CN104293757A

CN104293757A - Aldolase and encoding gene and application thereof

Info

Publication number: CN104293757A
Application number: CN201410521149.4A
Authority: CN
Inventors: 金志华; 吴志革; 金庆超; 杨郁; 蔡伟; 张鑫红; 戎凯
Original assignee: Ningbo Institute of Technology of ZJU
Current assignee: Ningbo Heheng Bioengineering Co., Ltd.
Priority date: 2014-09-30
Filing date: 2014-09-30
Publication date: 2015-01-21
Anticipated expiration: 2034-09-30
Also published as: CN104293757B

Abstract

The invention discloses aldolase and an encoding gene and application thereof. The aldolase has an amino acid sequence shown in SEQ ID No.1, and a base sequence of the encoding gene is shown in SEQ ID No.2. The application refers to application of the aldolase in production of statin medicine intermediates. On the basis of wild aldolase of thermophilic bacteria, the encoding gene is subjected to both random mutation and site-specific mutagenesis to obtain combined mutation gene. According to the aldolase expressed by the combined mutation gene, the substrate degeneration resistance of the wild aldolase is retained and the catalytic activity is greatly improved. When the aldolase is used for catalyzing aldol condensation between acetaldehyde and chloroacetaldehyde, the catalytic efficiency is improved by three times compared with that of the wild aldolase. Therefore, the aldolase has good industrial application prospects.

Description

A kind of zymohexase and encoding gene thereof and application

Technical field

The invention belongs to technical field of molecular biology, be specifically related to a kind of zymohexase and encoding gene thereof and application.

Background technology

Statins is reducing blood-fat key agents, all with side chain (3R in its structure, 5S)-dihydroxy ester structure, this structure can be used as first hydroxyl glutaryl coenzyme A (HMG-CoA) reductase inhibitor, HMG-CoA is suppressed to transform to the reductibility of mevalonic acid, and then reduce the level of low density lipoprotein cholesterol, thus reach the object reducing blood fat.

Although statins chemical structure is relatively simple, its preparation process difficulty is comparatively large, and reason has two, and one is that side chain (3R, 5S)-dihydroxy ester has 2 chiral centres, is difficult to be completed by single step reaction; Two is that the enantiomeric excess of side chain (3R, 5S)-dihydroxy ester in Statins molecule (e.e.) and diastereomeric excess (d.e.) value are greater than 99.5% and 99% respectively, makes the fractionation of enantiomorph more difficult.These factors all cause statins side chain synthetic yield lower, and cost is higher.

Biocatalysis is the important channel solving current problem.At present, have been found that biological enzyme has catalytic effect to aldol reaction, wherein studying is colibacillary zymohexase DERA comparatively widely, its can catalysis generate have two chiral centres product, such as can catalysis acetaldehyde and monochloroacetaldehyde generation aldol reaction, generate the chloro-(3R of 6-, 5S)-dihydroxyl aldehyde, 6-chloro-(3R, 5S)-dihydroxyl aldehyde can turn to (4R by automatic ring, 6S)-2,4-dihydroxyl-6 chloromethyl pyrans:

(4R, 6S)-2,4-dihydroxyl-6 chloromethyl pyrans can be used as statins side chain synthesis an important intermediate.But due to colibacillary zymohexase, to there is catalysis aldehyde contracting reactive behavior not high, the problems such as anti-substrate sex change ability, 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 Sch ü rmann, Michael Wolberg, Iris Hilker, Ruud Luiten, Marcel Wubbolts and Daniel Mink.Biotechnol.J.2006, 1, 537 – 548) orthogenesis is carried out to colibacillary zymohexase, although the zymohexase enzyme activity after sudden change and anti-substrate sex change ability increase, but required higher acetaldehyde and monochloroacetaldehyde concentration in industrial production relatively, its anti-acetaldehyde and monochloroacetaldehyde sex change ability still more limited, still be difficult to the demand adapting to scale operation.

Zymohexase expressed by thermophile bacteria Pyrobaculum aerophilum, its anti-substrate sex change ability is stronger, but its catalytic activity is lower, if its catalytic activity can be improved on the basis keeping its anti-substrate sex change ability, will greatly reduce the difficulty preparing statins.

Summary of the invention

The invention provides a kind of zymohexase, this zymohexase has high catalytic activity, and anti-substrate sex change is very capable.

A kind of zymohexase, its aminoacid sequence is as shown in SEQ ID No.1.

Present invention also offers a kind of gene of described zymohexase of encoding, the base sequence of this gene is as shown in SEQ ID No.2.

The encoding gene of the present invention to thermophile bacteria (Pyrobaculum aerophilum) wild-type zymohexase suddenlys change, the codon (ATT) of 174 coding Isoleucines is sported the codon (GTT) of encode valine, the codon (GTG) of 187 encode valine is sported the codon (ATT) of coding Isoleucine.

The preparation method of described zymohexase is:

(1) with the encoding gene of thermophile bacteria (Pyrobaculum aerophilum) wild-type zymohexase for template, carry out fallibility PC amplification, with amplified fragments build recombinant vectors, obtain mutant gene libraries;

(2) by mutant gene libraries transformed competence colibacillus cell, mutated library is built;

(3) utilize inductor induced mutation library to express, obtain expression library;

(4) enzyme analysis alive and sequencing are carried out to expression library, screening object mutator gene;

Pass through aforesaid method, the present invention has first filtered out two object mutator genes, the point mutation of these two object mutator genes occurs in 174 and 187 respectively, the zymohexase that these two object mutator genes are expressed, the zymohexase that its catalytic activity is produced than wild-type zymohexase and the recombinant bacterial strain containing other mutator genes all increases.

(5) based on above-mentioned purpose mutator gene, carry out rite-directed mutagenesis, obtain double mutant gene;

On the basis of step (4), the present invention for template, carries out rite-directed mutagenesis to its 187 with the object mutator gene of 174, obtains the double mutant gene occurring in 174,187; With the object mutator gene of 187 for template, carry out rite-directed mutagenesis to its 174, also can obtain the double mutant gene occurring in 174,187.

(6) build the recombinant bacterial strain containing double mutant gene, after inducing culture, separation and purification is carried out to the crude enzyme liquid obtained, obtain zymohexase.

The zymohexase that this double mutant gene is expressed, the zymohexase that its catalytic activity is produced than the gene of sudden change separately increases again, obtains zymohexase of the present invention thus.

In the present invention, the primer carrying out 174 rite-directed mutagenesises used is:

Upstream primer: 5 '-GGAGAGGGCGGCGGCAGTTGCCCGCTACATAAAAGAG-3 ' (SEQ ID No.4);

Downstream primer: 5 '-CTCTTTTATGTAGCGGGCAACTGCCGCCGCCCTCTCC-3 ' (SEQ ID No.5);

The primer carrying out 187 rite-directed mutagenesises used is:

Upstream primer: 5 '-GGTATAGACTGGGGATTAAAATGGCGGGGGGGATTAGG-3 ' (SEQ ID No.6);

Downstream primer: 5 '-CCTAATCCCCCCCGCCATTTTAATCCCCAGTCTATACC-3 ' (SEQ ID No.7).

Present invention also offers expression unit, recombinant vectors or the transformant containing described gene.

As preferably, the promotor of described expression unit is T7 promotor, lac promotor or araBAD promotor.Under the effect of these promotors, zymohexase directly can realize soluble-expression in born of the same parents in e. coli host cell.

The initial carrier of described recombinant vectors can select PET21a, PET28a, PET30a.

As preferably, host cell is intestinal bacteria.

Present invention also offers the application of described zymohexase in production statins drug midbody.As do not made specified otherwise, statins drug midbody described in the present invention refers to (4R, 6S)-2,4-dihydroxyl-6 chloromethyl pyrans.

Zymohexase of the present invention has not only possessed the anti-substrate sex change ability of wild-type zymohexase, and its catalytic activity also improves greatly; Utilize this enzyme catalysis acetaldehyde and monochloroacetaldehyde generation aldol reaction, its catalytic efficiency improves 3 times than wild-type zymohexase, than document (Directed evolution of an industrial biocatalyst:2-deoxy-D-ribose 5-phosphate aldolase.Stefan Jennewein, Martin Sch ü rmann, Michael Wolberg, Iris Hilker, Ruud Luiten, Marcel Wubbolts and Daniel Mink.Biotechnol.J.2006,1,537 – 548) disclosed in intestinal bacteria Aldolase mutant improve 8 times.

Compared with prior art, beneficial effect of the present invention is:

The present invention is on the basis of thermophile bacteria (Pyrobaculum aerophilum) wild-type zymohexase, random mutation superposition rite-directed mutagenesis is carried out to its encoding gene, obtain double mutant gene, the zymohexase that this double mutant gene is expressed not only has possessed the anti-substrate sex change ability of wild-type zymohexase, and its catalytic activity also improves greatly; Utilize this enzyme catalysis acetaldehyde and monochloroacetaldehyde generation aldol reaction, its catalytic efficiency improves 3 times than wild-type zymohexase; Show that zymohexase 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 invention _pyathe electrophoresis detection result figure of (I174V, V187I);

Fig. 3 is plasmid pET28a (+)-DERA being built with double mutant gene of the present invention _pyathe gene mapping of (I174V, V187I);

The electrophoresis detection result figure that Fig. 4 is plasmid shown in Fig. 3;

Fig. 5 produces by recombinant bacterial strain of the present invention the electrophoresis detection result figure of crude enzyme liquid;

Wherein, Marker represents protein molecular weight standard; DERA _pya(I174V, V187I) represents crude enzyme liquid that recombinant bacterial strain produces;

Fig. 6 is DERA _pyathe mass spectrum of product 6-chloro-(3R, 5S)-dihydroxyl aldehyde in (I174V, V187I) catalyst system;

Fig. 7 is DERA _pyathe mass spectrum of product (4R, 6S)-2,4-dihydroxyl-6 chloromethyl pyrans in (I174V, V187I) catalyst system;

Wherein, Abundance represents abundance, and m/z represents mass-to-charge ratio.

Embodiment

Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.

The preparation of embodiment 1 pair sudden change zymohexase

1, the encoding gene of thermophile bacteria wild-type zymohexase is synthesized

According to the coding gene sequence of thermophile bacteria wild-type zymohexase disclosed on NCBI, entrust this coding gene sequence of Shanghai Jierui Biology Engineering Co., Ltd's synthetic, its base sequence is as shown in SEQ ID N0.3.

2, mutator gene qualification and plasmid order-checking

1. with the encoding gene of thermophile bacteria (Pyrobaculum aerophilum) wild-type zymohexase for template, carry out fallibility pcr amplification.

Wherein, PCR reaction system is: pure water 21.5 μ L; 10 × buffer (Mg ²⁺free), 5 μ L; Upstream primer (concentration 10 μMs) 1 μ L; Downstream primer (concentration 10 μMs) 1 μ L; DNTP (2.5mM each), 4 μ L; Mgcl ₂(25mM), 14 μ L; MnCl ₂(5mM), 1.5 μ L; Template, 1 μ L; Taq polymerase, 1 μ L; Cumulative volume is 50 μ L.

PCR condition is: enter amplification cycles after 94 DEG C of sex change 5min, i.e. 94 DEG C of sex change 30s, 57 DEG C of annealing 30s, and 72 DEG C extend 1min, circulate 30 times altogether, and last 72 DEG C extend 10min.The sequence of PCR primer is:

Upstream primer: 5 '-CTA gAATTCaTGATACATTTAGTAGACTACGCGC-3 ' (underscore place is EcoRI restriction enzyme site) (SEQ ID No.8);

Downstream primer: 5 '-GTT cTCGAGcTATAGAAGAGCCTCTGGGGTGG-3 ' (underscore place is XhoI restriction enzyme site) (SEQ ID No.9).

2. the fragment above-mentioned amplification obtained, by EcoRI and XhoI double digestion, is connected to pET28a (+) carrier (adopting DNA ligation kit Ver.2.1 test kit), obtains mutant gene libraries;

3. by said mutation gene library transformation of E. coli (E.coli) BL21 (DE3) competent cell, be applied on the LB agar plate containing 50 μ g/ml kantlex, cultivate 15 hours, obtain mutant strain library for 37 DEG C;

4. said mutation strain clone is transferred in 24 orifice plates that 400 μ L substratum are housed and cultivates, inducing culture 12 hours at 1mM IPTG is 25 DEG C, obtain expression library;

5. thalline is collected, with the resuspended thalline of 300 μ L 0.1M sodium acetate soln, ultrasonication, is transferred to broken cytosol in 24 orifice plates, add 200mM acetaldehyde and 100mM monochloroacetaldehyde, 25 DEG C are reacted 2 hours, add the acetonitrile that nonuploid is long-pending, centrifugal segregation protein, by the chloro-(3R of product 6-in each reaction system of gas chromatographic detection, 5S)-dihydroxyl aldehyde, (4R, 6S)-2,4-content of dihydroxyl-6 chloromethyl pyrans.

Screening obtains two mutant that products collection efficiency is improved thus, wherein, and the DERA of mutant (I174V) _pyain gene, the codon (ATT) of 174 coding Isoleucines sports the codon (GTT) of encode valine, the DERA of mutant (V187I) _pyain gene, the codon (GTG) of 187 encode valine sports the codon (ATT) of coding Isoleucine.

3, the preparation of double mutant gene

Sequence according to SEQ ID No.3, design rite-directed mutagenesis primer, carries out rite-directed mutagenesis.The principle schematic of rite-directed mutagenesis is as Fig. 1.

Wherein, the primer carrying out suddenling change at the 174th is:

Upstream 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 be:

Upstream primer: 5 '-GGTATAGACTGGGGATTAAAATGGCGGGGGGGATTAGG-3 ' (SEQ ID No.6);

With the single mutation gene of 174 for template, utilize and carry out rite-directed mutagenesis PCR at the 187th primer carrying out suddenling change, result has found a double mutant gene (electrophoresis detection the results are shown in Figure 2) from recording gene, 174th amino acids residue codon usage of this genes encoding sports α-amino-isovaleric acid (GTT) by Isoleucine (ATT), the amino-acid residue codon of 187 sports Isoleucine (ATT) by α-amino-isovaleric acid (GTG), and therefore the zymohexase of this genes encoding is two mutant enzymes.The aminoacid sequence of two mutant enzyme is as shown in SEQ.No.1.With the single mutation gene of 187 for template, utilize the primer carrying out suddenling change at the 174th to carry out rite-directed mutagenesis PCR, identical result can be obtained.

PCR reaction system is: pure water 38.5 μ L; Pfu buffer 5 μ L; Upstream 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: enter amplification cycles after 94 DEG C of sex change 5min, i.e. 94 DEG C of sex change 30s, 57 DEG C of annealing 30s, and 68 DEG C extend 6min, circulate 20 times altogether, and last 68 DEG C extend 10min.

4, the expression of zymohexase and preparation

Respectively with single mutation gene DERA _pya(I174V), single mutation gene DERA _pya(V187I), single mutation gene DERA _pya(A67G), double mutant gene DERA _pya(I174V, V187I), wild type gene DERA _pya, intestinal bacteria zymohexase mutator gene is (see document: Directed evolution of an industrial biocatalyst:2-deoxy-D-ribose 5-phosphate aldolase.Stefan Jennewein, Martin Sch ü rmann, Michael Wolberg, Iris Hilker, Ruud Luiten, Marcel Wubbolts and Daniel Mink.Biotechnol.J.2006,1,537 – 548) build plasmid: pET28a (+)-DERA _pya(I174V), pET28a (+)-DERA _pya(V187I), pET28a (+)-DERA _pya(A67G), pET28a (+)-DERA _pya(I174V, V187I) (its plasmid map is shown in Fig. 3, and electrophoresis detection the results are shown in Figure 4), pET28a (+)-DERA _pya, pET28a (+)-DERA _eco(F200I), plasmid is imported e. coli bl21 (DE3) competent cell respectively, obtain the recombinant bacterial strain of expressing zymohexase.It is overnight incubation in the test tube of 50 μ g/ml kantlex that picking thalline contains final concentration in 2m1.

Being transferred to by above-mentioned incubated overnight liquid and being equipped with in 200ml TB nutrient solution (containing 50 μ g/ml kantlex) shaking flask, is 37 DEG C in temperature, and rotating speed is be cultured to OD under 200rpm condition ₆₀₀value is about 0.6-1, and add the IPTG induction of final concentration 1mM, be 25 DEG C in temperature, rotating speed is overnight induction under 200rpm condition.

Collect the bacterium liquid that fermentation is complete, the centrifugal 10min of 6000rpm, abandons supernatant, and the ice-cold PBS of thalline washs 1 time, containing pET28a (+)-DERA _eco(F200I) Imidazole-HCl (pH7.0) of recombinant bacterial strain bacterium liquid 40ml 0.1M is resuspended, the sodium-acetate buffer (pH6.0) of other recombinant bacterial strain bacterium liquid 40ml0.1M is resuspended, height crushes born of the same parents, the centrifugal 10min of 10000rpm, get supernatant, obtain each crude enzyme liquid.Wherein, containing two mutant enzyme DERA _pyathe electrophoresis detection of the crude enzyme liquid of (I174V, V187I) the results are shown in Figure 5.

Embodiment 2 utilizes zymohexase to synthesize statins drug midbody

The each crude enzyme liquid synthesis statins drug midbody utilizing embodiment 1 the 5th part to prepare respectively, except crude enzyme liquid kind is different with damping fluid, other conditions are all identical.Reaction process is as follows:

In 50ml reaction system, add 38.1ml damping fluid, 10ml crude enzyme liquid, 200mM acetaldehyde and 100mM monochloroacetaldehyde, 25 DEG C, catalyzed reaction 12h under 700rpm condition;

Wherein, containing pET28a (+)-DERA _eco(F200I) crude enzyme liquid that recombinant bacterial strain produces adopts Imidazole-HCl (pH7.0) to make damping fluid, and crude enzyme liquid that other recombinant bacterial strains produce adopts 0.1M sodium-acetate (pH6.0) to make damping fluid.

After reaction terminates, add the acetone of two volumes, the centrifugal 10min of 10000rpm, gets supernatant, rotary evaporation falls acetone, extraction into ethyl acetate 3 times, and combined ethyl acetate layer, revolves steaming, obtain the oily liquids of brown color, gas chromatography mass spectrometry detects, and testing conditions 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, 2min, HP-5 post at 250 DEG C.The mass spectral results of product is shown in Fig. 6 and Fig. 7, and in each reaction system, the concentration (peak area accounting) of product (4R, 6S)-2,4-dihydroxyl-6 chloromethyl pyrans is in table 1.

From Fig. 6 and Fig. 7, the mass spectrum of two products (A Highly Productive consistent with the report of document, Whole-Cell DERA Chemoenzymatic Process for Production of Key Lactonized Side-Chain Intermediates in Statin Synthesis.Matej Os ˇ laj, Je ' ro^e Cluzeau, Damir Orkic ', Gregor Kopitar, Peter Mrak, Zdenko C ˇ asar.Plos one, 2013,8 (5), e62250).

Table 1

From table 1, two mutant enzyme DERA _pyathe product of (I174V, V187I) comparatively wild-type improves 3 times, and comparatively intestinal bacteria DERAEco (F200I) improves about 8 times, comparatively single mutation enzyme DERA _pya(I174V) improve 1.7 times, comparatively single mutation enzyme DERA _pya(V187I) improve 1.3 times.It is pointed out that the sudden change of not all sites all can improve DERA _pyacatalytic activity, as mutant DERA _pya(A67G) catalytic activity approximately only has 92% of wild-type, slightly reduces.

Claims

1. a zymohexase, is characterized in that, aminoacid sequence is as shown in SEQ ID No.1.

2. encode the gene of zymohexase as claimed in claim 1, it is characterized in that, base sequence is as shown in SEQ ID No.2.

3. one kind contains the expression unit of gene as claimed in claim 2.

4. express unit as claimed in claim 3, it is characterized in that, promotor is T7 promotor, lac promotor or araBAD promotor.

5. one kind contains the recombinant vectors of gene as claimed in claim 2.

6. one kind contains the transformant of gene as claimed in claim 2.

7. transformant as claimed in claim 6, it is characterized in that, host cell is intestinal bacteria.

8. the application of zymohexase in production statins drug midbody as claimed in claim 1.