CN102533876A - Reductase and genes thereof, as well as recombinase and preparation method and application hereof - Google Patents
Reductase and genes thereof, as well as recombinase and preparation method and application hereof Download PDFInfo
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Abstract
The invention discloses reductase and genes thereof, recombinant plasmid and recombinant transformant containing the genes, recombinase and a preparation method thereof, and application of the reductase or the recombinase as catalysts for asymmetric reduction of prochiral carbonyl compound to prepare optically active chiral alcohols. The reductase or the recombinase comes from bacillus sp. CGMCC NO.2549 and can be used as catalysts for the asymmetric reduction of prochiral carbonyl compound to prepare various optically active chiral alcohols with high catalyst efficiency, strong stereoselectivity, mild applicable reaction conditions and environmentally-friendly property. Compared with a whole cell of bacillus sp. CGMCC NO.2549, the reductase disclosed by the invention is better in catalyst effect and wider in applicability of substrates, and has a better industrial application and development prospect.
Description
The application is dividing an application of " a kind of reductase enzyme and gene thereof, recombinase and preparation method and application ", application number are 201010527409.0, the applying date is on October 29th, 2010 patented claim for denomination of invention.
Technical field
The invention belongs to technical field of bioengineering; Be specifically related to a kind of reductase enzyme and gene thereof; And the recombinant expression vector and the recombinant expressed transformant that contain this gene; With and the preparation method of recombinase and this recombinase, and this reductase enzyme or its recombinase as catalyzer at asymmetric reduction prochirality carbonyl compound with the application in the preparation optical activity chirality alcohol.
Background technology
The optical activity chirality alcohol that contains the specific function group is the important chirality building block of synthetic medicine, agricultural chemicals and other fine chemicals.The researchist has developed multiple optical activity chirality alcohol synthetic method, comprises kinetic resolution and asymmetric synthesis.Wherein, utilize the approach of the asymmetric reduction synthesis of optically active chiral alcohol of prochirality carbonyl compound, but the productive rate of realization theory 100% is the important method of producing optical activity chirality alcohol.Chemists have been found that the chiral metal compound can be used as the asymmetric reduction that catalyzer is used for carbonyl; Although this chemical process has been used to industrial production, yet this method operation easier is big, severe reaction conditions; And may residual heavy metal in the product, so its application be restricted.Biocatalysis method not only reaction conditions is gentle, environmentally friendly; Regioselectivity and stereoselectivity with height; And avoided heavy-metal residual in the product; Just remedied the weak point of chemical process, therefore the application of carbonyl asymmetric reduction reaction in the chiral alcohol asymmetric synthesis of biocatalysis in recent years more and more comes into one's own.
Oxydo-reductase is a big fermentoid of catalysis biological oxidation/reduction reaction; Be widely used in compounds such as asymmetric synthesis optical activity chirality medicine and functional steroid, therefore develop the oxydo-reductase with actual application value is an important content of biological technical field always.Aldehyde ketone reductase enzyme (AKR) and short-chain alcohol dehydrogenase (SDR) are two big important superfamilies of carbonyl reduction enzyme family.Wherein the AKR superfamily can be divided into 15 families, comprises about 150 members.Comparatively speaking, SDR superfamily member number is comparatively huge, existing at present more than 46000 member.Mostly the aldehyde ketone reductase enzyme is NADPH dependent form, is generally single subunit structure, and the subunit size is generally about 35～40kDa; And the short-chain alcohol dehydrogenase majority is made up of two subunits or four subunits, and the subunit size is generally about 27kDa.The reductase enzyme source is very extensive, ubiquity in mikrobe.For example, comprise in the full genome of bread yeast 49 the coding reductase enzymes opening code-reading frame, wherein the gene of 18 reductase enzymes by clonal expression and be used for ketone ester asymmetric reduction (J.Am.Chem.S ℃., 2004,126:12827-12832).In addition, from many other mikrobes, also separate having obtained having stereoselective reductase enzyme, some quilt is clonal expression further, and is applied to biocatalysis field.For example, Kita etc. (J.Mol.Catal.B:Enzym., 1999,6:305-313) from shadow yeast (Sporobolomyces salmonicolor) but separate the reductase enzyme obtain three kinds of catalysis 4-chloroacetyl acetacetic ester asymmetric reductions the AKU4429; (Appl.Microbiol.Biotehnol. such as Itoh; 2004; 66:53-62) a kind of 'beta '-ketoester reductase enzyme (AKR3E1) among penicillium spp (Penicillium citrinum) IF04631 has been carried out heterogenous expression, and be applied in the production of (S)-4-bromo-3-beta-hydroxymethyl butyrate.
Yet the shared ratio of reduction enzyme catalyzer that is applied to industry at present still not enough total amount 14%.How to specific substrate find have high catalytic activity and optionally biological catalyst be the vital problem in this field.Information biology and recombinant gene being combined, develop the new bio catalyzer of excellent performance quickly and efficiently, is the effective way of the biological asymmetric reduction technology of development.(Appl.Microbiol.Biotechnol. such as Yamamoto; 2003; 61:133-139) utilize these means to clone from the FabG similar enzyme in the reorganization reductase enzyme FabG in the subtilis and other the multiple mikrobes, in order to the asymmetric reduction of catalysis 4-chloroacetyl acetacetic ester.In addition, some other is from the structure and the also existing research report of character of the reductase enzyme of subtilis.As subtilis reductase enzyme YtbE to multiple aldehyde compound have reducing activity (Protein Science, 2009,18:1792-1800); Reductase enzyme YueD to dibenzoyl have reducing activity (J.Biotechnol., 2002,96:157-169).But whether these three kinds can be used for the asymmetric reduction of other carbonyl compound of catalysis from the reductase enzyme of subtilis, still do not have bibliographical information so far.
Present known genus bacillus (Bacillus sp.) ECU0013, but the multiple aryl ketones asymmetric reduction of i.e. CGMCC No.2549 catalysis generates optical activity chirality secondary alcohol (Bioresour.Technol., 2010,101:1054-1059; Patent of invention CN101372677B).But when putting in order cell as catalyzer,, cause overall catalytic activity not high, thereby limited the advantage performance of biological catalyst because production of enzyme is very low with this wild strain.
Summary of the invention
Technical problem to be solved by this invention is to make overall catalytic activity not high to production of enzyme is low during as aryl ketones asymmetric reduction catalyzer with the whole cell of wild genus bacillus (Bacillus sp.) CGMCC No.2549; The defective that asymmetric reduction substrate spectrum is narrower; And provide a kind of and have that excellent asymmetry catalysis is active, wide, the environment amenable reductase enzyme of substrate suitability and gene thereof; And the recombinant expression vector and the recombinant expressed transformant that contain this gene; With and the preparation method of recombinase and this recombinase, and the application of this reductase enzyme or its recombinase.
The present invention through following technical proposals to solve the problems of the technologies described above:
The aminoacid sequence of reductase enzyme of the present invention is shown in SEQ ID No.2, SEQ ID No.4 or SEQ ID No.6 in the sequence table.It is that this reductase enzyme of NADPH type is that example describes that the present invention relies on type with the coenzyme that derives from genus bacillus (Bacillus sp.) ECU0013.Among the present invention, described genus bacillus (Bacillus sp.) ECU0013 is preserved in Chinese common micro-organisms culture presevation administrative center at present, and preserving number is CGMCC No.2549.This bacterial strain screens from soil and obtains, and in patent of invention (CN101372677B), detailed description is arranged.
The invention still further relates to a kind of reductase gene, it is that (1) its base sequence is shown in SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5 in the sequence table; Or (2) its protein of encoding and forming by the aminoacid sequence shown in SEQ ID No.2, SEQ ID No.4 or the SEQ ID No.6 in the sequence table.
Reductase gene of the present invention can derive from genus bacillus (Bacillus sp.) CGMCC No.2549; Concrete preparation method can be: according to the gene order design synthetic primer of reductase enzyme YtbE, FabG and the YueD of the subtilis of including among the Genbank (Bacillus subtilis) 168; Genomic dna with genus bacillus (Bacillus sp.) CGMCC No.2549 is a template then; Utilize polymerase chain reaction (PCR) to carry out gene amplification; Obtain three complete reductase enzyme full-length gene orders, be respectively:
The gene of base sequence shown in SEQ ID No.1 in the sequence table, called after BCR I, total length 843bp.Wherein, its encoding sequence (CDS) ends from 840 bases of the 1st base to the, and initiator codon is ATG, and terminator codon is TAA.This sequence intronless, the aminoacid sequence of its encoded protein matter is shown in SEQ ID No.2 in the sequence table.
The gene of base sequence shown in SEQ ID No.3 in the sequence table, called after BCR II, total length 741bp.Wherein, its encoding sequence (CDS) ends from 738 bases of the 1st base to the, and initiator codon is ATG, and terminator codon is TAA.This sequence intronless, the aminoacid sequence of its encoded protein matter is shown in SEQ ID No.4 in the sequence table.
The gene of base sequence shown in SEQ ID No.5 in the sequence table, called after BCR III, total length 732bp.Wherein, its encoding sequence (CDS) ends from 729 bases of the 1st base to the, and initiator codon is ATG, and terminator codon is TAG.This sequence intronless, the aminoacid sequence of its encoded protein matter is shown in SEQ ID No.6 in the sequence table.
As is known to the person skilled in the art, the base sequence of reductase gene of the present invention also can be proteinic other any base sequences that coding is made up of the aminoacid sequence shown in SEQ ID No.2, SEQ ID No.4 or the SEQ ID No.6 in the sequence table.
The present invention relates to the recombinant expression vector of the nucleotide sequence that comprises reductase gene of the present invention in addition.It can be connected in the nucleotide sequence of reductase gene of the present invention to make up on the various carriers and form through this area ordinary method.Described carrier can be the conventional various carriers in this area, like commercially available plasmid, clay, phage or virus vector etc., and preferred pET28a.Preferable; Can make recombinant expression vector of the present invention through following method: will be connected with carrier pMD-18T through the reductase gene product of pcr amplification gained; Form cloning vector pBCRI-18T, pBCRII-18T or pBCRIII-18T; Afterwards with cloning vector and expression vector pET28a with restriction enzyme BamHI and XhoI double digestion; Form the complementary sticky end, connect through the T4DNA ligase enzyme again, form the recombinant expression plasmid pET-BCRI, pET-BCRII or the pET-BCRIII that contain reductase gene of the present invention.
The invention further relates to the recombinant expressed transformant that comprises reductase gene of the present invention or its recombinant expression vector.It can make through recombinant expression vector of the present invention is converted in the host microorganism.Described host microorganism can be the conventional various host microorganisms in this area, as long as can satisfy duplicating voluntarily that recombinant expression vector can be stable, and entrained reductase gene of the present invention can be got final product by effective expression.The preferred intestinal bacteria of the present invention, more preferably ETEC (E.coli) BL21 (DE3) or ETEC (E.coli) DH5 α.Aforementioned recombinant expression plasmid pET-BCRI, pET-BCRII or pET-BCRIII are converted among ETEC (E.coli) BL21 (DE3); Get final product the preferred engineering strain of the present invention, i.e. ETEC (E.coli) BL21 (DE3)/pET-BCRI, ETEC (E.coli) BL21 (DE3)/pET-BCRII or ETEC (E.coli) BL21 (DE3)/pET-BCRIII.
The present invention also further relates to a kind of preparation method of the reductase enzyme of recombinating, and it comprises the steps: to cultivate recombinant expressed transformant of the present invention, obtains the reorganization reductase enzyme.Wherein, described recombinant expressed transformant is ditto given an account of and is continued, and can obtain through recombinant expression vector of the present invention is converted into host microorganism.
Wherein, but any substratum that makes the transformant growth and produce reductase enzyme of the present invention in used substratum this area in the recombinant expressed transformant of described cultivation, for bacterial strain; Preferred LB substratum: peptone 10g/L; Yeast extract paste 5g/L, NaCl 10g/L, pH 7.0.Cultural method and culture condition do not have special limitation, can carry out appropriate selection with the different of factor such as cultural methods by this area general knowledge according to host type, as long as transformant can be grown and produce reductase enzyme.Other culture transformation body concrete operations all can be undertaken by this area routine operation.For bacterial strain; Preferred following method: the recombination bacillus coli (the recombinant expressed transformant of preferred ETEC of the present invention (E.coli) BL21 (DE3)) that will express reductase gene of the present invention is seeded in the LB substratum that contains penbritin and cultivates, as the optical density(OD) OD of nutrient solution
600When reaching 0.5-0.7 (preferred 0.6), be under the inducing of sec.-propyl-β-D-sulfo-galactopyranoside (IPTG) of 0.1-1mmol/L (preferred 0.5mmol/L) at final concentration, can efficiently express reorganization reductase enzyme of the present invention.
The present invention further relates to a kind of reorganization reductase enzyme again, the reorganization reductase enzyme that it makes for (1) as stated above; Or the reorganization reductase enzyme of (2) aminoacid sequence shown in SEQ ID No.2, SEQ ID No.4 or SEQ ID No.6 in the sequence table.It is the NADPH type that the coenzyme of reorganization reductase enzyme of the present invention relies on type.
The present invention further relate to reductase enzyme of the present invention or its recombinase as catalyzer at asymmetric reduction prochirality carbonyl compound with the application in the preparation optical activity chirality alcohol.
Preferable, described application is carried out as follows: in the phosphate buffered saline buffer of pH 6-8, at Hexose phosphate dehydrogenase, glucose and NADP
+Existence under, reductase enzyme of the present invention or the reorganization reductase enzyme effect under, the prochirality carbonyl compound is carried out asymmetric reduction reaction, make optical activity chirality alcohol.
In the above-mentioned application, each condition of described asymmetric reduction reaction can be selected by the normal condition of this type of reaction of this area, preferably as follows:
What described prochirality carbonyl compound was preferable is aryl ketones compounds or ketone ester compounds.What the aryl in the described aryl ketones was preferable is phenyl or heterocyclic base, as contains 1～2 heteroatomic five yuan or hexa-atomic heterocyclic base that is selected from N, S and O, preferred pyridyl.The present invention is preferably suc as formula the prochirality carbonyl compound shown in 1,2,3 or 4:
Wherein, R
1For-H ,-Cl or-Br, R
1Substituted position is ortho position, a position or the contraposition of phenyl;
R
2For-CH
3,-CH
2Cl ,-CH
2Br or-CF
3
R
3Be 2-pyridyl, 3-pyridyl or 4-pyridyl;
R
4For-CH
3,-CH
2Cl ,-CH
2Br or-CF
3
R
5For-CH
3, o-Cl-C
6H
5-or-(CH
2)
2C
6H
5
That the concentration of described prochirality carbonyl compound in reaction solution is preferable is 1～15mmol/L.The consumption of reductase enzyme of the present invention or reorganization reductase enzyme is a catalytically effective amount, and preferable is 1～10U/L (embodiment 4 is seen in enzyme activity determination method and definition).What the consumption of Hexose phosphate dehydrogenase was preferable is 10～100U/L (embodiment 4 is seen in enzyme activity determination method and definition).That the consumption of glucose is preferable is 5～50g/L.NADP
+Consumption preferable be 0.5～1.0mmol/L.Described phosphate buffered saline buffer can be the conventional phosphate buffered saline buffer in this area, like phosphoric acid-sodium phosphate buffer.That the concentration of phosphate buffered saline buffer is preferable is 0.05～0.1mol/L, and described concentration is meant the total concn of conjugate acid and base in the buffered soln.What described asymmetric reduction reaction was preferable carries out under oscillating condition.What the temperature of described asymmetric reduction reaction was preferable is 20～35 ℃.What the time of described asymmetric reduction reaction was preferable is as the criterion to react completely, and is generally 1～12 hour.After asymmetric reduction reaction finishes, can from reaction solution, extract (S)-or (R)-type chiral alcohol product by this area ordinary method.
On the basis that meets this area general knowledge, above-mentioned each optimum condition, but arbitrary combination promptly get each preferred embodiments of the present invention.
Agents useful for same of the present invention and raw material are all commercially available to be got.
Positive progressive effect of the present invention is: it is pure to prepare multiple optical activity chirality in asymmetric reduction prochirality carbonyl compound that reductase enzyme of the present invention or its recombinase can be used as catalyst applications; Its catalytic efficiency (is high, stereoselectivity is strong, and the reaction conditions that is suitable for is gentle, environmentally friendly.Compare with the whole cell of wild-type genus bacillus (Bacillus sp.) CGMCC No.2549, reductase enzyme catalytic effect of the present invention is better, and the substrate suitability is wider, has the better industrial application development prospect.
Description of drawings
Fig. 1 is the pcr amplification electrophoretogram of gene BCRI, BCRII and BCRIII, wherein, and 1, DNA Marker (Marker II, sky, Beijing root biochemical technology ltd); 2, the pcr amplification product of gene BCRIII; 3, the pcr amplification product of gene BCRI; 4, the pcr amplification product of gene BCRII.
Fig. 2 is that the double digestion of BamHI and the XhoI of cloned plasmids pBCRI-18T, pBCRII-18T and pBCRIII-18T is analyzed collection of illustrative plates, wherein, and 1, pBCRI-18T/BamHI+XhoI; 2, pBCRII-18T/BamHI+XhoI; 3, pBCRIII-18T/BamHI+XhoI; 4, DNA Marker (Marker IV, sky, Beijing root biochemical technology ltd).
Fig. 3 is the bacterium liquid pcr amplification electrophorogram of ETEC (E.coli) BL21 (DE3)/pET-BCRI, ETEC (E.coli) BL21 (DE3)/pET-BCRII; Wherein, 1～6, the bacterium liquid pcr amplification electrophorogram of ETEC (E.coli) BL21 (DE3)/pET-BCRI; 7～12, the bacterium liquid pcr amplification electrophorogram of ETEC (E.coli) BL21 (DE3)/pET-BCRII; 13, DNA Marker (Marker II, sky, Beijing root biochemical technology ltd).
Fig. 4 is the bacterium liquid pcr amplification electrophorogram of ETEC (E.coli) BL21 (DE3)/pET-BCRIII, wherein, and 1～6, the bacterium liquid pcr amplification electrophorogram of ETEC (E.coli) BL21 (DE3)/pET-BCRIII; 7, DNA Marker (Marker IV, sky, Beijing root biochemical technology ltd).
Fig. 5 is the structure synoptic diagram of recombinant expression plasmid pET-BCRI.
Fig. 6 is the structure synoptic diagram of recombinant expression plasmid pET-BCRII.
Fig. 7 is the structure synoptic diagram of recombinant expression plasmid pET-BCRIII.
Fig. 8 is the polyacrylate hydrogel electrophorogram of reorganization reductase enzyme BCRI, BCRII and BCRIII.
Embodiment
Mode through embodiment further specifies the present invention below, but does not therefore limit the present invention among the described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example according to ordinary method and condition, or is selected according to catalogue.
Material source in the following example is:
Plasmid pMD18-T is available from Dalian TaKaRa company.
Expression plasmid pET28a is available from Shanghai Novagen company.
E.coli DH5 α and E.coli BL21 (DE3) competent cell, 2 * Taq PCR MasterMix, sepharose DNA reclaim test kit available from sky, Beijing root biochemical technology ltd.
The clone of embodiment 1 reductase gene
Gene order according to reductase enzyme YtbE, FabG and the YueD of the subtilis of having included among the Genbank (Bacillus subtilis) 168 is a foundation, and design PCR primer is following:
Primer 1 (amplification BCR I gene):
Upstream primer: CGC
GGATCCATGACAACACATTTACAAGCAAAAG
Downstream primer: CCG
GTCGAGTTAAAAATCAAAGTTGTCCGGATC
Primer 2 (amplification BCR II gene):
Upstream primer: CGC
GGATCCATGCTTAATGATAAAACGGCT
Downstream primer: CCG
GTCGAGTTACATCACCATTCCGCC
Primer 3 (amplification BCR III gene):
Upstream primer: CGC
GGATCCATGGAACTTTATATCATCACCGGAG
Downstream primer: CCG
GTCGAGCTACAAAAACTCTTTAATATCATAAATGCG
Wherein, the upstream primer underscore partly is the BmHI restriction enzyme site, and the downstream primer underscore partly is the XhoI restriction enzyme site.
Genomic dna with genus bacillus (Bacillus sp.) CGMCC No.2549 is a template, carries out pcr amplification.The PCR system is: 2 * Taq PCR MasterMix, 15 μ l, each 1 μ l (0.3 μ mol/L) of upstream primer and downstream primer, dna profiling 1 μ l (0.1 μ g) and ddH
2O 12 μ l.The pcr amplification step is: (1) 95 ℃, and preparatory sex change 5min; (2) 94 ℃, sex change 45s; (3) 60 ℃ of annealing 1min; (4) 72 ℃ are extended 1min; Repeat 35 times step (2)～(4); (5) 72 ℃ are continued to extend 10min, are cooled to 4 ℃.The PCR product utilizes sepharose DNA to reclaim test kit and reclaims the interval target stripe (Fig. 1) of 700～900bp through the agarose gel electrophoresis purifying.Obtain the reductase enzyme full-length gene order of three complete genus bacillus (Bacillus sp.) CGMCC No.2549, through dna sequencing, respectively as follows:
The preparation of embodiment 2 recombinant expression vectors (plasmid) and recombinant expressed transformant
The reductase gene of embodiment 1 gained is connected with the pMD-18T carrier, makes up cloned plasmids pBCRI-18T, pBCRII-18T and pBCRIII-18T respectively.Be converted into ETEC (E.coli) DH5 α competent cell afterwards respectively; Through bacterium colony PCR screening positive clone; Extract plasmid, at 37 ℃ with restriction enzyme BamHI and XhoI double digestion 12h, through the agarose gel electrophoresis purifying; Utilize sepharose DNA to reclaim test kit and reclaim target fragment, prove and contain correct insertion fragment (Fig. 2).With target fragment under the effect of T4DNA ligase enzyme, with the same plasmid pET28a after BamHI cuts with the XhoI enzyme, 4 ℃ down connection spend the night and obtain recombinant expression plasmid pET-BCRI, pET-B CRII and pET-B CRIII.
Respectively above-mentioned recombinant expression plasmid is transformed in ETEC (E.coli) the DH5 α competent cell; Positive recombinant chou is screened containing on the resistant panel of kantlex; The picking mono-clonal; Cultivate the reorganization bacterium, treat to extract plasmid behind the plasmid amplification, be converted into again in ETEC (E.coli) BL21 (DE3) competent cell; Conversion fluid is applied on the LB flat board that contains kantlex; Be inverted overnight cultures for 37 ℃, promptly obtain positive recombinant conversion body ETEC (E.coli) BL21 (DE3)/pET-BCRI, ETEC (E.coli) BL21 (DE3)/pET-BCRII and ETEC E.coli BL21 (DE3)/pET-BCRIII respectively, bacterium colony PCR verifies positive colony (Fig. 3 and 4).
The expression of embodiment 3 reorganization reductase enzymes
With 3 kinds of recombination bacillus colis of embodiment 2 gained, be seeded in the LB substratum that contains penbritin respectively, 37 ℃ of shaking culture are spent the night; 50ml LB substratum (peptone 10g/L is equipped with in inoculum size access by 1% (v/v); Yeast extract paste 5g/L, NaCl 10g/L, pH 7.0) the 250ml triangular flask in; Put 37 ℃, the cultivation of 180rpm shaking table, as the OD of nutrient solution
600Reach at 0.6 o'clock, the IPTG that adds final concentration and be 0.5mmol/L is as inductor, 25 ℃ induce 12h after; With medium centrifugal, collecting cell, and use the saline water washed twice; The resting cell of gained is suspended in the damping fluid of pH 7.0; Ultrasonication in ice bath, centrifugal collection supernatant, be the reorganization reductase enzyme crude enzyme liquid.Crude enzyme liquid is through polyacrylamide gel electrophoresis map analysis (Fig. 8), and three recombinant protein major parts exist with soluble form.Polyacrylamide gel electrophoresis figure gets through the BandScan software analysis respectively; The target protein reductase enzyme BCRI that expresses account for the crude enzyme liquid total protein 55%, the target protein reductase enzyme BCRII that expresses accounts for 58% of crude enzyme liquid total protein, the target protein reductase enzyme BCRIII of expression accounts for 63% of crude enzyme liquid total protein.
The mensuration of embodiment 4 reorganization reductase enzymes and Hexose phosphate dehydrogenase vigor
Utilize spectrophotometer to detect the change calculations reductase enzyme of 340nm light absorption value and the vigor of Hexose phosphate dehydrogenase.
Reorganization reductase vitality measuring method is following: in 1ml reaction system (100mmol/L sodium phosphate buffer, pH 7.0), add the 2mmol/L2-chloro-acetophenone; 0.05mmol/L NADPH; 30 ℃ of insulations add an amount of enzyme liquid after 2 minutes, shake up rapidly, detect the variation of 340nm place light absorption value.
The Hexose phosphate dehydrogenase vigour-testing method is following: in 1ml reaction system (100mmol/L sodium phosphate buffer, pH 7.0), add 2mmol/L glucose, 0.05mmol/L NADP
+, 30 ℃ of insulations add an amount of enzyme liquid after 2 minutes, shake up rapidly, detect the variation of 340nm place light absorption value.
The calculation formula of enzyme activity is: enzyme activity (U)=EW * V * 10
3/ (6220 * l); In the formula, EW is the variation of 340nm place absorbancy in the 1min; V is the volume of reaction solution, Unit; 6220 is molar extinction coefficient, the L/ of unit (molcm); L is an optical path length, the cm of unit.
The work of reductase enzyme per unit enzyme is defined as under these conditions, the enzyme amount that PM catalyzed oxidation 1 μ molNADPH is required.The enzyme work of reorganization reductase enzyme BCRI, BCRII and BCRIII is respectively 0.017U/mg albumen, 0.28U/mg albumen and 0.14U/mg albumen.
The work of per unit Hexose phosphate dehydrogenase enzyme is defined as under these conditions, PM catalytic reduction 1 μ molNADP
+Required enzyme amount.
The asymmetric reduction of embodiment 5-19 reorganization reductase enzyme BCR I catalysis of carbonyl compound
At 0.4ml phosphoric acid-sodium phosphate buffer (100mmol/L; PH 7.0) the middle BCR I enzyme liquid of 0.002U and the Hexose phosphate dehydrogenase of 0.04U of adding; Adding aromatic ketone that final concentration is 2mmol/L (embodiment 5-13) or final concentration respectively is the ketone ester (embodiment 14-19) of 10mmol/L, adds the NADP that final concentration is 0.5mmol/L again
+Glucose with 50g/L.At 30 ℃, 1100rpm oscillatory reaction certain hour.Reaction finishes the back and extracts with equal-volume ETHYLE ACETATE; Extracted twice; Combining extraction liquid; Add anhydrous sodium sulfate drying and spend the night, with the ee value of gc (chiral capillary column CP-Chirasil-DEXCB or Beta-DEX 120) or liquid chromatography (chirality OD-H post) assay determination substrate conversion efficiency and reduzate.The result sees table 1.
Product ee value concrete analysis condition is following:
Embodiment 5-17 and 19 uses the gas chromatographic analysis chirality, carrier gas nitrogen, and 280 ℃ of injector temperatures, 280 ℃ of detector temperatures, other conditions are following:
Embodiment 5: chiral capillary column Beta-DEX 120,130 ℃ of column temperatures;
Embodiment 6: chiral capillary column Beta-DEX 120,150 ℃ of column temperatures;
Embodiment 7: chiral capillary column Beta-DEX 120,160 ℃ of column temperatures;
Embodiment 8: chiral capillary column CP-Chirasil-DEX CB, 130 ℃ of column temperatures;
Embodiment 9: chiral capillary column Beta-DEX 120,160 ℃ of column temperatures;
Embodiment 10: chiral capillary column Beta-DEX 120,170 ℃ of column temperatures;
Embodiment 11: chiral capillary column Beta-DEX 120,110 ℃ of column temperatures;
Embodiment 14: chiral capillary column CP-Chirasil-DEX CB, and column temperature is kept 2min for 90 ℃, and 1 ℃/min rises to 120 ℃;
Embodiment 15: acetylate, and chiral capillary column CP-Chirasil-DEX CB, column temperature is kept 2min for 110 ℃, and 2 ℃/min rises to 126 ℃ and keeps 2min;
Embodiment 16: chiral capillary column CP-Chirasil-DEX CB, 130 ℃ of column temperatures;
Embodiment 17: chiral capillary column CP-Chirasil-DEX CB, 70 ℃ of column temperatures;
Embodiment 19: chiral capillary column CP-Chirasil-DEX CB, 160 ℃ of column temperatures.
Embodiment 18 uses liquid-phase chromatographic analysis chirality, chirality OD-H post, moving phase: normal hexane/Virahol=97/3, flow velocity 1ml/min, detector wavelength 254nm.
The result of table 1 reorganization reductase enzyme BCR I catalysis of carbonyl compound asymmetric reduction reaction
Embodiment 20-33 reorganization reductase enzyme BCR II catalysis of carbonyl compound asymmetric reduction
At 0.4ml phosphoric acid-sodium phosphate buffer (100mmol/L; PH 7.0) the middle BCR II enzyme liquid of 0.002U and the Hexose phosphate dehydrogenase of 0.04U of adding; Adding aromatic ketone that final concentration is 2mmol/L (embodiment 20-28) or final concentration respectively is the ketone ester (embodiment 29-33) of 10mmol/L, adds the NADP that final concentration is 0.5mmol/L again
+Glucose with 50g/L.At 30 ℃, 1100rpm jolting reaction certain hour.Reaction finishes the back and extracts with equal-volume ETHYLE ACETATE; Extracted twice; Combining extraction liquid adds anhydrous sodium sulfate drying and spends the night, with the ee value of gc (chiral capillary column CP-Chirasil-DEXCB) or liquid chromatography (chirality OD-H post) assay determination substrate conversion efficiency and reduzate.The result sees table 2.
Product ee value concrete analysis condition is following: embodiment 29: acetylate, and gc (chiral capillary column CP-Chirasil-DEX CB), column temperature is kept 2min for 110 ℃, and 5 ℃/min rises to 180 ℃.Other products ee value analysis condition is with described in the embodiment 5～19.
The result of table 2 reorganization reductase enzyme BCR II catalysis of carbonyl compound asymmetric reduction
The asymmetric reduction of embodiment 34-48 reorganization reductase enzyme BCR III catalysis of carbonyl compound
At 0.4ml phosphoric acid-sodium phosphate buffer (100mmol/L; PH 7.0) the middle BCR III enzyme liquid of 0.002U and the Hexose phosphate dehydrogenase of 0.04U of adding; Adding aromatic ketone that final concentration is 2mmol/L (embodiment 34-42) or final concentration respectively is the ketone ester (embodiment 43-48) of 10mmol/L, adds the NADP that final concentration is 0.5mmol/L again
+Glucose with 50g/L.At 30 ℃, 1100rpm jolting reaction certain hour.Reaction finishes the back and extracts with equal-volume ETHYLE ACETATE; Extracted twice; Combining extraction liquid adds anhydrous sodium sulfate drying and spends the night, with the ee value of gc (chiral capillary column CP-Chirasil-DEXCB) or liquid chromatography (chirality OD-H post) assay determination substrate conversion efficiency and reduzate.The result sees table 3.Product ee value analysis condition is with embodiment 5～33.
The result of table 3 reorganization reductase enzyme BCR III catalysis of carbonyl compound asymmetric reduction
Claims (8)
- A reductase enzyme as catalyzer at asymmetric reduction prochirality carbonyl compound with the application in the preparation optical activity chirality alcohol; The aminoacid sequence of described reductase enzyme is shown in SEQ ID No.2 in the sequence table.
- 2. application as claimed in claim 1; It is characterized in that: described application is carried out as follows: in the phosphate buffered saline buffer of pH 6-8; In the presence of Hexose phosphate dehydrogenase, glucose and NADP+; Under the effect of reductase enzyme as claimed in claim 1, the prochirality carbonyl compound is carried out asymmetric reduction reaction, make optical activity chirality alcohol.
- 3. according to claim 1 or claim 2 application, it is characterized in that: described prochirality carbonyl compound is aryl ketones compounds or ketone ester compounds.
- 4. application as claimed in claim 3 is characterized in that: the aryl in the described aryl ketones is phenyl or heterocyclic base.
- 5. application as claimed in claim 4 is characterized in that: described heterocyclic base is to contain 1～2 heteroatomic five yuan or hexa-atomic heterocyclic base that is selected from N, S and O.
- 6. application as claimed in claim 5 is characterized in that: described heterocyclic base is a pyridyl.
- 7. application as claimed in claim 3 is characterized in that: described prochirality carbonyl compound is suc as formula the prochirality carbonyl compound shown in 1,2,3 or 4:
Formula 1 formula 2 formulas 3 formulas 4Wherein, R 1For-H ,-Cl or-Br, R 1Substituted position is the contraposition of phenyl;R 2For-CH 3,-CH 2Cl ,-CH 2Br or-CF 3R 3Be 2-pyridyl, 3-pyridyl or 4-pyridyl;R 4For-CH 3,-CH 2Cl ,-CH 2Br or-CF 3R 5For-CH 3, o-Cl-C 6H 5-or-(CH 2) 2C 6H 5 - 8. like each described application of claim 3～7, it is characterized in that: the concentration of described prochirality carbonyl compound in reaction solution is 1～15mmol/L; The consumption of described reductase enzyme or reorganization reductase enzyme is 1～10U/L; The consumption of Hexose phosphate dehydrogenase is 10～100U/L; The consumption of described glucose is 5～50g/L; Described NADP +Consumption be 0.5～1.0mmol/L; The concentration of described phosphate buffered saline buffer is 0.05～0.1mol/L; The temperature of described asymmetric reduction reaction is 20～35 ℃; The time of described asymmetric reduction reaction is as the criterion to react completely.
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| BARBE,V.: "NP_390783.1", 《GENBANK》 * |
| JIAN LEI: "Structural and biochemical analyses of YvgN and YtbE from Bacillus subtilis", 《PROTEIN SCIENCE》 * |
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