MXPA99004513A - ENZYMATIC PROCESS FOR THE PREPARATION OF CEPHALOSPORANIC 7$b(g)-(4-CARBOXYBUTANAMIDE) ACID BY MEANS OF THE MODIFIED ENZYME D-AMINOACID OXIDASE OF TRIGONOPSIS VARIABILIS - Google Patents
ENZYMATIC PROCESS FOR THE PREPARATION OF CEPHALOSPORANIC 7$b(g)-(4-CARBOXYBUTANAMIDE) ACID BY MEANS OF THE MODIFIED ENZYME D-AMINOACID OXIDASE OF TRIGONOPSIS VARIABILISInfo
- Publication number
- MXPA99004513A MXPA99004513A MXPA/A/1999/004513A MX9904513A MXPA99004513A MX PA99004513 A MXPA99004513 A MX PA99004513A MX 9904513 A MX9904513 A MX 9904513A MX PA99004513 A MXPA99004513 A MX PA99004513A
- Authority
- MX
- Mexico
- Prior art keywords
- enzyme
- leu
- gly
- amino acid
- ser
- Prior art date
Links
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- UKKNTTCNGZLJEX-UHFFFAOYSA-N γ-glutamyl-Serine Chemical compound NC(=O)CCC(N)C(=O)NC(CO)C(O)=O UKKNTTCNGZLJEX-UHFFFAOYSA-N 0.000 description 1
Abstract
Enzymatic process for the preparation of cephalosporanic 7&bgr;-(4-carboxybutanamide) acid by using the modified enzyme D-aminoacid oxidase of Trigonopsis variabilis produced in Escherichia coli. The process for the expression of the enzyme comprises:(I) isolating the DNA corresponding to the gene which codes for the enzyme D-aminoacide oxidase;(II) removing the intron which is contained in said gene;(III) inserting the DNA fragment obtained into the plasmide which is capable of replication in Escherichia coli;(IV) fusing at the extremity 5'of the structural region of the gene a synthetic assembler which contains a nucleotide sequence which codes for six histidines;(V) transforming a strain of Escherichia coli with the resulting recombinant plasmide;(VI) cultivating the transformed cells of Escherichia coli;and (VII) recovering the enzyme D-aminoacid oxidase of the former cultivation operation through affinity chromatography.
Description
AN ENZYMATIC PROCEDURE FOR PREPARING ACID 7 ETA- (4- CA BOXIBUTANAMIDO) CEFALOSPORANICO USING THE D-AMINOACIDO ENZYME OXIDASE OF TRIGONOPSIS MODIFIED VARIABILIS PRODUCED IN ESCHERICHIA COLI
FIELD OF THE INVENTION
The present invention relates to an enzymatic process for the preparation of 7β ~ (4-carboxy-butanamido) cephalosporanic acid. More particularly ,. describes a method to isolate the gene that codes for an enzyme with D-amino acid oxidase activity through the use of recombinant DNA techniques, the cloning of said gene into a microorganism of the genus Escherichia, the modification of said enzyme by engineering techniques of proteins, the overproduction of said enzyme modified by fermentation in said microorganism and the extraction of the modified enzyme for the preparation of 7β- (4-carboxybutanamido) cephalosporinic acid. This acid is an intermediate compound for the preparation of 7-amino cephalosporinic acid, which in turn is a known intermediate for the preparation of a wide variety of antibacterial agents of the cephalosporin family.
REF .: 30154 BACKGROUND OF THE INVENTION
For the production of 7β- (4-carboxybutanamido) cephai-sporic acid, also called glutaryl-7-aminocephalosporanic acid (hereinafter referred to as GL-7ACA) from cephalosporin C, the use of the enzyme D-amino acid oxidase is known. (hereinafter referred to as DAO) from various microorganisms such as Trigonopsis variabilis (Biochem. Biophys., Res. Commun. (1993) 31: 709), Rhodotorula gracilis (J. Biol. Chem. (1994) 269: 17809) and Fusarium. Solani (J. Biochem. (1990) 108: 1063). The production of DAO through the use of these microorganisms has many drawbacks. On the one hand, the production level of DAO activity is very low, and on the other, along with said enzyme, other undesirable enzymatic activities such as esterases and catalases are present. The former degrade the GL-7ACA acid, decreasing the yield and therefore increasing the purification process. The second ones destroy the hydrogen peroxide necessary in the catalysis and force the addition of this compound, which also makes the process more expensive and at the same time produces a loss of activity of the enzyme, shortening its possibilities of reuse. To avoid such enzymatic contamination, it is necessary to purify the DAO activity, which makes the enzymatic process of obtaining GL-7ACA from cephalosporin C considerably more difficult and has recently described a method for isolating the gene coding for DAO in T. variahilis and expressing it in E. coli and in T. variahili (Japanese Patent Application Laid-Open No. 71180/1988; European Pat.ent Application No. 93202219.7, Publication No. 0583817A2). On the other hand, it has also been donated and expressed in E. coil and Achremonium chrysogenum and the gene coding for the DAO activity of F. solani (Japanese Patent Application Laid-Open No. 2000181/1990, J. Biochem. (1990) 108 : 1063; Bio / Technology (1991) 9: 188) and more recently the gene coding for DAO has been donated and expressed in E. ooli in R. gracilis (Spanish Patent P9600906). Given the great industrial interest in the production of GL-7ACA for the availability of a purified DAO activity, the objective of the present invention was focused on the production of an enzyme with DAO activity that was easily purifiable. In this sense, it is known that one of the most effective ways for the purification of a protein is the use of affinity chromatography (Sassenfeld, H.M. (1990) Trends Biocechnol, 8:88). For this it is necessary to find a chromatographic support that allows the union and / or selective elution of the protein of interest. Said chromatographic support must contain a recognition molecule or ligand for said protein in such a way that the interaction between the ligand and it is sufficiently specific and strong to allow selective elution of the protein. The development of an affinity chromatographic support is not simple and to date no process has been described that would allow purification by affinity in a single step of enzymatic DAO activities. On the other handIt is known that certain genetic engineering procedures allow the modification of proteins improving certain properties that facilitate their purification. Thus, different systems for modifying the structure of a protein have been developed to allow its purification by affinity (Sii, D. and Sadana, A. (1991) J.
Biotechnol. 19:83; Narayanan, S.R. and Crane, L. J.
(1990) Trends Biotechnol. 8:12; Scouten,. H. (1991) Curr. Opinion Biotechnol. 2:37; ñassenfeld, H. M. (1990) Trends Biotechnol. 8:88). In essence, these modifications consist of fusing to the protein a polypeptide that possesses specific properties of interaction with a determined chromatographic support and that therefore confers to the fusion protein the capacity to be purified- by affinity chromatography. One of these modifications consists in fusing to the protein in question a polyhistidine sequence that confers to the fusion protein the property of being able to be purified by affinity using a support containing divalent metal ions (Arnols, FH (1991) Bio / Technology 9 : 151; Hochuli et al. (1988) Bio / Technology 6: 1321). Although obtaining fusion proteins to improve the purification properties is in principle a simple and effective technique, its biggest drawback lies in the fact that the modification of a protein in its primary structure entails changes that can have an important reflection in its secondary, tertiary and quaternary structures. These structural changes can affect the protein to the point of losing its functionality totally or partially, turning it into a useless protein for the function that had been foreseen. Therefore, obtaining a fusion protein always entails great uncertainty since the final result is largely unpredictable and especially when the first fusion experiment is carried out, that is, when the result of previous mergers is not known. It is in this uncertainty of the final result that lies in the novelty of the process of obtaining a fusion protein, since currently it is not possible to predict with certainty what will happen when a protein fusion experiment is carried out. No procedure has been described in the scientific literature to produce the genetically modified T. variabilis DAO enzyme in such a way that it can be purified in a single step and that it can be actively overproduced in either E. coli or another microorganism.
DETAILED DESCRIPTION OF THE INVENTION
For the description of this invention, we start from the yeast T. variabilis ATCC 20931 as a donor of deoxyribonucleic acid (hereinafter referred to as DNA). Once the genomic DNA of the yeast (which contains the gene with the genetic information relative to the production of the DAO, also referred to as the dao gene) was obtained, it was used to construct a library in E. coli using the vector phage? -GEM12. The analysis of the library was carried out using standard hybridization techniques using synthetic oligonucleotides designed as a function of regions of similarity found in three different DAOs. In this manner, a series of recombinant E. coli clones containing a DNA fragment of T. variabilis which codes for the dao gene were isolated. The DNA fragment thus obtained was subcloned into a plasmid vector obtained from an E. coli strain. The recombinant vector was used to obtain the sequence of the DNA fragment containing the dao gene of T. Variabilis (SEQ ID NO: 1). The analysis of this sequence allowed to characterize the dao gene, which is structured in two exons and one intron. As the DNA fragment previously obtained
which contains the genomic sequence of the dao gene of T. variabilis possesses an intron, can not be used directly for its expression in E. coli. For this, we proceeded to obtain a dao gene lacking this intron. For this, an amplification of the dao gene was carried out
by PCR using synthetic oligonucleotides. The first one was designed in such a way as to contain the following elements: a ribosome binding site, a translation initiation site, the complete sequence of the first exon and the first nucleotides of the
end 5 'of the second exon. The second oligonucleotide contained the sequence complementary to the 3 'end corresponding to the second exon of the dao gene, including a translation stop codon. In these synthetic oligonucleotides were also included different
? D restriction sites useful for the cloning of DNA fragments. A new DNA fragment was obtained which, once isolated, was cloned into a plasmid vector of E. coli (Figure 1). Using the restriction targets created above, the DNA fragment containing the complete dao gene without the intron was subsequently subcloned into different plasmid vectors of E. coli that possess promoters that allow hyperexpression of genes in this host bacterium. The evaluation of the DAO activity produced by the different clones was carried out by colorimetric techniques and by HPLC chromatography. In this manner recombinant E. coli clones were obtained which produced a large amount of active DAO enzyme. Next, the dao gene was modified by adding a nucleotide sequence that codes for a polyhistidine, for which a plasmid containing the dao gene was digested with a restriction enzyme that cut at the initiation codon of the translation and the ends of DNA resulting from digestion were made blunt with the Klenow fragment of DNA polymerase I from E. coli, then ligated in the presence of a synthetic cenector containing the codons of pelihistidine, generating a plasmid containing the gene Dao modified at the 5 'end of its coding region (SEQ ID NO: 2) The recombinant plasmid thus generated was transformed into an E. coli strain and selected by hybridization techniques using the oligonucleotides of the polyhistidine linker as a probe. The modified dao gene was sequenced to verify that the desired fusion had been correctly produced, then the production of D was studied. AO modified with polyhistidine (hereinafter referred to as hisDAO) using, as hosts of the plasmids previously constructed, different E. coli strains. In this way it was found that the hisDAO enzyme was active. For the production of hisDAO using the previously selected recombinant E. coli clones, they are grown in a medium containing a carbon source, a nitrogen source and mineral salts. The incubation temperature is between 18 ° C and 37 ° C and the pH must be maintained between 5 and 9. For small-scale fermentation, flasks of different volumes, from 50 ml to 1,000 ml, can be used, with an amount of medium between 10% and 50% of the volume of the flask. The duration of the fermentation can range between 12 and 90 hours. The production of DAO by the recombinant microorganism can be improved if suitable culture conditions are chosen to maintain the stability of the recombinant vectors, which is achieved by the addition to the culture medium of the antibiotics for which the recombinant vector containing the gene present a marker of resistance (ampicillin, chloramphenicol, kanamycin, tetracycline, etc.). This, in addition to stabilizing the production, avoids the contamination of the culture medium with other undesirable microorganisms and also eliminates the strains that, due to having lost the recombinant vector, have stopped producing DAO. The hisDAO producing recombinant cells were separated from the culture medium by centrifugation and subsequently broken or permeabilized by chemical, enzymatic or mechanical methods. To obtain a more pure enzymatic extract, hisDAO was purified in a single step by affinity chromatography on Co2 + -IDA columns. For this, the crude enzymatic extract was loaded onto a Co2 + -IDA resin and the contaminating proteins were eluted by washing with 20 mM phosphate buffer pH 7.0 containing 0.2 M NaCl. Then the hisDAO enzyme was eluted by washing with 10 mM imidazole. If, instead of Co2 + -IDA, it is used as a chromatographic support Cu2 + -IDA or Zn2 + -IDA, the hisDAO enzyme is strongly bound to the matrix in an active form without being able to be eluted with high concentrations of imidazole. In this way the support, conveniently washed to remove contaminating proteins, can be used as an immobilized enzyme system. Using purified hisDAO enzyme from recombinant clones of E. coli expressing the chimeric gene, GL-7ACA was obtained from cephalosporin C. Enzymatic extracts obtained from chromatography on Co2 + -ID support dialysates and concentrates can also immobilized by reacting them with other suitable inert solid supports, being able to cyclically use immobilized hisDAO. The novelty of this invention lies in the fact that it is the first time that the DAO enzyme of the yeast T. variabilis, modified as hisDAO, can be expressed in an active form in a prokaryotic microorganism as E. coli and purified in a single step by affinity chromatography. Additionally, an increase in DAO production has been achieved with respect to the amount obtained in T. variabilis, which facilitates the industrial scale use of this enzyme. The possibility of producing hisDAO in different strains of E. coli lacking undesirable enzymes such as catalase or esterases, as well as the possibility of improving its stability and catalytic properties through new modifications by protein engineering techniques are other aspects that increase the concept of novelty of this patent. The present invention, without limitation, will be illustrated in more detail in the Examples described below.
EXAMPLE 1
1. - Test of the DAO activity
The DAO activity assay was performed according to a previously described procedure (J. Biol. Chem. (1967) 242: 3957), using D-phenylglycine (25 mM) as substrate. The incubation was carried out in 50 mM phosphate buffer pH 8.0 for 15 to 30 minutes, stopping the reaction with 1/10 volume of pure acetic acid. The variation of the D.O. at 252 nm it determines the activity of the enzyme, taking into account that 89.77 nmol of the benzoyl formic acid that originates in the reaction have a D.0. 2 of 1.0.
2. - Preparation of DNA vectors and E. cells
coli competent for the transformation.
The plasmid vectors pBluescript I KS (+) (Apr) (Stratagene) and pKK233.2 (Apr) (Pharmacia) were prepared following the alkaline lysis method (Sambrook et al. (1989) Molecular Cloning: A laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA). For this, the strains of E. coli carrying the aforementioned plasmids were incubated for 16 hours with agitation in an orbital shaker at 250 r.p.m. and 37 ° C in 500 ml of LB with 100 μg / ml of ampicillin. The plasmid DNA obtained by this method was purified by centrifugation in CsCl gradient. The competent cells of E. coli TG1 and E. coli DH5 were obtained by the RbCi method (Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA) .
3. - Preparation of the donor DNA that possesses the genetic information related to the production of DAO
The strain T. variabilis ATCC 20931 was cultured in the YMPG medium (malt extract 0.3%, yeast extract 0.3% peptone 0.5% and glucose 1%). Incubation was continued for 36 hours (D.0.66o - 5.0) with agitation in an orbital shaker at 250 r.p.m. and at a temperature of 30 ° C. The cells were then pelleted, washed and used with zymolyase and the DNA was extracted following a previously described procedure (Sherman et al (1986) Laboratory Course for Methods in Yeast Genetics, Coid Spring Harbor Laboratory, Cold Spring Harbor, New York). To achieve greater purity the DNA was treated with RNase, extracted several times with phenol and chloroform-isoamylalcohol 24/1 (CIA) and precipitated with isopropanol. The precipitated DNA was washed with 100% ethanol and 70% ethanol and was "dissolved in the buffer 10 mM Tris-HCl pH 7.5 containing 1 mM EDTA (TE buffer).
EXAMPLE 2
1. - Construction of a T. variabilis library
Obtaining the total DNA of strain T. variabilis ATCC 20931 was carried out as described in section 3 of example 1. A total of 300 μg of said total DNA was partially digested with 20 units of Sau3AI in a reaction volume of 600 μl at 37 ° C and 3 aliquots of 200 μl were collected at 45 seconds, 1 minute and 2 minutes respectively, stopping digestion with EDT? 20 mM cold. After checking the digestions in a gcl of 0.7% agarose, they were mixed, heated at 68 ° C for 10 minutes, allowed to cool slowly to room temperature and placed on a gradient of sucrose (10-40%) of 38 ml . Said gradient was centrifuged at 26,000 r.p.m. for 24 hours at 15 ° C, collecting aliquots of 0.5 ml, 10 μl of which were analyzed on a 0.4% agarose gel. Aliquots whose? ND ranged between 18 and 22 kb were mixed and diluted with distilled water to approximately 10% sucrose. The DNA was then precipitated with ethanol, resuspended in 50 μl of a TE buffer and 3 μl of the latter solution was analyzed on a 0.4% agarose gel. In said gel it was found that the size of the DNA fragments was correct and that their concentration was approximately 50 ng / μl. In parallel, the bacteriophage ADM-GEM12 (Promega) was prepared by previously described procedures with slight modifications (Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA). For this, the strain E. coli NM538 (Promega) was grown for 10 hours in NZCYM-0.2% maltose and its D.O. at 600 nm. The culture volume corresponding to 3 x 10 9 cells was centrifuged at 4,000 r.p.m. for 10 minutes at 4 ° C in a tabletop centrifuge and resuspended in 1.2 ml of SM buffer. To these cells were added 3 x 107 plaque forming units (pfu) of phage? -GEM12 and the mixture was incubated for 30 minutes at 37 ° C without agitation. With 200 μl of the infected cells, each of the flasks (500 ml with 100 ml of NZCYM-0.2% maltose medium) was inoculated preheated to 37 ° C. Said flasks were incubated at 37 ° C until the culture appeared lysate (5-6 hours). The lysates were treated with DNase (1 μg / ml) and RNase (2 μg / ml) for 45 minutes at room temperature. Then 5.8 grams of NaCl were added per 100 ml of lysate and the mixture was kept for 60 minutes on ice. At the end of this time the lysate was filtered to remove the cell debris and after adding 20 ml of 50% PEG-6000 for each 100 ml of lysate, the mixture was kept 60 minutes on ice and centrifuged at 10,000 xg for 20 minutes at 4 minutes. ° C. The pellet was resuspended in 1 ml of TE buffer and extracted 2 times with CIA to remove the remains of PEC-6000 without breaking the phage. Subsequently, it was extracted several times with neutral phenol, one with phenol-CIA and one with CIA. The aqueous phase was brought to 0.5 M NaCl (with 4 M NaCl) and the DNA was precipitated with two volumes of ethanol at -2 ° C. Once centrifuged for 20 minutes at 4 ° C and 12,000 r.p.m. in a mini-centrifuge, the precipitated DNA was washed with 70% ethanol, dried and resuspended in 50 μl of TE buffer. 50 μg of bacteriophage DNA were digested with BainHI and Xbal endonucleases at 37 ° C for 2 hours. The double digestion was extracted with phenol-CIA and CIA, precipitated with ethanol and resuspended in 50 μl of TE buffer. After collecting an aliquot of 2 μl, MgCl2 was added to the rest up to 10 μM and incubated at 42 ° C for 1 hour to favor the recoilment of the vector arms by their cohesive ends. Again, a fraction of 2 μl was collected and analyzed with the previous one in a 0.5% agarose gel. Once the correct coagulation was verified by the cohesive ends, the mixture was placed on a gradient (10-40%) of 38 ml sucrose. In this case, the heating of the DNA at 68 ° C was prevented before being placed in the gradient, since this would lead to the separation of the cohesive ends of the phage. The gradient was centrifuged at 26,000 r.p.m. for 24 hours at 15 ° C, then collecting in aliquots of 0.5 ml. After analyzing 15 μl of each of them in a 0.5% agarose gel, those lacking the central fragment or "stuffer" were mixed and diluted with distilled water to about 10% sucrose. The DNA was precipitated with ethanol, resuspended in 50 μl of TE and 2 μl of the latter solution were visualized on an agarose gel (0.5%) to confirm the absence of the central fragment and estimate that its approximate concentration was 100 ng / μl. Subsequently, a series of ligations was performed using 0.25 μg of insert and vector quantities between 0.25 and 0.75 μg, varying the insert / vector ratio. The reactions were incubated at 12-14 ° C for 16 hours. After checking on a 0.4% agarose gel the appearance of DNA fragments (caused by ligation) larger than that of the vector or insert, all the ligation reactions were mixed, precipitated with ethanol and resuspended in 4 μl of buffer of ligation. The encapsidation of the recombinant phage DNA originated after ligation was performed with the packaging in vitro extracts Packagene (Promega). With the result of the encapsidation reaction resuspended in 500 μl of SM, E. coli NM538 infections were performed to titrate the number of phage present and E. coli NM539 (Promega) in order to determine the percentage of recornbinant phage. E. coli NM539 is a lysogenic strain of phage P2 and only causes lysis plaques when the phage infecting it lacks the central dispensable region. The constructed library contained about 200,000 pfu and about 85% of the phages were carriers of an exogenous DNA fragment. Once these calculations are made, E. coli NM539 was infected and the entire genetic library was spread on 150 mm diameter Petri dishes.
2. - Identification of the clones that contain the dao gene
The complete library was transferred to nitrocellulose filters and the positive phage selection process was carried out according to a previously described hybridization procedure with slight modifications (Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA). The process was initiated with the prehybridization of the nitrocellulose filters by incubation at 42 ° C for 3 hours in hybridization buffer. Hybridization was carried out by removing the buffer used in the prehybridization and introducing new hybridization buffer together with 10 pmol of the OA oligonucleotides (5 '-TCTTGTCCTCGACACC-3') and OB (5'-GACGTGATTGTCAACTG-3 ') labeled at its 5 end ' with
32 P by polynucleotide kinase from phage T4 and [? - P] ATP (ICN Biochemicals) by standard procedures (Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA ). The filters were incubated at 42 ° C for 16 hours and washed several times for 20 minutes at room temperature in 2 X SSC - 0.1% SDS, followed by two other washings at the same time at the hybridization temperature in 0.1 X SSC buffer. 0.1% SDS. Finally, the nitrocellulose filters were exposed with Hyperfilm-MP (Amersha) under amplifying screens at -70 ° C for 48 hours. Once the process of prehybridization, hybridization, washing and autoradiography was completed, 63 clones were selected that originated "positive signals with the two OA and OB probes, only 9 of the positive lysis plates were collected individually with the help of a Pasteur pipette and each one of them was resuspended in 1 ml of SM plus 50 μl of chloroform, then the phages present in said solution were titrated, for which it was necessary to dilute 5,000 times said phage so that when carrying out the infection with 20 μl of it, the lysis plates per Petri dish will oscillate between 500 and 1000. Once the content of each Petri dish was transferred to its corresponding nitrocellulose filter, the latter hybridized again with the same OA and OB pathways. The autoradiography showed that between 20 50% of the phages from each Petri dish generated a positive signal, therefore, it became necessary to purify each of the positive phages through a t erce hybridization cycle. To do this, those positive lysis plates that were more isolated from the rest or those that were surrounded by positive lysis plates were collected with Pasteur pipette and resuspended in 1 ml of SM plus 50 μl of chloroform. After diluting this phage solution 100 times and infecting it with 15 μl of it, titers of about 300 lysis plates were obtained per Petri dish. Once processed in identical conditions to the previous two cycles, it was achieved that 100% of the fages of each Petri dish were positive. In this way, 9 independent lysis plates were purified. Each lysis plate was resuspended in 100 μl of SM plus 10 μl of chloroform and with 2 μl of this solution confluent lysis plates were obtained in order to amplify said positive phages in solid medium. After collecting the upper agarose layer and resuspending it in 5 ml of SM, solutions with an approximate concentration of 107 pfu / μl were obtained for each of the positive phages. The above-mentioned oligonucleotides OA and OB were also used as a probe to determine by the Southern method (Sambreok et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA) of genomic DNA that included the dao gene of T. variahili. For this purpose, a hybridization with the digested DNA with the endonucleases (Pharmacia) of restriction BamHl, EcoRl, Hindl? L, Kpnl, Pstl, Pvull was carried out under the conditions described below., Xbal and Xhol and fractionated in agarose gel. The agarose gel in which DNA fragments were separated by their molecular size was incubated sequentially at room temperature and with gentle agitation for 15 minutes in 0.25 M HCl, 1 hour in denaturing solution and 1 hour in neutralizing solution. The gel was then placed on a block of Whatman 3 MM filter papers soaked in 10 x SSC and a BA85 nitrocellulose filter (0.45 μm) (Schleicher and Schuell) of the dimensions of the gel and soaked in 2 x SSC was placed on it. avoiding the formation of bubbles. Two sheets of Whatman 3MM paper, embedded in 2 x SSC, were placed on the nitrocellulose filter, 8-10 centimeters of dry filter paper of the same dimensions were placed on top of it and above all, a weight of around 500 grams. The transfer process was maintained for 16 hours. Once the DNA was transferred, the nitrocellulose filter was carefully immersed in 6 x SSC for 5 minutes, allowed to dry at room temperature for 1 hour and incubated between two sheets of Whatman 3MM paper at 80 ° C with an additional 3 hours to get the fixation of the DNA to the filter. Then, it was prehybridized and hybridized under the same conditions described above for the screening of the library. The result of autoradiography showed the appearance of specific bands of hybridization for each of the digestions. Specifically, the size of said bands was as follows: 10.2 kb for EcoRI digestion,
3. 7 kb for the BamHl, 3.5 kb for the Hindlll, 11.6 kb for the Kpnl, 11.3 for the Psti, 4.8 kb for the PvuII,
2. 8 for the Xbal and 4.7 kb for the Xhol.
3. Cloning of the DNA fragment that codes for the gene
After purifying the phage DNA as indicated in section 1 of Example 2 for the bacteriophage. ? -GEM12, a Sal digestion was performed identifying a band of 9.2 kb that was subcloned in the plasmid pBluescript I KS (+) (Stratagene) digested with Sali using DNA ligase from phage T4 (Amersham), ATP and the buffer recommended by the suppliers of the enzyme. The resulting ligation mixture was incubated for 5 hours at 12 ° C and used to transform competent E. coli TG1 cells (Amersham). Transformants were selected in solid LB medium to which ampicillin (100 μg / ml), X-gal (40 μg / ml) and 0.2 mM IPTG had been added. Among the clones that had the white selection phenotype, the plasmid pALT1 was isolated. Next, using the Seuthern technique and the OB path, the dao gene was located in a 3.7 kb BamHl fragment included in the pALTl plasmid. This fragment .BamHl was subcloned into the plasmid pBluescript I KS (+) (Stratagene) digested with BamHl. Thus plasmids pALT2 and pALT3 were isolated, which contain the BamHI fragment of 3.7 kb in both orientations (Figure 1).
4. - Sequencing of the fragment containing the dao gene
The nucleotide sequence of the fragment contained in the plasmid pALT2 was determined on the same plasmid by a previously described method (Sanger et al. (1977) Proc. Nati. Acá. Sci. USA 74, 5463-5464) using the T7 DNA polymera .se Kit (Pharmacia) and [35S] dATP. This sequence is shown in SEQ ID NO: 1.
Sequence analyzes and their comparison with other known sequences in the international data banks (GeneBank / EMBL) indicated that the cloned fragment encoded for the dao gene of T. variahilis.
EXAMPLE 3
1. - Elimination by PCR of the intron of the dao gene
A sample of 0.15 μg of plasmid pALT2 DNA was mixed with 10 μl (25 μM) of each of the following nucleotides: DAOl (5 '-CATGCCATGGCTAAAATCGTTGTTATTGGGGCCGGTGTTGCCGGTTTAAC-3') which encodes the complete sequence of the first exon and the first nucleotides from the 5 'end of the second exon and contained a Ncol restriction site; and DA02 (5'-CCCAAGCTTCTAAAGGTTTGGACGAG-3 ') which contained a Hind restriction site? 1 and the 3 'end sequence corresponding to the second exon of the dao gene including a translation termination codon. To this mixture were added 2.5 units of Taq polymerase (Perkin-Elmer) together with the appropriate buffer recommended by the suppliers and the preparation was subjected to an amplification process in a PCR apparatus (Gene-ATAQ, Pharmacia) using 30 cycles, each of which consisted of: 95 ° C (1 minute), 50 ° C (2 minutes) and 72 ° C (2.5 minutes). The result of the amplification was visualized by staining with ethidium bromide after electrophoresis in a 1% agarose gel. The DNA fragment obtained by PCR, of a size of approximately 0.9 kb, was purified by extraction of the agarose gel using β-agarase (Biolabs) following the manufacturer's recommendations. 0.2 μg of the purified fragment was ligated with 1 μg of the vector M13tgl30 (Amersham) digested with the Hincll enzyme (Pharmacia) by the phage T4 DNA ligase (Amersham) in the presence of ATP using "the buffer recommended by the supplier. the M13DAO phage was generated (Figure 1) which was subsequently sequenced to verify that the intron had been adequately eliminated.The nucleotide sequence of the fragment contained in phage M13DAO was determined on the same recombinant phage by a previously described method (Sanger et al.
(1977) Proc. Nati Ac. Sci. USA 74: 5463-5464) using the T7 DNA polymerase Kit (Pharmacia) and [35S] dATP. The nucleotide sequence revealed that the donor fragment possessed 0.9 kb and contained the sequence of the dao gene of T. variabais without the intron.
2. - Subcloning of the dao gene lacking intron in PKK233.2.
A sample of 0.1 μg of plasmid pKK233.2 DNA was digested with the restriction endonucleases Ncol and Hindlll (Pharmacia) at 37 ° C, in a buffer recommended by the suppliers, for 1 hour and heated for 10 minutes at 65 ° C. C to stop the reaction. The digested plasmid was mixed with 0.2 μg of the M13DA0 phage digested with the same restriction endonucleases indicated above and both D? As were ligated by the enzyme AD? T4 phage ligase
(Amersham) in the presence of ATP using the buffer recommended by the supplier. The resulting ligation mixture was used to transform competent E. coil TG1 cells. The transformants were isolated in LB medium with ampicillin (100 μg / ml). By means of this procedure, a clone was obtained which contained the plasmid recoipbinaiite pKDA03 (Figure 1) which possesses the fragment of AD? of 0.9 kb resulting from the PCR amplification inserted between the Ncol and Hi ndl ll sites of the plasmid pKK233.2, that is, expressed under the control of the trc promoter.
EXAMPLE 4
1. - Design of a chimeric DAO enzyme containing a tail of 6 histidines.
To insert the polyhistidine tail into the amino-terminal end of the DAO enzyme of T. variabilis, 0.1 pg of plasmid pKDA03 was digested with the restriction endonuclease Ncol (Pharmacia) at 37 ° C for 1 hour under the recommended conditions by the supplier and heated for 10 minutes at 65 ° C to stop the reaction. The plasmid thus digested was treated with the Klenow fragment of the AD? E. coli polymerase I (Pharmacia) according to the instructions recommended by the supplier and then the sample was heated for 10 minutes at 65 ° C to stop the reaction. The resulting linearized plasmid was ligated to a fragment of AD? which contains the nucleotide sequence encoding 6 histidine residues, by the enzyme AD? T4 phage ligase (Amersham) in the presence of ATP using the buffer recommended by the supplier. The coding fragment of the 6 histidine residues was obtained by a mixture of 1.5 μg of two complementary oligonucleotides called HIS1 (5 '-CATCATCACCACCATCACTT-3') and HIS2 (5'-AAGTGATGGTGGTGATGATG-3 ') which was subsequently heated to 100 °. C for 5 minutes and cooled to room temperature to hybridize to form a double-stranded DNA fragment. The resulting ligation mixture was used to transform competent E. coil TG1 cells. The transformants were isolated in LB medium with ampicillin (100 μg / ml). By means of this procedure, a site containing the recombinant plasmid pKDAOIIIS possessing the dao gene linked to a sequence of 18 nucleotides coding for 6 residues of histidine at its 5 'end was obtained. To check that the PKDAOHIS plasmid (Figure 1) contained the expected chimeric construct, the sequence on the same recombinant plasmid was determined by a previously described method (Sanger et al. (1977) Proc. Nati. Acá. Sci. USA 74: 5463- 5464) using the T7 DNA polymerase Kit (Pharmacia) and [35S] dATP. The sequence obtained is shown in SEQ ID NO: 2. The strain E. coli TG1 transformed with the plasmid pKDAOHIS was fermented in the LB medium for 20 hours at 25 ° C and 250 r.p.m. The cells were then harvested by centrifugation at 5,000 xg for 10 minutes, broken by sonication and their DAO activity tested as and described in section 1 of Example 1. The DAO activity obtained by this procedure was 350 U / mg of protein. In this way it was found that the DAO chimeric enzyme with the histidine chain (hisDAO) thus obtained was active. Furthermore, by SDS-polyacrylamide gel electrophoresis it was found that, as expected, the hisDAO enzyme was slightly larger than the native DAO enzyme. The strain E. coli TG1 transformed with the plasmid pKDAOHIS has been deposited in the Spanish Collection of Type Cultures (CECT), located in the Department of Microbiology of the Faculty of Biological Sciences of the University of Valencia, 46100 Burjasot (Valencia), 10.06.97, with the deposit number CECT4888.
EXAMPLE 5
i. - Preparation of metal agarose-chelate support
To a suspension of 7 g of CL6B agarose in 57 ml of a 0.1 N NaOH solution containing 340 mg of NaBH, a mixture of 5.7 ml of epichlorohydrin and 11.4 ml of ethylene glycol dimethyl ether is added slowly and the suspension is kept under gentle stirring. 25 ° C for 4 hours. The agarose-epoxide thus obtained is washed with abundant distilled water and added to a solution consisting of 2.5 ml of 2 M sodium iminodiacetate and 19 ml of 0.1 M sodium bicarbonate buffer pH 11. This suspension is kept under gentle agitation for 12 hours at 25 ° C. Finally, the support is washed with distilled water and resuspended in an aqueous solution containing the desired metal salt (5 mg / ml). The metallic agarose-chelate support thus formed is washed with abundant water and is ready for later use. When CoCl2 is added as a metal salt, a cobalt agarose-chelate support is obtained.
2. - Purification of the hisDAO chimeric enzyme in the cobalt agarose-chelate support.
The column containing the cobalt agarose-chelate support is equilibrated with 20 mM sodium phosphate buffer (pH 7.0) and 0.2 M NaCl. In this column the soluble cell extract obtained as described in the previous example is loaded from cells of E. coli TG1 transformed with the pKDAOHIS plasmid. Once the extract is loaded, the column is washed abundantly with the same equilibration buffer, thus eliminating all the proteins of the extract except hisDAO, which remains retained in the column. The hisDAO enzyme is then eluted by using a 20 mM sodium phosphate pH 7.0 buffer containing 10 mM imidazole. Fractions containing the enzyme hisDAO are reined and dialyzed against 20 mM sodium phosphate buffer pH 7.0. The enzyme (9000 U / mg) thus prepared has a degree of purity greater than 90% and is ready to be used for the transformation of cephalosporin C into GL-7ACA.
DETAILED DESCRIPTION OF THE FILMS
Figure 1.- Construction process of plasmid pKDAOHIS. The dao gene, devoid of intimacy and with a Ncol site in the ATG that codes for the first methionine of the protein, was obtained by PCR from the plasmid pALT2, then the fragment amplified by PCR was subcloned in the Hincll site. of the M13tgl30 vector originating M13DAO A Ncol-HindIII fragment obtained from M13DAO was subcloned into the NcoI-JindIII sites of the plasmid? KK233.2 giving rise to pKDA03 This plasmid possesses the dao gene expressed under the control of the E.sub.3 promoter. Lastly, the D? A fragment encoding the polyhistidine tail at the 5 'end of the dao gene was introduced generating the pKDAOHIS plasmid.
LIST OF SEQUENCES
< 110 > ANTIBIOTICS, S.A.U. < 120 > AN ENZYMATIC PROCEDURE FOR PREPARING 7ß- (4-C? RBOXIBUT? NAMIDO) CEF? LOSOR? NIC ACID USING THE
ENZYME D-AMINOACIDO OXIDASE OF TRIGONOPSIS
MODIFIED VARIABILIS PRODUCED IN ESCHERICHIA
COLI ". <130> PCT-47 <150> ES 9702008 <151> 25.09.97 160> 2 <210> SEQ ID NO: 1 <211> 3668 base pairs <<212> DNA <213 Trigonopsis variabiiis <220> <221> CDS <222> (1481 ... 1503, 1543 ... 2506) <223> <220> <g> < 221 > Intron < 222 > 1504 ... 1442 < 220 > < 221 > CDS < 222 > 2952 ... 3668 < 223 > ORF 1 < 400 > GGATCCTTAC GAGGAAGATC TGATAATGGA GAATTCTCTC GCTTTATGCC ATGACTTGCA 60 GGTTCCTCGG GTAATGGAAC CACTGACAAA TCGGCTGCTT GAGGTTTTGT TCCCTTCAAC 120 ACTTCATCTT CCAAGGTTCT AATTCGTAGT TTCTTCTCAT TCAATAAAGC TGCAAACCGC 180 TCCAACATTT GTAGATCATT ATTCTTTGTC TTCACGGCAA CTGTATCTAG TTCCTTCTGA 240 AGGTCCTTCG TTTCTTTGGT TAGTAGATCT ACCATGCCAG TTAAGCGATC AATCTCCTCT 300 TGGACCTCCT TCTTGAGCGA TAGCTCTGAC CAGAACCAAT CAAAAAGAAC ACCAGGATCG 360 GTACATGTTC CTTGTCGGGA TAGGGACAGT GATCCCAAAG TAATTGATAG ATCCTGAACT 420 TTCTGGG TAA TCGAGATAAT AACCGAACTC AAATCTTGTG ATGGCTTGGC GTTCAATTGA 480 ATGTTTTCGC TAGAAAGCCT CGGACTCTTA CGTTGGTCTT CATCTGTAAG CGACCGCGTG 540 AACAGTTGTT GGAATAAGTC ATTCCATTCA CTTTCACTTT GAGGACTTCT ATTTGACCGT 600 AGATCTTTAA TTGACTCATT TCCAGTTACT GAAAGTTAGC TGGAAGCTTT CAACATGTCC 660 TCACCAGAGG TTTGATAGAG GTCTAACGAT GGCTTCATAC AGGCTGTGAA TGTGATTGTT 720 TCGCCAGTCT CAACTCTGAC GATCAAACGC TTAGATCCAC CAATCTCAAT ACCGAACGAG 780 TTAATTCGAC TCATTGCTCA ATATGATTGA TCGCGCGGGA TGACATCGAA GGTGACAACC 840 GTACGCGAAA GATGCGTGAC GATAAGGACA ACGACTAAGG GAGTAGATGG ACTGGGGAGT 900 GAAGGAAATG TGAGACTAGA GAAAAGCCAC TGACTGAGAG TAAAACAGCC ATGATTAGAC 960 AATCAGCCAT GACAGCACTA TAACGTGATA TGATAAGTAA GGCTCTGTTG CCCGCTGACG 1020 GCCAACGGCT GACGGCCAAC TTGATGATTC TACCACAAAA AATCATACGA GAAGTCAACG 1080 AAAAGTCCTT AGTTTGGAAT TCCAGACATG GCAGAATTTA ACGGCCACTA CAGTTGGCCG 1140 TTCGTAAACG AGACAAGTGA CTCATGGCAG CACCGTCTCA GTCCACCGGT CTAAAGCACT 1200 TGGTGCCAGA TGAATTTGGA AACTGTCACC TTATAGAATT ACTTTTGGAT AGTTTTTGTA 1260 ACCCTGGAGA CTTGTAAGCC TGACTCAGTT GACTCATCGG CGAAAGCTTC CTAT CTTGGA 1320 GCTAAGATCG CCTGATCGTT TTGCCCTACT TATCTTGGTT GCATGAGTTG GCCGGTCAGA 1380 GCCGCATTCT AGCCAAAGGG TTATAGCGTT ACACTCTTGA TAGGCAAATC CGTGCTCGGA 1440 TTATATATAA GGCAAAAGTC ATG GCT AAA GATTCAACGG ATCAATAAAA ATC GTT 1495 Met Ala Val Lys lie GTT ATT GG May 1 GTAAGTGCCT TGATACCAGA CGGCTGACAT TTGTTTAG T GGT GCC 1548
Vai lie Gly Ala Gly 10 GTT GCC GGT TTA ACT ACA GCT CTT CAA CTT CTT CGT AAA GGA CAT GAG 1596 Val Ala Gly Leu Thr Thr Ala Leu Gln Leu Leu Arg Lys Gly His Glu 15 20 25 GTT ACA ATT GTG TCC GAG TTT ACG CCC GGT GAT CTT AGT ATC GGA TAT 1644 Val Thr lie Val Ser Glu Phe Thr Pro Gly Asp Leu Ser lie Gly Tyr 30 35 40 ACC TCG CCT TGG GCA GGT GCC AAC TGG CTC ACA TTT TAC GAT GGA GGC 1692 Thr Ser Pro Trp Wing Gly Wing Asn Trp Leu Thr Phe Tyr? Sp Cly Gly 45 50 55 AAG TTA GCC GAC TAC GAT GCC GTC TCT TAT CCT ATC TTG CGA GAG CTG 1740 Lys Leu Wing Asp Tyr Asp Aia Vai Ser Tyr Pro lie Leu Arg Glu Leu 60 65 70 GCT CGA AGC AGC CCC GAG GCT GGA ATT CGA CTC ATC AAC CAA CGC TCC 1788 Wing Arg Ser Ser Pro Glu Wing Gly lie Arg Leu lie Asn Gln Arg Ser 75 80 85 90 CAT GTT CTC AAG CGT GAT CTT CCT AAA CTG GAA GGT CCC ATG TCG GCC 1836 His Val Leu Lys Arg Asp Leu Pro Lys Leu Glu Gly Wing Met Wing 95 100 105 ATC TGT CAA CGC AAC CCC TGG TTC AAA AAC ACA GTC GAT TCT TTC GAG 1884 lie Cys Gln Arg Asn Pro Trp Phe Lys Ace n Thr Val Asp Ser Phe Glu 110 115 120 3 ATT ATC GAG GAC AGG TCC AGG ATT GTC CAC GAT GAT GTG GCT TAT CTA 1932
He He Glu Asp Arg Ser Arg He Val His Asp Asp Val Wing Tyr Leu 125 130 135 GTC GAA TT GCT TCC GTT TGT ATC CAC ACC GGA GTC TAC TTG AAC TGG 1980
Val Glu Phe Ala Ser Val Cys Tl? His Thr Gly Val Tyr Leu Asn Trp 140 145 150 CTG ATG TCC CAA TGC TTA TCG CTC GGC GCC ACG GTG GTT AAA CGT CGA 2028
Leu Met Ser Gln Cys Leu Ser Leu Gly Wing Thr Val Val Lys Arg Arg 155 160 165 170 GTG AAC CAT ATC AAG GAT GCC AAT TTA CTA CAC TCC TCA GGA TCA CGC 2076
Val Asn His He Lys Asp Wing Asn Leu Leu His Ser Ser Gly Ser Arg 175 180 185 CCC GAC GTG ATT GTC AAC TGT AGT GGT CTC TTT GCC CGG TC TTG GGA 2124
Pro Asp Val He Val Asn Cys Ser Gly Leu Phe Wing Arg Phe Leu Gly 190 195 200 GGC GTC GAG AAG AAG ATG TAC CCT ATT CGA GGA CAA GTC CTT 217 GTC?
Gly Val Glu Asp Lys Lys Met Tyr Pro He Arg Gly Gln Val Val Leu 205 210 215 GTT CGA AAC CT CTT CCT TTT ATG GCC CC rpm TCC AGC ACT CCT GAA 2220
Val Arg Asn Ser Leu Pro Phe Met Wing Being Phe Ser Ser Thr Pro Glu 220 225 230 AAA GAA AAT GAA GAC GAA GCT CTA TAT ATC ATG ACC CGA TTC GAT GGT 2268
Lys Glu Asn Glu Asp Glu Wing Leu Tyr He Met Met Thr Arg Phe Asp Gly 235 240 245 250 ACT TCT ATC ATT GGC GGT TGT TTC CAA CCC AAC AAC TGG TCA TCC GAA 2316
Thr Ser He Gly Gly Cly Phe Gln Pro Asn Asn Trp Be Ser Glu 255 260 265 CCC GAT CCT TCT CTC ACC CAT CGA ATC CTG TCT AGA GCC CTC GAC 'CGA 2364 Pro Asp Pro Ser Leu Thr His Arg He Leu Ser Arg Ala Leu Asp Arg 270 275 280 TTC CCG GAA CTG ACC AAA GAT GGC CCT CTT GAC ATT GTG CGC GAA TGC 2412 Phe Pro Glu Leu Thr Lys Asp Gly Pro Leu Asp He Val Arg Glu Cys 285 290 295 GTT GGC CAC CGT CCT GGT AGA GAG GGC GGT CCC CGA GT? GAA TTA GAG 2460 Val Gly His Arg Pro Gly Arg Glu Gly Gly Pro Arg Val Glu Leu Glu 300 305 310 AAG ATC CCC GGC GTT GGC TTT GTT GTC CAT AAC TAT GGT GCC GCC GGT 2508 Lys He Pro Gly Val Gly Phe Val Val His Asn Tyr Gly Wing Wing Gly 315 320 325 330 GCT GGT TAC CAG TCC TCT TAC GGC ATG GCT GAT GAA GCT GTT TCT TAC 2556 Aia Gly Tyr Gln Ser Ser Tyr Gly Met Wing Asp Glu Wing Val Ser Tyr 335 340 345 GTC GAA AGA GCT CTT ACT CGT CCA AAC CTT TAGAAATCAT GTATACAATT 2606 Val Glu Arg Ala Leu Thr Arg Pro Asn Leu 350 355 ATTCTCTCTC TATAAATCTA ATTTTTTTGT GTGGTCTAAT ATTCGTAAAC ACGTCGCAGT 2666 CGTCTATGTC GCCCTCGTCA CCGTGTCCAA AGTCGTAAAG TGACTGATTG CAATTGCGAC 2726 AACACGTGAC TCGACCTGCC TCCTTACCTC CCATCAACAA CAAAAGAAGC TGGCTAAGAT 2786 AGAGGTCTGT TGACGAGCAC TCGTAAGAAC GGCAAACATA GAAAGGAGGC TCTATAATTA 2846 CCTGGAAACT GTGTTATATA CTATCACTAG TGAGGGTGAG TGATTAGAAG CAAGGGGACT 2906 AGAATACTGA CATGGATAGA GATCCAGGAG CCTTATAAAT AATCA ATG AAT ACA ATT 2963 Met Asn Thr He GAT TTG GAA TCT TGG GAT GAT GAT CCA GAT TTC GCA GA T GAC TTT GAG 3011 Asp Leu Glu Ser Trp Asp Asp Asp Pro Asp Phe Wing Asp Asp Phe Glu 360 365 370 375 AAT TTA AAG ACT CCA GCT CCT ACG TT TAT GAA GCC AAC GAT GAG ATT 3059
Asn Leu Lys Thr Pro Wing Pro Thr Phe Tyr Glu Wing Asn Asp Glu He 380 385 390 AGG GAT GAA GAA GAA GAG GAT GAT TTT TTC TCT CAA GAT TC GAG TTG 3107
? rg Asp Gly Glu Glu Glu Asp Asp Phe Phe Ser Gln Asp Phe Glu Leu 395 400 405 GAT CAT AAG AAT ACA CTC GGA CGA CAG AAC AAG ATT TCG ACT AGT CAC 3155
Asp Asp Lys Asn Thr Leu Gly Arg Gln Asn Lys He Ser Thr Ser His 410 415 420 CTA AAG TCC GCG AGT CAG GAA CAA GCA GAG ACC CC TTT CGT GAC TCG 3203
Leu Lys Ser Wing Be Gln Glu Gln Wing Glu Thr Ser Phe Arg Asp Ser 425 430 435 AAC GCT GGC GTC AAT GCT TTC AGC TGC GGG ACT ATA AAA GCC TTA GGA 3251
Asn Ala Gly Val Asn Ala Phe Ser Cys Gly Thr He Lys Ala Leu Gly 440 445 450 455 AAG AAT AGG ATG ACG ACG GTG GAA GAG AAG TGG GAA AAG GAA GTT AGA 3299
Lys Asn Arg Mer Thr Thr Val Glu Glu Lys Trp Glu Lys Glu Val Arg 460 465 470 CGC GAT CAA ATT GGG TTC AAT GAA GCT ACT CTT AGA GCT CAT GAG ACT 3347
Arg? Sp Gln He Gly Phe Asn Glu Wing Thr Leu Arg Wing His Glu Thr 475 480 485 ACC AGA GAA TGG TTA AAA TCC CAG ACT GGC GAA GCT GGG ACT AAA AGC 3395
Thr Arg Glu Trp Leu Lys Ser Gin Thr Gly Glu Wing Gly Thr Lys Ser 490 495 500 AAG GTC TTT AGC CCA ATT CTC GAC GGA TCA TTC TGA GAA CCG TTT 3443
Lys Val Phe Pro Pro He Leu Asp Gly Ser Phe Ghe Pro Pro Leu 505 510 515 GAA TCT AAA GTC AGG CGT TAT CAT TCA CCC CGG AAA CAG GCA CCT CCT 3491 Glu Ser Lys Val Arg Arg Tyr Hia Ser Pro Arg Lya Gln Ala Pro Pro 520 525 530 CCT CCT CCT GAC GAT TTT TCA GAT GCA TTT GAA CTA TCT ACA GAA GAG 3539 Pro Pro Pro Asp Aap Phe Ser Asp Wing Phß Glu Lßu Ser Thr Glu Glu 535 540 545 550 CC? CTG AAA TTA AAA GTC CAA CCA GTT CAA CCT CAT ATG ACG CCT GCT 3587 Pro Leu Lys Leu Lys Val Gln Pro Val Gln Pro His Met Thr Pro Ala 555 560 565 CTG AGT GAT AAT GAT CTA TGG GGT GAG GAA TCT ATT GGT GTG CGA CGA 3635 Leu Ser Asp Asn Asp Leu Trp Gly Glu Glu Be He Gly Val? Rg? Rg 570 575 580 GGA GGC AGG GAC TCG TCA AGT ATG GGA GGA TCC 3668
Gly Gly Arg Asp Being Ser Met Met Gly Gly Ser 585 590
< 210 > SEQ ID NO: 2 < 211 > 1128 base pairs < 212 > DNA < 213 > Trigonopsis variabilis < 220 > < 221 > CDS < 222 > 16 ... 1107 < 223 > dao gene with a coia of 6 His in 5 'CACAGGAAAC AGACC ATG CAT CAT CAC CAC CAT CAC TTC ATG GCT AAA ATC 51 Met His His His His His His His Phe Met Ala Lys He 1 5 10 GTT GTT ATT GGG GCC GGT GTT GCC GGT TTA ACT ACA GCT CTT CAA CTT 99
Val Val He Gly Wing Gly Val Wing Gly Leu Thr Thr Wing Leu Gln Leu 15 20 25 CTT CGT AAA GGA CAT GAG GTT ACA ATT GTG TCC GAG TTT ACG CCC GGT 147
Leu Arg Lys Gly His Glu Val Thr He Val Ser Glu Phe Thr Pro Gly 30 35 40 GAT CTT AGT ATC GGA TAT ACC TCG CCT TGG GCA GGT GCC AAC TGG CTC 195
Asp Leu Ser He Gly Tyr Thr Ser Pro Trp Wing Gly Wing Asn Trp Leu 45 50 55 60 ACA TTT TAC GAT GGA GGC AAG TTA GCC GAC TAC GAT GCC GTC TCT TAT 243
Thr Phe Tyr Asp Gly Gly Lys Leu Wing Asp Tyr Asp Wing Val Ser Tyr 65 70 75 CCT ATC TTG CGA GAG CTG GCT CGA AGC AGC CCC GAG GCT GGA ATT CGA 291
Pro He Leu Arg Glu Leu Wing Arg Ser Ser Pro Glu Wing Gly He Arg 80 85 90 CTC ATC AAC CAA CGC TCC CAT GTT CTC AAG CGT GAT CTT CCT ??? CTC 339
Leu He Asn Gln Arg Ser His Val Leu Lys Arg Asp Leu Pro Lys Leu 95 100 105 GAA GGT GCC ATG TCG GCC ATC TGT CAA CGC AAC CCC TGG TTC AAA AAC 387
Glu Gly Ala Met Be Ala He Cys Gln Arg Asn Pro Trp Phe Lys Asn 110 115 120 ACA GTC GAT TCT TTC GAG ATT ATC GAG GAC AGG TCC AGG ATT GTC CAC 435
Thr Val Asp Ser Phe Glu He He Glu Asp Arg Ser Arg He Val His 125 130 135 140 GAT GAT GTG GCT TAT CTA GTC GAA TTT GCT TCC GTT TGT ATC CAC ACC 483
Asp Asp Val Ala. Tyr Leu Val Glu Phe Ala Ser Val Cys He His Thr 145 150 155 GGA GTC TAC TTG AAC TGG CTG ATG TCC CAA TGC TTA TCG CTC GGC GCC 531
Gly Val Tyr Leu Asn Trp Leu Met Ser Gln Cys Leu Ser Leu Gly Wing 160 165 170 ACG GTG GTT AAA CGT CGA GTG AAC CAT ATC AAG GAT GCC AAT TTA CTA 579
Thr Val Val Lys? Rg Arg Vai Asn His He Lys Asp Wing Asn Leu Leu 175 180 185 CAC TCC TCA GGA TCA CGC CCC GAC GTG ATT GTC? AC TGT AGT GGT CTC 627
His Ser Ser Gly Ser Arg Pro Asp Val He Val Asn Cys Ser Gly Leu 190 195 200 TT GCC CGG TTC TTG GGA GGC GTC GAG GAC AAG AAG ATG TAC CCT ATT 675
Phe Wing Arg Phe Leu Gly Gly Val Glu Asp Lys Lys Met Tyr Pro He 205 210 215 220 CGA GGA CAA GTC GTC CTT GTT CGA AAC CT CTT CCT TTT ATG GCC CC 723
Arg Gly Gln Val Val Leu Val Arg Asn Ser Leu Pro Phe Met Wing Ser 225 230 235 1 T i TCC AGC ACT CCT GAA AAA GAA AAT GAA GAC GAA GCT CTA TAT ATC 771
Phe Ser Ser Thr Pro Giu Lys Glu Asn Glu Asp Glu Wing Leu Tyr He 240 245 250 ATG ACC CGA TC GAT GGT ACT TCT ATC ATT GGC GGT TGT TTC CAA CCC 819
Met Thr Arg Phe Asp Gly Thr Ser He He Gly Gly Cys Phe Gln Pro 255 260 265 AAC AAC TGG TCA TCC GAA CCC GAT CCT TCT CTC ACC CAT CGA ATC CTG 867
Asn Asn Trp Ser Ser Glu Pro Asp Pro Ser Leu Thr His Arg He Leu 270 275 280 TCT AGA GCC CTC GAC CGA TTC CCG GAA CTG ACC AAA GAT GGC CCT CTT 915 Ser Arg Ala Leu Asp Arg Phe Pro Glu Leu Thr Lys Asp Gly Pro Leu 285 290 295 300 GAC ATT GTG CGC GAA TGC GTT GGC CAC CGT CCT GGT AGA GAG GGC GGT 963 Asp He Val Arg Glu Cys Val Gly His Arg Pro Gly Arg Glu Gly Gly 305 310 315 CCC CGA GTA GAA TTA GAG AAG ATC CCC GGC GTT GGC TT GTT GTC CAT 1011 Pro Arg Val Glu Leu Glu Lys He Pro Gly Val Gly Phe Val Val His 320 325 330 AAC TAT GGT GCC GCC GGT GCT TAC CAG TCC TCT TAC GGC ATG GCT 1059 Asn Tyr Gly Ala Wing Gly Wing Gly Tyr Gln Being Ser Tyr Gly Met Wing 335 340 345 GAT GAA GCT GTT TCT TAC GTC GAA AGA GCT CTT ACT CGT CCA AAC CTT 1107 Asp Glu Wing Vai Ser Tyr Val Glu Arg Wing Leu Thr Arg Pro Asn Leu 350 355 360 364 TAGAAGCTTG GCTGTTTTGG C 1128
It is noted that in relation to this date the best method known by the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention. Having described the invention as above, the content of the following is claimed as property.
Claims (8)
1. A process for producing modified D-amino acid oxidase of Trigonopsis variabilis, in a non-human host organism, characterized by the following operations: (a) isolating the DNA of the gene encoding the D-amino acid oxidase activity of any strain of microorganism producing said enzyme; (b) eliminating the intron containing said gene by a method based on the use of synthetic oligonucleotides and the polymerase chain reaction (PCR); (c) insert the DNA fragment obtained in (b) in a plasmid that is capable of replicating in the host organism; (d) fusing at the 5 'end of the structural region of the intron-free gene encoding the D-amino acid oxidase activity a synthetic assembler containing a nucleotide sequence encoding six histidines; (e) transforming the host organism with the recombinant plasmid resulting from (d); (f) culturing the transformed cells of the host organism obtained in (e) in a suitable culture medium and under conditions that allow the production of D-amino acid oxidase; (g) recovering the D-amino acid oxidase enzyme from the culture of the operation (f) by affinity chromatography.
2. A method according to claim 1, characterized in that the gene coding for the enzyme D-amino oxidase is that of Trigonopsis variabilis.
3. A method according to claim 1 or 2, characterized in that the non-human host organism is Escherichia coli.
4. A method according to any of the preceding claims, characterized in that the affinity chromatography used to recover the modified enzyme is based on a support based on divalent cations.
5. The D-amino acid oxidase enzyme characterized in that it is modified, produced and purified in accordance with the method of any of the preceding claims.
6. Process for the use of the enzyme D-amino acid oxidase of claim 5, in the enzymatic synthesis of 7β- (4-carboxybutanamido) cephalosporanic acid.
7. Method of use according to claim 6, characterized by reacting in an aqueous medium cephalosporin C with the enzyme D-amino acid oxidase.
8. A method of use according to claims 6 and 7, characterized in that the enzymatic reaction is carried out with the immobilized D-amino acid oxidase enzyme.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
ES9702008 | 1997-09-25 |
Publications (1)
Publication Number | Publication Date |
---|---|
MXPA99004513A true MXPA99004513A (en) | 2000-02-02 |
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