CN105671025A - D-alanyl-D-alanine carboxypeptidase dacB gene and application - Google Patents
D-alanyl-D-alanine carboxypeptidase dacB gene and application Download PDFInfo
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- CN105671025A CN105671025A CN201610123619.0A CN201610123619A CN105671025A CN 105671025 A CN105671025 A CN 105671025A CN 201610123619 A CN201610123619 A CN 201610123619A CN 105671025 A CN105671025 A CN 105671025A
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Abstract
The invention relates to the field of gene engineering, and in particular relates to a D-alanyl-D-alanine carboxypeptidase dacB gene and application. The invention provides a D-alanyl-D-alanine carboxypeptidase dacB from Escherichia coli BL21(DE3), wherein the amino acid sequence is as shown in the sequence 1; and the invention provides an ecoding gene dacB for encoding the dacB. The D-alanyl-D-alanine carboxypeptidase can remove the last tail end D-alanine (D-Ala) from peptidoglycan pentapeptide to cause the condition that a donor in a transpeptidation reaction is unavailable, thereby controlling the netty cross-linking level of the peptidoglycan, and improving the E.coli protein extracellular secretion level.
Description
Technical field
Invention relates to genetically engineered field, specifically, the present invention relates to a kind of D-alanyl-D-alanine carboxypeptidase dacB and gene thereof and application.
Background technology
Peptidoglycan is by dissacharide units, and four peptide tails also have peptide bridge to be polymerized to obtain multilayer net macromolecular structure. The skeleton of peptidoglycan layer is formed by connecting by β-1,4 glycosidic link by N-Acetyl-D-glucosamine and-acetylmuramic acid, is cross-linked by peptide chain, forms stable reticulated structure, constitute the cell walls of whole bacterium surface like this between sugar chain. As the main component of cell walls, the maintenance of permeability of the composition of peptidoglycan and the integrity of cellularstructure, adventitia, the resist drying ability of bacterium and thermotolerance etc. are all closely related. D-alanyl-D-alanine the carboxypeptidase of dacB genetic expression can remove last end D-Ala from peptidoglycan pentapeptide, causes donor in transpeptidation reaction unavailable, it is possible to the cross-linked network level of control peptidoglycan, thus changes the permeability of cell.
Intestinal bacteria (Escherichiacoli) expression system is protein expression system the most frequently used in current genetically engineered, has the advantages such as simple to operate, with low cost, it is possible to produce target protein in rapid large-scale. But in the expression process of E.coli, major part albumen is secreted into periplasmic space under the guiding of signal peptide, intermediate can be caused to gather in a large number, the production of protein is caused obstruction.
Summary of the invention
It is an object of the invention to provide a kind of can improve E.coli exocytosis level D-alanyl-D-alanine carboxypeptidase, its aminoacid sequence, the gene encoding this D-alanyl-D-alanine carboxypeptidase, the recombinant plasmid containing this gene, the recombinant bacterial strain containing this recombinant vectors, this D-alanyl-D-alanine carboxypeptidase application.
Specifically comprise:
The present invention provides a kind of D-alanyl-D-alanine carboxypeptidase dacB, and its aminoacid sequence is as shown in sequence 1.
Sequence 1:
MRFSRFIIGLTSCIAFSVQAANVDEYVTQLPAGANLALMVQKVGASAPAIDYHSQQMALPASTQKVITALAALIQLGPDFRFTTTLETKGNVENGVLKGDLVARFGADPTLKRQDIRNMVATLKKSGVNQIDGNVLIDTSIFASHDKAPGWPWNDMTQCFSAPPAAAIVDRNCFSVSLYSAQKPGDMAFIRVASYYPVTMFSQVRTLPRGSAEAQYCELDVVPGDLNRFTLTGCLPQRSEPLPLAFAVQDGASYAGAILKDELKQAGITWSGTLLRQTQVNEPGTVVASKQSAPLHDLLKIMLKKSDNMIADTVFRMIGHARFNVPGTWRAGSDAVRQILRQQAGVDIGNTIIADGSGLSRHNLIAPATMMQVLQYIAQHDNELNFISMLPLAGYDGSLQYRAGLHQAGVDGKVSAKTGSLQGVYNLAGFITTASGQRMAFVQYLSGYAVEPADQRNRRIPLVRFESRLYKDIYQNN
Wherein, this enzyme genes encoding 477 amino acid, its theoretical molecular is 51.81kDa.
The present invention provides the gene dacB encoding above-mentioned D-alanyl-D-alanine carboxypeptidase, and concrete, the gene order of this enzyme is as shown in sequence 2.
Sequence 2:
ATGCGATTTTCCAGATTTATCATCGGATTGACCAGCTGTATAGCGTTCAGTGTTCAGGCCGCGAATGTTGATGAGTACGTTACTCAACTCCCCGCTGGTGCCAACCTTGCCCTGATGGTGCAAAAAGTCGGCGCGTCGGCCCCCGCTATTGATTACCACAGTCAGCAGATGGCGCTGCCCGCCAGTACGCAGAAAGTGATTACTGCGCTGGCGGCGTTGATTCAACTCGGCCCCGATTTTCGTTTTACCACGACGCTTGAAACCAAAGGCAATGTGGAAAACGGCGTACTTAAGGGTGACTTAGTGGCGCGATTTGGTGCCGATCCGACGTTAAAACGTCAGGATATTCGCAATATGGTCGCGACTTTGAAAAAATCTGGCGTCAACCAAATCGATGGCAATGTGTTGATAGATACCTCCATTTTCGCCAGCCACGATAAAGCCCCCGGCTGGCCATGGAATGACATGACACAATGCTTTAGCGCTCCGCCTGCCGCCGCCATAGTTGACCGCAACTGTTTCTCCGTCTCGCTCTACAGTGCCCAAAAACCCGGTGATATGGCCTTTATACGCGTGGCATCTTATTACCCCGTTACGATGTTCAGCCAGGTACGCACCCTCCCCCGTGGTTCTGCCGAAGCGCAATACTGCGAACTGGATGTGGTGCCAGGCGACCTGAACCGCTTTACGCTGACGGGATGCCTGCCACAACGTTCTGAGCCGCTCCCGTTGGCTTTTGCCGTACAGGATGGAGCCAGCTATGCCGGGGCAATTCTGAAAGATGAGTTAAAACAGGCGGGTATCACCTGGAGTGGAACACTGCTGCGCCAGACTCAGGTTAACGAACCTGGAACGGTAGTTGCCAGTAAACAGTCGGCCCCGCTGCACGATCTGCTTAAGATTATGCTGAAAAAGTCAGACAACATGATCGCCGATACGGTTTTCCGCATGATAGGCCATGCGCGCTTCAATGTGCCTGGAACATGGCGGGCCGGGTCGGACGCCGTGCGTCAGATCCTGCGCCAGCAAGCCGGTGTCGATATTGGAAACACCATTATTGCCGATGGTTCAGGGCTTTCGCGGCATAACCTGATTGCCCCCGCCACCATGATGCAGGTGCTGCAATACATTGCCCAACACGACAATGAACTTAACTTTATCTCCATGCTGCCGCTGGCGGGCTATGACGGCTCTTTGCAGTACCGTGCAGGTCTGCATCAGGCGGGCGTGGATGGCAAAGTCTCAGCGAAAACCGGTTCGTTGCAGGGGGTATATAACCTGGCGGGATTCATTACCACAGCGAGCGGGCAACGAATGGCGTTTGTGCAATATCTTTCTGGCTATGCAGTAGAACCGGCGGATCAGCGTAATCGCCGTATTCCGTTAGTGCGTTTCGAAAGCCGTTTGTATAAAGATATTTATCAGAACAATTAG
The present invention has cloned D-alanyl-D-alanine carboxypeptidase gene dacB by PCR method, and DNA complete sequence analysis result shows, the encoding gene dacB total length 1434bp of D-alanyl-D-alanine carboxypeptidase.
Above-mentioned D-alanyl-D-alanine carboxypeptidase gene dacB sequence and the aminoacid sequence derived are carried out BLAST comparison, this gene and the D-alanyl-D-alanine carboxypeptidase sequence similarity the highest (99%) deriving from E.colistr.K-12substr.MG1655 in GenBank.
Present invention also offers the recombinant plasmid comprising above-mentioned D-alanyl-D-alanine carboxypeptidase gene dacB, it is pRSFDuet-dacB. The D-of the present invention alanyl-D-alanine carboxypeptidase gene is inserted into the corresponding restriction site of expression vector, its nucleotide sequence is connected with expression regulation sequence, as one of the present invention most preferably experimental program, preferred D-alanyl-D-alanine carboxypeptidase gene is inserted into plasmid pRSFDuet, the downstream making this nucleotide sequence be positioned at T7lac promotor also regulates and controls by it, obtains restructuring E.coli expression plasmid pRSFDuet-dacB.
Present invention also offers the application bacterial strain of above-mentioned D-alanyl-D-alanine carboxypeptidase gene dacB, it is preferable that described bacterial strain is E.coli.
The present invention can strengthen E.coli intracellular protein and arrive outside born of the same parents, promotes that it is secreted, reduces it at intracellular accumulation, it is provided that a kind of D-alanyl-D-alanine carboxypeptidase and aminoacid sequence, gene order improving E.coli albumen exocytosis level.
Accompanying drawing explanation
Fig. 1 is D-alanyl-D-alanine carboxypeptidase recombinant plasmid pRSFDuet-dacB collection of illustrative plates.
Fig. 2 is the overexpression of D-alanyl-D-alanine carboxypeptidase
Fig. 3 is that overexpression D-alanyl-D-alanine carboxypeptidase is on the impact of E.coli exocytosis alpha-galactosidase.
Embodiment
The clone of embodiment 1:E.coliBL21 (DE3) D-alanyl-D-alanine carboxypeptidase gene dacB
PCR-based obtains the DNA sequence dna of D-alanyl-D-alanine carboxypeptidase dacB, the gene fragment of D-alanyl-D-alanine carboxypeptidase is connected with expression vector pRSFDuet, obtains recombinant plasmid pRSFDuet-dacB (Fig. 1). Then it is transformed in E.coliBL21 (DE3) competent cell, obtains recombinant bacterial strain E.coliBL21 (DE3)/dacB.
Embodiment 2:D-alanyl-D-alanine carboxypeptidase enzyme is lived
The restructuring bacterium that will have built, inoculation 250mL shake-flask culture, after inductor (IPTG) is induced, collects E.coli cell, adopts sonioation method broken cells. After high speed centrifugation, collect broken wall supernatant, the carboxypeptidase enzyme activity in sampling and measuring broken wall supernatant. After calculating overexpression by mensuration enzyme activity, the ratio enzyme activity of carboxypeptidase dacB-Δ p in born of the same parents is 2.65U/g (Fig. 2), and control group carboxypeptidase is only 1.51U/g than enzyme activity.
Embodiment 3: overexpression dacB is on the impact of E.coli exocytosis alpha-galactosidase
In E.coli during overexpression carboxypeptidase dacB, after induction 10h, the level of E.coli exocytosis alpha-galactosidase significantly improves, and the outer alpha-galactosidase enzyme vigor of born of the same parents reaches 6.80U/g (Fig. 3). But after not having the control strain induction fermentation 10h of any carboxypeptidase of overexpression, the enzyme activity of the alpha-galactosidase recorded outside E.coli born of the same parents is only 1.82U/g.
Claims (5)
1. D-alanyl-D-alanine carboxypeptidase dacB, it is characterised in that: the protein being made up of aminoacid sequence shown in sequence 1.
2. D-alanyl-D-alanine carboxypeptidase gene dacB, it is characterised in that, encode D-according to claim 1 alanyl-D-alanine carboxypeptidase dacB.
3. D-alanyl-D-alanine carboxypeptidase gene dacB according to claim 2, it is characterised in that: its DNA sequence dna is as shown in sequence 2.
4. comprise the recombinant plasmid of the alanyl of the D-described in Claims 2 or 3-D-alanine carboxypeptidase gene dacB.
5. recombinant plasmid according to claim 4, it is characterised in that, described carrier is pRSFDuet.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610123619.0A CN105671025A (en) | 2016-03-04 | 2016-03-04 | D-alanyl-D-alanine carboxypeptidase dacB gene and application |
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| CN201610123619.0A CN105671025A (en) | 2016-03-04 | 2016-03-04 | D-alanyl-D-alanine carboxypeptidase dacB gene and application |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101115832A (en) * | 2004-11-26 | 2008-01-30 | 协和发酵工业株式会社 | Industrially useful microorganism |
| CN102186974A (en) * | 2008-08-19 | 2011-09-14 | 巴斯夫植物科学有限公司 | Plants with increased yield by increasing or generating one or more activities in a plant or a part thereof |
| CN104862329A (en) * | 2015-04-23 | 2015-08-26 | 上海工业生物技术研发中心 | L-threonine genetic engineering production bacteria |
-
2016
- 2016-03-04 CN CN201610123619.0A patent/CN105671025A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101115832A (en) * | 2004-11-26 | 2008-01-30 | 协和发酵工业株式会社 | Industrially useful microorganism |
| CN102186974A (en) * | 2008-08-19 | 2011-09-14 | 巴斯夫植物科学有限公司 | Plants with increased yield by increasing or generating one or more activities in a plant or a part thereof |
| CN104862329A (en) * | 2015-04-23 | 2015-08-26 | 上海工业生物技术研发中心 | L-threonine genetic engineering production bacteria |
Non-Patent Citations (3)
| Title |
|---|
| ALEXANDER J F, EGAN等: "Activities and regulation of peptidoglycan synthases.", 《PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY OF LONDON. SERIES B, BIOLOGICAL SCIENCES》 * |
| MARIA-ROSARIO BAQUERO等: "dacD, an Escherichia coli Gene Encoding a Novel Penicillin-Binding Protein (PBP6b) with DD-Carboxypeptidase Activity", 《AMERICAN SOCIETY FOR MICROBIOLOGY》 * |
| WC HUNG等: "Association of a D-Alanyl-D-Alanine Carboxypeptidase Gene with the Formation of Aberrantly Shaped Cells during the Induction of Viable but Nonculturable Vibrio parahaemolyticus", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
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Application publication date: 20160615 |