CN103060281A - Fusion protein as well as coding gene and application thereof - Google Patents
Fusion protein as well as coding gene and application thereof Download PDFInfo
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- CN103060281A CN103060281A CN2013100287740A CN201310028774A CN103060281A CN 103060281 A CN103060281 A CN 103060281A CN 2013100287740 A CN2013100287740 A CN 2013100287740A CN 201310028774 A CN201310028774 A CN 201310028774A CN 103060281 A CN103060281 A CN 103060281A
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Abstract
The invention discloses a fusion protein as well as a coding gene thereof and application thereof. The fusion protein provided by the invention is protein which is obtained by in series connecting the connecting peptides of ethanol dehydrogenase and formate dehydrogenase. The ethanol dehydrogenase is from lactobacillus brevis and the formate dehydrogenase is from candida boidinii; the connecting peptides are rigid structures Linker (EAAAK). According to the experiment in the invention, the invention provides the fusion protein which is obtained by in series connecting the connecting peptides of ethanol dehydrogenase and formate dehydrogenase, wherein enzyme activity of the formate dehydrogenase is higher than the activity for independently expressing the formate dehydrogenase after the formate dehydrogenase is expressed in escherichia coli; moreover, the stability and heat stability of the ethanol dehydrogenase in organic substrate are also improved. The construction of the fusion protein provides important reference for the improvement to the preparation method for chiral alcohol and chiral hydroxy compound.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of fusion rotein and encoding gene thereof and application.
Background technology
Chiral alcohol with specific function group occupies critical role in the chiral drug preparation, it be multiple antiviral, antimycotic, antitumor and treat the important intermediate of the chiral drug of neural system, the disease such as cardiovascular.Therefore, the maturation of chiral alcohol synthetic method has positive promoter action to chiral drug synthetic with improving.
Enzyme catalysis method be more than 100 times of organic catalyst efficient generally speaking, and the purity of product is very high because to have a catalytic efficiency high, reaction conditions is gentle, generally just can react at ambient temperature, therefore, in recent years enzyme catalysis method in the preparation research of chiral alcohol by extensive concern.Ethanol dehydrogenase has the advantage that catalytic efficiency is high and the product enantiomeric purity is high and is widely used in the Study on Transformation of chiral alcohol, this enzyme belongs to a member of the oxydo-reductase superfamily (SDRs) of short chain, be homotetramer, the molecular weight of each monomer is 26.6kD, each monomer contains 251 amino acid, and catalysis produces specific R-configuration product.But owing to the reduced coenzyme that needs in this enzyme catalysis process in the consumption system, the extra interpolation of reduced coenzyme significantly increases reaction cost.Therefore the method that reducibility coenzyme is regenerated in the Development System becomes the key of research.
Summary of the invention
An object of the present invention is to provide a kind of fusion rotein and encoding gene thereof.
Fusion rotein provided by the invention is the protein that ethanol dehydrogenase and hydrogenlyase are obtained by the connection peptides series connection.
In the above-mentioned fusion rotein, described connection peptides is (EAAAK)
2, being rigid structure linker, its aminoacid sequence is that sequence 2 is from the terminal 365-374 of N ' position.
In the above-mentioned fusion rotein, described ethanol dehydrogenase derives from short lactobacillus (Lac tobacillus brevis), and described hydrogenlyase derives from Candida boidinii (Candida boidinii);
Described fusion rotein is following (a) or (b) specifically:
(a) protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 2;
(b) with the aminoacid sequence shown in the sequence in the sequence table 2 through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and have ethanol dehydrogenase and the two enzymes of hydrogenlyase are lived by the derivative protein of sequence 2.
The gene of above-mentioned fusion rotein of encoding also is the scope of protection of the invention.
Said gene is any dna molecular in following (1)-(3):
(1) encoding sequence is the dna molecular shown in the sequence 1 in the sequence table;
(2) the dna sequence dna hybridization that limits with (1) under stringent condition and coding have the dna molecular of ethanol dehydrogenase and the two enzyme living proteins of hydrogenlyase;
(3) dna sequence dna that limits with (1) has 70% at least, have at least 75%, have at least 80%, have at least 85%, have at least 90%, have at least 95%, have at least 96%, have at least 97%, have at least 98% or have at least a dna molecular that 99% homology and coding have ethanol dehydrogenase and the two enzyme living proteins of hydrogenlyase.
Recombinant vectors, expression cassette, transgenic cell line or the recombinant bacterium that contains said gene also is the scope of protection of the invention.
Above-mentioned recombinant vectors is that the gene of above-mentioned fusion rotein inserts in the expression vector, obtains expressing the recombinant vectors of above-mentioned fusion rotein.In an embodiment of the present invention, expression vector is pETDuet-1, and recombinant vectors pETDuet-FDH-linker-ADH is specially the carrier that obtains between the NcoI of the 1 insertion expression vector pETDuet-1 of sequence in the sequence table and XhoI restriction enzyme site.
Above-mentioned recombinant bacterium is described recombinant vectors to be imported in the Host Strains recombinant bacterium that obtains.
The application in improving hydrogenlyase enzymic activity or preparation ethanol dehydrogenase and/or hydrogenlyase of above-mentioned fusion rotein, said gene or above-mentioned recombinant vectors, expression cassette, transgenic cell line or recombinant bacterium also is the scope of protection of the invention.
Another object of the present invention provides a kind of method for preparing ethanol dehydrogenase and/or hydrogenlyase.
Method provided by the invention, the above-mentioned recombinant bacterium that comprises the steps: to ferment is collected tunning, namely obtains ethanol dehydrogenase and/or hydrogenlyase.
Of the present invention experimental results show that, the invention provides a kind of fusion rotein that is obtained by the connection peptides series connection by ethanol dehydrogenase and hydrogenlyase, this albumen is behind expression in escherichia coli, the activity that has kept two kinds of enzymes, and the enzymic activity of the hydrogenlyase in the fusion rotein is high during than single expression, stability and thermostability the situation when all being better than single expression of the ethanol dehydrogenase after the fusion in organic substrates.The raising of the enzymic activity that this fusion rotein shows and enzyme stability and the thermostability in organic substrates strengthens, point out this enzyme when catalysis prepares chiral alcohol and chiral hydroxyl group compound, to have the more catalytic effect of advantage, for the improvement of chiral alcohol and chiral hydroxyl group compounds process for production thereof provides reference.
Description of drawings
Fig. 1 is recombinant bacterium abduction delivering SDS-PAGE analytical results
Fig. 2 is the residual activity result of ADH under the different methyl phenyl ketone concentration
Fig. 3 is the residual activity result of ADH under the differing temps
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The acquisition of embodiment 1, fusion rotein and encoding gene thereof
Ethanol dehydrogenase (ADH) and the Candida boidinii Candida boidinii in the short lactobacillus that catalytic activity is high and enantio-selectivity is strong (Lactobacillus brevis) source selected in this research) hydrogenlyase (FDH) of originating, according to e. coli codon preferences principle, carry out respectively codon optimized to two enzyme genes.To between ethanol dehydrogenase and hydrogenlyase, add rigid structure linker(EAAAK)
2, obtaining fusion protein F DH-linker-ADH, the aminoacid sequence of this fusion rotein is the sequence 2 in the sequence table.The encoding gene of this fusion rotein is the sequence 1 in the sequence table.
The application of embodiment 2, fusion rotein and encoding gene thereof
One, the structure of two enzyme co-expression carriers
1, the structure of recombinant vectors pETDuet-FDH-ADH
The carrier pGH-FDH (contain the FDH sequence, total order is classified sequence 3 as) that will contain FDH cuts with NdeI and Xhol enzyme; The 1164bp enzyme that obtains is cut product and the 5364bp co-expression carrier pETDuet-1(Novagen, the 71146-3 that cut through same enzyme) the skeleton connection, obtain intermediate carrier pETDuet-FDH;
The carrier pGH-ADH (contain the ADH sequence, total order is classified sequence 4 as) that will contain ADH cuts with BamHI and NotI enzyme; The 768bp enzyme that obtains is cut product and is connected with the 6484bp intermediate carrier pETDuet-FDH skeleton of cutting through same enzyme, obtains recombinant vectors pETDuet-FDH-ADH(and cuts evaluation correctly through enzyme).
2, the structure of recombinant vectors pETDuet-FDH-linker-ADH
Take pGH-FDH as template, with FDHF:GACCATGGGCAAAATCGTTCTGGTTCTG, FLAR:TTTGGCCGCTGCTTCTTTGGCCGCTGCTTCTTTTT TGTCGTGTT is primer, obtains the PCR product of 1124bp; Take pGH-ADH as template, take FLAF:GCAGCGGCCAAAGAAGCAGCGGCCAAATCTAACCGTCTGGACADHR:GTCT CGAGTTACTGAGCGGTGTAAC as primer, obtain the PCR product of 791bp;
Take above-mentioned two PCR products as template, be that primer merges PCR with FDHF and ADHR, obtain 1896bpPCR product (having the Nucleotide shown in the sequence 1 in the sequence table); The 1896bpPCR product is cut with NcoI and XhoI enzyme, and the enzyme that obtains is cut product and is connected with the co-expression carrier pETDuet-1 that cuts through same enzyme, obtains recombinant vectors pETDuet-FDH-linker-ADH.
Through order-checking, this recombinant vectors is the carrier that obtains between the NcoI of the 1 insertion expression vector pETDuet-1 of sequence in the sequence table and XhoI restriction enzyme site.
Two, the structure of recombinant bacterium and abduction delivering fusion rotein
1, the structure of recombinant bacterium
Above-mentioned one two kinds of recombinant vectors pETDuet-FDH-ADH that obtain and pETDuet-FDH-linker-ADH are transformed respectively among the intestinal bacteria Rosseta (DE3), obtain Rosseta (DE3)/pETDuet-FDH-ADH and Rosseta (DE3)/pETDuet-FDH-l inker-ADH.
2, recombinant bacterium abduction delivering fusion rotein
Rosseta (DE3) after transforming/pETDuet-FDH-ADH and Rosseta (DE3)/pETDuet-FDH-linker-ADH are coated with respectively Amp
+Flat board, respectively then the picking positive transformant press 1:100 and is inoculated in 37 ℃ of overnight incubation, and 37 ℃, 200r/min shaking culture are 3h approximately, adds IPTG to final concentration 1mmol/L, induces 8h for 20 ℃, and centrifugal collection bacterial sediment is for subsequent use.The PBS washed twice, resuspended with PBS, with the centrifugal 30min of 12000r/min after the bacterial sediment ultrasonication, collect respectively supernatant liquor and the precipitation of every kind of bacterium, protein ingredient is carried out SDS-PAGE analyze.
The result as shown in Figure 1,1 for Rosseta (DE3)/pETDuet-FDH-linker-ADH (supernatant), 2 for Rosseta (DE3)/pETDuet-FDH-linker-ADH (precipitation), 3 be the marker of 10-230KDa for Rosseta (DE3)/pETDuet-FDH-ADH (supernatant), 4 is Rosseta (DE3)/pETDuet-FDH-ADH (precipitation), M; Can find out, molecular weight being arranged in 1 and 2 is the expression of the fusion protein F DH-linker-ADH of 66.6KDa, and the fusion rotein content in supernatant after the series connection of two enzyme is higher, illustrate that the fusion rotein that two enzymes are connected can realize solubility expression.And Rosseta (DE3)/pETDuet-FDH-ADH is not amalgamation and expression, but two kinds of enzymes are expressed respectively simultaneously.
The centrifugal supernatant liquor that obtains was FDH-linker-ADH crude enzyme liquid and FDH-ADH crude enzyme liquid after above-mentioned ultrasonication was described.
Three, the fusion rotein enzymic activity detects
1, the fusion rotein enzyme assay after two enzyme series connection
Be the activity of the fusion rotein of two enzyme series connection in the checking recombinant bacterium, the supernatant liquor of collecting after the above-mentioned ultrasonication carried out following enzyme activity assay:
The mensuration of alcohol dehydrogenase enzyme (ADH) alive: the enzyme reaction system comprises 100mmol/L buffer solution of potassium phosphate (pH value 7.0), 0.1mmol/LNADH, 1mMMgCl
2, 50mmol/L methyl phenyl ketone and 100ul ultrasonication crude enzyme liquid; Measure the lower liter of (surveying 60s) light absorption value at the 340nm place.By typical curve y=0.011x+0.003(R
2=1) calculates the amount that reaction consumes NADH.Enzyme unit alive: it is the enzyme U of unit alive that per minute consumes the needed enzyme amount of 1 μ molNADH.
The mensuration of hydrogenlyase enzyme (FDH) alive: the enzyme reaction system comprises 100mmol/L buffer solution of potassium phosphate (pH value 7.0), 0.5mmol/L NAD
+, 100mmol/L sodium formiate and 100ul ultrasonication crude enzyme liquid; Measure the rising of (surveying 60s) light absorption value at the 340nm place.By typical curve y=0.011x+0.003(R
2=1) calculates the amount that reaction generates NADH.Enzyme unit alive: it is the enzyme U of unit alive that per minute produces the needed enzyme amount of 1 μ mol NADH.
The result as shown in Table 1 and Table 2, fusion rotein after the series connection has the activity of ethanol dehydrogenase and hydrogenlyase, the activity of ADH slightly descends behind the tandem expression, and the activity of FDH significantly improves before merging, and may be by after the series connection of rigid structure connection peptides, and the C end of FDH originally irregular space structure has formed the oriented structure that is easier to active performance, in addition, because ADH is homotetramer albumen, so its activity is subjected to the impact of amalgamation and expression larger, demonstrates activity and slightly descends.The activity that still can keep preferably two enzymes after the two enzyme series connection of above presentation of results.
Table 1 is alcohol dehydrogenase enzyme measurement result alive
Can find out, Rosseta (DE3)/pETDUet-FDH-Linker-ADH has the catalytic activity of two kinds of enzymes, and the FDH activity is improved.
2, stability and the thermostability of ADH in organic substrates is improved
Be the characteristic of the ADH of performance Main Function in the fusion rotein of two enzyme series connection in the checking recombinant bacterium, the above-mentioned two FDH-linker-ADH crude enzyme liquids that obtain carried out respectively enzyme activity assay under different concentration of substrate and the differing temps with the FDH-ADH crude enzyme liquid:
Stability analysis in the organic substrates: the methyl phenyl ketone of crude enzyme liquid and different concns (20,40,80mmol/L) is mixed, and after 30 ° of C were hatched 2.5h, the method by above-mentioned 1 was analyzed the ADH activity of residual enzyme.
Thermal stability analysis: with crude enzyme liquid at a certain temperature (50,60,70 ℃) place immediately 5min on ice after hatching 10min, the method by above-mentioned 1 is analyzed the ADH activity of residual enzyme.
Stability result in the organic substrates can find out as shown in Figure 2, the ADH relative enzyme work of FDH-linker-ADH crude enzyme liquid 20,40, under the 80mmol/L methyl phenyl ketone is respectively 80.4,67.0,39.2(%) (with the 5mm methyl phenyl ketone time activity count 100%);
The ADH relative enzyme work of FDH-ADH crude enzyme liquid crude enzyme liquid 20,40, under the 80mmol/L methyl phenyl ketone is respectively 65.0,51.1,22.6(%) (with the 5mm methyl phenyl ketone time activity count 100%);
Thermal stability analysis result can find out as shown in Figure 3, the FDH-linker-ADH crude enzyme liquid is respectively 56.8,51.4 in the relative enzyme work of the ADH under 50,60,70 ℃, 30.5(%) (enzymic activity under with 30 ℃ counts 100%).
FDH-ADH crude enzyme liquid crude enzyme liquid is respectively 53.6,33.3 in the relative enzyme work of the ADH under 50,60,70 ℃, 20.0(%) (enzymic activity under with 30 ℃ counts 100%).
Found out by the result, although the ADH enzymic activity after the series connection slightly descends, but stability and the thermostability of ADH in organic substrates is high during all than single expression among the FDH-LinkerADH, illustrates that the two enzyme tandem expression systems that make up have strengthened stability in organic substrates and reached stability to temperature.
Claims (10)
1. fusion rotein is the protein that ethanol dehydrogenase and hydrogenlyase are obtained by the connection peptides series connection.
2. fusion rotein according to claim 1, it is characterized in that: the aminoacid sequence of described connection peptides is that sequence 2 is from the terminal 365-374 of N ' position.
3. fusion rotein according to claim 1 and 2 is characterized in that:
Described ethanol dehydrogenase derives from short lactobacillus (Lactobacillusb revis), and described hydrogenlyase derives from Candida boidinii (Candida boidinii);
Described fusion rotein is following (a) or (b) specifically:
(a) protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 2;
(b) with the aminoacid sequence shown in the sequence in the sequence table 2 through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and have ethanol dehydrogenase and the two enzymes of hydrogenlyase are lived by the derivative protein of sequence 2.
4. the gene of arbitrary described fusion rotein among the coding claim 1-3.
5. gene as claimed in claim 4 is characterized in that: described gene is any dna molecular in following (1)-(3):
(1) encoding sequence is the dna molecular shown in the sequence 1 in the sequence table;
(2) the dna sequence dna hybridization that limits with (1) under stringent condition and coding have the dna molecular of ethanol dehydrogenase and the two enzyme living proteins of hydrogenlyase;
(3) dna sequence dna that limits with (1) has 70% at least, have at least 75%, have at least 80%, have at least 85%, have at least 90%, have at least 95%, have at least 96%, have at least 97%, have at least 98% or have at least a dna molecular that 99% homology and coding have ethanol dehydrogenase and the two enzyme living proteins of hydrogenlyase.
6. the recombinant vectors, expression cassette, transgenic cell line or the recombinant bacterium that contain claim 4 or 5 described genes.
7. recombinant vectors as claimed in claim 6 is characterized in that:
Described recombinant vectors is that the gene of arbitrary described fusion rotein among the claim 1-3 is inserted in the expression vector, obtains expressing the recombinant vectors that profit requires arbitrary described fusion rotein among the 1-3.
8. recombinant bacterium as claimed in claim 6 is characterized in that: described recombinant bacterium is for importing described recombinant vectors in the Host Strains recombinant bacterium that obtains.
9. arbitrary described fusion rotein, claim 4 or 5 described genes or the described recombinant vectors of claim 6, expression cassette, transgenic cell line or the recombinant bacterium application in improving hydrogenlyase enzymic activity or raising ethanol dehydrogenase thermotolerance or raising ethanol dehydrogenase stable or preparation ethanol dehydrogenase and/or the hydrogenlyase in organic substrates among the claim 1-3.
10. method for preparing ethanol dehydrogenase and/or hydrogenlyase, comprise the steps: to ferment claim 6 or 8 described recombinant bacteriums are collected tunning, namely obtain ethanol dehydrogenase and/or hydrogenlyase.
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| CN106188309A (en) * | 2016-07-14 | 2016-12-07 | 北京师范大学 | Anticoagulant fusion proteins HA and application thereof |
| CN112851750A (en) * | 2021-02-20 | 2021-05-28 | 中国标准化研究院 | Connecting peptide, fusion protein containing connecting peptide and application of fusion protein |
| CN116904409A (en) * | 2023-04-17 | 2023-10-20 | 杭州力文所生物科技有限公司 | FDH mutant with improved protein soluble expression and encoding gene thereof |
| CN117603924A (en) * | 2023-04-25 | 2024-02-27 | 杭州力文所生物科技有限公司 | Formate dehydrogenase mutant with improved protein solubility expression and application thereof |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107299074B (en) * | 2017-08-30 | 2020-06-16 | 山东省科学院生态研究所 | Construction method and application of formate dehydrogenase engineering strain |
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- 2013-01-25 CN CN201310028774.0A patent/CN103060281B/en not_active Expired - Fee Related
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106188309A (en) * | 2016-07-14 | 2016-12-07 | 北京师范大学 | Anticoagulant fusion proteins HA and application thereof |
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| CN112851750A (en) * | 2021-02-20 | 2021-05-28 | 中国标准化研究院 | Connecting peptide, fusion protein containing connecting peptide and application of fusion protein |
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| CN114539428B (en) * | 2021-02-20 | 2023-02-28 | 中国标准化研究院 | Fusion protein and application thereof |
| CN116904409A (en) * | 2023-04-17 | 2023-10-20 | 杭州力文所生物科技有限公司 | FDH mutant with improved protein soluble expression and encoding gene thereof |
| CN116904409B (en) * | 2023-04-17 | 2024-04-05 | 杭州力文所生物科技有限公司 | FDH mutant with improved protein soluble expression and encoding gene thereof |
| CN117603924A (en) * | 2023-04-25 | 2024-02-27 | 杭州力文所生物科技有限公司 | Formate dehydrogenase mutant with improved protein solubility expression and application thereof |
| CN117603924B (en) * | 2023-04-25 | 2024-05-17 | 杭州力文所生物科技有限公司 | Formate dehydrogenase mutant with improved protein solubility expression and application thereof |
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