CN107841489B - Clostridium sardinieri 7 α -hydroxysteroid dehydrogenase mutant K179M - Google Patents
Clostridium sardinieri 7 α -hydroxysteroid dehydrogenase mutant K179M Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a Clostridium sarmentosum 7 α -hydroxysteroid dehydrogenase mutant K179M, the amino acid sequence of which is shown as SEQ ID NO. 2, wherein the 179 th amino acid of 7 α -hydroxysteroid dehydrogenase with the nucleic acid sequence of SEQ ID NO. 1 is obtained by changing lysine into methionine.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to clostridium sardine 7 α -hydroxysteroid dehydrogenase mutant K179M.
Background
Although chemical methods have been successful, they have disadvantages of limited catalyst types and numbers, low stereoselectivity, expensive auxiliary reagents and difficulty in recovery, and enzymatic reactions have high efficiency, chemoselectivity, regioselectivity and stereoselectivity.A Hydroxyl Steroid Dehydrogenase (HSDH) -mediated enzymatic reaction has relatively strict stereoselectivity and "not strict" substrate specificity.for example, scientists have begun to attempt to synthesize deoxycholic acid (ursodeoxycholic acid, UDCA) by joint epimerization of 7 α -, 7 β -HSDH produced by microorganisms as early as the eighties of the twentieth century, and the conversion process of free taurocholic acid-can also catalyze the conversion of Chenodeoxycholic acid (Taurochenodeoxycholic acid, DCTUoxocholic acid, DCTUOXA) into taurocholic acid (DCTUOXA).
The substrate of HSDH is not limited to steroids, and the HSDH can catalyze carbonyl asymmetric reduction of alkyl substituted monocyclic ketones, bicyclic ketones and other substances, which is reported in documents, however, the HSDH with higher activity is a basic guarantee for further application in the field of biotransformation, in recent years, researchers have gradually recognized the great application potential of 7 α -and 7 β -HSDH in the field of biotransformation, at present, the existing 7 α -HSDH in the field has 5 total parts, which are respectively from Bacteroides fragilism, Clostridium sciindens, Clostridium sordidii, Clostridium annum and Escherichia coli, and the biotransformation system constructed by the above two-enzyme coupling not only overcomes the problem of coenzyme circulation, but also realizes the hydroxyl epimerization in a specific chemical region in an oxidation and reduction one-pot manner.
The low thermal stability of the enzyme is one of the main factors limiting the industrial application of the enzyme, so how to improve the enzyme activity and the enzyme stability and develop the enzyme with good enzyme activity and stability is a problem which needs to be solved urgently at present in the field.
Disclosure of Invention
In order to meet the requirements in the field, the invention provides the clostridium sardiniformis 7 α -hydroxysteroid dehydrogenase (CA7 α -HSDH) mutant which has higher catalytic efficiency, can be used for bioconversion of various substrates and has great application value in industrial production.
The technical scheme of the invention is as follows:
a method for improving the enzyme activity and stability of wild clostridium sardiniforme 7 α -hydroxysteroid dehydrogenase is characterized in that the 179 th lysine of the wild clostridium sardiniforme 7 α -hydroxysteroid dehydrogenase is mutated into methionine.
The amino acid sequence of the wild clostridium sardine 7 α -hydroxysteroid dehydrogenase is shown in SEQ ID NO: 1, and the enzyme activity and stability of the mutant of the clostridium sardine 7 α -hydroxysteroid dehydrogenase obtained after mutation are obviously improved compared with those of the wild clostridium sardine 7 α -hydroxysteroid dehydrogenase.
The clostridium sardinifolium 7 α -hydroxysteroid dehydrogenase mutant K179M is characterized in that the amino acid sequence is shown as SEQ ID NO. 2.
A gene encoding said clostridium sardiniformis 7 α -hydroxysteroid dehydrogenase mutant K179M.
The nucleotide sequence of the gene is shown as SEQ ID NO: 3, respectively.
An expression cassette comprising said gene.
A vector comprising said gene, and/or said expression cassette.
A recombinant cell comprising said gene, and/or said expression cassette, and/or said vector.
A catalyst, characterized in that the effective component of the catalyst comprises the clostridium sardinifolium 7 α -hydroxysteroid dehydrogenase mutant K179M.
The catalyst also comprises other reagents which can improve the catalytic efficiency or increase the stability of the enzyme when being used together with the clostridium sardiniformis 7 α -hydroxysteroid dehydrogenase mutant K179M.
A method for realizing carbonyl asymmetric reduction of chemical substances, which is characterized in that the clostridium sardinifolium 7 α -hydroxysteroid dehydrogenase mutant K179M is used, and/or the catalyst and a reaction substrate are subjected to catalytic reaction at the temperature of 20-50 ℃ and the pH value of 6.0-11.0.
The first aspect of the present invention provides a method for improving the enzyme activity and stability of wild clostridium sardiniformis 7 α -hydroxysteroid dehydrogenase, wherein the 179 th lysine of the wild clostridium sardiniformis 7 α -hydroxysteroid dehydrogenase is mutated into methionine.
In a specific embodiment, the amino acid sequence of the wild clostridium sardine 7 α -hydroxysteroid dehydrogenase is shown in SEQ ID NO: 1, and the enzyme activity and stability of the mutant of the clostridium sardine 7 α -hydroxysteroid dehydrogenase enzyme obtained after mutation are obviously improved compared with those of the wild clostridium sardine 7 α -hydroxysteroid dehydrogenase.
The invention discloses a Clostridium sarmentosum 7 α -hydroxysteroid dehydrogenase (CA7 α -HSDH) mutant in a second aspect, which is characterized in that the amino acid sequence is shown as SEQ ID NO. 2, and the 179 th amino acid of 7 α -HSDH with the amino acid sequence being SEQ ID NO. 1 is obtained by changing Lys into Met.
"mutant" as used herein refers to a mutant of an enzyme, i.e., a mutated enzyme, which may also be referred to as a "mutant enzyme", as is conventional in the art. Specifically, the modified form of an enzyme in which a single site is artificially modified does not mean a cell or other organism carrying the mutant enzyme.
The invention changes 1 amino acid residue by lysine (K) mutation at 179 position of wild clostridium sardine 7 α -hydroxysteroid dehydrogenase into methionine, so that the activity and stability of the enzyme are obviously improved.
In another aspect, the present invention also claims a gene encoding the above-described Clostridium sardinieri 7 α -hydroxysteroid dehydrogenase CA7 α -HSDH mutant.
Further, the nucleotide sequence of the gene is shown as SEQ ID NO: 3, respectively.
It is clear in the art that, due to the degeneracy of the codons, the expressed proteins are identical (identical amino acid sequence, identical structure, identical function) although the nucleotide sequences differ. The degeneracy of the codon only causes the difference of the expression amount and the purity of the expressed protein, thereby influencing the purification. Based on the disclosure of the present invention, the skilled in the art can perform routine adjustment and selection according to the codon preference of escherichia coli, optimize codons to reduce the expression of the hybrid protein, and further improve the expression purity of the target protein (the enzyme mutant of the present invention).
In a fourth aspect, the present invention provides an expression cassette comprising the gene described above.
In a fifth aspect, the present invention provides a vector comprising the above gene or expression cassette.
In a sixth aspect, the present invention provides a recombinant cell comprising the above gene or expression cassette or vector.
In a seventh aspect, the present invention provides a method for producing the above-mentioned CA7 α -HSDH mutant, which comprises culturing the above-mentioned recombinant cell under conditions that allow successful induction of protein expression, and isolating the CA7 α -HSDH mutant.
In an eighth aspect, the present invention provides a catalyst, wherein the active ingredient comprises the above CA7 α -HSDH mutant.
The catalyst is characterized by also comprising other reagents which can improve the catalytic efficiency or increase the stability of the enzyme when being used together with the CA7 α -HSDH mutant.
The ninth aspect of the present invention provides a method for realizing asymmetric carbonyl reduction of a chemical substance, which comprises catalytically reacting the above CA7 α -HSDH mutant or any of the above catalysts with a substrate at 20-50 ℃ and pH 6.0-11.0.
"CA 7 α -HSDH" herein refers to Clostridium sarmentosum 7 α -hydroxysteroid dehydrogenase.
The invention firstly compares the similarities and differences of wild CA7 α -HSDH and homologous enzyme protein from a primary structure to a high-level structure in a multi-angle multilayer system, determines that the site influencing the enzymological property of CA7 α -HSDH is 179 th amino acid-lysine, then changes the lysine into methionine by codon substitution, and obtains the gene of the CA7 α -HSDH K179M mutant by utilizing the whole gene synthesis technology.
The vector provided by the invention can be a cloning vector, comprises a CA7 α -HSDH K179M mutant gene and other elements required by plasmid replication, or an expression vector, comprises a CA7 α -HSDH K179M mutant gene and other elements capable of enabling protein to be successfully expressed, in some embodiments, the expression vector is pGEX-6p-2 vector inserted with the CA7 α -HSDH-K179M mutant gene.
The recombinant cell provided by the invention can be a recombinant cell containing a cloning vector, such as E.coli DH5 α, and the intracellular CA7 α -HSDH K179M mutant gene is replicated by culturing the cell, or a cell containing an expression vector is cultured under a proper condition, for example, a proper amount of IPTG is added, and the expression of the CA7 α -HSDH K179M mutant protein is induced at 16 ℃.
The invention provides a catalyst, the active ingredient of which comprises a CA7 α -HSDHK179M mutant, the catalyst can also be used together with other suitable catalysts, thereby improving the enzyme catalysis efficiency or carrying out two catalytic reactions in the same reaction system.
The CA7 α -HSDHK179M mutant can catalyze the asymmetric reduction reaction of carbonyl under the conditions of 20-50 ℃ and pH 6.0-11.0.
Drawings
FIG. 1.7 α -, 7 β -HSDH combined with transformation of TCDCA to prepare TUDCA.
FIG. 2. agarose gel electrophoresis results of construction of K179M mutant gene by overlappinging PCR (overlap PCR); wherein M is a DNA Marker; 1 and 2 overlapping PCR products with K179M.
FIG. 3 shows the result of plasmid double restriction enzyme identification; wherein M is a DNA Marker; 1 and 2 are recombinant plasmids after enzyme digestion.
FIG. 4 is an SDS-PAGE electrophoresis of Clostridium sarmentosum 7 α -hydroxysteroid dehydrogenase mutant K179M, wherein M is a protein molecular weight standard (Marker), and 1 and 2 are K179M mutant proteins.
FIG. 5 shows the result of sequence alignment between the K179M gene and Clostridium sarlandicum 7 α -HSDH gene, wherein WT (only two sequences of WT and 179 in FIG. 5, without Ca7 column-HSDH) is the nucleotide sequence of the existing Clostridium sarlandicum 7 α -HSDH gene, and 179 is the nucleotide sequence of the 7 α -HSDH mutant prepared by the present invention.
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 main reagents are as follows:
pGEX-6p-2 is a known vector and is stored in the laboratory;
coli BL21 cells were stored in this laboratory;
lysis buffer, prepared from 10mM PBS with pH7.3 containing PMSF 0.1mM and leupeptin Leupepptin 0.5 mg/mL;
glutaminone Sepharose 4B, purchased from GE Healthcare, cat No.: 10223836, respectively;
PreScission Protease, purchased from GenScript, Cat. No.: z02799-100;
BCA kit, purchased from Beyotime, cat # s: p0006;
TCDCA, purchased from carbofuran technologies, cat # k: 330776, respectively;
plasmid extraction kit, purchased from OMEGA, cat # s: d6943;
the biochemical reagents not specifically described in the following examples are conventional in the art, and are commercially available or formulated according to conventional methods in the art.
Example 1 preparation of CA7 α -HSDH mutant
1. Mutant gene synthesis
Original sequence of wild type CA7 α -HSDH gene sequence (see patent CN102827848A publication text) after codon optimization, and the nucleotide sequence is shown as SEQ ID NO. 4.
By comparing the difference and the homology of the wild type CA7 α -HSDH and the homologous enzyme protein in a multi-layer system from a primary structure to a high-level structure, the site influencing the enzymological properties is the 179 th amino acid of the wild type CA7 α -HSDH, the amino acid is lysine, and the corresponding nucleotide sequence is the 535-537 th codon.
The codon 535-537 of the wild-type CA7 α -HSDH gene sequence was changed from AAA to ATG, and the original lysine was replaced by methionine to obtain the CA7 α -HSDH mutant named CA7 α -HSDH K179M mutant, the nucleotide sequence of which is shown as SEQ ID NO. 3 and the amino acid sequence of which is shown as SEQ ID NO. 2.
2. Construction of vectors
2.1K179M was constructed by overlapping PCR with the following cloning sites: BamHI and NotI, the primers used are as follows:
the method for obtaining K179M mutant gene comprises the steps of obtaining mutant gene by overlapping PCR method, and obtaining PCR template, wherein the PCR template is CA7 α -HSDH plasmid (pGEX-6p-1/CA 7 α -HSDH) after codon optimization
The first PCR reaction system and conditions:
PCR reaction system 1:
PCR reaction System 2:
and (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 5 min; 40 cycles, including: denaturation at 98 ℃ for 10 seconds, annealing at 60 ℃ for 15 seconds, and extension at 72 ℃ for 15 seconds; after the reaction was complete, the extension was carried out at 72 ℃ for 10 minutes.
The second PCR reaction system and conditions:
and (3) PCR reaction system:
and (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 5 min; 5 cycles, including: denaturation at 98 ℃ for 10 seconds, annealing at 60 ℃ for 15 seconds, and extension at 72 ℃ for 15 seconds. Here, overlapping PCR is involved, without the step of extension for 10 min.
Third PCR system and conditions: to the second reaction system, 1.5. mu.L of the forward primer and 1. mu.L of the reverse primer were added. The PCR reaction was performed under the following conditions.
And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 5 min; 3 cycles, including: denaturation at 98 ℃ for 10 seconds, annealing at 60 ℃ for 15 seconds, and extension at 72 ℃ for 15 seconds; after the reaction was complete, the extension was carried out at 72 ℃ for 10 minutes.
Agarose gel electrophoresis was used to detect the overlapping PCR result, and as shown in FIG. 2, the K179M mutant gene was obtained.
2.2 cleavage and ligation reactions
The K179M mutant gene obtained by the overlap PCR and pGEX-6p-2 construct were double-digested with BamHI and NotI restriction enzymes, respectively, and then ligated according to the following system and conditions.
A connection system:
the reaction conditions are 16 ℃ for overnight ligation, and a ligation product pGEX-6p-2/CA 7 α -HSDH K179M is obtained.
2.3 ligation products transformed E.coli DH5 α competent cells
1) Coli DH5 α was thawed on ice.
2) The ligation system obtained in step 2.2 was added to the thawed e.coli DH5 α competence and placed on ice for 30 minutes.
3) Heat treatment at 42 deg.C for 90 s.
4) Ice for 2 minutes.
5) LB medium 600. mu.L was added, the temperature of the shaker was 37 ℃ and the shaking speed of the shaker was 150rpm for 45 minutes.
6) Draw 200. mu.L of coated Amp+Resistant LB plate medium.
7) The culture was carried out overnight at 37 ℃.
2.4 Single colony amplification culture
Picking single colony and inoculating Amp+In LB medium, the temperature of the shaker was 37 ℃ and the shaking speed of the shaker was 220 rpm. OD600When the concentration is approximately equal to 1.0, the bacteria are obtained for plasmid extraction by centrifugation at 8000rpm for 5 minutes.
2.5 extraction of plasmids
Plasmids were extracted according to the instructions of the OMEGAPlasmid Mini Kit I (cat # D6943).
2.6 double restriction enzyme identification
A double enzyme digestion identification system:
reaction conditions are as follows: the enzyme was cleaved at 37 ℃ for 1.5 hours.
The results of the double digestion are shown in FIG. 3.
2.7 sequencing confirmation
And (3) selecting the recombinant plasmid with correct double enzyme digestion identification, sending the recombinant plasmid to TAKARA (China, Dalian) company for sequencing, and taking the recombinant plasmid with the correct sequencing result as an expression vector of the K179M mutant.
3. GST fusion heterologous expression of enzyme proteins
(1) Coli BL21 cells transformed with the plasmid
a. BL21 competent cells were removed at-80 ℃ and placed on ice.
b. Purified pGEX-6p-2/CA 7 α -HSDH K179M expression vector was added in 2. mu.L and left on ice for 30 minutes.
c.42 ℃ for 90 seconds.
d. The mixture was left on ice for 2 minutes.
e. Resuscitate, add 600. mu. LLB medium, 37 ℃, 150rpm, 45 minutes.
f. Aspirate 200. mu.L of Medium and spread on Amp+LB plate medium.
g.37 ℃ overnight.
(2) Protein expression and purification
a. The strain was inoculated into a sterile LB medium with a final ampicillin concentration of 50. mu.g/mL, cultured at 37 ℃ and 180 rpm.
b. When OD 600. apprxeq.0.8, IPTG was added to a final concentration of 0.2mM and induced overnight at 16 ℃ (12 h). At a speed of 8000rpm,
the cells were collected for 5 minutes.
c. Resuspend the thallus according to the proportion of adding 30mL lysine buffer into 1L culture system, and break the thallus by ultrasound until the thallus is clear. 12000rpm, 20 minutes. And taking the supernatant.
d. The supernatant was bound to Glutathione Sepharose 4B using 5mL of the filler per liter of the culture system and binding at 4 ℃ for 2 hours. The suspension was gently inverted vertically.
e. After the completion of the binding, the filler was precipitated at 500rpm for 5 minutes. The packing was washed 3-5 column volumes with 4 ℃ pre-cooled PBS. Removing the hybrid protein.
f. Adding PreScission Protease enzyme digestion buffer solution and adding PreScission Protease enzyme.
g.4 ℃ overnight. After the enzyme digestion is finished, the supernatant is discharged from the chromatographic column.
h. And performing SDS-PAGE on the obtained sample to identify the molecular weight and purity of the sample, and testing the concentration of the purified protein by using a BCA kit.
The results are shown in FIG. 4, and the SDS-PAGE result shows that the K179M mutant is successfully expressed in a soluble way, and the protein has a single band after one-step affinity chromatography.
Example 2 detection of enzyme Activity of K179M mutant
And (3) enzyme activity determination:
to the cuvette were added 1958uL of 50mM Tris-HCl (pH 8.0), 20uL of 50mM NADP at room temperature+The solution was mixed with 2uL of the K179M enzyme protein solution obtained in example 1, and then zeroed, and finally 20uL of 50mM TCDCA solution was added, and mixing and timing were started. The change in light absorption at 340nm was read once per minute.
Meanwhile, the enzyme activity of CA7 α -HSDH was measured in the same manner as described above.
The results show that the mutant K179M of the invention catalyzes the epimerization of the hydroxyl at position 7 of TCDCA, leading it to generate tauro-7-ketolithocholic acid (T7K-LCA), which is an intermediate of TUDCA, the specific activities of the K179M enzyme protein of the invention and Clostridium sarmentosum 7 α -HSDH enzyme protein to TCDCA are shown in Table 1.
Specific Activity (U/mg) definition: under the above reaction conditions, the enzyme activity unit is contained per mg of protein.
The results are shown in Table 1, where the CA7 α -HSDH K179M mutant is in NADP+As a coenzyme, the specific activity of the catalytic conversion TCDCA is 2.9 times that of the wild-type CA7 α -HSDH.
TABLE 1 comparison of enzyme specific Activity of K179M mutant with wild-type CA7 α -HSDH for catalysis of TCDCA
Example 5K 179M mutant thermostability Studies
The enzyme sample (without glycerol) was left in a metal bath at 50 ℃ for 20 minutes, and the residual enzyme activity was measured according to the method for measuring the enzyme activity in example 2. Each sample was subjected to not less than 3 replicates.
The result is shown in Table 2, after heat treatment for 20 minutes, the activity of wild CA7 α -HSDH enzyme is rapidly reduced, only 32.33% of the enzyme activity is retained, and K179M still retains 45.09% of the enzyme activity, which shows that compared with the wild CA7 α -HSDHK179M, the wild CA7 α -HSDH enzyme has better heat stability and is more suitable for industrial application.
TABLE 2 comparison of the thermal stability of wild-type CA7 α -HSDH with 7 α -HSDHJ-1-1
Sequence listing
<110> university of Chongqing
<120> Clostridium sardinieri 7 α -hydroxysteroid dehydrogenase mutant K179M
<130>P1730479CN-CN-CQD-CQ
<141>2017-11-14
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Met Leu Ala Leu Gly Gly Leu Val Ala Ile Val Thr Ser Ser Thr Ala
1 5 10 15
Gly Ile Gly Ala Ala Ser Ala Gly Ala Leu Ala Leu Gly Gly Ala Leu
20 25 30
Val Thr Leu Ala Ala Ala Ser Gly Gly Leu Ala Ala Gly Val Ile Ala
35 40 45
Ala Ile Leu Leu Gly Gly Gly Val Ala Leu Pro Val Thr Pro Ala Ala
50 55 60
Ala Gly Gly Gly Thr Thr Thr Ser Met Val Gly Leu Val Ala Gly Ala
65 70 75 80
Gly Gly Ala Ile Ala Ile Leu Val Ala Ala Thr Gly Gly Thr Ala Val
85 90 95
Ala Leu Ala Leu Ala Leu Thr Ala Gly Ala Thr Ala Gly Pro Pro Ala
100 105 110
Ile Leu Leu Ala Ala Val Gly Ser Val Thr Leu Pro Ala Leu Ala Ala
115 120 125
Ile Pro His Met Gly Leu Val Gly Gly Gly Ser Ile Val Ala Ile Ser
130 135 140
Thr Ile Gly Ser Val Val Pro Ala Ile Ser Ala Ile Ala Thr Cys Val
145 150 155 160
Ser Leu Ser Ala Ile Ala Ser Leu Thr Gly Ala Ile Ala Leu Gly Thr
165 170 175
Ala Ala Leu Ala Ile Ala Cys Ala Ala Val Leu Pro Gly Leu Ile Gly
180 185 190
Thr Ala Ala Ala Leu Gly Ala Met Thr Ala Gly Pro Ala Ala Ser Pro
195 200 205
Leu Gly His Val Pro Leu Ala Ala Val Gly Ala Pro Gly Ala Ile Ala
210 215 220
Ala Ala Val Leu Thr Thr Ala Ser Ala Ala Ser Gly Thr Val Thr Gly
225 230 235 240
Met Ile His Gly Val Ala Gly Gly Pro Ala Leu Gly Thr Pro Gly Thr
245 250 255
Ser Gly Thr Cys Pro Ala
260
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Met Leu Ala Leu Gly Gly Leu Val Ala Ile Val Thr Ser Ser Thr Ala
1 5 10 15
Gly Ile Gly Ala Ala Ser Ala Gly Ala Leu Ala Leu Gly Gly Ala Leu
20 25 30
Val Thr Leu Ala Ala Ala Ser Gly Gly Leu Ala Ala Gly Val Ile Ala
35 40 45
Ala Ile Leu Leu Gly Gly Gly Val Ala Leu Pro Val Thr Pro Ala Ala
50 55 60
Ala Gly Gly Gly Thr Thr Thr Ser Met Val Gly Leu Val Ala Gly Ala
65 70 75 80
Gly Gly Ala Ile Ala Ile Leu Val Ala Ala Thr Gly Gly Thr Ala Val
85 90 95
Ala Leu Ala Leu Ala Leu Thr Ala Gly Ala Thr Ala Gly Pro Pro Ala
100 105 110
Ile Leu Leu Ala Ala Val Gly Ser Val Thr Leu Pro Ala Leu Ala Ala
115 120 125
Ile Pro His Met Gly Leu Val Gly Gly Gly Ser Ile Val Ala Ile Ser
130 135 140
Thr Ile Gly Ser Val Val Pro Ala Ile Ser Ala Ile Ala Thr Cys Val
145 150 155 160
Ser Leu Ser Ala Ile Ala Ser Leu Thr Gly Ala Ile Ala Leu Gly Thr
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Ala Ala Met Ala Ile Ala Cys Ala Ala Val Leu Pro Gly Leu Ile Gly
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Thr Ala Ala Ala Leu Gly Ala Met Thr Ala Gly Pro Ala Ala Ser Pro
195 200 205
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210 215 220
Ala Ala Val Leu Thr Thr Ala Ser Ala Ala Ser Gly Thr Val Thr Gly
225 230 235 240
Met Ile His Gly Val Ala Gly Gly Pro Ala Leu Gly Thr Pro Gly Thr
245 250 255
Ser Gly Thr Cys Pro Ala
260
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atgaaacgcc tggaaggcaa agtggcaatt gtgaccagct ctactcgtgg cattggccgt 60
gcatctgcag aagcactggc aaaagaaggt gctctggtgt atctggcagc acgtagcgag 120
gaactggcta atgaagttat tgcagatatt aaaaaacagg gtggcgtggc taaatttgtt 180
tactttaatg ctcgtgaaga agaaacttac actagcatgg tggaaaaagt tgctgaagct 240
gaaggccgca ttgatattct ggttaataac tacggtggca ccaatgttaa tctggataaa 300
aacctgactg ctggcgatac cgatgaattc tttcgcattc tgaaagataa cgttcagagc 360
gtgtacctgc cggcaaaagc tgctattccg catatggaaa aagtgggcgg tggcagcatt 420
gttaatatca gcactattgg cagcgttgtt ccggatatta gccgcattgc ttactgcgtg 480
agcaaaagcg ctattaactc tctgactcag aacattgcac tgcagtatgc acgcatgaat 540
atccgctgca atgcagtgct gccgggtctg attggcactc gcgcagcact ggaaaatatg 600
actgatgaat ttcgcgacag cttcctgggc catgttccgc tgaatcgcgt gggccgcccg 660
gaagatattg caaatgcagt tctgtactat gcctctgatg atagcggtta tgtgaccggc 720
atgattcatg aagttgcagg cggttttgca ctgggcactc cgcagtatag cgaatactgc 780
ccgcgctaa 789
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atgaaacgcc tggaaggcaa agtggcaatt gtgaccagct ctactcgtgg cattggccgt 60
gcatctgcag aagcactggc aaaagaaggt gctctggtgt atctggcagc acgtagcgag 120
gaactggcta atgaagttat tgcagatatt aaaaaacagg gtggcgtggc taaatttgtt 180
tactttaatg ctcgtgaaga agaaacttac actagcatgg tggaaaaagt tgctgaagct 240
gaaggccgca ttgatattct ggttaataac tacggtggca ccaatgttaa tctggataaa 300
aacctgactg ctggcgatac cgatgaattc tttcgcattc tgaaagataa cgttcagagc 360
gtgtacctgc cggcaaaagc tgctattccg catatggaaa aagtgggcgg tggcagcatt 420
gttaatatca gcactattgg cagcgttgtt ccggatatta gccgcattgc ttactgcgtg 480
agcaaaagcg ctattaactc tctgactcag aacattgcac tgcagtatgc acgcaaaaat 540
atccgctgca atgcagtgct gccgggtctg attggcactc gcgcagcact ggaaaatatg 600
actgatgaat ttcgcgacag cttcctgggc catgttccgc tgaatcgcgt gggccgcccg 660
gaagatattg caaatgcagt tctgtactat gcctctgatg atagcggtta tgtgaccggc 720
atgattcatg aagttgcagg cggttttgca ctgggcactc cgcagtatag cgaatactgc 780
ccgcgctaa 789
<210>5
<211>26
<212>DNA
<213>Artificial Sequence
<400>5
cgggatccat gaaacgcctg gaaggc 26
<210>6
<211>31
<212>DNA
<213>Artificial Sequence
<400>6
cttttgcggc cgcttagcgc gggcagtatt c 31
<210>7
<211>27
<212>DNA
<213>Artificial Sequence
<400>7
tgcagtatgc acgcatgaat atccgct 27
<210>8
<211>28
<212>DNA
<213>Artificial Sequence
<400>8
attgcagcgg atattcatgc gtgcatac 28
Claims (11)
1. A method for improving the enzyme activity and stability of wild clostridium sardinifolium 7 α -hydroxysteroid dehydrogenase is characterized in that
The 179 th lysine of the wild type clostridium sardiniformis 7 α -hydroxysteroid dehydrogenase is mutated into methionine, and the amino acid sequence of the wild type clostridium sardiniformis 7 α -hydroxysteroid dehydrogenase is shown as SEQ ID NO: 1.
2. The method according to claim 1, wherein the mutant clostridium sarDilum 7 α -hydroxysteroid dehydrogenase enzyme obtained after the mutation has significantly improved enzyme activity and stability compared with the wild type clostridium sardiniformis 7 α -hydroxysteroid dehydrogenase.
3. A Clostridium sardinieri 7 α -hydroxysteroid dehydrogenase mutant K179M has an amino acid sequence as shown in
SEQ ID NO: 2, respectively.
4. A gene encoding clostridium sardinieri 7 α -hydroxysteroid dehydrogenase mutant K179M according to claim 3.
5. The gene of claim 4, wherein the nucleotide sequence is as set forth in SEQ ID NO: 3, respectively.
6. An expression cassette comprising the gene of claim 4.
7. A vector comprising the gene of claim 4 and/or the expression cassette of claim 6.
8. A recombinant cell comprising the gene of claim 4, and/or the expression cassette of claim 6, and/or the vector of claim 7.
9. A catalyst, characterized in that the active ingredient thereof comprises Clostridium sarmentosum 7 α -hydroxysteroid dehydrogenase mutant K179M according to claim 3.
10. The catalyst of claim 9, further comprising an additional agent that increases the efficiency or stability of the enzyme when used simultaneously with the clostridium sardinieri 7 α -hydroxysteroid dehydrogenase mutant K179M of claim 3.
11. A method for achieving carbonyl asymmetric reduction of a chemical substance, characterized in that clostridium sardinieri 7 α -hydroxysteroid dehydrogenase mutant K179M according to claim 3 and/or a catalyst according to claim 9 or 10 is/are used to catalyze a reaction with a reaction substrate under the conditions of 20-50 ° C, pH 6.0.0-11.0.
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| CN109182284B (en) * | 2018-09-28 | 2020-12-04 | 湖南福来格生物技术有限公司 | 7 beta-hydroxysteroid dehydrogenase mutant, coding sequence, recombinant expression vector, genetic engineering bacteria and application |
| CN113462665B (en) * | 2021-06-30 | 2023-03-10 | 中山百灵生物技术股份有限公司 | 7 alpha-HSDH enzyme mutant and coding gene and application thereof |
| CN113604446B (en) * | 2021-08-16 | 2023-04-07 | 重庆大学 | Mutant R16Q of 7 alpha-hydroxysteroid dehydrogenase St-2-2 |
| CN113430183B (en) * | 2021-08-16 | 2022-08-23 | 重庆大学 | Mutants of 7 alpha-hydroxysteroid dehydrogenase St-2-2T15G, T15S and T15A |
| CN114250205B (en) * | 2021-12-28 | 2022-11-11 | 宋建芳 | 7 alpha-hydroxysteroid dehydrogenase mutant with high thermal stability and application thereof |
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| EP3174977B1 (en) * | 2014-07-29 | 2019-04-10 | Pharmazell GmbH | 7beta-hydroxysteroid dehydrogenase mutants and method for producing ursodeoxycholic acid |
| KR102471639B1 (en) * | 2014-08-12 | 2022-11-25 | 파마젤 게엠베하 | 3-alpha-hydroxysteroid dehydrogenase mutants and process for the preparation of ursodeoxycholic acid |
| CN106282138B (en) * | 2016-09-22 | 2019-10-08 | 重庆大学 | Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase mutant T145S |
| CN106434582B (en) * | 2016-09-22 | 2019-10-08 | 重庆大学 | Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase mutant R194A |
| CN106701708B (en) * | 2017-01-05 | 2019-07-26 | 重庆大学 | 7alpha-Hydroxysteroid dehydrogenase gene Y1-a-1 |
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