CN106282138A - Clostridium sardiniense 7 α hydroxysteroid dehydrogenase mutant T145S - Google Patents

Abstract

The present invention relates to hydroxysteroid dehydrogenase, it is specifically related to a kind of Clostridium sardiniense 7 α hydroxysteroid dehydrogenase mutant T145S, its aminoacid sequence as shown in SEQ ID NO:2, be aminoacid sequence be that the 145th amino acids of the 7 α hydroxysteroid dehydrogenases of SEQ ID NO:1 is become Ser gained from Thr.This mutant is to substrate TCDCA and NADP⁺Catalytic efficiency be 4.85 times of wild type, during bioconversion CDCA/TCDCA obtains UDCA/TUDCA, there is huge application potential.

Description

Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase mutant T145S

Technical field

The present invention relates to hydroxysteroid dehydrogenase, be specifically related to a kind of Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase Mutant T145S.

Background technology

The asymmetric reduction of carbonyl is always one of focus of chemical reaction research.Although chemical method has been achieved with at present Certain achievement, but chemical method often exists, and catalyst type and Limited Number, stereo selectivity be the highest, auxiliary reagent The shortcoming such as expensive and be not easily recycled.And enzymatic reaction not only has high efficiency, chemo-selective, regioselectivity and also has height Stereo selectivity.The enzymatic that hydroxysteroid dehydrogenase (Hydroxysteroid dehydrogenase, HSDH) mediates is anti- Should have the substrate specificity that relatively stringent stereo selectivity is strict with " no ".Such as, as far back as the early 1980s Scientist just have begun to attempt 7 α that utilize microorganism to produce-, 7 β-HSDH associating epimerisms convert chenodeoxycholic acid (Chenodeoxycholic acid, CDCA) synthesis 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 tauroursodeoxycholic 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 materials such as ketone, bicyclo-ketone.The outstanding catalysis quality that HSDH is had determines it and turns at biology Change field has bigger application potential.But, active higher HSDH variant is that it is applied further at field of bioconversion Basic guarantee.In recent years, scientific research personnel gradually recognized 7 α-, 7 β-HSDH field of bioconversion had huge should Use potentiality.At present, in GenBank registration function it has been acknowledged that 7 α-HSDH have 5, they are respectively from Bacteroides fragilis、Clostridium scindens、Clostridium sordellii、Clostridium Absonum and Escherichia coli；From Clostridium absonum and Collinsellaaerofaciens 7 β-HSDH gene is the most successfully cloned.The bioconversion system that above-mentioned dual-enzyme coupling builds not only overcomes coenzyme circulation difficulty Topic, also achieve oxidation, reduce " cooking-pot type " carry out the hydroxyl epimerism in specified chemical region.

The activity of enzyme is relatively low is one of principal element limiting its commercial Application, almost all of native enzyme be required for through The requirement of commercial Application is can be only achieved after transformation.Wherein, Clostridium sardiniense 7 α-HSDH (Clostridium absonum7 α- HSDH, CA 7 α-HSDH) there is long research history and wide application prospect.In addition the crystal structure of this enzyme has solved Analysis, so it is significant to obtain the mutant that catalytic efficiency significantly improves by the way of rationality is transformed.

Summary of the invention

For meeting the demand that above-mentioned field exists, the present invention provides a kind of Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase Mutant, has higher catalytic efficiency, can be used for the bioconversion of multiple substrate, has huge application valency in the industrial production Value.

The technical scheme that the present invention is claimed is as follows:

A kind of Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase mutant, it is characterised in that its aminoacid sequence such as SEQ Shown in ID NO:2, be aminoacid sequence be the 7alpha-Hydroxysteroid dehydrogenase of SEQ ID NO:1 the 145th amino acids by Thr becomes Ser gained.

Encode the gene of above-mentioned Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase mutant.

Described gene, it is characterised in that its nucleotide sequence is as shown in SEQ ID NO:3.

A kind of expression cassette, it is characterised in that comprise said gene.

A kind of carrier, it is characterised in that comprise said gene or expression cassette.

A kind of reconstitution cell, it is characterised in that comprise said gene or expression cassette or carrier.

A kind of method preparing above-mentioned Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase mutant, it is characterised in that at energy Enough success induced proteins cultivate above-mentioned reconstitution cell under conditions of expressing and isolated Clostridium sardiniense 7 alpha-hydroxysteroid takes off Hydrogen enzyme mutant.

A kind of catalyst, it is characterised in that its effective ingredient comprises above-mentioned Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase Mutant.Described catalyst, it is characterised in that also include and above-mentioned Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase mutant Enzyme catalysis efficiency can be improved simultaneously when using or increase other reagent of enzyme stability.

A kind of method of carbonyl asymmetric reduction realizing chemical substance, it is characterised in that use above-mentioned Clostridium sardiniense 7 Alpha-hydroxysteroid dehydrogenase mutant or arbitrary above-mentioned catalyst and reaction substrate 15-37 DEG C, under the conditions of pH 6.0-11.0 Carry out catalytic reaction.

First the present invention compares wild type CA 7 α-HSDH with same from primary structure to the many series of strata of higher structure multi-angle The similarities and differences of source pheron, it is determined that the site affecting CA 7 α-HSDH zymologic property is the 145th amino acids threonine.So Replaced by codon afterwards and threonine is become serine, and utilize full genome synthetic technology to obtain CA 7 α-HSDH T145S The gene of mutant.Finally by the GST fusion expression vector the quiding gene engineering bacteria E.coli that build mutant gene Abduction delivering in BL21, it is thus achieved that CA 7 α-HSDH T145S mutant zymoprotein.By measuring the enzyme under different concentration of substrate Promote initial velocity of reaction, utilize Michaelis-Menten equation to be calculated CA 7 α-HSDH wild type and the enzyme kinetic analysis of T145S mutant Parameter.Result shows, T145S mutant is to substrate TCDCA and NADP⁺Catalytic efficiency be 4.85 times of wild type, this sudden change Body has huge application potential during bioconversion CDCA/TCDCA obtains UDCA/TUDCA.

The carrier that the present invention provides, can be cloning vehicle, comprise CA 7 α-HSDH T145S mutant gene and plasmid Other element needed for duplication；Can also be expression vector, comprise CA 7 α-HSDH T145S mutant gene and egg can be made Other element of white successful expression.In certain embodiments, described expression vector is for inserting CA 7 α-HSDH T145S sudden change The pGEX-6p-1 carrier of body gene.

The reconstitution cell that the present invention provides, can be the reconstitution cell comprising cloning vehicle, such as E.coli DH5 α, logical Crossing cultivation cell makes intracellular CA 7 α-HSDH T145S mutant gene replicate；Can also be comprise expression vector thin Born of the same parents, cultivate cell under suitable condition, such as, add appropriate IPTG, 16 DEG C of induction CA 7 α-HSDH T145S mutants The expression of albumen.

The present invention provides a kind of catalyst, and its effective ingredient includes CA 7 α-HSDH T145S mutant.Described catalyst Can also use with other catalyst being suitable for simultaneously, thus improve enzyme catalysis efficiency or first the most laggard in same reaction system Two kinds of catalytic reactions of row.

The CA 7 α-HSDH T145S mutant of the present invention, 15-37 DEG C, can under the reaction condition of pH 6.0-11.0 Catalysis TCDCA C₇Alpha-hydroxy carbonyl asymmetric reduction reaction.Its optimal reactive temperature is 37 DEG C, and optimum pH is 10.5.

Accompanying drawing explanation

Fig. 1 .7 α-, 7 β-HSDH combine conversion CDCA prepare UDCA schematic diagram.

Fig. 2. the SDS-PAGE of Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase mutant T145S；

Wherein, M is protein molecular weight standard (Marker)；1 is T145S mutant protein.

The standard curve of Fig. 3 .NADPH；

Wherein, abscissa is the concentration of NADPH solution, and vertical coordinate is the NADPH solution of each concentration light at 340nm Absorption value.

Detailed description of the invention

The present invention is further described below in conjunction with specific embodiment, it is to be understood that, following embodiment is only used as explaining And explanation, limit the scope of the present invention never in any form.

Main agents:

PGEX-6p-1 is known carrier, and this laboratory preserves；

E.coli BL21 cell is that this laboratory preserves；

Lysis buffer, prepares and obtains, and the PBS of 10mM pH7.3 contains PMSF 0.1mM, leupeptin Leupeptin 0.5mg/mL；

Glutathione Sepharose 4B, buys from GE Healthcare, article No.: 10223836；

PreScission Protease, buys from GenScript company, article No.: Z02799-100；

BCA test kit, buys from Beyotime company, article No.: P0006；

TCDCA, buys from lark prestige scientific & technical corporation, article No.: 330776；

Biological chemical reagent not specified in following example is this area conventional reagent, commercially available or press Obtain according to the preparation of this area conventional method.

The preparation of embodiment 1. Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase mutant

1. mutant gene synthesis

Original series: the wild type CA 7 α-HSDH gene order after codon optimized (sees patent CN102827848A Publication), its nucleotide sequence is as shown in SEQ ID NO:4.

By comparing wild type CA 7 α-HSDH and homology enzyme egg from the many series of strata of the multi-angle of primary structure to higher structure The white similarities and differences, it is determined that affect the 145th amino acids that site is wild type CA 7 α-HSDH of zymologic property, described aminoacid For threonine, corresponding nucleotides sequence is classified as 433-435 bit codon.

Codon at the 433-435 of wild type CA 7 α-HSDH gene order is changed to AGT by ACT, by original Threonine replaces to serine, obtains CA 7 α-HSDH mutant, named CA 7 α-HSDH T145S mutant, its nucleotide Sequence is as shown in SEQ ID NO:3, and aminoacid sequence is as shown in SEQ ID NO:2.

5 ' the ends in CA 7 α-HSDH T145S mutant gene sequence introduce restriction enzyme site BamHI, and 3 ' ends introduce enzyme action Site NotI, for inserting pGEX-6p-by double digestion reaction and coupled reaction by CA 7 α-HSDH T145S mutant gene Corresponding site in 1 carrier, then send Sangon Biotech company carry out full genome synthesis and carry out sequence verification.Sangon The plasmid of the CA 7 α-HSDHT145S/pGEX-6p-1 that Biotech company directly provides and CA7 α-HSDHT145S/pGEX-6p- 1/DH5 α glycerol stock.

2. the GST of pheron merges heterogenous expression

(1) Plastid transformation E.coli BL21 cell

A.-80 DEG C is taken out BL21 competent cell E.coli place on ice.

B. add expression plasmid pGEX-6p-1/CA 7 α-HSDH T145S2 μ L after purification, place 30min on ice.

C. heat shock 42 DEG C, 90s.

Place 2min the most on ice.

E. recover, add 600 μ L LB culture medium, 37 DEG C, 150rpm, 45min.

F. draw 200 μ L culture medium and coat Amp⁺In LB plating medium.

G.37 DEG C overnight incubation.

(2) protein expression and purification

A. inoculating in sterile LB medium by strain, the final concentration of 50 μ g/mL of ampicillin, 37 DEG C, 180rpm trains Support.

B., when OD600 ≈ 0.8, the IPTG of final concentration of 0.2mM, 16 DEG C of overnight induction (12h) are added.8000rpm, 5min collects thalline.

C. add the resuspended thalline of ratio of 30mL Lysis buffer in 1L cultivating system, carrying out ultrasonic bacteria breaking is to clarification. 12000rpm, 20min.Take supernatant.

D. supernatant is combined with Glutathione Sepharose 4B, and the ratio that filler uses is that every liter of cultivating system uses 5mL filler, 4 DEG C combine 2h.The most vertically overturn suspendible.

E. after combining, 500rpm, 5min precipitation filling.Filler rinses 3-5 column volume with 4 DEG C of pre-cooling PBS.

Remove foreign protein.

F. add PreScission Protease enzyme cutting buffering liquid, add PreScission Protease enzyme.

G.4 DEG C enzyme action is overnight.After enzyme action, supernatant is released from chromatographic column.

H. gained sample is carried out SDS-PAGE, identify its molecular size range and purity, BCA test kit test purifying protein Concentration.

Result is as in figure 2 it is shown, the display T145S mutant solubility expression success of SDS-PAGE testing result, and through one After step affinity chromatograph, the band of albumen is single.

Embodiment 2.T145S mutant enzyme kinetic analysis parametric measurement

The making of 1.NADPH standard curve

Reaction buffer (50mM Tris-HCl, 200mM NaCl, pH 8.0) is utilized to prepare 0.1,0.2,0.3 and respectively The NADPH solution of 0.4mM.After returning to zero by above-mentioned blank solvent, the NADPH solution of each concentration is separately added into 2mL cuvette In, measure absorbance value OD at 340nm₃₄₀.With the concentration of NADPH solution as abscissa, corresponding absorbance value is vertical coordinate, Draw standard curve.

Result is as shown in Figure 3, it is thus achieved that standard curve equation be y=2.7868x-0.0002, R²=0.9999.

2. enzyme activity determination

The cuvette of 2mL volume is sequentially added into 958 μ L reaction buffers (50mM Tris-HCl, 200mM NaCl, PH 8.0), the NADP of 20 μ L 50mM⁺, the CA 7 α-HSDH T145S mutant (2.46mg/ for preparing of 2 μ L embodiments 1 ML), the substrate TCDCA of 20 μ L 50mM is added after zeroing.During enzyme kinetic analysis data determination, the change model of the final concentration of substrate Enclose for 0.1-10mM.

In 25 DEG C, at 340nm, record the light Change of absorption in 1min, and calculate product according to the standard curve of NADPH Growing amount.Double reciprocal by Lineweaver-Burk by Michaelis-Menten equation (formula 1) equal sign two side draw double (formula 2) reciprocal Graphing method obtains K_m、k_cat；And by comparing k_cat/K_mValue weighs the catalytic efficiency change of mutant.

$v=\frac{V_{max}\·\[S\]}{K_m+\[S\]}---\left(1\right)$

$\frac{1}{v}=\frac{K_m}{V_{max}}\·\frac{1}{\[S\]}+\frac{1}{V_{max}}---\left(2\right)$

Result is as shown in table 1, and CA 7 α-HSDH T145S mutant catalysis TCDCA is changed into cattle sulphur-7-Ketolithocholsaeure (T- Efficiency 7-KLCA) is 4.85 times of wild type CA 7 α-HSDH.

The enzyme kinetic analysis parameter of table 1. wild type CA 7 α-HSDH and T145S mutant catalysis TCDCA

Claims (10)

1. a Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase mutant, it is characterised in that its aminoacid sequence such as SEQ ID Shown in NO:2, be aminoacid sequence be that the 145th amino acids of the 7alpha-Hydroxysteroid dehydrogenase of SEQ ID NO:1 is become by Thr For Ser gained.

2. the gene of coding Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase mutant described in claim 1.

Gene the most according to claim 2, it is characterised in that its nucleotide sequence is as shown in SEQ ID NO:3.

4. an expression cassette, it is characterised in that comprise the gene described in claim 2.

5. a carrier, it is characterised in that comprise the gene described in claim 2 or the expression cassette described in claim 4.

6. a reconstitution cell, it is characterised in that comprise the gene described in claim 2 or the expression cassette described in claim 4 Or the carrier described in claim 5.

7. the method preparing Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase mutant described in claim 1, its feature It is, the reconstitution cell described in claim 6 isolated Sadinia can cultivated under conditions of success induced protein expression Island clostridium 7alpha-Hydroxysteroid dehydrogenase mutant.

8. a catalyst, it is characterised in that its effective ingredient comprises the Clostridium sardiniense 7 Alpha-hydroxy class described in claim 1 Sterin dehydrogenase mutant.

Catalyst the most according to claim 8, it is characterised in that also include and the Clostridium sardiniense 7 described in claim 1 Enzyme catalysis efficiency can be improved simultaneously when alpha-hydroxysteroid dehydrogenase mutant uses or increase other reagent of enzyme stability.

10. the method for the carbonyl asymmetric reduction realizing chemical substance, it is characterised in that use described in claim 1 Catalyst described in Clostridium sardiniense 7alpha-Hydroxysteroid dehydrogenase mutant or claim 8 or 9 and reaction substrate are at 15- 37 DEG C, carry out catalytic reaction under the conditions of pH 6.0-11.0.