CN1448506A - Mutation penicillin ring enlargement enzyme and process preparing 7-ADCA using same - Google Patents
Mutation penicillin ring enlargement enzyme and process preparing 7-ADCA using same Download PDFInfo
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Abstract
The present invention is mutant expandase with even higher activity to benzyl penicillin used for preparing phenylacetyl-7-aminodeacetoxy cephalosporanic acid (7-ADCA). The mutant expandase has one or several amino acid substitutes selected from M73T, S79E, V275I, L277K, C281Y, G300V, N304K, I305L and I305M.
Description
Technical field
The present invention relates to penicillin G is had the penicillin ring enlargement enzyme (expandase) of the sudden change of high substrate specificity, express the reconstitution cell of the ring enlargement enzyme of this sudden change, and prepare 7-amino with the ring enlargement enzyme of this sudden change and go acetoxyl cephalosporonic acid (7-aminodeacetoxycephalosporanic acid, method 7-ACDA).
Background technology
It is preparation frequent also microbiotic cephalosporins of life-time service in humans and animals that 7-amino removes acetoxyl cephalosporonic acid (7-ADCA), one of important intermediate of Cephalexin Monohydrate Micro/Compacted, Cephradine and S 578.The industrial preparative method of 7-ADCA mainly comprises two steps: with penicillin G chemistry ring expansion is phenylacetyl-7-ADCA, and the fracture of the enzymatic side chain of phenylacetyl-7-ADCA.Yet the chemical reaction of ring expansion is complicated and expensive, and byproduct and organic solvent (as pyridine and hydrogen bromide) are poisonous to environment.Therefore, press for enzymatic reaction and replace this chemical reaction.
Report a kind of natural enzyme, gone acetoxyl cephalosporin synthase (deacetoxycephalosporain C synthase, DAOCS, or ring enlargement enzyme) may be responsible for the catalysis of ring expansion.Streptomyces (as band spillikin streptomycete, living dyadic streptomycete, streptomyces chartreusis) can produce ring enlargement enzyme.As described in EP-A-0341892, ring enlargement enzyme can obtain from band spillikin streptomycete, and has been cloned.Chemistry and functional property to ring enlargement enzyme have been carried out excellent research, see EP-A-0366354.Regrettably, natural ring enlargement enzyme is lower than (Rollins, M.J. etc. to normal substrate penicillin N to the substrate specificity of penicillin G, Can.J.Microbiol.34:1196-1202 (1998) and Crawford, L. etc., Bio/Technology, 13:58-62 (1995)).Penicillin G can obtain from commercial channels cheaply.On the contrary, penicillin N is more expensive, and is difficult for obtaining.In addition, even with the penicillin N ring expansion, its side chain can not easily be removed.Therefore, still use the chemosynthesis of 7-ADCA in the industrial preparation, rather than enzymatic is synthetic.
There are many applications to relate to and generate 7-ADCA by ring enlargement enzyme.USP 5,731, and 165 have described by the enzymatic ring expansion activity to penicillin G, with the penicillium chrysogenum transformant bacterial strain of expressing ring enlargement enzyme, and the method for preparation and recovery 7-ADCA.USP 5,559,005 discloses with having a ring enlargement enzyme actively, can accept the Penicllium chrysogenum bacteria strain of hexanedioyl-6-amino-penicillanic acid (hexanedioyl-6-APA) as the conversion of substrate, the biological method of preparation 7-amino-cephalosporonic acid (7-ACA) or 7-ADCA.Yet because external, hexanedioyl-6-APA and penicillin G all are the bad substrates of natural ring enlargement enzyme, and therefore when using in vivo, it unlikely has high ring expansion efficient.
Recently, (Biochemical and BiophysicalResearch Communications, 287:507-513 (2001)) such as Chih H.S. discloses a kind of DAOCS of sudden change, and it comprises the amino-acid substitution of N304L.USP5,919,680 have described a kind of ring enlargement enzyme, and the aminoacid sequence from natural ring enlargement enzyme that it has change causes the substrate specificity that changes.In this patent, mentioned the several amino acid site, in the miscellany of penicillin G and penicillin N, show the higher penicillin G and the specific activity of penicillin N by the ring enlargement enzyme that changes the sudden change that one or more amino acid of mentioning produce, but it has the independent activity to penicillin G and penicillin N that is lower than the wild-type ring enlargement enzyme.Therefore, still need to develop the penicillin ring enlargement enzyme that penicillin G is had the sudden change of higher substrate specificity and enzymatic activity.
Summary of the invention
The invention provides the ring enlargement enzyme of sudden change, its ring expansion specific activity wild-type ring enlargement enzyme to penicillin G is high 2 to 32 times.
An object of the present invention is to provide the penicillin ring enlargement enzyme of sudden change, it is included in the amino-acid substitution in one or more residues site, described residue site is corresponding to the residue site of wild-type ring enlargement enzyme, it is selected from methionine(Met) 73, Serine 79, Xie Ansuan 275, leucine 277, halfcystine 281, glycine 300, l-asparagine 304 and Isoleucine 305, and condition is that the amino-acid substitution in the residue site of l-asparagine 304 is not N304L.Specifically, the invention provides the penicillin ring enlargement enzyme of sudden change, it comprises the specific amino acids displacement of one or more M73T of being selected from, S79E, V275I, L277K, C281Y, G300V, N304K, I305L and I305M, and wherein the residue site of amino-acid substitution is corresponding to the residue site of wild-type.
Another object of the present invention provides the isolated nucleic acid molecule of the penicillin ring enlargement enzyme of encoding mutant.
Another object of the present invention provides the recombinant vectors that comprises nucleic acid molecule of the present invention and be connected in its adjusting sequence through operation.
A further object of the present invention provides the reconstitution cell that comprises nucleic acid molecule of the present invention.
On the other hand, the invention provides the method for the penicillin ring enlargement enzyme that produces sudden change.In one embodiment of the invention, this method comprises expresses nucleic acid molecule of the present invention, and reclaims the penicillin ring enlargement enzyme of sudden change.In another embodiment of the present invention, this method comprises cultivates the penicillin ring enlargement enzyme of reconstitution cell of the present invention with the expression sudden change, and reclaims the penicillin ring enlargement enzyme of sudden change from cell culture.
On the other hand, the invention provides the method for preparing 7-ADCA, described method comprises with the penicillin ring enlargement enzyme of sudden change handles penicillin G with preparation phenylacetyl-7-ADCA, then phenylacetyl-7-ADCA is gone acidylate with preparation 7-ADCA.
More on the one hand, the invention provides the method for preparing 7-ADCA, said method comprising the steps of: (a) under the condition of the penicillin ring enlargement enzyme that is suitable for producing penicillin G and expressing sudden change, cultivate the product penicillin G cell that transforms with nucleic acid molecule of the present invention, so that the ring enlargement enzyme ring expansion that penicillin G is suddenlyd change, and preparation phenylacetyl-7-ADCA; (b) phenylacetyl-7-ADCA is gone acidylate with preparation 7-ADCA.
To understand the present invention fully from following embodiment and description of drawings.
Description of drawings
Fig. 1 is the SDS-PAGE result of the DAOCS of purifying of the present invention.Swimming lane 1 to 9 is respectively the DAOCS of the purifying of molecular weight marker and mutant YS5, YS8, YS11, YS12, YS16, YS49, YS53 and YS59.Each DAOCS application of sample amount is 5 μ g.
Fig. 2 is the SDS-PAGE result of the DAOCS of purifying of the present invention.Swimming lane 1 and 2 is represented the DAOCS of the purifying of molecular weight marker and YS67 mutant respectively.The application of sample amount of DAOCS is 5 μ g.
Fig. 3 is the SDS-PAGE result of the DAOCS of purifying of the present invention.Swimming lane 1 is a molecular weight marker.The sample that swimming lane 2 to 5 representatives and the present invention have nothing to do. Swimming lane 6 and 7 is represented the DAOCS of the purifying of SC29 and SC39 mutant respectively.The application of sample amount of DAOCS is 5 μ g.
Fig. 4 is the SDS-PAGE result of the DAOCS of purifying of the present invention.Swimming lane 1 is represented molecular weight marker.The sample that swimming lane 2 to 5 representatives and the present invention have nothing to do.Swimming lane 6 to 9 is represented the DAOCS of the purifying of YS98, YS108, YS115 and YS125 respectively.The application of sample amount of DAOCS is 5 μ g.
Embodiment
Main aspect of the present invention provides the penicillin ring enlargement enzyme of sudden change, it has better substrate specificity to penicillin G, wherein the penicillin ring enlargement enzyme of this sudden change is included in the amino-acid substitution in one or more residues site, described residue site is corresponding to the residue site of wild-type ring enlargement enzyme, it is selected from methionine(Met) 73, Serine 79, Xie Ansuan 275, leucine 277, halfcystine 281, glycine 300, l-asparagine 304 and Isoleucine 305, and condition is that the amino-acid substitution in the residue site of l-asparagine 304 is not N304L.More particularly, the invention provides the penicillin ring enlargement enzyme of sudden change, it comprises the amino-acid substitution of one or more M73T of being selected from, S79E, V275I, L277K, C281Y, G300V, N304K, I305L and I305M.
Term used herein " wild-type penicillin ring enlargement enzyme " refers to from the natural penicillin ring enlargement enzyme of streptomyces acquisition.Preferably, this wild-type ring enlargement enzyme obtains from band spillikin streptomycete.Natural penicillin ring enlargement enzyme and corresponding gene (cefE gene) thereof characterize well and describe in prior art (for example EP-A-0366354 and EP-A-0341892).Those skilled in the art can easily obtain the nucleotide sequence and the amino acid sequence corresponding of wild-type penicillin ring enlargement enzyme from prior art.
The penicillin ring enlargement enzyme of sudden change of the present invention comprises its function equivalent.As used herein, " function equivalent " of the penicillin ring enlargement enzyme of sudden change may comprise other amino acid mutation (for example lack, add or replace) in the site that is positioned at except that above-mentioned site, wherein said other amino acid mutation causes reticent the change, does not therefore influence the function (for example enzymic activity) of the penicillin ring enlargement enzyme of sudden change basically.In addition, in the penicillin ring enlargement enzyme " function equivalent " of sudden change, specific amino-acid substitution (promptly being selected from M73T, S79E, V275I, L277K, C281Y, G300V, N304K, I305L and I305M) can with reticent other amino-acid substitution exchange that changes that causes of similar characteristics.For example, penicillin ring enlargement enzyme with sudden change of " S79D " amino-acid substitution is the function equivalent of penicillin ring enlargement enzyme with sudden change of " S79E " amino-acid substitution, because amino acid D (aspartic acid) and E (L-glutamic acid) are classified as acidic amino acid and similar performance.
On the other hand, the invention provides the isolated nucleic acid molecule of the penicillin ring enlargement enzyme of coding sudden change of the present invention.Isolated nucleic acid molecule of the present invention obtains by the nucleic acid mutation that makes the encoding wild type penicillin ring enlargement enzyme.The conventional sudden change technology of bringing out is being known in the art, and as with gamma-radiation or uviolizing, or handles mutagenic compound such as azanol and ethyl methane (ethylmethane), or site-directed mutagenesis with mutagenic compound.Those skilled in the art can select the mutating technology that suits and the nucleic acid molecule that obtains to suddenly change.The nucleic acid molecule of sudden change can further be cloned and be selected according to their biological activity.Be used for obtaining the more detailed technology of isolated nucleic acid molecule of the present invention, comprise mutagenesis, clone and bioactivity screening, will in following examples, describe.
According to the present invention, isolated nucleic acid molecule can be inserted carrier to form recombinant vectors.Term used herein " carrier " refers to nucleic acid molecule, and it can be that purpose is carried and shifted this nucleic acid fragment and enters host cell to express or to duplicate interested nucleic acid fragment.Specifically, carrier refers to plasmid, clay, phage or virus.Usually, interested nucleic acid fragment is connected to through operation to be regulated on the sequence, so that for example when introducing host cell, this nucleic acid fragment can be expressed under the control of adjusting sequence.Regulating sequence can comprise, for example promoter sequence (for example cytomegalovirus (CMV) promotor, simian virus 40 (SV40) early promoter and T7 promotor), replication orgin and other adjusting sequence (for example SD sequence and terminator sequence).Preferably, interested nucleic acid fragment can be connected in another nucleic acid fragment, to produce fusion polypeptide (for example fusion polypeptide of histidine mark), is beneficial to purge process subsequently.Identifying and selecting the method for adjusting sequence is well known to a person skilled in the art, and extensive description is arranged in the literature.Those skilled in the art can easily make up this recombinant vectors according to this specification sheets and known technology.
Recombinant vectors of the present invention can be introduced host cell, to produce the penicillin ring enlargement enzyme of sudden change.Therefore, the reconstitution cell that transforms with recombinant vectors within the scope of the invention.This reconstitution cell can be prokaryotic cell prokaryocyte (for example bacterium) or eukaryotic cell (for example fungi, animal and plant cell).Especially, reconstitution cell of the present invention is to produce the penicillin G cell, and preferably the Penicllium chrysogenum mycetocyte is as mentioned below, and this cell can be used for producing in vivo 7-ADCA.Many transformation technologies, the transfection, fat transfection and the microinjection that mediate as calcium chloride processing, calcium-PEG process, electroporation, DEAE-dextrin are having detailed description in a lot of documents.Those skilled in the art can select suitable technique according to host cell and the character that will introduce the carrier of host cell.
The present invention also provides the method for the penicillin ring enlargement enzyme that produces sudden change.Can under the condition of the penicillin ring enlargement enzyme that is suitable for expressing sudden change, cultivate above-mentioned reconstitution cell, reclaim then and the expressed ring enlargement enzyme of purifying.It will be understood to those of skill in the art that recovery and purification process are having extensive description in a lot of reference, and be not limited to, for example, various chromatographic techniques (for example HPLC or affinity column).
Top gene engineering method of mentioning such as DNA mutagenesis, clone, vector construction, conversion, protein expression, and purifying can be finished by those skilled in the art, its can referring to, for example, Molecular Cloing:A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, Sambrook, J., E.F.Frisch and T.Maniatis write, (1989).
The invention still further relates to the method for preparing 7-ADCA, described method comprises with the penicillin ring enlargement enzyme of sudden change of the present invention handles penicillin G with preparation phenylacetyl-7-ADCA, and goes acidylate with preparation 7-ADCA phenylacetyl-7-ADCA.Especially, express and reclaim the ring enlargement enzyme of sudden change as mentioned above, add the substrate penicillin G then.Mixture in insulation (for example under 30 ℃ temperature) under the condition of the enzymic activity of the ring enlargement enzyme that is suitable for this sudden change, so that carry out ring expansion by the enzyme of sudden change, is converted into phenylacetyl-7-ADCA with the substrate penicillin G.Preferably, for example, can be by simple solvent extraction and purifying phenylacetyl-7-ADCA, handle with suitable enzyme (for example penicillin amidase, as described in EP-A-0453047) then, removing the phenylacetyl side chain, and obtain the 7-ADCA of expectation.On the other hand, the substrate penicillin G directly can be added in the cell culture of reconstitution cell of the ring enlargement enzyme of expressing sudden change of the present invention.Then, expressed ring enlargement enzyme and penicillin G reaction, and be translated into phenylacetyl-7-ADCA, then phenylacetyl-7-ADCA is gone acidylate to produce the 7-ADCA of expectation.
Have been found that the product penicillin G cell (for example penicillium chrysogenum) that transforms with the ring enlargement enzyme encoding gene can produce phenylacetyl-7-ADCA in vivo.Therefore, another aspect of the present invention provides with the method for producing penicillin G cell preparation 7-ADCA, said method comprising the steps of: (a) under the condition of the ring enlargement enzyme that is suitable for producing penicillin G and expressing sudden change of the present invention, cultivate the product penicillin G cell that transforms with recombinant vectors of the present invention, so that the ring enlargement enzyme ring expansion that penicillin G is suddenlyd change, and produce phenylacetyl-7-ADCA; (b) phenylacetyl-7-ADCA is gone acidylate to produce 7-ADCA.Term used herein " produces the penicillin G cell " and refers under general condition to produce naturally the cell of penicillin G, and need not any genetic engineering technique (for example transforming).Preferably, producing the penicillin G cell is the Penicllium chrysogenum mycetocyte.Can randomly pass through filtration and extraction step purifying phenylacetyl-7-ADCA from step (a).Detailed process, as this transformation and fermentation, and the purifying of the phenylacetyl-7-ADCA that is produced describes in prior art, as US 5,919,680 and EP 5,731,165, these two pieces of documents are incorporated herein for referencial use.
Embodiment
With reference to following examples, it is clear that the present invention will become.Following embodiment only provides in the explanation mode, rather than any limitation of the invention.Material
Except that specifying, all pharmaceutical chemicalss are all available from Merk company.Bacterial strain band spillikin streptomycete is ordered the ﹠amp from Culture Collection; Research Center (Taiwan).Oligonucleotide is synthetic by Genset (Singapore Biotech).Dna sequence dna is analyzed by Mission Biotech (Taiwan).Liquid chromatography-mass spectrography analysis is undertaken by Protech Laboratory (Taiwan).Penicillin G is from HarbinPharmaceutical Co. (China).C
14-penicillin G is ordered from Moravek.Penicillin N, go acetoxyl cephalosporin and cynnematin G synthetic in this laboratory.Other material and provide company as follows: the enzyme (Promega) that is used for the DNA operation; ZeroBlunt TOPO PCR clones test kit (Invitrogen); Dna gel extraction agent box; GFX Micro Plasmid Prep Kit and FPLC equipment and post (Amersham Pharmacia Biotech Inc); PCR clean up-M (Viogene); PET24a, pET30a, BL21 (DE3) and Tuner cell (Novagen); Bradford reagent, 30% acrylamide/bisacrylamide solution, and PAGE equipment (Bio-Rad); HPLC device (LC-10AT, Sil-10AD, SPD-10A; Shimadu); C
18Post (250 * 4.6mm, 5 μ; Hypersil); HPLC data analysis software (Scientific Information ServiceCorporation); Pefabloc SC, and leupeptin (Roche)
Embodiment 1 random mutagenesis
The cefE gene fragment is downcut from pYB4, pYB4 is the pET24a main chain that BamHI-Hind IIIcefE inserts that has that carries the spillikin streptomycete with Zero Blunt TOPOPCR Cloning test kit clone, this cefE gene fragment was handled 2 hours at 65 ℃ with the 0.8M azanol, purify with PCR Clean up-M test kit, connection is back to the main chain carrier again.The cefE storehouse that will suddenly change by electroporation is transformed into BL21 (DE3), selects transformant on the flat board that contains 50 μ l/mL kantlex.Make the transformant of sudden change accept the active screening that improves.Embodiment 2 active improvement are screened
The transformant of sudden change is grown in 96 orifice plates, has 56.7 μ l to contain the LB substratum of kantlex in each hole.Add 0.1mM IPTG with at 2 hours indirect induction DAOCA of 30 ℃ of vibrations to each hole then, then vibration 1 hour in addition after adding 7 μ l 100mg/ml N,O-Diacetylmuramidases.The active following mensuration of DAOCA: add 30 μ l test mixing thing (500mM Mops/pH 7.0,18mM FeSO
4, 40mM xitix, 25.6mM α-Tong Wuersuan and an amount of penicillin G), 30 ℃ of insulations 1 hour of vibrating in addition down.The gained mixture is added on the thick paper disc of 8mm, and be put into inoculation and have on the biological assay flat board of intestinal bacteria ESS bacterial strain (the beta-lactam hypersensitivity mutant strain of being so kind as to give by doctor Demain), as Cho H. etc., Proc.Natl.Acad.Sci.USA, 95:11544-11548 (1998) is described.Selection has the transformant of the clear district band bigger than the control strain of not sudden change, and the activity of accepting further to be undertaken by TLC is proved conclusively.After the active improvement screening, it is that the NdeI-Hind III in the identical carrier (pET24a) inserts model (version) that the cefE that suddenlys change is controlled, and recombinant chou such as YS5 (V275I), YS53 (C281Y) and YS59 (S79E) are selected.
Also develop the method that is used for separating penicillin G and cynnematin G, be used for the biological assay screening.Silica-gel plate 60 F254 are used as solid phase, and liquid phase is a chloroform: acetone: the mixture of acetic acid=6: 5: 0.5 (volume ratio).With C
14In the standard test that the penicillin G of mark is used for carrying out with 20 μ g cell extracts (with Bio-Rad Bradford test kit, being standard quantitative with BSA), add ethanol after, mixture is without centrifugal and directly put on the TLC plate.Embodiment 3 site-directed mutagenesises
From band spillikin streptomycete PCR clone wild-type ring enlargement enzyme gene (being cefE), and be inserted into pET30a NdeI-Hind III site, resulting plasmid is named as pYS16.The rite-directed mutagenesis type that Quick Change Mutagenesis Kit is used to produce pYS16.Use Swiss-Pdb Viewer (V3.7b2) program,, select the mutational site based on the crystalline structure (ValegardK. etc., Nature, 394:805-809 (1998)) of DAOCS by the residue in 10 around the selection active centre.At first change each site into the Ala residue, change the positively charged residue then into, hydrophobic residue and sulfur-bearing residue.Specification sheets design primer according to manufacturers.By the plasmid that contains gene is carried out the mutant that dna sequencing is confirmed gained, and the preparation cell crude extract, carry out the DAOCS determination of activity.Embodiment 4 DAOCS determinations of activity
BL21 (DE3) transformant contains grow overnight in the LB substratum of 50 μ g/ml kantlex at 5ml under 30 ℃.This culture is used to inoculate the same substratum of 100ml, and 30 ℃ of growths 1 hour.Add 0.1mM IPTG then, with 30 ℃ of indirect induction DAOCS 4 hours.Collecting cell is with buffer A (50mMMops/pH7.5,1mM PMSF, 1mM DTT and 0.5 μ g/ml leupeptin) washing, ultrasonic (VCX750; Sonics ﹠amp; Materials, U.S.) dissolving, at 15000xg centrifugal 15 minutes.This supernatant liquor is used as cell extract.
To penicillin G, 20 μ l cell extracts are joined in the standard 200 μ l test reaction liquid, this solution contains 50mM Mops/pH 7.5,4mM xitix, 1mMFeSO
4, 4mM α-pentanone diacid and 7mM penicillin G, and 30 ℃ of insulations 20 minutes.By adding 200 μ l ethanol termination reactions, then centrifugal 5 minutes at 15000xg.By the HPLC concentration of cynnematin G in the clear liquid analytically, sample introduction 40 μ l.Moving phase is the 25mM potassiumphosphate/pH 6.5 that contains 19% acetonitrile, and flow velocity is 1ml/min.Detect at the 215nm place, the retention time of cynnematin G is 8.5 minutes, and the retention time of penicillin G is 12.5 minutes, and retention time is that the molecular weight of 8.5 minutes product is 370 in reacting by LC-MS affirmation DAOCS.Obtain rite-directed mutagenesis type such as YS49 (L277K), YS8 (I305L), YS11 (I305M) and Y12 (N304K), they have higher ring expansion activity to penicillin G than existing bacterial strain YS16.The amino acid whose various combination of said mutation also makes up by site-directed mutagenesis.Embodiment 5 DAOCS purifying
The cell extract (2ml) of mutant is added with on buffer B (50mMMops/pH 7.5,1mM DTT, 0.4mM pefabloc SC, 0.5 μ g/ml leupeptin) the equilibrated HiTrap Q post (5ml).Wash post with the 20ml buffer B then, then the buffer B that contains 120mM NaCl with 25ml is further washed, and DAOCS is eluted by the buffer B that 20ml contains 150mM NaCl.Merge the part (10ml) that contains DAOCS, with UFl5 device (NMWL 5K; Millipore) concentrate, and be loaded on Hiload Superdex 75 (16/60) posts, this post is through the flow velocity pre-equilibration of buffer B with 1ml/min.Detect by SDS-PAGE, the purity of the DAOCS that is purified into like this is higher than 90%, sees Fig. 1 and Fig. 2, immediately it is stored among 1mM DTT and the 2mg/ml BSA in-80 ℃.The kinetic determination of the DAOCS of embodiment 6 purifying
Carry out standard penicillin G mensuration according to kinetic determination (Kinetic assay), but use the DAOCS of 20 μ g purifying, and the concentration of α-pentanone diacid is reduced to 1mM.Obtain kinetic parameter by Hans-Woolf Plot from parallel three parts of tests.For using the mainly mensuration of substrate of penicillin N conduct, the reaction solution that with cumulative volume is 240 μ l was 30 ℃ of insulations 10 minutes, and this reaction solution contains 50mM Mepes/pH 7.5,0.4mM xitix, 0.1mM FeSO
4, 0.1mM α-pentanone diacid, the DAOCS of 0.1mM penicillin N and 0.5 μ g purifying adds isopyknic 10mM EDTA/pH7.5 then, and this mixture is with UFl5 device (NMWL 5K; Millipore) ultrafiltration.Filtrate is analyzed by HPLC, uses 25mM potassiumphosphate/pH 6.5 as moving phase.The retention time of DAOC and penicillin N was respectively 12 minutes and 13.5 minutes.Kinetic parameter the results are shown in table 1, the wherein definition of parameter and calculate at Lehninger etc., Principles of Biochemistry, 2.sup.nd Ed.WorthPublishers, New York (1993), it is incorporated herein for referencial use.Table 1: kinetic parameter bacterial strain Km (mM) K of penicillin N and penicillin G
Cat(S
-1) K
Cat/ Km
(M
-1S
-1) penicillin N YS16 (wild type) 0.014 ± 0.006 0.307 ± 0.038 22000YS5 (V275I) 0.012 ± 0.003 0.252 ± 0.020 20000YS8 (I305L) 0.006 ± 0.002 0.284 ± 0.030 44000YS11 (I305M) 0.012 ± 0.001 0.310 ± 0.004 26000YS12 (N304K) 0.004 ± 0.001 0.366 ± 0.023 92000YS125 (N304L) 0.018 ± 0.004 0.415 ± 0.063 23000YS49 (L277K) 0.011 ± 0.005 0.220 ± 0.042 20000YS53 (C281Y) 0.006 ± 0.001 0.273 ± 0.014 47000YS59 (S79E) 0.009 ± 0.003 0.178 ± 0.019 20000YS115 (M73T) 0.006 ± 0.003 0.239 ± 0.009 40000YS67 (V275I ﹠ amp; I305M) 0.013 ± 0.005 0.316 ± 0.032 24000 benzyl penicillin YS16 (wild type), 2.58 ± 0.22 0.0302 ± 0.0007 12YS5 (V275I), 1.68 ± 0.20 0.0335 ± 0.0008 20YS8 (I305L), 0.66 ± 0.07 0.0506 ± 0.0010 77YS11 (I305M), 0.75 ± 0.04 0.0968 ± 0.0013 129YS12 (N304K), 0.22 ± 0.03 0.0376 ± 0.0002 171YS125 (N304L), 0.55 ± 0.12 0.0268 ± 0.0004 49YS49 (L277K), 0.72 ± 0.02 0.0343 ± 0.0005 48YS53 (C281Y), 0.68 ± 0.34 0.0496 ± 0.022 73YS59 (S79E), 0.75 ± 0.02 0.0210 ± 0.0002 28YS115 (M73T), 0.74 ± 0.16 0.0418 ± 0.0014 56YS67 (V275I ﹠ amp; I305M) 0.25 ± 0.08 0.0972 ± 0.025 389
As shown in table 1, from k
Cat/ Km (M
-1S
-1) parameter as can be seen, all mutants are all high 2 to 32 times than wild-type ring enlargement enzyme to the ring expansion activity of penicillin G.Comparatively speaking, except mutant YS12, these mutants do not cause deriving from the noticeable change of the kinetic parameter of penicillin N, and the ring expansion specific activity wild-type YS16 of YS12 is high 4 times.The YS125 (N304L) of (on seeing) such as YS12 more of the present invention (N304K) and Chih H.S., YS125 is high 4 times to the ring expansion specific activity wild-type ring enlargement enzyme of penicillin G, and YS12 is high 14 times to the ring expansion specific activity wild-type of penicillin G.
The relative reactivity of each mutant sees Table 2, and it calculates according to (on seeing) such as Chih H.S., with the activity of wild-type DAOCS as 100%.Measure the same standard test of other condition with the 1mM penicillin G.Table 2: compare with wild-type, relative reactivity DAOCS bacterial strain mutational site relative reactivity (%) the YS16---100YS67 275I of the mutant of the combination of purifying, 305M 500YS74 275I, 304K, 305L 300YS81 275I, 281Y, 305M 1290YS88 79E, 275I, 281Y 430YS94 281Y, 304K, 305M 650YS96 79E, 275I, 305M 1110YS100 79E, 275I, 305L 470YS76 79E, 275I, 281Y, 305L 250YS108 300V 410SC29 275I, 281Y, 300V 620SC39 73T, 281Y 610
Claims (14)
1. Tu Bian penicillin ring enlargement enzyme, it is included in the amino-acid substitution in one or more residues site, described residue site is corresponding to the residue site of wild-type ring enlargement enzyme, it is selected from methionine(Met) 73, Serine 79, Xie Ansuan 275, leucine 277, halfcystine 281, glycine 300, l-asparagine 304 and Isoleucine 305, and condition is that the amino-acid substitution in the residue site of l-asparagine 304 is not N304L.
2. the penicillin ring enlargement enzyme of the sudden change of claim 1, wherein the wild-type ring enlargement enzyme obtains from band spillikin streptomycete.
3. the penicillin ring enlargement enzyme of the sudden change of claim 1, it is included in the amino-acid substitution that is selected from M73T, S79E, V275I, L277K, C281Y, G300V, N304K, I305L and I305M in one or more residues site.
4. the isolated nucleic acid molecule of the penicillin ring enlargement enzyme of the sudden change of the claim 1 of encoding.
5. comprise the isolated nucleic acid molecule of claim 4 and regulate the recombinant vectors of sequence.
6. use the reconstitution cell of the nucleic acid molecule conversion of claim 4.
7. the reconstitution cell of claim 6, it is to produce the penicillin G cell.
8. the reconstitution cell of claim 7, it is the Penicllium chrysogenum mycetocyte.
9. produce the method for the penicillin ring enlargement enzyme of sudden change, described method comprises the nucleic acid molecule of expressing claim 4, and the step that reclaims the penicillin ring enlargement enzyme of sudden change.
10. produce the method for the penicillin ring enlargement enzyme of sudden change, described method comprises the reconstitution cell of cultivating claim 6 expressing the penicillin ring enlargement enzyme of sudden change, and the step that reclaims the penicillin ring enlargement enzyme that suddenlys change from cell culture.
11. prepare the method that 7-amino removes acetoxyl cephalosporonic acid (7-ADCA); described method comprises with the penicillin ring enlargement enzyme of the sudden change of claim 1 handles penicillin G with preparation phenylacetyl-7-ADCA, and phenylacetyl-7-ADCA is gone the step of acidylate with preparation 7-ADCA.
12. prepare the method for 7-ADCA, said method comprising the steps of: (a) under the condition of the penicillin ring enlargement enzyme that is suitable for producing penicillin G and expressing sudden change, cultivate product penicillin G cell with the nucleic acid molecule conversion of claim 4, so that the ring enlargement enzyme ring expansion that penicillin G is suddenlyd change, and preparation phenylacetyl-7-ADCA; (b) phenylacetyl-7-ADCA is gone acidylate with preparation 7-ADCA.
13. the method for claim 12, wherein producing the penicillin G cell is the Penicllium chrysogenum mycetocyte.
14. the method for claim 12 is wherein by the phenylacetyl-7-ADCA that filters and extraction step recycling step (a) prepares.
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| WO2005103261A1 (en) * | 2004-04-22 | 2005-11-03 | Orchid Chemicals & Pharmaceuticals Ltd. | Modified expandase enzyme and its use |
| CN105154489A (en) * | 2015-08-25 | 2015-12-16 | 浙江昂利康制药股份有限公司 | Method for preparing cephalosporin intermediates by aid of enzymatic processes |
| WO2017181809A1 (en) * | 2016-04-18 | 2017-10-26 | 百瑞全球有限公司 | Penicillin expandase mutant, dna encoding mutant, and reagent kit containing mutant and use thereof |
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2002
- 2002-04-01 CN CN02108779A patent/CN1448506A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005103261A1 (en) * | 2004-04-22 | 2005-11-03 | Orchid Chemicals & Pharmaceuticals Ltd. | Modified expandase enzyme and its use |
| JP2007533316A (en) * | 2004-04-22 | 2007-11-22 | オーキッド ケミカルズ アンド ファーマシューティカルズ リミテッド | Modified expandase enzyme and its use |
| CN105154489A (en) * | 2015-08-25 | 2015-12-16 | 浙江昂利康制药股份有限公司 | Method for preparing cephalosporin intermediates by aid of enzymatic processes |
| CN105154489B (en) * | 2015-08-25 | 2018-12-21 | 浙江昂利康制药股份有限公司 | A kind of method that enzyme process prepares cephalosporin intermediate |
| WO2017181809A1 (en) * | 2016-04-18 | 2017-10-26 | 百瑞全球有限公司 | Penicillin expandase mutant, dna encoding mutant, and reagent kit containing mutant and use thereof |
| CN107304418A (en) * | 2016-04-18 | 2017-10-31 | 百瑞全球有限公司 | Penicillin ring enlargement enzyme mutant, the DNA for encoding the mutant, the kit containing the mutant and its application |
| US10988742B2 (en) | 2016-04-18 | 2021-04-27 | Bioright Worldwide Co., Ltd. | Penicillin expandase mutants, DNA coding the mutants, reagent kit containing the mutants and the application |
| CN107304418B (en) * | 2016-04-18 | 2022-08-26 | 百瑞全球有限公司 | Penicillin expandase mutant, DNA encoding the mutant, kit containing the mutant and use thereof |
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