CN104293757B - A kind of aldolase and encoding gene thereof and application - Google Patents
A kind of aldolase and encoding gene thereof and application Download PDFInfo
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Abstract
The invention discloses a kind of aldolase and encoding gene thereof and application.The aminoacid sequence of aldolase is as shown in SEQ ID No.1, and the base sequence of its encoding gene is as shown in SEQ ID No.2.Described application is the application in producing statins drug midbody of this aldolase.The present invention is on the basis of Thermophilic Bacteria wild type aldolase, its encoding gene is carried out random mutation superposition rite-directed mutagenesis, obtaining double mutant gene, the aldolase that this double mutant gene is expressed not only has possessed the anti-substrate degeneration ability of wild type aldolase, and its catalysis activity is also greatly improved；Utilizing this enzyme catalysis acetaldehyde and 2-Chloro-1-ethanal generation aldol reaction, its catalytic efficiency improves 3 times than wild type aldolase；Show that the aldolase of the present invention has good prospects for commercial application.
Description
Technical field
The invention belongs to technical field of molecular biology, be specifically related to a kind of aldolase and encoding gene thereof and application.
Background technology
Statins is blood fat reducing key agents, all with side chain (3R, 5S)-dihydroxy ester structure in its structure, and should
Structure can suppress HMG-CoA going back to mevalonic acid as first hydroxyl glutaryl coenzyme A (HMG-CoA) reductase inhibitor
Originality converts, and then reduces the level of low-density lipoprotein cholesterol, thus reduces the purpose of blood fat.
Although statins chemical constitution is relatively easy, but its preparation process difficulty is relatively big, and reason has two, and one is side
Chain (3R, 5S)-dihydroxy ester has 2 chiral centres, it is difficult to completed by single step reaction；Two is side chain (3R, 5S)-dihydroxy
Ester enantiomeric excess (e.e.) in Statins molecule and diastereomeric excess (d.e.) value are respectively greater than 99.5% and 99%,
The fractionation making enantiomer is relatively difficult.It is relatively low that these factors result in statins side chain synthetic yield, relatively costly.
Living things catalysis is to solve the important channel of current problem.At present, it has been found that have enzyme to aldol reaction
Having catalytic effect, wherein studying relatively broad is colibacillary aldolase DERA, and it can be catalyzed generation and have two chiralitys
Center product, such as can be catalyzed acetaldehyde and 2-Chloro-1-ethanal generation aldol reaction, generate chloro-(3R, the 5S)-dihydroxy of 6-
Aldehyde, 6-chloro-(3R, 5S)-dihydroxy aldehyde can turn to (4R, 6S)-2 by automatic ring, 4-dihydroxy-6 chloromethyl pyrans:
(4R, 6S)-2,4-dihydroxy-6 chloromethyl pyrans can be as an important centre of statins side chain synthesis
Body.But due to problems such as colibacillary aldolase existence catalysis al reactivity are the highest, anti-substrate degeneration ability, it is difficult to
Adapt to large-scale industrial production.
Document (Directed evolution of an industrial biocatalyst:2-deoxy-D-ribose
5-phosphate aldolase.Stefan Jennewein,Martin Schürmann,Michael Wolberg,Iris
Hilker,Ruud Luiten,Marcel Wubbolts and Daniel Mink.Biotechnol.J.2006,1,537–
548) colibacillary aldolase is oriented evolution, although the aldolase enzyme activity after sudden change has with anti-substrate degeneration ability
Improved, but relatively in commercial production for required higher acetaldehyde and 2-Chloro-1-ethanal concentration, its anti-acetaldehyde and 2-Chloro-1-ethanal degeneration
Ability still ratio is relatively limited, is still difficult in adapt to the demand of large-scale production.
Aldolase expressed by Thermophilic Bacteria Pyrobaculum aerophilum, its anti-substrate degeneration ability is relatively strong, but its
Catalysis activity is relatively low, if its catalysis activity can be improved on the basis of keeping its anti-substrate degeneration ability, will be substantially reduced preparation
The difficulty of statins.
Summary of the invention
The invention provides a kind of aldolase, this aldolase has high catalytic activity, and anti-substrate degeneration is very capable.
A kind of aldolase, its aminoacid sequence is as shown in SEQ ID No.1.
Present invention also offers a kind of gene encoding described aldolase, the base sequence of this gene such as SEQ ID No.2
Shown in.
The encoding gene of Thermophilic Bacteria (Pyrobaculum aerophilum) wild type aldolase is suddenlyd change by the present invention,
The codon (ATT) of 174 coding isoleucine is sported the codon (GTT) of coding valine, by 187 coding figured silk fabrics ammonia
The codon (GTG) of acid sports the codon (ATT) of coding isoleucine.
The preparation method of described aldolase is:
(1) with the encoding gene of Thermophilic Bacteria (Pyrobaculum aerophilum) wild type aldolase as template, carry out
Fallibility PC expands, and builds recombinant vector with amplified fragments, it is thus achieved that mutant gene libraries；
(2) by mutant gene libraries transformed competence colibacillus cell, mutated library is built；
(3) inducer induced mutation library is utilized to express, it is thus achieved that expression library；
(4) expression library is carried out enzyme analysis alive and sequencing, screens purpose mutant gene；
By said method, the present invention has first filtered out two purpose mutant genes, the point of the two purpose mutant gene
Sudden change is respectively occurring at 174 and 187, and the aldolase that the two purpose mutant gene is expressed, it is catalyzed specific activity wild type aldehyde
The aldolase that contracting enzyme and the recombination engineering containing other mutant genes are produced all increases.
(5) based on above-mentioned purpose mutant gene, rite-directed mutagenesis is carried out, it is thus achieved that double mutant gene；
On the basis of step (4), its 187, with the purpose mutant gene of 174 as template, are pinpointed by the present invention
Sudden change, it is thus achieved that occur at 174, the double mutant gene of 187；With the purpose mutant genes of 187 as template, to its 174
Carry out rite-directed mutagenesis, also can obtain generation at 174, the double mutant gene of 187.
(6) build the recombination engineering containing double mutant gene, after inducing culture, the crude enzyme liquid obtained is separated pure
Change, obtain aldolase.
The aldolase that this double mutant gene is expressed, the aldolase that the gene that its catalysis specific activity is individually suddenlyd change is produced is the most
Improve, be derived from the aldolase of the present invention.
In the present invention, carrying out the primer used by 174 rite-directed mutagenesises is:
Forward primer: 5 '-GGAGAGGGCGGCGGCAGTTGCCCGCTACATAAAAGAG-3 ' (SEQ ID No.4)；
Downstream primer: 5 '-CTCTTTTATGTAGCGGGCAACTGCCGCCGCCCTCTCC-3 ' (SEQ ID No.5)；
Carrying out the primer used by 187 rite-directed mutagenesises is:
Forward primer: 5 '-GGTATAGACTGGGGATTAAAATGGCGGGGGGGATTAGG-3 ' (SEQ ID No.6)；
Downstream primer: 5 '-CCTAATCCCCCCCGCCATTTTAATCCCCAGTCTATACC-3 ' (SEQ ID No.7).
Present invention also offers expression unit, recombinant vector or the transformant containing described gene.
As preferably, the promoter of described expression unit is T7 promoter, lac promoter or araBAD promoter.At this
Under the effect of a little promoteres, aldolase directly can realize intracellular soluble-expression in e. coli host cell.
The initial carrier of described recombinant vector can be selected for PET21a, PET28a, PET30a.
As preferably, host cell is escherichia coli.
Present invention also offers the application in producing statins drug midbody of the described aldolase.As do not made special theory
Bright, heretofore described statins drug midbody refers to (4R, 6S)-2,4-dihydroxy-6 chloromethyl pyrans.
The aldolase of the present invention has not only possessed the anti-substrate degeneration ability of wild type aldolase, and its catalysis activity is the most significantly
Improve；Utilizing this enzyme catalysis acetaldehyde and 2-Chloro-1-ethanal generation aldol reaction, its catalytic efficiency improves 3 than wild type aldolase
Times, than document (Directed evolution of an industrial biocatalyst:2-deoxy-D-ribose 5-
phosphate aldolase.Stefan Jennewein,Martin Schürmann,Michael Wolberg,Iris
Hilker,Ruud Luiten,Marcel Wubbolts and Daniel Mink.Biotechnol.J.2006,1,537–
548) the escherichia coli Aldolase mutant disclosed in improves 8 times.
Compared with prior art, the invention have the benefit that
The present invention, on the basis of Thermophilic Bacteria (Pyrobaculum aerophilum) wild type aldolase, encodes base to it
Because carrying out random mutation superposition rite-directed mutagenesis, it is thus achieved that double mutant gene, the aldolase that this double mutant gene is expressed not only is possessed
The anti-substrate degeneration ability of wild type aldolase, its catalysis activity is also greatly improved；This enzyme catalysis acetaldehyde and 2-Chloro-1-ethanal is utilized to send out
Raw aldol reaction, its catalytic efficiency improves 3 times than wild type aldolase；Show that the aldolase of the present invention has good
Prospects for commercial application.
Accompanying drawing explanation
Fig. 1 is the principle schematic of rite-directed mutagenesis；
Fig. 2 is double mutant gene DERA of the present inventionpyaThe electrophoresis detection result figure of (I174V, V187I)；
Fig. 3 be built with double mutant gene of the present invention plasmid pET28a (+)-DERApyaThe gene of (I174V, V187I)
Collection of illustrative plates；
Fig. 4 is the electrophoresis detection result figure of plasmid shown in Fig. 3；
The electrophoresis detection result figure that Fig. 5 is the produced crude enzyme liquid of recombination engineering of the present invention；
Wherein, Marker represents protein molecular weight standard；DERApya(I174V, V187I) represents that recombination engineering is produced
Crude enzyme liquid；
Fig. 6 is DERApyaThe mass spectrum of product 6-chloro-(3R, 5S)-dihydroxy aldehyde in (I174V, V187I) catalyst system and catalyzing；
Fig. 7 is DERApyaProduct (4R, 6S)-2,4-dihydroxy-6 chloromethyl pyrans in (I174V, V187I) catalyst system and catalyzing
Mass spectrum；
Wherein, Abundance represents that abundance, m/z represent mass-to-charge ratio.
Detailed description of the invention
With detailed description of the invention, the present invention is described in further detail below in conjunction with the accompanying drawings.
The preparation of embodiment 1 pair sudden change aldolase
1, the encoding gene of Thermophilic Bacteria wild type aldolase is synthesized
According to the coding gene sequence of Thermophilic Bacteria wild type aldolase disclosed on NCBI, entrust Shanghai JaRa biological engineering
This coding gene sequence of company limited's synthetic, its base sequence is as shown in SEQ ID N0.3.
2, mutant gene is identified and plasmid order-checking
1. with the encoding gene of Thermophilic Bacteria (Pyrobaculum aerophilum) wild type aldolase as template, carry out easily
Wrong PCR amplification.
Wherein, PCR reaction system is: pure water 21.5 μ L；10×buffer(Mg2+Free), 5 μ L；Forward primer (concentration
10μM)1μL；Downstream primer (concentration 10 μMs) 1 μ L；DNTP (2.5mM each), 4 μ L；Mgcl2(25mM), 14 μ L；MnCl2
(5mM), 1.5 μ L；Template, 1 μ L；Taq polymerase, 1 μ L；Cumulative volume is 50 μ L.
PCR condition is: entering amplification cycles, i.e. 94 DEG C degeneration 30s after 94 DEG C of degeneration 5min, 57 DEG C of annealing 30s, 72 DEG C are prolonged
Stretching 1min, altogether circulation 30 times, last 72 DEG C extend 10min.The sequence of PCR primer is:
Forward primer: 5 '-CTAGAATTCATGATACATTTAGTAGACTACGCGC-3 ' (is EcoRI enzyme action at underscore
Site) (SEQ ID No.8)；
Downstream primer: 5 '-GTTCTCGAGCTATAGAAGAGCCTCTGGGGTGG-3 ' (is XhoI enzyme action position at underscore
Point) (SEQ ID No.9).
2. the fragment above-mentioned amplification obtained pass through EcoRI and XhoI double digestion, be connected to pET28a (+) carrier (use
DNA ligation kit Ver.2.1 test kit), obtain mutant gene libraries；
3. said mutation gene library is converted escherichia coli (E.coli) BL21 (DE3) competent cell, be applied to contain
Have on the LB agar plate of 50 μ g/ml kanamycin, cultivate 15 hours, obtain mutant strain library for 37 DEG C；
4. it is transferred to said mutation strain clone in 24 orifice plates equipped with 400 μ L culture medium cultivate, 1mM IPTG 25 DEG C
Lower inducing culture 12 hours, it is thus achieved that expression library；
5. collect thalline, with the 300 μ L resuspended thalline of 0.1M sodium acetate solution, ultrasonication, broken cytosol is transferred to 24 holes
In plate, adding 200mM acetaldehyde and 100mM 2-Chloro-1-ethanal, 25 DEG C are reacted 2 hours, add the acetonitrile that nonuploid is long-pending, centrifugal segregation albumen
Matter, by product 6-chloro-(3R, 5S)-dihydroxy aldehyde, (4R, 6S)-2,4-dihydroxy-6 in each reaction system of gas chromatographic detection
The content of chloromethyl pyrans.
Thus screening obtains two mutants making products collection efficiency improve, wherein, the DERA of mutant (I174V)pyaGene
In 174 coding isoleucine codons (ATT) sport coding valine codon (GTT), mutant (V187I)
DERApyaIn gene, the codon (GTG) of 187 coding valines sports the codon (ATT) of coding isoleucine.
3, the preparation of double mutant gene
According to the sequence shown in SEQ ID No.3, design rite-directed mutagenesis primer, carry out rite-directed mutagenesis.Rite-directed mutagenesis former
Reason schematic diagram such as Fig. 1.
Wherein, the primer carrying out suddenling change at the 174th is:
Forward primer: 5 '-GGAGAGGGCGGCGGCAGTTGCCCGCTACATAAAAGAG-3 ' (SEQ ID No.4)；
Downstream primer: 5 '-CTCTTTTATGTAGCGGGCAACTGCCGCCGCCCTCTCC-3 ' (SEQ ID No.5)；
At the 187th primer carrying out suddenling change it is:
Forward primer: 5 '-GGTATAGACTGGGGATTAAAATGGCGGGGGGGATTAGG-3 ' (SEQ ID No.6)；
Downstream primer: 5 '-CCTAATCCCCCCCGCCATTTTAATCCCCAGTCTATACC-3 ' (SEQ ID No.7).
With the single mutation genes of 174 as template, utilize and carry out rite-directed mutagenesis PCR at the 187th primer carrying out suddenling change,
Result is found that a double mutant gene (electrophoresis detection result is shown in Fig. 2) from recording gene, the 174th ammonia of this gene code
Base acid residue codon usage is sported valine (GTT) by isoleucine (ATT), and the amino acid residue codon of 187 is by figured silk fabrics ammonia
Acid (GTG) sports isoleucine (ATT), and therefore the aldolase of this gene code is double mutant enzymes.The aminoacid of double mutant enzymes
Sequence is as shown in SEQ.No.1.With the single mutation genes of 187 as template, utilize and carry out at the 174th primer carrying out suddenling change
Rite-directed mutagenesis PCR, available identical result.
PCR reaction system is: pure water 38.5 μ L；Pfu buffer 5μL；Forward primer (concentration 10 μMs) 1 μ L；Downstream
Primer (concentration 10 μMs) 1 μ L；DNTP (2.5mM each), 4 μ L；Template, 1 μ L；Pfu, 0.5 μ L；Cumulative volume is 50 μ L.
PCR condition is: entering amplification cycles, i.e. 94 DEG C degeneration 30s after 94 DEG C of degeneration 5min, 57 DEG C of annealing 30s, 68 DEG C are prolonged
Stretching 6min, altogether circulation 20 times, last 68 DEG C extend 10min.
4, the expression of aldolase and preparation
Respectively with single mutation gene DERAPya(I174V), single mutation gene DERAPya(V187I), single mutation gene
DERAPya(A67G), double mutant gene DERAPya(I174V, V187I), wild type gene DERAPya, escherichia coli aldolase dash forward
Become gene and (see document: Directed evolution of an industrial biocatalyst:2-deoxy-D-
ribose 5-phosphate aldolase.Stefan Jennewein,Martin Schürmann,Michael
Wolberg,Iris Hilker,Ruud Luiten,Marcel Wubbolts and Daniel
Mink.Biotechnol.J.2006,1,537 548) structure plasmid: pET28a (+)-DERAPya(I174V)、pET28a(+)-
DERAPya(V187I)、pET28a(+)-DERAPya(A67G)、pET28a(+)-DERAPya(I174V, V187I) (its plasmid map
See that Fig. 3, electrophoresis detection result are shown in Fig. 4), pET28a (+)-DERAPya、pET28a(+)-DERAEco(F200I), respectively by plasmid
Import e. coli bl21 (DE3) competent cell, obtain expressing the recombination engineering of aldolase.Picking thalline contains in 2m1
Overnight incubation in the test tube of final concentration of 50 μ g/ml kanamycin.
Above-mentioned incubated overnight liquid is transferred to equipped with in 200ml TB culture fluid (containing 50 μ g/ml kanamycin) shaking flask,
Being 37 DEG C in temperature, rotating speed is to cultivate to OD under the conditions of 200rpm600Value is that about 0.6-1, the IPTG adding final concentration 1mM lure
Leading, be 25 DEG C in temperature, rotating speed is overnight induction under the conditions of 200rpm.
Collecting the bacterium solution that fermentation is complete, 6000rpm is centrifuged 10min, abandons supernatant, and the ice-cold PBS of thalline washs 1 time,
Containing pET28a (+)-DERAEco(F200I) recombination engineering bacterium solution Imidazole-HCl (pH7.0) weight of 40ml 0.1M
Outstanding, the sodium-acetate buffer (pH6.0) of other recombination engineering bacterium solution 40ml0.1M is resuspended, and height crushes born of the same parents, 10000rpm from
Heart 10min, takes supernatant, obtains each crude enzyme liquid.Wherein, containing double mutant enzyme DERAPyaThe electricity of the crude enzyme liquid of (I174V, V187I)
Swimming testing result is shown in Fig. 5.
Embodiment 2 utilizes aldolase to synthesize statins drug midbody
It is utilized respectively each crude enzyme liquid synthesis statins drug midbody prepared by embodiment 1 the 5th part, except crude enzyme liquid kind
Outside different with buffer, other conditions are the most identical.Course of reaction is as follows:
In 50ml reaction system, addition 38.1ml buffer, 10ml crude enzyme liquid, 200mM acetaldehyde and 100mM 2-Chloro-1-ethanal,
25 DEG C, catalytic reaction 12h under the conditions of 700rpm；
Wherein, containing pET28a (+)-DERAEco(F200I) the produced crude enzyme liquid of recombination engineering uses Imidazole-HCl
(pH7.0) making buffer, the produced crude enzyme liquid of other recombination engineerings uses 0.1M sodium acetate (pH6.0) to make buffer.
After reaction terminates, adding the acetone of two volumes, 10000rpm is centrifuged 10min, takes supernatant, and rotary evaporation falls third
Ketone, ethyl acetate extracts 3 times, combined ethyl acetate layer, and rotation is steamed, and obtains the oily liquids of brown color, and gas chromatography mass spectrometry detects, detection
Conditional parameter is: sample introduction 280 DEG C；Detector 280 DEG C；Column temperature: 2min at 100 DEG C, is warming up to 250 DEG C with 10 DEG C/min, 250 DEG C
Lower 2min, HP-5 post.The mass spectral results of product is shown in Fig. 6 and Fig. 7, product (4R, 6S)-2 in each reaction system, 4-dihydroxy-6 chlorine
The concentration (peak area accounting) of Methylpyrane is shown in Table 1.
From Fig. 6 and Fig. 7, the mass spectrum of two products consistent with the report of document (A Highly Productive,
Whole-Cell DERA Chemoenzymatic Process for Production of Key Lactonized Side-
Chain Intermediates in Statin Synthesis.Matej Osˇlaj,Je′ro^e Cluzeau,Damir
Orkic′,Gregor Kopitar,Peter Mrak,Zdenko Cˇasar.Plos one,2013,8(5),e62250)。
Table 1
From table 1, double mutant enzyme DERAPyaThe product of (I174V, V187I) relatively wild type improves 3 times, relatively large intestine bar
Bacterium DERAEco (F200I) improves about 8 times, relatively single mutation enzyme DERAPya(I174V) improve 1.7 times, relatively single mutation enzyme
DERAPya(V187I) improve 1.3 times.It is pointed out that the sudden change of not all sites all can improve DERAPyaCatalysis
Activity, such as mutant DERAPya(A67G) the 92% of catalysis activity only about wild type, slightly reduces.
Claims (8)
1. an aldolase, it is characterised in that aminoacid sequence is as shown in SEQ ID No.1.
2. the gene encoding aldolase as claimed in claim 1, it is characterised in that base sequence such as SEQ ID No.2 institute
Show.
3. the expression unit containing gene as claimed in claim 2.
Express unit the most as claimed in claim 3, it is characterised in that promoter is T7 promoter, lac promoter or araBAD
Promoter.
5. the recombinant vector containing gene as claimed in claim 2.
6. the transformant containing gene as claimed in claim 2.
7. transformant as claimed in claim 6, it is characterised in that host cell is escherichia coli.
8. aldolase application in producing statins drug midbody as claimed in claim 1.
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Effective date of registration: 20181128 Address after: 315800 No. 18 Workshop of Jingang Pioneering Base, Southern Binhai New Area, Ningbo, Ningbo City, Zhejiang Province Patentee after: Ningbo Heheng Bioengineering Co., Ltd. Address before: 315100 No. 1 Qian Hunan Road, Ningbo City, Zhejiang Province Patentee before: Ningbo Institute of Technology, Zhejiang University |
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