CN102559722B

CN102559722B - Heatproof aldolase gene with high enzyme activity, aldolase, carrier, engineering bacteria and application

Info

Publication number: CN102559722B
Application number: CN201210064027.8A
Authority: CN
Inventors: 谢恬; 王秋岩; 杜鹏飞
Original assignee: Hangzhou Normal University
Current assignee: Hangzhou Normal University
Priority date: 2012-03-12
Filing date: 2012-03-12
Publication date: 2017-05-17
Anticipated expiration: 2032-03-12
Also published as: CN102559722A

Abstract

The invention provides an aldolase gene, aldolase coded by the gene, a carrier containing the gene, engineering bacteria and application of the gene. The gene is from Pyrococcus kodakaraensis. The nucleotide sequence of the aldolase gene (DERA) is shown in the SEQ ID NO.1 and the amino acid sequence of aldolase is shown in the SEQ ID NO.2. Aldolase can be used for synthesizing 2,4,6-tri-deoxyhexamethylose compounds, is environment-friendly and has broad application prospect.

Description

A kind of high enzyme activity, heat-resisting aldolase gene, enzyme, carrier, engineering bacteria and application

(1) technical field

The present invention relates to a kind of aldolase gene, and the aldolase of the gene code, and the carrier containing the gene, Engineering bacteria and its application.

(2) background technology

Aldolase can be widely applied in organic chemical synthesis as the enzyme of catalysis carbon carbon atom solid bonding, it Easily asymmetry catalysis synthesis can be carried out to big complicated molecule direction by starting point of small molecule, this process is in carbon aquation Compound is especially prominent for meaning in the reaction of substrate.Using the high regioselectivity of enzyme, can easily avoid to carbohydrate In active hydroxyl protection, more prominent is be catalyzed carbon-carbon bond synthesize while aldolase can introduce new chirality Center.The exactly this ability that can efficiently introduce chiral carbon atom so that aldolase has in drug molecule synthesis field It is widely applied, compared with chemical technology, has a clear superiority, such as：The use of poisonous and harmful substance is avoided, synthesis can be normal Carry out under the conditions of normal temperature and pressure.From the angle for substituting traditional chemical synthesis technique catalyst, the enzymatic reaction has very strong Green attribute, to the Green Chemistry of traditional chemical technique important in inhibiting is transformed, thus the enzyme has broad application prospects.

(3) content of the invention

It is an object of the invention to provide a kind of aldolase gene, the aldolase of the gene code, and containing the gene Carrier, engineering bacteria and its application.

The technical solution used in the present invention is：

A kind of aldolase gene (DERA) from Pyrococcus kodakaraensis, its nucleotide sequence is such as Shown in SEQ ID NO.1：

atgaacaaac gtgaaattgc acgttacatt gatcagacca acctgaaacc atacgcgact 60

aaagaagata tcatcaagct gtgtgatgaa gcgatcgaat acggctttta cgccgtttgc 120

gtgaacccgt accgtgtgaa gctggcgaaa gactatctgc gtgaaaagaa tgctgatgtt 180

aaagtagcat ctgttatcgg tttcccgctg ggcgccaccc cgaccgaagt taaagttttc 240

gaagccaaac gtgctctgga agatggtgca gacgagctgg acatggttat caacatcggc 300

gcgctgaaag ataaagacta tgaatatgtt aagaacgaca tcgcagaagt ggttaaagta 360

gcgcacgaac gcggcgctaa agtgaaagta attatcgaaa cttgctacct gaccgaagag 420

gagaaagtga aagcgtgtga gctggccaaa gaggcgggtg ctgacttcgt taagactagc 480

accggtttcg gtaccggtgg tgctaccgtg gaagacgtgc gtctgatgcg caaagtggtt 540

ggcccggaaa tgggcgtaaa agcagctggt ggtattcgca cctatgaaca ggctctggag 600

atgatcgaag caggtgcgaa ccgtatcggc acctcctctg gcgtaaaaat tgtagaaggt 660

gcgccggacg aactcgag 678

Due to the particularity of nucleotide sequence, any SEQ NO：The variant of polynucleotide shown in 1, as long as itself and the multinuclear Thuja acid has more than 90% homology, belongs to the row of the scope of the present invention.The variant of the polynucleotide refers to a kind of tool There is the polynucleotide sequence that one or more nucleotide change.The variant of this polynucleotide can make raw allelic variant or non- Raw variant, including substitution variants, Deletion variants and insert variation.As known in the art, allelic variant is The alternative forms of one polynucleotide, it is probably the replacement of a multiple nucleotide, disappearance or inserts, but will not be from substantial Change the function of the aminoacid of its coding.

A kind of high enzyme activity, heat-resisting aldolase, are encoded by the aldolase gene.Specifically, the aldolase aminoacid Sequence is as shown in SEQ ID NO.2：

Met Asn Lys Arg Glu Ile Ala Arg Tyr Ile Asp Gln Thr Asn Leu Lys

1 5 10 15

Pro Tyr Ala Thr Lys Glu Asp Ile Ile Lys Leu Cys Asp Glu Ala Ile

20 25 30

Glu Tyr Gly Phe Tyr Ala Val Cys Val Asn Pro Tyr Arg Val Lys Leu

35 40 45

Ala Lys Asp Tyr Leu Arg Glu Lys Asn Ala Asp Val Lys Val Ala Ser

50 55 60

Val Ile Gly Phe Pro Leu Gly Ala Thr Pro Thr Glu Val Lys Val Phe

65 70 75 80

Glu Ala Lys Arg Ala Leu Glu Asp Gly Ala Asp Glu Leu Asp Met Val

85 90 95

Ile Asn Ile Gly Ala Leu Lys Asp Lys Asp Tyr Glu Tyr Val Lys Asn

100 105 110

Asp Ile Ala Glu Val Val Lys Val Ala His Glu Arg Gly Ala Lys Val

115 120 125

Lys Val Ile Ile Glu Thr Cys Tyr Leu Thr Glu Glu Glu Lys Val Lys

130 135 140

Ala Cys Glu Leu Ala Lys Glu Ala Gly Ala Asp Phe Val Lys Thr Ser

145 150 155 160

Thr Gly Phe Gly Thr Gly Gly Ala Thr Val Glu Asp Val Arg Leu Met

165 170 175

Arg Lys Val Val Gly Pro Glu Met Gly Val Lys Ala Ala Gly Gly Ile

180 185 190

Arg Thr Tyr Glu Gln Ala Leu Glu Met Ile Glu Ala Gly Ala Asn Arg

195 200 205

Ile Gly Thr Ser Ser Gly Val Lys Ile Val Glu Gly Ala Pro Asp Glu

210 215 220

Leu Glu

225

The experiment proved that, the protein of said structure has high temperature resistant, high enzyme than characteristic living.

Due to the particularity of aminoacid sequence, it is any containing the fragment of the polypeptide of aminoacid sequence shown in SEQ NO.2 or its Variant, such as its examples of conservative variations, bioactive fragment or derivant, as long as the fragment or polypeptide variants of the polypeptide and aforementioned amino Acid sequence homology belongs to the row of the scope of the present invention more than 95%.Specifically, the change may include aminoacid sequence The disappearance of aminoacid, insertion or replacement in row；Wherein, for the conservative of variant sexually revises, the aminoacid replaced has and original The structure or chemical property of amino acid similarity, such as replaces isoleucine with leucine, and variant also can change with non-conservation, such as Glycine is replaced with tryptophan.

The invention further relates to a kind of recombinant vector containing the aldolase gene, and recombinant vector conversion is obtained Recombination engineering bacteria.

The invention further relates to application of the described aldolase gene in Prepare restructuring aldolase.

Specifically, described application is：The recombinant vector containing the aldolase gene is built, the recombinant vector is turned Change into escherichia coli, the recombination engineering bacteria of acquisition carries out inducing culture (generally with IPTG as derivant), culture fluid point From obtain containing restructuring aldolase somatic cells.

The present invention is characterized by there is provided the aminoacid shown in the nucleotide sequence shown in SEQ NO.1 and SEQ NO.2 Sequence, in the case of the known nucleotide sequence and aminoacid sequence, the acquisition of the nucleotide sequence and aminoacid sequence, with And related recombinant vector, the acquisition of genetic engineering bacterium, it is to those skilled in the art obvious.

The high enzyme activity of the present invention, heat-resisting aldolase can be obtained as follows：

1st, the cDNA synthesis of DERA full length genes and clone：According to the nucleotide sequence of DERA genes, full genome conjunction is carried out Into the aldolase gene of the nucleotide sequence shown in acquisition SEQ ID NO.1.

2nd, the structure of the expression vector containing genes of interest：The aldolase gene that step 1 is obtained is cloned into into intermediate carrier.

3rd, the recombinant expression carrier that step 2 is obtained is proceeded in host cell, such as escherichia coli (Escherichia Coli) BL 21, under conditions of expression aldolase is adapted to, cultivate the host cell.

4th, aldolase is isolated from the culture of step 3.

The beneficial effects are mainly as follows：There is provided a kind of aldolase, and encode the gene of the aldolase, carry Body, engineering bacteria and its application, the aldolase can be used for the synthesis of 2,4,6- tri- deoxyhexamethylose compounds, environmental protection, using front Scape is wide.

(4) illustrate

Fig. 1 is the aldolase expression of results (M of Pyrococcus kodakaraensis：marker；2：Whole cell albumen； 3：DERA).

Fig. 2 is NADH light absorption value change curves at 340nm in DERA cracking DRI -5- phosphate ester reactions.

Fig. 3 is the relative activity of DERA after treatment of different temperature.

(5) specific embodiment

With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in This：

Embodiment 1：CDNA synthesis and the clone of DERA full length genes

The nucleotide sequence of the DERA genes from Pyrococcus kodakaraensis in NCBI, hands in sea Sheng Gong biotechnologies company limited carries out full genome synthesis, successfully obtains the sequence of nucleotide shown in SEQ ID NO.1.Root According to the DERA gene coded sequences (SEQ ID NO.1) of Pyrococcus kodakaraensis, design amplifies complete coding The primer of reading frame, and introduce restriction endonuclease sites respectively on upstream and downstream primer, so as to expression vector.With gene The product of synthesis is template, Jing after PCR amplifications, the DERA of Pyrococcus kodakaraensis is cloned into into intermediate carrier PET303/CT (is purchased from Invitrogen companies), the expression vector identified under the premise of ensureing that reading frame is correct, then will It is proceeded in E.Coil BL21, obtains engineering bacteria E.Coil BL21/pET 303CT/DERA.

Embodiment 2：The expression of DERA albumen

By the E.Coil BL21/pET 303CT/DERA obtained in embodiment 1 in the g/mL ammonia benzyl penicillium sp of μ containing 100mL100 Incubator overnight culture in the LB fluid mediums of element.Pour the bacterium solution after 10ml incubated overnight into 1L 100 μ g/mL ammonia benzyl penicillium sp The LB fluid medium cultures of element, until bacterium solution OD₆₀₀The final concentration of 0.5mM of IPTG, 28 DEG C of inductions are added when reaching 0.6~0.8 After 16h, thalline is collected by centrifugation, thalline makes its resuspension, sonicated cells with 25ml phosphate buffer pH7.4.In centrifuging and taking Clearly, affinity chromatography carries out purification and obtains the pure enzymes of DERA after the supernatant containing DERA is filtered with 0.22 μm of cellulose acetate sheets. Comprehensive BCA methods pheron concentration mensuration data, the expression of DERA enzymes reaches 56mg/L (Fig. 1) in LB culture medium.

Embodiment 3：The specific enzyme activity power of DERA is determined

The present invention with DRI -5- phosphate esters as substrate, under room temperature condition, the molecule bottom of DERA catalytic pyrolysiss one Thing solution obtains a molecule glyceraldehyde 3-phosphate, its in the presence of NADH and triose-phosphate isomerase, by glycerol triphosphoric acid Dehydrogenase catalyzed, each molecule glyceraldehyde 3-phosphate is catalyzed one molecule NADH of consumption.By measuring NADH in 340nm wavelength UV absorbs to detect kinetic curve to analyze the natural substrate degrading activity for determining DERA.The consumption correspondence one of one molecule NADH The decomposition of molecule DRI -5- phosphate esters.

Concrete operations are as follows：Enzyme liquid is made into 100mM Triethanolamine buffers (pH8.0) dissolving DERA, is added to substrate Mixed solution (0.1mM NADH, 0.4mM DRI -5- phosphate esters, 11U/mL triose-phosphate isomerases, 4U/mL GPDH), light absorption value at 340nm is determined, measure per second is once.Calculated according to the data of 100 seconds before curve The speed of NADH extinction value changes is：-0.024min^-1(Fig. 2).Enzyme amount used by primary first-order equation is 5 μ L, and enzyme concentration Jing BCA methods are surveyed It is set to 12.5ng/ μ L, is according to the specific activity of enzyme that data above calculates DERA：31.27U/mg, with higher enzyme than living Power.

Embodiment 4：The heat stability of DERA

The enzyme liquids that are made into of DERA will be dissolved respectively at 20 DEG C, 30 DEG C, 40 DEG C with 100mM Triethanolamine buffers (pH8.0), 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C, temperature bath 10min, mixes afterwards, in 50 with reactant liquor described in embodiment 3 at 100 DEG C Decomposition vigor is determined at DEG C.The decomposition vigor determined under the same conditions with untreated DERA obtains each temperature as 100% DERA after process decomposes vigor relatively.As shown in figure 3, as a result showing the DERA from Pyrococcus kodakaraensis Enzyme has higher heat stability.

SEQUENCE LISTING

<110>Hangzhou Pedagogic University

<120>A kind of high enzyme activity, heat-resisting aldolase gene, enzyme, carrier, engineering bacteria and application

<130>

<160> 2

<170> PatentIn version 3.4

<210> 1

<211> 678

<212> DNA

<213> Pyrococcus kodakaraensis

<400> 1

atgaacaaac gtgaaattgc acgttacatt gatcagacca acctgaaacc atacgcgact 60

aaagaagata tcatcaagct gtgtgatgaa gcgatcgaat acggctttta cgccgtttgc 120

gtgaacccgt accgtgtgaa gctggcgaaa gactatctgc gtgaaaagaa tgctgatgtt 180

aaagtagcat ctgttatcgg tttcccgctg ggcgccaccc cgaccgaagt taaagttttc 240

gaagccaaac gtgctctgga agatggtgca gacgagctgg acatggttat caacatcggc 300

gcgctgaaag ataaagacta tgaatatgtt aagaacgaca tcgcagaagt ggttaaagta 360

gcgcacgaac gcggcgctaa agtgaaagta attatcgaaa cttgctacct gaccgaagag 420

gagaaagtga aagcgtgtga gctggccaaa gaggcgggtg ctgacttcgt taagactagc 480

accggtttcg gtaccggtgg tgctaccgtg gaagacgtgc gtctgatgcg caaagtggtt 540

ggcccggaaa tgggcgtaaa agcagctggt ggtattcgca cctatgaaca ggctctggag 600

atgatcgaag caggtgcgaa ccgtatcggc acctcctctg gcgtaaaaat tgtagaaggt 660

gcgccggacg aactcgag 678

<210> 2

<211> 226

<212> PRT

<213> Pyrococcus kodakaraensis

<400> 2

Met Asn Lys Arg Glu Ile Ala Arg Tyr Ile Asp Gln Thr Asn Leu Lys

1 5 10 15

Pro Tyr Ala Thr Lys Glu Asp Ile Ile Lys Leu Cys Asp Glu Ala Ile

20 25 30

Glu Tyr Gly Phe Tyr Ala Val Cys Val Asn Pro Tyr Arg Val Lys Leu

35 40 45

Ala Lys Asp Tyr Leu Arg Glu Lys Asn Ala Asp Val Lys Val Ala Ser

50 55 60

Val Ile Gly Phe Pro Leu Gly Ala Thr Pro Thr Glu Val Lys Val Phe

65 70 75 80

Glu Ala Lys Arg Ala Leu Glu Asp Gly Ala Asp Glu Leu Asp Met Val

85 90 95

Ile Asn Ile Gly Ala Leu Lys Asp Lys Asp Tyr Glu Tyr Val Lys Asn

100 105 110

Asp Ile Ala Glu Val Val Lys Val Ala His Glu Arg Gly Ala Lys Val

115 120 125

Lys Val Ile Ile Glu Thr Cys Tyr Leu Thr Glu Glu Glu Lys Val Lys

130 135 140

Ala Cys Glu Leu Ala Lys Glu Ala Gly Ala Asp Phe Val Lys Thr Ser

145 150 155 160

Thr Gly Phe Gly Thr Gly Gly Ala Thr Val Glu Asp Val Arg Leu Met

165 170 175

Arg Lys Val Val Gly Pro Glu Met Gly Val Lys Ala Ala Gly Gly Ile

180 185 190

Arg Thr Tyr Glu Gln Ala Leu Glu Met Ile Glu Ala Gly Ala Asn Arg

195 200 205

Ile Gly Thr Ser Ser Gly Val Lys Ile Val Glu Gly Ala Pro Asp Glu

210 215 220

Leu Glu

225

Claims

1. a kind of aldolase gene from Pyrococcus kodakaraensis, its nucleotide sequence such as SEQ ID Shown in NO.1.

2. a kind of recombinant vector containing aldolase gene described in claim 1, it is characterised in that the recombinant vector is to pass through By SEQ ID NO:The DERA of the Pyrococcus kodakaraensis shown in 1 is cloned in intermediate carrier pET303/CT and makes It is standby to form.

3. it is a kind of that the recombination engineering bacteria E.Coil BL21/ for obtaining are converted with recombinant vector described in claim 2 PET303CT/DERA, it is characterised in that the recombination engineering bacteria E.Coil BL21/pET303CT/DERA are by inciting somebody to action It is prepared from the recombinant vector Transformed E .Coil BL21.

4. application of the aldolase gene as claimed in claim 1 in Prepare restructuring aldolase.

5. application as claimed in claim 4, it is characterised in that described application is：By SEQ ID NO:Shown in 1 The DERA of Pyrococcus kodakaraensis is cloned in intermediate carrier pET303/CT, and structure is obtained containing the al The recombinant vector of enzyme gene；The recombinant vector is converted into escherichia coli E.Coil BL21, the recombination engineering of acquisition Bacterium E.Coil BL21/pET303CT/DERA；Recombination engineering bacteria E.Coil BL21/pET303CT/DERA are being contained Incubator overnight culture in the LB fluid mediums of 100mL100 μ g/mL ampicillin, the bacterium solution after 10ml incubated overnight is fallen Enter the LB fluid medium cultures of 1L100 μ g/mL ampicillin, until bacterium solution OD₆₀₀IPTG ends are added when reaching 0.6~0.8 Concentration is 0.5mM, after 28 DEG C of induction 16h, thalline is collected by centrifugation, and thalline makes its resuspension with 25ml phosphate buffer pH7.4, surpasses Sound smudge cellses；Centrifuging and taking supernatant, affinity chromatography enters after the supernatant containing DERA is filtered with 0.22 μm of cellulose acetate sheets Row purification obtains the pure enzymes of DERA.