CN103525796A - Recombinase with activity of glutaryl-7-aminocephalosporanic acid acylase and D-amino acid oxidase as well as preparation method and application thereof - Google Patents
Recombinase with activity of glutaryl-7-aminocephalosporanic acid acylase and D-amino acid oxidase as well as preparation method and application thereof Download PDFInfo
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- C12N9/0022—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
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- C12P35/00—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin
- C12P35/02—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin by desacylation of the substituent in the 7 position
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- C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
- C12Y305/01093—Glutaryl-7-aminocephalosporanic-acid acylase (3.5.1.93)
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Abstract
The invention discloses a double-functional recombinase with the activity of glutaryl-7-aminocephalosporanic acid acylase and D-amino acid oxidase. The gene sequence of the recombinase and a gene sequence defined by SEQ ID No.2 have more than 90% of homology and are used for coding proteins with the same functions; the gene sequence of an expression vector of a recombinase coding gene and a gene sequence shown in SEQ ID No.1 have more than 90% of homology and are used for coding gene sequences of the proteins with the same functions. The expression production and the separation and purification of an enzyme catalyst and the catalytic hydrolysis process of deacetoxyvinblastine cephalosporin C are simplified in a 7-ADCA (glutaryl-7-aminocephalosporanic acid) production process.
Description
Technical field
The present invention relates to a kind of recombinase with glutaryl-7-amidocephalosporanic acid acylase and D-AAO activity and preparation method thereof and in single stage method, prepare the application in 7-aminodesacetoxycephalosporanic acid with this recombinase.
Background technology
7-aminodesacetoxycephalosporanic acid (7-ADCA) is semi-synthetic intermediate and the raw material of now widely used cephalosporin analog antibiotic medicine, in the production of clinical medicine, has significant application value.7-ADCA mainly contains the synthetic and two kinds of methods of chemosynthesis of biocatalysis and obtains.Chemical synthesis is to obtain through a few step reactions of peroxidation, expansion and hydrolysis from penicillin G, although raw material is cheap and easy to get, and complex process, use a large amount of toxic reagents, not only cause constant product quality lower, cost is higher, and produces a large amount of refuses to environment serious harm.Use that biological enzyme is synthetic replaces chemosynthesis to produce 7-ADCA, have that technique is simple, efficient, an advantage such as safety non-pollution and constant product quality, its research is subject to the common attention of industrial community and academia always.
Minimum and the comparatively ripe biological enzyme technique of cost that at present industrial community is used is to take tunning DAOC as raw material, take two step enzyme methods that D-AAO (DAO) and Glularyl-7-amino-cephalo-alkanoic acid acylase (GLA) be catalyzer.The oxidized deamination of DAOC under DAO catalysis; generate corresponding alfa-ketone group-hexanedioyl-7-ADCA and hydrogen peroxide; then under hydrogen peroxide effect, oxidative decarboxylation generates glutaryl-7-ADCA (GL-7-ADCA), then generates 7-ADCA through the de-glutaryl-side chain of GLA catalytic hydrolysis.This method is the comparatively practical enzyme catalysis operational path of current production 7-ADCA, two kinds of biological enzyme DAO that use and GLA can obtain through biotechnology fermentation by cultivating different bacterial classifications, external DAO and the GAL of greater activity unit of having obtained by suitable gene recombination and protein engineering means, and realize industrialization, although domestic, there have been research and the production of involved enzyme, but be only applied in the preparation of 7-amino-cephalosporanic acid (7-ACA), for 7-ADCA its to be still faced with activity lower, the problem such as product yield that less stable causes is lower, in addition, this two step enzyme method techniques need to be cultivated respectively and express two kinds of bacterial strains, then carry out separation and the purifying of enzyme, finally unit operation carries out enzymic catalytic reaction in two steps, in process, also need control and monitor according to different conditions, so develop more economical, easy 7-ADCA enzyme catalysis synthesis technique and relevant enzyme preparation have become and have improved China's cephalosporins medicine intermediate production technology level, promote the reality of microbiotic industrial repositioning upgrading to need.
Applied molecular biology breeding strain and genetic engineering technique, develop a kind of enzyme that can an one-step hydrolysis removes the amino acid side chain of DAOC (DAOC), is best means and the research and development focus that improves at present 7-ADCA enzymatic production process.With DAOC acylase, carrying out direct catalytic hydrolysis D-alfa-aminoadipic acid side chain is one of method; but up to the present; relevant DAOC acylase comprises through improved recombinases of range gene engineering means such as rite-directed mutagenesis and orthogenesiss; its catalytic hydrolysis vigor is all still very low, far can not meet the needs of Industrial Catalysis.Another kind method is in same reactor, to recur the reaction of D-amino-acid oxidase and glutaryl hydrolysis reaction, and one kettle way is produced 7-ADCA.In preparation 7-ACA process, there is the report of similar production technique at present.Comprise and add respectively the enzyme or the cell that produce DAO and GLA activity to carry out one pot reaction, but because of the most applicable condition of two kinds of enzymes is inconsistent and reaction system in the interference of by product hydrogen peroxide, activity and the stability of enzyme are reduced, and cause transforming the accumulation of stagnation and intermediate product.
After successfully cloning the gene that two kinds of enzymes of DAO and GLA are corresponding and its gene order fully understood, build the recombinant plasmid that merges two kinds of gene orders, transform plasmid and can give expression to the recombinase with two enzymic activitys, be so-called fusion gene and fusion rotein, it is that the coding region of two or more genes is joined end to end, be placed in the mosaic gene forming under same set of regulating and controlling sequence control, after appropriate expression system is expressed, can obtain the Multifunction enzyme together being formed by the amalgamation of difference in functionality albumen.This new enzyme being obtained by genetic engineering means can to two steps even multistep Catalytic processes improve, make it to become more simple, efficient.And, only carrying out single fermentation just can the DAO of accomplished two kinds of katalysis and the fusion protease of GLA, using this bifunctional enzyme also can make the two step enzyme method technique successful modifications of 7-ADCA is one pot of one-step reaction, makes biological enzyme preparation technology and biological enzyme reaction process realize further upgrading.
Summary of the invention
Technical problem to be solved by this invention is: provides a kind of and there is glutaryl-7-amidocephalosporanic acid acylase and D-AAO activity, and active and the high recombinase of stability.
For solving the problems of the technologies described above, the technical solution used in the present invention is: a kind of recombinase with glutaryl-7-amidocephalosporanic acid acylase and D-AAO activity, the encoding gene of described recombinase, the gene order that its gene order and SEQ ID NO.2 limit has 90% above homology (comprising homologous genes sequence), and the protein of coding identical function;
The expression vector of the described recombinase encoding gene with glutaryl-7-amidocephalosporanic acid acylase and D-AAO activity; the gene order of this expression vector and SEQ ID NO.1 have 90% above homology (comprising homologous genes sequence), and the gene order of coding identical function protein.
Second technical problem to be solved by this invention is: the preparation method that the recombinase of the recombination that a kind of fusion D-AAO (DAO) gene and glutaryl-remove acetyl oxygen Cephalosporin Acylases gene is provided.
For solving the problems of the technologies described above, technical scheme of the present invention is: the preparation method with the recombinase of Glularyl-7-amino-cephalo-alkanoic acid acylase (gla) and D-AAO (dao) vigor, the steps include: to utilize engineered means, glutaryl-7-amino-cephalo phytanic acid acylated enzyme gene (acy) and daao gene (daao) are placed in respectively under the regulation and control of strong promoter separately, build double gene coexpression plasmid, this double gene coexpression plasmid is through being transformed into intestinal bacteria and through cultivating, obtain expressing the fusion protease of possess Glularyl-7-amino-cephalo-alkanoic acid acylase (gla) and D-AAO (dao) vigor simultaneously.
Described glutaryl-7-amino-cephalo phytanic acid acylated enzyme gene sequence and SEQ ID NO.4 have 90% above homology (comprising homologous genes sequence), and the gene order of coding Glularyl-7-amino-cephalo-alkanoic acid acylase activity.
Described daao gene sequence and SEQ ID NO.3 have 90% above homology (comprising homologous genes sequence), and the gene order of encoding D-amino-acid oxidase enzymic activity.
Described glutaryl-7-amidocephalosporanic acid acylase and D-AAO co-expression plasmid, its coexpression promotor is T5.
The Host Strains of described glutaryl-7-amidocephalosporanic acid acylase and D-AAO co-expression plasmid, its Host Strains is a kind of in intestinal bacteria E.coli BL21, intestinal bacteria M15, escherichia coli DH5a and intestinal bacteria W3110 preferably.
The 3rd technical problem to be solved by this invention is: a kind of application in above-mentioned recombinase catalysis DAOC (DAOC) direct production 7-amino based desacetoxycephalosporanic acid (7-ADCA) is provided.
For solving the 3rd technical problem, the technical solution used in the present invention is: the application of recombinase in the preparation of 7-amino based desacetoxycephalosporanic acid: will have the crude enzyme liquid of the recombinase of glutaryl-7-amidocephalosporanic acid acylase and D-AAO activity, add in phosphate buffer solution, add again DAOC, stirring reaction under 32 ± 5 ℃ of temperature condition, with alkali lye, maintain reaction in pH=7.5 ± 0.5, HPLC detection reaction process, treat that system pH no longer changes, stopped reaction, the 7-amino based desacetoxycephalosporanic acid of purifying to obtain.
Beneficial effect of the present invention: express respectively independent production with two kinds of traditional enzymes and catalyze and synthesize 7-ADCA technique with two step enzyme methods and compare, can, in producing the process of 7-ADCA, the catalytic hydrolysis process of Expression product, separation and purification and the DAOC of enzyme catalyst be all simplified.
By genetic engineering means, transform downstream process, for the upgrading of the enzyme catalysis production technique of 7-ADCA provides new approaches, can accelerate the industrialization paces by DAOC direct production 7-ADCA.
Embodiment:
Embodiment 1
The structure of the recombinant plasmid pET-GLD that contains recombinase encoding gene (gla-dao is called for short GLD).
The enzyme relating in the present embodiment is cut, connects, is transformed and express the methods such as preparation and is conventional molecular biology experiment operation, can be with reference to relevant teaching and research books.
1) with restriction enzyme EcoRI and HindIII, the plasmid pET-GLA that comprises SEQ ID NO.4 sequence is carried out to complete degestion, and reclaim this acylase gene fragment that enzyme is cut rear generation.
2) with restriction enzyme EcoRI and HindIII digested plasmid pET28b (Novagen).After digestion reaction finishes, use respectively phenol and phenol/chloroform (1:1) extracting enzyme liquid, to remove the foreign protein in reaction solution, then add the dehydrated alcohol of two volumes to precipitate DNA, filtration collecting precipitation is laid equal stress on and is dissolved in distilled water.
3) mix the chain pET28b carrier DNA obtaining in the gla gene fragment obtaining in step 1 and step 2, add T4DNA ligase enzyme, in 16 ℃ of reactions 12 hours.
4) mixed solution after connecting in step 3 is distinguished after extracting through phenol and phenol/chloroform (1:1), add the dehydrated alcohol of 1/3 volume pH7.5 Spirit of Mindererus and 2 times of volumes to precipitate DNA wherein, collecting precipitation is heavily dissolved in 5 μ L distilled water, get 1 μ L and be transformed into E.coli BL21 competent cell, containing on 50mg/L spectinomycin LB substratum, obtaining transformant.Select transformant and carry out enzyme and cut evaluation, determine in the plasmid DNA of this transformant and contain external source gla gene fragment, plasmid called after pBLG.
5) with restriction enzyme NheI and NotI, plasmid pBLG is carried out to complete degestion, reclaim the acylase gene fragment that enzyme is cut rear generation.
6) the recombinant plasmid pET-DAO that comprises SEQ ID NO.4 sequence with restriction enzyme SpeI and NotI digestion.After digestion reaction finishes, use respectively phenol and phenol/chloroform (1:1) extracting enzyme liquid, to remove the foreign protein in reaction solution, then add the dehydrated alcohol of two volumes to precipitate DNA, filtration collecting precipitation is laid equal stress on and is dissolved in distilled water.
7) the gla acylase gene fragment that mixing is obtained by step 5) and step 6) respectively and the pET-DAO vector plasmid DNA of open chain, add T4DNA ligase enzyme in 16 ℃ of reactions 12 hours.
8) mixed solution after connecting in step 7) is distinguished after extracting through phenol and phenol/chloroform (1:1), add the p7.5 Spirit of Mindererus of 1/3 volume and the dehydrated alcohol of 2 times of volumes precipitation DNA wherein, collecting precipitation is heavily dissolved in 5 μ L distilled water, get 1 μ L and be transformed into E.coli BL21 competent cell, containing on 50mg/L spectinomycin LB substratum, obtaining transformant.Select transformant and carry out enzyme and cut evaluation, determine in the plasmid DNA of this transformant and contain external source gla gene fragment, the plasmid called after pET-GLD that obtains.
Embodiment 2
Merge the expression of enzyme:
By recombinant plasmid pET-GLD transformed host cell E.coli BL21, obtain merging the expression strain E.coli BL21/pET-GLD of enzyme.
Recombinant bacterium is inoculated in LB liquid nutrient medium, 37 ℃ of shaking table overnight incubation, with 5% inoculum size, be transferred in the shaking flask of 50mL LB substratum again, continue to cultivate 2 hours, add 0.5mM IPTG, in 28 ℃, induce, after 20 hours, centrifugal collection thalline, ultrasonication in the phosphate buffer solution of pH=7.5, the centrifugal bacterial chip of removing, clear enzyme solution in collection.
Embodiment 3
The direct catalysis DAOC of recombinase is produced 7-ADCA
By the crude enzyme liquid extracting abduction delivering in embodiment 2 and preliminary, add 50mL phosphate buffer solution (0.1M, pH=8.0) in, then add DAOC to total concn 6g/L, 500 turn/min stirring reactions under 30 ℃ of water bath condition, with alkali lye, maintain reaction at pH=7.5, HPLC detection reaction process, treats that system pH no longer changes, stopped reaction, transformation efficiency is greater than 97%, 7-ADCA yield 67%.
Claims (8)
1. a recombinase with glutaryl-7-amidocephalosporanic acid acylase and D-AAO activity; it is characterized in that: the encoding gene of described recombinase; the gene order that its gene order and SEQ ID NO.2 limit has 90% above homology, and the protein of coding identical function.
2. a kind of recombinase with glutaryl-7-amidocephalosporanic acid acylase and D-AAO activity according to claim 1; it is characterized in that: the expression vector gene order of described recombinase encoding gene and SEQ ID NO.1 have 90% above homology, and the gene order of coding identical function protein.
3. a kind of preparation method with the recombinase of glutaryl-7-amidocephalosporanic acid acylase and D-AAO activity claimed in claim 1; the steps include: glutaryl-7-amino-cephalo phytanic acid acylated enzyme gene and daao gene to be placed in respectively under the regulation and control of promotor separately; build double gene coexpression plasmid; this double gene coexpression plasmid is through being transformed into intestinal bacteria; through cultivating, obtain expressing the recombinase that possesses Glularyl-7-amino-cephalo-alkanoic acid acylase and D-AAO vigor more simultaneously.
4. a kind of preparation method with the recombinase of glutaryl-7-amidocephalosporanic acid acylase and D-AAO activity according to claim 3; it is characterized in that: described glutaryl-7-amino-cephalo phytanic acid acylated enzyme gene sequence and SEQ ID NO.4 have 90% above homology, and the gene order of coding Glularyl-7-amino-cephalo-alkanoic acid acylase activity.
5. a kind of preparation method with the recombinase of glutaryl-7-amidocephalosporanic acid acylase and D-AAO activity according to claim 3; it is characterized in that: described daao gene sequence and SEQ ID NO.3 have 90% above homology, and the gene order of encoding D-amino-acid oxidase enzymic activity.
6. according to a kind of preparation method with the recombinase of glutaryl-7-amidocephalosporanic acid acylase and D-AAO activity described in claim 4,4 or 5; it is characterized in that: described glutaryl-7-amidocephalosporanic acid acylase and D-AAO co-expression plasmid, its coexpression promotor is T5.
7. a kind of preparation method with the recombinase of glutaryl-7-amidocephalosporanic acid acylase and D-AAO activity according to claim 3, is characterized in that: described intestinal bacteria are a kind of in intestinal bacteria E.coli BL21, intestinal bacteria M15, escherichia coli DH5a and intestinal bacteria W3110.
8. the application of a kind of recombinase with glutaryl-7-amidocephalosporanic acid acylase and D-AAO activity claimed in claim 1 in the preparation of 7-amino based desacetoxycephalosporanic acid: will there is the crude enzyme liquid of the recombinase of glutaryl-7-amidocephalosporanic acid acylase and D-AAO activity, add in phosphate buffer solution, add again DAOC, stirring reaction under 32 ± 5 ℃ of temperature condition, with alkali lye, maintain reaction in pH=7.5 ± 0.5, HPLC detection reaction process, treat that system pH no longer changes, stopped reaction, the 7-aminodesacetoxycephalosporanic acid of purifying to obtain.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108410895A (en) * | 2018-01-23 | 2018-08-17 | 南华大学 | A method of it improving recombinant dna fragment and converts Escherichia coli efficiency |
Citations (3)
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|---|---|---|---|---|
| WO2000078989A1 (en) * | 1999-06-18 | 2000-12-28 | Antibioticos, S.A.U. | Method for producing 7-aminodesacetoxycephalosporanic acid (7-adca) |
| CN102154429A (en) * | 2010-12-28 | 2011-08-17 | 哈药集团制药总厂 | One-step enzymatic method for preparing 7-aminocephalosporanic acid |
| CN103014114A (en) * | 2012-12-27 | 2013-04-03 | 华北制药河北华民药业有限责任公司 | Method for preparing 7-aminocephalosporanic acid via enzymic method |
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2013
- 2013-10-18 CN CN201310495948.4A patent/CN103525796A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000078989A1 (en) * | 1999-06-18 | 2000-12-28 | Antibioticos, S.A.U. | Method for producing 7-aminodesacetoxycephalosporanic acid (7-adca) |
| CN102154429A (en) * | 2010-12-28 | 2011-08-17 | 哈药集团制药总厂 | One-step enzymatic method for preparing 7-aminocephalosporanic acid |
| CN103014114A (en) * | 2012-12-27 | 2013-04-03 | 华北制药河北华民药业有限责任公司 | Method for preparing 7-aminocephalosporanic acid via enzymic method |
Non-Patent Citations (1)
| Title |
|---|
| 刘越: "7-ADCA反应结晶过程研究", 《中国博士学位论文全文数据库》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108410895A (en) * | 2018-01-23 | 2018-08-17 | 南华大学 | A method of it improving recombinant dna fragment and converts Escherichia coli efficiency |
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