMTAAGSASPEYADAIKQFVESIPIGYSAMPEDQANAVSFLLSDKARFISGAVLFVDGGHDAMFRPDQY SEQ ID NO: 2, Amino acid sequence of wild-type 3a-hydroxysteroid dehydrogenase MGTYVVTGAASGIGKAVAEQISEEGHRVVAVDLRNTELTADLSDRESCKKVIEQILERAPEGLDGLVPCAGVGPDVARRELIPLVNYFAVVDLVEGLLAALQQRKGSIVLISSNSSQMMEYNASFMDAMLDDDRERALEVAADIGGQDAYGGSKQALARWMRRNNQNISRSGVRMNAIAPGHTETGMTAAGSASPEYADAIKQFVESIPIGYSAMPEDQANAVSFLLSDKARFISGAVLFVDGGHDAMFRPDQY SEQ ID NO: 3, Amino acid sequence of 3a-hydroxysteroid dehydrogenase mutant E139S and E140S MGTYVVTGAASGIGKAVAEQISEEGHRVVAVDLRNTELTADLSDRESCKKVIEQILERAPEGLDGLVPCAGVGPDVARRELIPLVNYFAVVDLVEGLLAALQQRKGSIVLISSNSSQMMEYNASFMDAMLDDDRERALSVAADIGGQDAYGGSKQALARWMRRNNQNISRSGVRMNAIAPGHTETGMTAAGSASPEYADAIKQFVESIPIGYSAMPEDQANAVSFLLSDKARFISGAVLFVDGGHDAMFRPDQYNO: 3 DNA coding sequence of 3a-hydroxysteroid dehydrogenase mutant E139S ATGGGTACCTACGTTGTTACCGGTGCTGCTTCTGGTATCGGTAAAGCTGTTGCTGAACAGATCTCTGAAGAAGGTCACCGTGTTGTTGCTGTTGACCTGCGTAACACCGAACTGACCGCTGACCTGTCTGACCGTGAATCTTGCAAAAAAGTTATCGAACAGATCCTGGAACGTGCTCCGGAAGGTCTGGACGGTCTGGTTCCGTGCGCTGGTGTTGGTCCGGACGTTGCTCGTCGTGAACTGATCCCGCTGGTTAACTACTTCGCTGTTGTTGACCTGGTTGAAGGTCTGCTGGCTGCTCTGCAGCAGCGTAAAGGTTCTATCGTTCTGATCTCTTCTAACTCTTCTCAGATGATGGAATACAACGCTTCTTTCATGGACGCTATGCTGGACGACGACCGTGAACGTGCTCTGTCTGTTGCTGCTGACATCGGTGGTCAGGACGCTTACGGTGGTTCTAAACAGGCTCTGGCTCGTTGGATGCGTCGTAACAACCAGAACATCTCTCGTTCTGGTGTTCGTATGAACGCTATCGCTCCGGGTCACACCGAAACCGGTATGACCGCTGCTGGTTCTGCTTCTCCGGAATACGCTGACGCTATCAAACAGTTCGTTGAATCTATCCCGATCGGTTACTCTGCTATGCCGGAAGACCAGGCTAACGCTGTTTCTTTCCTGCTGTCTGACAAAGCTCGTTTCATCTCTGGTGCTGTTCTGTTCGTTGACGGTGGTCACGACGCTATGTTCCGTCCGGACCAGTACTAA BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Comparative analysis chart of relative activity of 3a-hydroxysteroid dehydrogenase mutant E139S and wild type 3a-hydroxysteroid dehydrogenase described in the embodiments of the present application, wherein: A represents the comparison of catalytic TCDCA activity; B represents the comparison of catalytic TUDCA activity.

[0019] Figure 2SDS-PAGE analysis characterization chart of 3a-hydroxysteroid dehydrogenase mutant E139S described in the embodiments of the present application; wherein: M represents protein molecular weight standard (Marker); E139S represents mutant E139S. DETAILED DESCRIPTION

[0020] The present application will be described in detail below with reference to the accompanying drawings and specific embodiments. The following embodiments will help those skilled in the art to further understand the present application, but do not limit the present application in any form. It should be noted that for those skilled in the art, without departing from the concept of the present application, a number of changes and improvements can be made. These all belong to the protection scope of the present application.

[0021] The biochemical reagents not specifically described in the following examples are conventional reagents in the art, which can be commercially available or prepared according to conventional methods in the art.

[0022] Main reagents:

[0023] pGEX-6p-1 is a known vector, which is preserved in the laboratory;

[0024] E. coli BL21 cells are preserved in the laboratory;

[0025] Lysis buffer, prepared, 10 mM pH 7.3 PBS containing PMSF 0.1 mM, Leupeptin 0.5 mg / mL;

[0026] Glutathione Sepharose 4B, purchased from GE Healthcare;

[0027] PreScission Protease, purchased from GenScript Corporation.

[0028] The first group of embodiments, a 3a-hydroxysteroid dehydrogenase mutant E139S

[0029] This group of embodiments provides a 3a-hydroxysteroid dehydrogenase mutant E139S, whose amino acid sequence is shown in SEQ ID NO: 1, and the 139th amino acid of the wild-type 3a-hydroxysteroid dehydrogenase with amino acid sequence of SEQ ID NO: 2 is changed from Glu to Ser.

[0030] The second group of embodiments, a gene

[0031] This group of embodiments provides a coding gene of the 3a-hydroxysteroid dehydrogenase mutant E139S described in the first group of embodiments.

[0032] In a specific embodiment of the present application, the nucleotide sequence of the gene is shown as SEQ ID NO: 3.

[0033] A third group of embodiments, an expression cassette

[0034] The expression cassette of the present group of embodiments comprises the gene of the second group of embodiments.

[0035] A fourth group of embodiments, a vector

[0036] The vector of the present group of embodiments comprises the gene of the second group of embodiments or the expression cassette of the third group of embodiments.

[0037] A fifth group of embodiments, a recombinant cell

[0038] The recombinant cell of the present group of embodiments comprises the gene of the second group of embodiments, or the expression cassette of the third group of embodiments, or the vector of the fourth group of embodiments.

[0039] A sixth group of embodiments, a preparation method

[0040] The present group of embodiments provides a method for preparing the 3a-hydroxysteroid dehydrogenase mutant E139S of the first group of embodiments, which comprises culturing the recombinant cell of the fifth group of embodiments under conditions that can successfully induce protein expression and isolating the 3a-hydroxysteroid dehydrogenase mutant E139S.

[0041] A seventh group of embodiments, a catalyst

[0042] The present group of embodiments provides a catalyst, which comprises the 3a-hydroxysteroid dehydrogenase mutant E139S of the first group of embodiments as an effective component.

[0043] Preferably, the catalyst further comprises other reagents that can improve the catalytic efficiency of the enzyme or increase the stability of the enzyme when used simultaneously with the 3a-hydroxysteroid dehydrogenase mutant E139S of the first group of embodiments.

[0044] An eighth group of embodiments, a method for realizing asymmetric reduction of carbonyl of a chemical substance

[0045] The present group of embodiments provides a method for realizing asymmetric reduction of carbonyl of a chemical substance, which uses the 3a-hydroxysteroid dehydrogenase mutant E139S of the first group of embodiments or the catalyst of the seventh group of embodiments to catalyze the oxidation of the C3 position of TCDCA and TUDCA to generate Tauro-3-dehydro-CDCA and Tauro-3-dehydro-UDCA, respectively.

[0046] Experimental examples

[0047] Example 1.3 Gene design and acquisition of the α-hydroxysteroid dehydrogenase mutant E139S

[0048] By comparing and analyzing the sequences of previously reported 3α-hydroxysteroid dehydrogenases and considering the structural characteristics of the SDR superfamily gene sequences, the gene sequence of the 3α-hydroxysteroid dehydrogenase mutant E139S described in this invention was designed. The entire gene was synthesized by Shanghai Sangon Biotech Co., Ltd., and cloned into the pEGX-6p-1 vector with BamHI / XhoI restriction sites. This recombinant plasmid was then transformed into *E. coli* BL21. The procedure was as follows: *E. coli* BL21 competent cells were removed from the container at -80℃ and placed on ice; 2 μL of the pEGX-6p-1 / E139S recombinant plasmid was added and placed on ice for 30 min; heat shock was applied at 42℃ for 90 seconds; the cells were placed on ice for 2 minutes; the cells were then revived, and 600 μL of LB medium was added, and the culture was incubated at 37℃, 150 rpm for 45 min; 200 μL of the medium was spread onto LB agar plates containing ampicillin; the cells were incubated overnight at 37℃; single colonies were picked for expansion culture and preservation.

[0049] Example 2.3 Expression of the α-hydroxysteroid dehydrogenase mutant E139S in Escherichia coli BL21

[0050] Inoculate pEGX-6p-1 / E139S / BL21 bacterial strain into sterile LB medium and incubate at 37°C and 180 rpm; wait for OD... 600 When the concentration reaches approximately 0.8, add IPTG to a final concentration of 0.2 mM and induce at 16°C for 12 hours. Collect bacterial cells at 8000 rpm for 5 min; resuspend the bacterial cells at a ratio of 30 mL Lysis buffer per 1 L culture volume and sonicate until clear. Incubate at 12000 rpm for 20 min. Collect the supernatant; bind the supernatant to Glutathione Sepharose 4B at 4°C for 2 h. Gently invert the supernatant vertically to resuspend it; after binding, precipitate the packing material at 5000 rpm for 5 min. Wash the packing material with 3-5 column volumes of pre-cooled PBS at 4°C; add PreScission Protease digestion buffer, add PreScission Protease, and digest overnight at 4°C. After digestion, release the supernatant from the chromatography column.

[0051] The novel NAD(H)-dependent 3α-hydroxysteroid dehydrogenase mutant E139S provided by this invention is shown in SEQ ID NO: 1; the SDS-PAGE analysis of the 3α-hydroxysteroid dehydrogenase mutant E139S is as follows: Figure 2 As shown.

[0052] Experiment 3.3 Activity assay of α-hydroxysteroid dehydrogenase mutant E139S

[0053] Prepare a pH 7.5 50mM Tris-HCl buffer to test enzyme activity. The specific procedure is as follows: Add the reaction buffer and NAD2 to a 2mL cuvette sequentially. + The enzyme was deactivated, and substrate was added. The change in light absorption was recorded at 340 nm. The amount of product generated was calculated based on the NADH standard curve. Enzyme activity was calculated. An enzyme activity unit (U) is defined as the amount of enzyme required to convert 1 μmol of substrate per minute under the corresponding conditions. Wild-type 3α-hydroxysteroid dehydrogenase was heterologously expressed and its activity was detected using the above method.

[0054] like Figure 1 As shown, the results indicate that the 3α-hydroxysteroid dehydrogenase mutant E139S is effective against the substrate NAD. + The catalytic efficiency of TCDCA is 1.26 times that of the wild type, and it is also better for the substrate NAD. + The catalytic efficiency of TUDCA is 1.73 times that of the wild type.

Claims

1. A 3α-hydroxysteroid dehydrogenase mutant E139S, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

1.

2. The gene encoding the 3α-hydroxysteroid dehydrogenase mutant E139S as described in claim 1.

3. The gene according to claim 2, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:

3.

4. An expression box, characterized in that, It contains the gene described in claim 2.

5. A carrier, characterized in that, It comprises the gene of claim 2 or the expression cassette of claim 4.

6. A recombinant cell, characterized in that, It comprises the gene of claim 2, the expression cassette of claim 4, or the vector of claim 5.

7. A method for preparing the 3α-hydroxysteroid dehydrogenase mutant E139S according to claim 1, characterized in that, The recombinant cells of claim 6 were successfully cultured under conditions that induce protein expression, and the 3α-hydroxysteroid dehydrogenase mutant E139S was isolated.

8. A catalyst, characterized in that, Its active ingredient contains the 3α-hydroxysteroid dehydrogenase mutant E139S as described in claim 1.

9. The catalyst according to claim 8, characterized in that, The catalyst also includes other reagents that can improve enzyme catalytic efficiency or increase enzyme stability when used in conjunction with the 3α-hydroxysteroid dehydrogenase mutant E139S.

10. A method for realizing the biotransformation of chemical substances, characterized in that, Using the 3α-hydroxysteroid dehydrogenase mutant E139S as described in claim 1 or the catalyst as described in claim 8, the C3-α-hydroxyl group of taurochenodeoxycholic acid and tauroursodeoxycholic acid are oxidized to generate Tauro-3-dehydro-CDCA and Tauro-3-dehydro-UDCA, respectively.

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