CN106434582B - Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase mutant R194A - Google Patents
Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase mutant R194A Download PDFInfo
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Abstract
The present invention relates to hydroxysteroid dehydrogenases, more particularly to a kind of Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase mutant R194A, its amino acid sequence is that the 194th amino acids of the 7alpha-Hydroxysteroid dehydrogenase that amino acid sequence is SEQ ID NO:1 become Ala gained from Arg as shown in SEQ ID NO:2.The mutant is to substrate NADP+Catalytic efficiency with TCDCA is 3.34 times of wild type.R194A mutant of the invention has huge application potential in the industrial processes that bioconversion CDCA/TCDCA obtains UDCA/TUDCA.
Description
Technical field
The present invention relates to hydroxysteroid dehydrogenases, and in particular to a kind of Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase
Mutant R194A.
Background technique
The asymmetric reduction of carbonyl is always one of the hot spot for chemically reacting research.Although current chemical method has been achieved with
Certain achievement, but chemical method often there is catalyst type and Limited Number, stereoselectivity be high, auxiliary reagent
It is expensive and the disadvantages of be not easily recycled.And enzymatic reaction not only has high efficiency, chemo-selective, regioselectivity also and has height
Stereoselectivity.The enzymatic that hydroxysteroid dehydrogenase (Hydroxysteroid dehydrogenase, HSDH) mediates is anti-
There should be the substrate specificity of relatively stringent stereoselectivity and " not stringent ".For example, early in the early 1980s
Scientist just has begun 7 α-, 7 β-HSDH the joint epimerism conversion chenodeoxycholic acid for attempting to generate using microorganism
(Chenodeoxycholic acid, CDCA) synthesizes ursodesoxycholic acid (Ursodesoxycholic acid, UDCA), conversion
Process is as shown in Figure 1.And resolvase can be with catalyzed combination state bile acid --- Taurochenodeoxycholic Acid
(Taurochenodeoxycholic acid, TCDCA) is converted into Tauro ursodesoxy cholic acid (Tauroursodeoxycholic
Acid, TUDCA).
The substrate of HSDH is not solely restricted to steroid compound, and document report HSDH can also be catalyzed alkyl and replace monocycle
The carbonyl asymmetric reduction of the substances such as ketone, two cyclic ketones classes.Outstanding catalysis quality possessed by HSDH determines that it turns in biology
Change field has larger application potential.However, the higher HSDH variant of activity is that it is further applied in field of bioconversion
Basic guarantee.In recent years, scientific research personnel has gradually recognized that 7 α-, 7 β-HSDH are huge possessed by the field of bioconversion and has answered
Use potentiality.Currently, the 7 α-HSDH that the function of registering in GenBank has been acknowledged share 5, they are respectively from
Bacteroides fragilis、Clostridium scindens、Clostridium sordellii、Clostridium
Absonum and Escherichia coli；7 from Clostridium absonum and Collinsellaaerofaciens
β-HSDH gene has also successfully been cloned.It is difficult that the bioconversion system of above-mentioned dual-enzyme coupling building not only overcomes coenzyme circulation
Topic also achieves oxidation, reduction " cooking-pot type " carries out the hydroxyl epimerism in specified chemical region.
Enzyme activity it is lower be to limit one of principal element of its industrial application, almost all of native enzyme require by
It can be only achieved the requirement of industrial application after transformation.Wherein, 7 α-HSDH of Clostridium sardiniense (Clostridium absonum7 α-
7 α-HSDH of HSDH, CA) there is long research history and wide application prospect.In addition the crystal structure of the enzyme has solved
Analysis is of great significance so obtaining the mutant that catalytic efficiency significantly improves in such a way that rationality is transformed.
Summary of the invention
To meet demand existing for above-mentioned field, the present invention provides a kind of Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase
Mutant has higher catalytic efficiency, can be used for the bioconversion of a variety of substrates, has in the industrial production huge using valence
Value.
The claimed technical solution of the present invention is as follows:
A kind of Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase mutant, which is characterized in that its amino acid sequence such as SEQ
Shown in ID NO:2, be amino acid sequence be SEQ ID NO:1 7alpha-Hydroxysteroid dehydrogenase the 194th amino acids by
Arg becomes obtained by Ala.
Encode the gene of above-mentioned Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase mutant.
The gene, which is characterized in that its nucleotide sequence is as shown in SEQ ID NO:3.
A kind of expression cassette, which is characterized in that include said gene.
A kind of carrier, which is characterized in that include said gene or expression cassette.
A kind of recombinant cell, which is characterized in that include said gene or expression cassette or carrier.
A method of preparing above-mentioned Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase mutant, which is characterized in that in energy
Above-mentioned recombinant cell is cultivated under conditions of enough successful inducible protein expression and 7 alpha-hydroxysteroid of isolated Clostridium sardiniense is de-
Hydrogen enzyme mutant.
A kind of catalyst, which is characterized in that its effective component includes above-mentioned Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase
Mutant.
The catalyst, which is characterized in that further include and above-mentioned Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase mutant
It can be improved enzymatic efficiency when using simultaneously or increase other reagents of enzyme stability.
A method of realizing the carbonyl asymmetric reduction of chemical substance, which is characterized in that use above-mentioned Clostridium sardiniense 7
Alpha-hydroxysteroid dehydrogenase mutant or any above-mentioned catalyst and reaction substrate are under the conditions of 15-37 DEG C, pH 6.0-11.0
Carry out catalysis reaction.
The present invention compares 7 α-HSDH of wild type CA and same from primary structure to higher structure multi-angle multilayer system first
The similarities and differences of source zymoprotein, it is determined that the site for influencing 7 α-HSDH zymologic property of CA is the 194th amino acids --- arginine.So
Being replaced afterwards by codon becomes alanine for arginine, and obtains 7 α-HSDH R194A of CA using full genome synthetic technology
The gene of mutant.Finally by the GST fusion expression vector and quiding gene engineering bacteria E.coli of building mutant gene
Inducing expression in BL21 obtains 7 α-HSDH R194A mutant zymoprotein of CA.By measuring the enzyme under different concentration of substrate
Promote initial velocity of reaction, the enzyme kinetic analysis of 7 α-HSDH wild type and R194A mutant of CA has been calculated using Michaelis-Menten equation
Parameter.The result shows that R194A mutant is to substrate NADP+Catalytic efficiency with TCDCA is 3.34 times of wild type.The present invention
R194A mutant bioconversion CDCA/TCDCA obtain UDCA/TUDCA industrial processes in have huge application
Potentiality.
Carrier provided by the invention, can be cloning vector, include 7 α-HSDH R194A mutant gene of CA and plasmid
Other elements needed for duplication；It is also possible to expression vector, comprising 7 α-HSDH R194A mutant gene of CA and egg can be made
Other elements of white successful expression.In some embodiments, the expression vector is to insert 7 α-HSDH R194A of CA mutation
The pGEX-6p-1 carrier of body gene.
Recombinant cell provided by the invention can be the recombinant cell comprising cloning vector, such as E.coli DH5 α, lead to
Crossing culture cell replicates intracellular 7 α-HSDH R194A mutant gene of CA；It is also possible to thin comprising expression vector
Born of the same parents cultivate cell under suitable condition, for example, suitable IPTG is added, 16 DEG C of 7 α-HSDH R194A mutant of induction CA
The expression of albumen.
The present invention provides a kind of catalyst, and effective component includes 7 α-HSDH R194A mutant of CA.The catalyst
Can also be used simultaneously with other suitable catalyst, thus improve enzymatic efficiency or in same reaction system first it is laggard
Two kinds of catalysis reactions of row.
7 α-HSDH R194A mutant of CA of the invention, can be catalyzed carbonyl under the conditions of 15-37 DEG C, pH 6.0-11.0
Base asymmetric reduction reaction.
Detailed description of the invention
Fig. 1 .7 α-, 7 β-HSDH joint conversion CDCA prepare UDCA.
The agarose gel electrophoresis results of Fig. 2 .Overlapping PCR (over-lap PCR) building R194A mutant gene；
Wherein, M is DNA Marker；1 and 2 be the overlapping PCR product of R194A.
Fig. 3 plasmid double digestion qualification result；
Wherein, M is DNA Marker；1 and 2 be the recombinant plasmid after digestion.
The SDS-PAGE electrophoresis of Fig. 4 Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase mutant R194A；
Wherein, M is protein molecular weight standard (Marker)；1 is R194A mutant protein.
The standard curve of Fig. 5 .NADPH；
Wherein, abscissa is the concentration of NADPH solution, and ordinate is light of the NADPH solution of each concentration at 340nm
Absorption value.
Specific embodiment
The present invention is further described combined with specific embodiments below, it is to be understood that, following embodiments are only as explanation
And explanation, it does not limit the scope of the invention in any way.
Main agents:
PGEX-6p-1 is known carrier, this laboratory saves；
E.coli BL21 cell is the preservation of this laboratory；
Lysis buffer is prepared and is obtained, and the PBS of 10mM pH7.3 contains PMSF 0.1mM, leupeptin Leupeptin
0.5mg/mL；
Glutathione Sepharose 4B is bought from GE Healthcare, article No.: 10223836；
PreScission Protease is bought from GenScript company, article No.: Z02799-100；
BCA kit is bought from Beyotime company, article No.: P0006；
TCDCA is bought from lark prestige scientific & technical corporation, article No.: 330776；
Plasmid extraction kit is purchased from OMEGA company, article No.: D6943；
Not specified biological chemical reagent is this field conventional reagent in following embodiment, commercially available or press
It prepares and obtains according to conventional method in that art.
The preparation of 1. Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase mutant of embodiment
1. mutant gene synthesizes
Original series: the 7 α-HSDH gene order of wild type CA after codon optimization is (referring to patent CN102827848A
Publication), nucleotide sequence is as shown in SEQ ID NO:4.
Compare 7 α-HSDH of wild type CA and homology enzyme egg by the multi-angle multilayer system from primary structure to higher structure
The white similarities and differences, it is determined that the site for influencing zymologic property is the 194th amino acids of 7 α-HSDH of wild type CA, the amino acid
For arginine, corresponding nucleotides sequence is classified as 580-582 bit codon.
Codon at the 580-582 of 7 α-HSDH gene order of wild type CA is changed to GCC by CGC, it will be original
Arginine is substituted for alanine, obtains 7 α-HSDH mutant of CA, is named as 7 α-HSDH R194A mutant of CA, nucleotide
Sequence is as shown in SEQ ID NO:3, and amino acid sequence is as shown in SEQ ID NO:2.
2. the building of carrier
2.1 R194A mutant genes pass through overlapping PCR method construct, clone restriction enzyme site are as follows: BamHI and
NotI, the primer are as follows:
Upstream primer are as follows: 5'-CGGGATCCATGAAACGCCTGGAAGGC-3'；
Downstream primer are as follows: 5'-CTTTTGCGGCCGCTTAGCGCGGGCAGTATTC-3'.
Upstream mutant primer are as follows: 5'-CTGATTGGCACTGCCGCAGCACTGGAA-3'；
Downstream mutant primer are as follows: 5'-TTCCAGTGCTGCGGCAGTGCCAATCAG-3'.
The PCR method of Arg194Ala mutant gene: mutation base is obtained using over-lap PCR (overlapping PCR) method
Cause；DNA profiling used in PCR: the 7 α-HSDH plasmid of CA (7 α-HSDH of pGEX-6p-1/CA) after codon optimization, is to pass through
BamHI and NotI double enzyme digestion reaction and connection reaction are constructed and are obtained.
First time PCR system and condition:
System 1:
System 2:
Condition:
Second of PCR system and condition:
System:
Condition:
Third time PCR system and condition: upstream primer, each 1 μ L of downstream primer are added into second of reaction system.Using
The following conditions complete PCR reaction.
Condition:
Agarose gel electrophoresis detects overlapping PCR as a result, as shown in Fig. 2, having obtained R194A mutant base
Cause.
2.2 digestions and connection reaction
Using BamHI and NotI restriction enzyme respectively to over-lap PCR obtain R194A mutant gene and
PGEX-6p-1 carrier carries out double digestion, is then attached according to following system and condition.
Linked system:
3 μ L of R194A mutant gene
pGEX-6p-1 3μL
6 μ L of DNA ligase mixture
Reaction condition: 16 DEG C of connection 1.5h obtain 7 α-HSDH R194A of connection product pGEX-6p-1/CA.
2.3 connection product Transformed E .coli DH5 α competent cells
1) competent cell E.coli DH5 α placement is melted on ice.
2) the resulting linked system of step 2.2 is added in the E.coli DH5 α competence melted, on ice 30min.
3) 42 DEG C of heat treatment 90s.
4) 2min on ice.
5) 600 μ L of LB culture medium is added, 37 DEG C of shaking table temperature, shaking table shakes fast 150rpm, time 45min.
6) 200 μ L are drawn and is coated with Amp+Resistance LB plating medium.
7) it is incubated overnight for 37 DEG C.
2.4 picking single colonies expand culture
Picking single colonie is inoculated with Amp+In LB culture medium, 37 DEG C of shaking table temperature, shaking table shakes fast 220rpm.OD600≈1.0
When, 8000rpm is centrifuged 5min and obtains thallus for plasmid extraction.
2.5 extract plasmid
It is operated by OMEGA Plasmid Mini Kit I (article No.: D6943) specification.
The identification of 2.6 double digestions
System:
Condition: 37 DEG C of digestion 3h.
Double digestion result is as shown in Figure 3.
2.7 sequencing confirmations
It chooses double digestion and identifies that correct recombinant plasmid send the sequencing of TAKARA (China, Dalian) company, sequencing result is correct
Expression vector of the recombinant plasmid as R194A mutant.
3. the GST of zymoprotein merges heterogenous expression
(1) plasmid Transformed E .coli BL21 cell
BL21 competent cell E.coli is taken out in a.-80 DEG C to place on ice.
B. 7 α-HSDH R194A expression vector of pGEX-6p-1/CA, 2 μ L after purification is added, places 30min on ice.
C.42 DEG C heat treatment 90s.
D. 2min is placed on ice.
E. it recovers, 600 μ L LB culture mediums of addition, 37 DEG C, 150rpm, 45min.
F. 200 μ L culture mediums are drawn and are coated on Amp+In LB plating medium.
G.37 DEG C overnight incubation.
(2) protein expression and purifying
A. strain is inoculated in sterile LB medium, the final concentration of 50 μ g/mL of ampicillin, 37 DEG C, 180rpm is trained
It supports.
B. when OD600 ≈ 0.8, the IPTG of final concentration of 0.2mM, 16 DEG C of overnight inductions (12h) are added.8000rpm,
5min collects thallus.
C. add the ratio of 30mL Lysis buffer that thallus, carrying out ultrasonic bacteria breaking to clarification is resuspended in 1L cultivating system.
12000rpm, 20min.Take supernatant.
D. for supernatant in conjunction with Glutathione Sepharose 4B, the ratio that filler uses is that every liter of cultivating system uses
5mL filler, 4 DEG C combine 2h.Gently vertical reverse suspension.
E. after combining, 500rpm, 5min precipitation filling.Filler rinses 3-5 column volume with 4 DEG C of pre-cooling PBS.Removal
Foreign protein.
F. PreScission Protease enzyme cutting buffering liquid is added, PreScission Protease enzyme is added.
G.4 DEG C digestion is stayed overnight.After digestion, supernatant is released from chromatographic column.
H. gained sample is subjected to SDS-PAGE, identifies its molecular size range and purity, BCA kit test purifying protein
Concentration.
As a result as shown in figure 4, SDS-PAGE testing result shows the success of R194A mutant solubility expression, and through one
The band of albumen is single after step affinity chromatography.
Embodiment 2.R194A mutant enzyme kinetic analysis parametric measurement
The production of 1.NADPH standard curve
Prepare 0.005 respectively using reaction buffer (50mM Tris-HCl, 200mM NaCl, pH 8.5), 0.01,
0.02, the NADPH solution of 0.03,0.04,0.06,0.08,0.1,0.2,0.3,0.4 and 0.5mM.It is returned to zero with above-mentioned blank solvent
The NADPH solution of each concentration is separately added into 2mL cuvette afterwards, measures absorbance value OD in 340nm340.It is molten with NADPH
The concentration of liquid is abscissa, and corresponding absorbance value is ordinate, draws standard curve.
As a result as shown in figure 5, the calibration curve equation formula obtained is y=2.5771x+0.0045, R2=0.9995.2. enzyme
Measurement living
958 μ L reaction buffers (50mM Tris-HCl, 200mM NaCl, pH are sequentially added in 2mL cuvette
8.5), the NADP of 20 μ L 50mM+7 α-HSDH the R194A of CA that solution, 2 μ L embodiments 1 are prepared is mutated liquid solution
The substrate TCDCA of 20 μ L 50mM is added in (1.77mg/mL) after zeroing.When enzyme kinetic analysis data determination, the final concentration of coenzyme
Variation range be 0.1-10mM.
The condition of enzymatic reaction is: in 20 DEG C, variation of the light absorption in 1min is recorded at 340nm, and according to NADPH
Standard curve calculate product production quantity.Passed through by Michaelis-Menten equation (formula 1) equal sign two double (formula 2) reciprocal side-draw
Lineweaver-Burk double-reciprocal plot method obtains Km、kcat；And by comparing kcat/KmValue measures the catalytic efficiency of mutant
Variation.
The results are shown in Table 1, and 7 α-HSDH R194A mutant of CA is catalyzed NADP using TCDCA as substrate+It is converted into
NADPH efficiency is 3.34 times of 7 α-HSDH of wild type CA.
1. wild type CA of table, 7 α-HSDH and R194A mutant are catalyzed NADP+Enzyme kinetic analysis parameter
Claims (10)
1. a kind of Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase mutant, which is characterized in that its amino acid sequence such as SEQ ID
It is that the 194th amino acids of the 7alpha-Hydroxysteroid dehydrogenase that amino acid sequence is SEQ ID NO:1 are become by Arg shown in NO:2
For obtained by Ala.
2. encoding the gene of Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase mutant described in claim 1.
3. gene according to claim 2, which is characterized in that its nucleotide sequence is as shown in SEQ ID NO:3.
4. a kind of expression cassette, which is characterized in that include gene as claimed in claim 2.
5. a kind of carrier, which is characterized in that include gene as claimed in claim 2 or expression cassette as claimed in claim 4.
6. a kind of recombinant cell, which is characterized in that include gene as claimed in claim 2 or expression cassette as claimed in claim 4
Or carrier described in claim 5.
7. a kind of method for preparing Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase mutant described in claim 1, feature
It is, cultivates recombinant cell as claimed in claim 6 and isolated Sadinia under conditions of can succeed inducible protein expression
Island clostridium 7alpha-Hydroxysteroid dehydrogenase mutant.
8. a kind of catalyst, which is characterized in that its effective component includes 7 Alpha-hydroxy class of Clostridium sardiniense described in claim 1
Sterol dehydrogenase mutant.
9. catalyst according to claim 8, which is characterized in that further include and Clostridium sardiniense 7 described in claim 1
Alpha-hydroxysteroid dehydrogenase mutant can be improved enzymatic efficiency or increase other reagents of enzyme stability when using simultaneously.
10. a kind of method for the carbonyl asymmetric reduction for realizing chemical substance, which is characterized in that using described in claim 1
Catalyst described in Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase mutant or claim 8 or 9 and reaction substrate are in 15-
37 DEG C, carry out catalysis reaction under the conditions of pH 6.0-11.0.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107841489B (en) * | 2017-11-14 | 2020-02-18 | 重庆大学 | Clostridium sardinieri 7 α -hydroxysteroid dehydrogenase mutant K179M |
| CN108034643B (en) * | 2017-12-18 | 2021-06-15 | 重庆大学 | 7 alpha-hydroxysteroid dehydrogenase and coding gene and application thereof |
| CN110387360B (en) * | 2019-06-18 | 2021-12-28 | 华东理工大学 | Hydroxysteroid dehydrogenase and application thereof in synthesis of ursodeoxycholic acid precursor |
| CN113430183B (en) * | 2021-08-16 | 2022-08-23 | 重庆大学 | Mutants of 7 alpha-hydroxysteroid dehydrogenase St-2-2T15G, T15S and T15A |
| CN113604446B (en) * | 2021-08-16 | 2023-04-07 | 重庆大学 | Mutant R16Q of 7 alpha-hydroxysteroid dehydrogenase St-2-2 |
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|---|---|---|---|---|
| CN102827847A (en) * | 2012-07-25 | 2012-12-19 | 上海凯宝药业股份有限公司 | Codon-optimized 7 beta-hydroxy steroid dehydrogenase gene |
| CN102827848A (en) * | 2012-07-25 | 2012-12-19 | 上海凯宝药业股份有限公司 | 7alpha-hydroxyl steroid dehydrogenase gene optimized by codon |
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- 2016-09-22 CN CN201610841870.0A patent/CN106434582B/en active Active
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102827847A (en) * | 2012-07-25 | 2012-12-19 | 上海凯宝药业股份有限公司 | Codon-optimized 7 beta-hydroxy steroid dehydrogenase gene |
| CN102827848A (en) * | 2012-07-25 | 2012-12-19 | 上海凯宝药业股份有限公司 | 7alpha-hydroxyl steroid dehydrogenase gene optimized by codon |
Non-Patent Citations (1)
| Title |
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| The three-dimensional structure of Clostridium absonum 7α-hydroxysteroid dehydrogenase: new insights into the conserved arginines for NADP(H) recognition;Deshuai Lou et al;《Scientific Reports》;20160310;第5页第3-4段，表1，摘要、第1页第二段 * |
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