WO2004111241A1 - Acylase de penicilline g - Google Patents

Acylase de penicilline g Download PDF

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WO2004111241A1
WO2004111241A1 PCT/EP2004/006423 EP2004006423W WO2004111241A1 WO 2004111241 A1 WO2004111241 A1 WO 2004111241A1 EP 2004006423 W EP2004006423 W EP 2004006423W WO 2004111241 A1 WO2004111241 A1 WO 2004111241A1
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acylase
polypeptide
polynucleotide
coli
seq
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PCT/EP2004/006423
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Esther Michèle GABOR
Dirk Barend Janssen
Erik Jan De Vries
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Stichting Voor De Technische Wetenschappen
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C12N9/84—Penicillin amidase (3.5.1.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P35/00—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin
    • C12P35/04—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin by acylation of the substituent in the 7 position
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P37/00—Preparation of compounds having a 4-thia-1-azabicyclo [3.2.0] heptane ring system, e.g. penicillin
    • C12P37/04—Preparation of compounds having a 4-thia-1-azabicyclo [3.2.0] heptane ring system, e.g. penicillin by acylation of the substituent in the 6 position
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01011—Penicillin amidase (3.5.1.11), i.e. penicillin-amidohydrolase

Definitions

  • the present invention relates to novel polypeptides and polynucleotides encoding such polypeptides and precursors thereof.
  • the polypeptides have been found to possess Penicillin G acylase activity.
  • the invention further relates to a process for the preparation of ⁇ -lactam antibiotics by using said novel polypeptides with penicillin acylase activity.
  • Penicillin acylases are a group of hydrolases originating from microorganisms, for example bacteria, that are capable of reversibly hydrolysing the 6- acyl group of penicillins or the 7-acyl group of cephalosporins to form the corresponding free amines without the ring structure of the penicillins or cephalosporins being destroyed.
  • Reaction diagram I illustrates a hydrolysis reaction.
  • a few examples of side chains R in the penicillin compound are phenylacetyl and phenoxyacetyl.
  • Penicillin G acylase is variously known as for example Penicillin G (PenG) amidase or Benzylpenicillin amidohydrolase (enzyme classification E.C. 3.5.1.11).
  • acylases In connection with ⁇ -lactam antibiotics these acylases usually are further specified as " ⁇ -lactam acylases" as not all amidases accept a ⁇ -lactam nucleus as an acceptor/donor moiety for the acyl group.
  • ⁇ -lactam acylases may be envisaged, based on their substrate specificity and molecular structure.
  • the substrate specificity of the acylase is determined by a side chain binding pocket at the enzyme which recognizes the side chain moiety of ⁇ -lactam molecules (R in Reaction diagram 1).
  • the acylases are not very specific for the moiety adjacent to the nitrogen atom of the amide group (this may be a ceph-3-em group, a penam group, an amino acid, a sugar, etc.
  • PenG acylases the preferred side chain moiety must be hydrophobic and is preferably phenylacetyl or (short) alkyl.
  • PenG acylase is used commercially to hydrolyse PenG or Cephalosporin G (CefG) to phenylacetic acid on the one hand and 6-APA or 7-ADCA, respectively, on the other hand. These latter compounds are the most important intermediates for the industrial production of semi- synthetic penicillins and cephalosporins.
  • substrates which are not hydrolyzed by PenG acylases are those with charged acyl moieties such as dicarboxylic acids: succinyl, glutaryl, adipyl and also amino-adipyl, the side-chain of Cephalosporin C (CefC).
  • Penicillin V acylases are highly specific for phenoxyacetyl, while ampicillin acylase prefers D-phenylglycine as a side chain.
  • Glutaryl-acylases deacylate glutaryl-7-ACA, which is prepared from CefC after enzymatic deamidation of the side chain with D-amino acid oxidase followed by chemical decarboxylation of the formed ketoadipyl derivative with peroxide, which is produced in the first step.
  • acylases have been reported to be capable of hydrolyzing cephalosporins (including the desacetoxy-derivative) with succinyl, glutaryl and adipyl as an acyl moiety and even in one case CefC to a very limited degree (for a review see EP-A- 322032, Merck).
  • acylases may also be classified based on molecular aspects:
  • Type-I acylases which are specific for Penicillin V, are composed of four identical subunits, each having a molecular weight of about 35 kDa.
  • Type-I I acylases all share a common molecular structure: these enzymes are heterodimers composed of a small subunit (a; 16-26 kDa) and a large subunit
  • Type-ll acylases may be further subdivided into two groups: o Type-I IA acylases comprising the PenG acylases; o Type-IIB acylases comprising the Glutaryl acylases.
  • Type III acylases are the Ampicillin acylases which have been reported to be dimers consisting of two identical subunits with a molecular weight of about 72 kDa.
  • PenG acylase is understood to be a Type ll-A acylase.
  • Type II acylases all share a common molecular structure.
  • the term "PenG acylase” is intended to mean prokaryotic Type ll-A acylase as well as its precursor (such as preenzyme and preproenzyme forms).
  • the Synthesis/Hydrolysis ratio (S/H ratio) is high.
  • S/H ratio is high and at the same time the enzymatic activity is also sufficiently high.
  • the S/H ratio is understood to be the molar ratio of synthesis product to hydrolysis product at a particular moment during the enzymatic reaction.
  • Synthesis product is understood to be the ⁇ -lactam antibiotic formed from the activated side chain and ⁇ -lactam nucleus.
  • Hydrolysis product is understood to be the corresponding acid of the activated side chain.
  • the S/H ratio is a function of, amongst other things, the concentration of the reactants, the molar ratio of activated side chain to ⁇ -lactam nucleus, the temperature, the pH and the enzyme. It is therefore important to indicate in what conditions an S/H ratio is determined.
  • a comparative experiment is carried out where the particular candidate is tested against a reference enzyme, preferably E.coli PenG acylase
  • the S/H ratio is a function of the conversion, amongst other things.
  • S/H ini can be determined with sufficient accuracy by carrying out the acylation reaction until a sufficiently high conversion is reached so that the products, in particular the hydrolysis product, can be measered accurately and then constructing a graph of S/H ini versus conversion and extrapolating it to 0 % conversion.
  • Sufficient data points means at least three data points, which should represent a difference in conversion of at least 0.5%.
  • Enzymatic activity can generally be defined as the molar quantity of reactant or product that is converted or synthesized per unit time and per quantity of dissolved or immobilised enzyme at a particular moment during the enzymatic acylation reaction.
  • enzymes are applied in immobilised form and the enzymatic activity is defined per quantity of immobilised enzyme.
  • the enzymatic activity per quantity of enzyme often is also indicated as specific activity of the particular enzyme. More specifically, in the context of the invention enzymatic activity is defined as the quantity of side chain donor (or activated side chain) that is converted in the acylation reaction per unit time and per quantity of dissolved or immobilised enzyme.
  • the enzymatic activity generally decrease overtime.
  • the enzymatic activity is a function of the conversion.
  • the enzymatic activities of different penicillin acylases are preferably compared at equal conversion. They are most usually compared at 0% conversion, the so-called initial enzymatic activity, which thus is a measure of enzymatic activity.
  • the initial enzymatic activity can be determined with sufficient accuracy by carrying out the acylation reaction until a certain conversion is reached and then constructing a graph of the enzymatic activity versus the conversion and extrapolating it to 0% conversion.
  • Sufficient data points means at least three data points.
  • the conversion to be achieved in an acylation reaction can be expressed as the molar quantity of ⁇ -lactam antibiotic formed in the acylation reaction at a particular moment during the reaction per molar quantity of reactant used, where the reactant may be either the ⁇ -lactam nucleus or the (activated) side chain.
  • Suitable reaction conditions for the performance of an enzymatic acylation reaction in which a mutated or unmutated penicillin acylase is used are known to one skilled in the art.
  • ⁇ -lactam antibiotic comprises all antibiotics that contain a penam or ceph-3-em ring system.
  • the best-known ⁇ -lactam antibiotics are the penicillins and cephalosporins.
  • penicillins are defined as compounds according to formula (II) and cephalosporins as compounds according to formula (III),
  • X represents S, O, C, S(O), or SO 2 ,
  • Ri represents a side chain, such as for example phenylacetyl, phenoxyacetyl, hydroxyphenylglycyl, phenylglycyl, dihydrophenylglycyl and derivatives thereof and acetyl, adipyl, glutaryl and derivatives thereof, -
  • R 2 and R 3 each independently may represent Ci, aliphatic or aromatic groups, optionally with one or more O, S or N atoms
  • R 4 represents OH, aliphatic or aromatic alcohols and derivatives thereof, optionally with one or more O, S or N atoms.
  • Side chains in the context of the present invention may include any suitable compounds that can be attached to the 6-penam or 7-cephem position of a ⁇ - lactam nucleus, resulting in an antibiotically active compound.
  • the aliphatic group in R 2 and/or R 3 preferably contains 1-4 C atoms, and is preferably a methyl group.
  • Semisynthetic penicillins or cephalosporins are prepared for example by acylating the 6-amino-group of 6-aminopenicillanic acid (6-APA) or a derivative thereof as shown in formula (IV), or the 7-amino group of 7- aminodesacetoxycephalosporanic acid (7-ADCA) or a derivative thereof as shown in formula (V), with the aid of an activated side chain and a penicillin acylase enzyme. Also 7-aminocephalosporanic acid (7-ACA)-nuclei can be acylated with the aid of penicillin acylases.
  • the present invention discloses a new PenG acylase (which is further indicated herein as S2) with enzymatic activities different from that of the wild type Pencillin G acylase of Escherichia coli (E. coli).
  • PenG acylases comprises mutation of the enzyme, thereby replacing amino acids involved in the biocatalytic process.
  • WO96/05318 and WO98/20120 disclose several alternatives to alter Penicillin acylase of E. coli by mutating the nucleotide sequence, which encodes the wild type enzyme.
  • Ser290 is an essential amino acid in penicillin acylase of E.coli.
  • the best mutant that is shown is ⁇ F57L which shows an improvement of S/H ratio of 14% at an enzymatic activity which is 71% of the wild type E.coli enzymatic activity (table 3, Alkema et al.) "
  • a general conclusion from the known attempts to improve the enzymatic characteristics of PenG acylase for use in the synthesis of synthetic antibiotics is that a higher S/H ratio seriously corrupts enzymatic activity which is reflected in lower synthesis rate per mg of acylase protein.
  • the present invention provides for a newly isolated PenG acylase (PAS2) of an unknown source, which surprisingly exhibits an improved S/H, ⁇ i as well as an essentially unaltered enzymatic activity with respect to the acyl donors, in particular amide acyl donors as compared to PenG acylase of E. coli.
  • PAS2 PenG acylase
  • Fig. 1 Composition of PAS2.
  • the positions of the signal peptide (pos. 1-24), the ⁇ - subunit (pos. 25-253, 25.5 kD), the spacer peptide (pos. 254-308) and the ⁇ - subunit (pos. 309-863, 62 kD) in the prepropolypeptide are indicated.
  • Fig. 2 Ampicillin synthesis by E. coli penicillin acylase. Formation of ampicillin (black squares) and D-phenylglycine (black circles) refers to the left y-axis, D- phenylglycine amide (white circles) and 6-APA (white squares) refer to the right y-axis.
  • Fig. 3 Ampicillin synthesis by PAS2. Formation of ampicillin (black squares) and D- phenylglycine (black circles) refers to the left y-axis, D-phenylglycine amide (white circles) and 6-APA (white squares) refer to the right y-axis.
  • Amoxicillin synthesis by E. coli penicillin acylase Formation of amoxicillin (black squares) and D-p-hydroxyphenylglycine (black circles) refers to the left y-axis, D- p-hydroxyphenylglycine amide (white circles) and 6-APA (white squares) refer to the right y-axis.
  • the present invention is concerned with an enzyme having PenG acylase activity and which is characterised by a) an initial Synthesis/Hydrolysis ratio (S/H ini ) of at least 50% above the SI m] of PenG acylase of E. coli and b) an initial rate of activated side chain conversion less that 20% below the level of
  • PenG acylase of E. coli wherein as an activated side chain precursor is used D-p-hydroxyphenylglycine amide (DHPGA) and wherein as a penam nucleophile is used 6-amino penicillanic acid.
  • DHPGA D-p-hydroxyphenylglycine amide
  • PenG acylase which conforms the above characteristics and which is characterised in that it comprises two peptides with amino acid sequences corresponding to positions 25 or 26 to 253 and positions 309 to 863 of SEQ ID NO: 2, respectively.
  • Peptide 25 or 26 to 253 shows similarity to the so-called ⁇ subunits and the peptide 309 to 863 shows similarity to the so-called ⁇ subunits of Type-IIA acylases.
  • S2 enzyme was tentatively classified as a Type-IIA acylase.
  • the sequence of S2 suggests that the enzyme is initially expressed as a preproprotein consisting of a signal sequence and the two subunits ( ⁇ and ⁇ ) that are separated by a spacer peptide.
  • the signal sequence directs the protein to the periplasm and is subsequently cleaved off. In this compartment, the spacer peptide is removed, probably by an autocatalytic process. To determine the exact cleavage points and masses of the subunits of the protein, a mass spectrum was recorded.
  • the preproprotein comprises a signal peptide, an ⁇ -subunit, a spacer peptide and a ⁇ -subunit, and may be characterised by SEQ ID NO: 2.
  • the present invention also relates to a polypeptide having PenG acylase activity and which shows at least 85 %, preferably 90%, more preferably 95%, most preferably 99% homology with SEQ ID NO 2.
  • the invention relates to a polynucleotide encoding an enzyme having Penicillin acylase activity and which is characterised by a. an initial Synthesis/Hydrolysis ratio (S/H ini )of at least 50% above the S/H ini of PenG acylase of E. coli and b. an initial rate of activated side chain conversion less that 20% below the level of PenG acylase of E. coli wherein as an activated side chain precursor is used D-p-hydroxyphenylglycine amide (DHPGA) and wherein as a penam nucleophile is used 6-amino penicillanic acid.
  • S/H ini Synthesis/Hydrolysis ratio
  • DHPGA D-p-hydroxyphenylglycine amide
  • the invention relates to a polynucleotide encoding a polypeptide comprising peptides having the amino acid sequences corresponding to positions about 23-27 to about 244-308 of SEQ ID NO: 2 and positions 309 to 863 of SEQ ID NO: 2 or amino acid sequences having at least 85 %, preferably 90%, more preferably 95%, most preferably 99% homology to said sequences.
  • the invention relates to a polynucleotide encoding a polypeptide comprising peptides having amino acid sequences corresponding to positions 25 to 253 and positions 309 to 863 of SEQ ID NO: 2 or corresponding to positions 26 to 253 and 309 to 863 of SEQ ID NO: 2.
  • these polynucleotides may further comprise polynucleotides encoding a signal peptide and a spacer peptide.
  • the present invention relates to a vector suitable for transforming an appropriate host cell comprising any of the polynucleotides described above.
  • this polynucleotide is operatively linked with regulatory sequences suitable for expression of said polynucleotide in an appropriate host cell.
  • the invention further comprises a recombinant host cell comprising a polynucleotide or a vector described above.
  • Suitable hosts are prokaryotic and eukaryotic organisms, preferably prokaryotic organisms, more preferably £. coli.
  • Vectors according to the present invention are vectors which are suitable for stabile transformation of the desired host, and which are capable of promoting synthesis of the desired protein in said host.
  • such recombinant host cell is capable of expressing and preferable also excreting an enzyme having PenG acylase activity and which is characterised by a. an initial Synthesis/Hydrolysis ratio (S/H ini )of at least 50% above the S/H in ⁇ of PenG acylase of £. coli and b. an initial rate of activated side chain conversion less that 20% below the level of PenG acylase of £ coli wherein as an activated side chain precursor is used D-p-hydroxyphenylglycine amide (DHPGA) and wherein as a penam nucleophile is used 6-amino penicillanic acid.
  • S/H ini an initial Synthesis/Hydrolysis ratio
  • DHPGA D-p-hydroxyphenylglycine amide
  • the invention also relates to a method for the preparation of a polypeptide comprising the steps of; transforming a host cell with a polynucleotide described above; culturing said host cell under conditions allowing expression and preferably also secretion of said polynucleotide; and - optionally purifying the encoded polypeptide from said cell or culture medium optionally formulating said purified polypeptide.
  • Transformation of the host with the desired polynucleotide or vector, culturing of the transformed host cell purification of the polypeptide and formulation of the polypeptide may be carried out by methods known in the art.
  • the enzymatic polypeptide is immobilised in order to be able to use the enzyme repeatedly in subsequent reaction cycles.
  • the present invention also relates to a method for the preparation of a ⁇ -lactam antibiotic, for example starting from 6-amino penicillanic acid wherein the above novel Penicillin acylase is used as a biocatalyst.
  • a method for the preparation of a ⁇ -lactam antibiotic for example starting from 6-amino penicillanic acid wherein the above novel Penicillin acylase is used as a biocatalyst.
  • the molar ratio of activated side chain to ⁇ -lactam nucleus i.e. the total quantity of activated side chain added divided by the total quantity of ⁇ -lactam nucleus added, both expressed in moles, may vary between wide limits.
  • the molar ratio is between 0.5 and 2.0, in particular between 0.7 and 1.8.
  • the temperature at which the enzymatic acylation reaction is carried out is generally lower than 40 °C, preferably between -5 and 35 °C.
  • the pH at which the enzymatic acylation reaction is carried out generally lies between 5.5 and 9.5, preferably between 6.0 and 9.0.
  • the reaction is almost completely terminated when the maximum conversion is all but reached.
  • a suitable embodiment to terminate the reaction is lowering the pH, preferably to a value between 4.0 and 6.3, in particular between 4.5 and 5.7.
  • Another suitable embodiment is lowering the temperature of the reaction mixture as soon as maximum conversion is achieved. A combination of both embodiments is also possible.
  • a decrease in pH can be accomplished for example by the addition of an acid.
  • Suitable acids are for example mineral acids, in particular sulphuric acid, hydrochloric acid solution or nitric acid and carboxylic acids, for example acetic acid, oxalic acid and citric acid.
  • An increase in pH can be accomplished for example by the addition of a base.
  • Suitable bases are for example inorganic bases, in particular ammonia, potassium hydroxide or sodium hydroxide solution and organic bases, for example triethylamine and D-phenylglycine amide. Preferably ammonia is applied.
  • the enzymatic acylation reaction can be carried out in water.
  • the reaction mixture may also contain an organic solvent or a mixture of organic solvents, preferably less than 30 vol.%.
  • organic solvents that may be applied are alcohols with 1-7 C-atoms, for example a monoalcohol, in particular methanol or ethanol; a diol, in particular ethylene glycol or a triol, in particular glycerol.
  • the enzymatic acylation reaction is preferably carried out as a batch process. If desired, it is also possible to carry out the reaction continuously.
  • Suitable examples of ⁇ -lactam nuclei that can be used in the process according to the invention are penicillin derivatives, for example 6-APA, and cephalosporin derivatives, for example a 7-aminocephalosporanic acid with or without a substituent on the 3-site, for example 7-ACA, 7-ADCA and 7-amino-3-chloro-ceph-3-em-4-carboxylic acid (7-ACCA) and 7-amino-3-chloro-8-oxo-1 -azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid.
  • an activated side chain in the (enzymatic) acylation reaction use may be made of for example phenylglycine in activated form, preferably a (primary, secondary or tertiary) amide or salt thereof or a lower alkyl (1-4C) ester, for example a methyl ester; as phenylglycines use may be made of for example substituted or unsubstituted phenylglycines, in particular phenylglycine, p-hydroxyphenylglycine, dihydrophenylglycine.
  • phenylglycine amide is used as activated side chain
  • ⁇ -lactam antibiotics that are preferably prepared by the process according to the invention are amoxicillin, ampicillin, cephalexin, cefadroxil, cephradine and cefaclor.
  • S2 gene a DNA fragment having a polynucleotide sequence according to SEQ ID NO 1 (S2 gene) was cloned behind the tightly regulatable P B AD promoter located on pBAD/Myc-HisA_ ⁇ /del.
  • P B AD promoter located on pBAD/Myc-HisA_ ⁇ /del.
  • two ⁇ /ctel sites located on the commercially available vector pBAD/Myc- HisA (Invitrogen) were removed and the ⁇ /col site present in the multiple doing site of the vector was changed to an Ndel recognition sequence. All changes were carried out with the QuickChange Site-Directed Mutagenesis Kit (Stratagene) according to the manual.
  • the S2 gene was cloned including its own signal sequence, using a forward PCR primer based on the 5'-end of the gene with an introduced Ndel restriction site (restriction site underlined, start codon bold),
  • PCR amplifications were carried out with pWO polymerase (Roche) under standard conditions as recommended by the supplier. PCR product and vector were digested with Nde ⁇ /Sal ⁇ and Nde ⁇ IXho ⁇ , respectively, and ligated with T4 ligase according to the instructions of the manufacturer (Roche).
  • the ligation mixture was transformed to electrocompetent £ coli TOP10 cells (Invitrogen), and the construct (pBADS2) was confirmed by sequencing.
  • a disadvantage of the pBAD system is its selection marker (AmpR), a ⁇ -lactamase, which interferes with synthesis/hydrolysis experiments of some ⁇ -lactam antibiotics, when periplasmatic extracts are used. Therefore, all further experiments were carried out with purified protein.
  • AmpR selection marker has been replaced by a chloramphenicol marker which allows also for the use of unpurified pS2
  • E. coli penicillin acylase (ECPA) was produced and purified as described before [Alkema, 2000, Protein Engineering 13:857-863].
  • the obtained enzyme solution was concentrated by ultrafiltration (Amicon bioseparations, YM 30 filter) before the enzyme was rebuffered in 50 mM potassium phosphate buffer (pH 7.0) with 5 % glycerol, using an Econo-pac 10DG column (BioRad).
  • E. coli TOP10 (pBADS2) cells were grown in LB [Sambrook, 1989] at 17°C with rotary shaking at 200 rpm. In order to induce protein expression from P B A D , the medium was supplied with 0.8 % arabinose after 2 d of growth. When the late exponential phase was reached, cells were harvested by cenfrifugation at 5,000 ⁇ g for 10 min (4°C). To prepare a periplasmatic extract, cells were resuspended in 1/10 of the original culture volume of ice-cold osmotic shock buffer (20 % sucrose, 100 mM Tris.HCI, 10 mM EDTA; pH 8.0) and centrifuged as described above.
  • ice-cold osmotic shock buffer (20 % sucrose, 100 mM Tris.HCI, 10 mM EDTA; pH 8.0
  • the solution was subjected to hydrophobic interaction chromatography, using a Resource Phe (Amersham Pharmacia Biotech) column, and eluted with a linear gradient of 1.5 M to 0 M (NH 4 ) 2 SO 4 in 20 mM potassium phosphate buffer (pH 7.0). S2 eluted at a concentration of 300 mM (NH 4 ) 2 SO 4 .
  • Enzyme containing fractions were pooled, concentrated and rebuffered as described for ECPA. Both enzymes were stored at - 20°C and could be defrosted several times without detectable loss of activity.
  • the amount of active enzyme was determined by titration with the irreversible inhibitor phenylmethyisulfonyl fluoride (Boehringer Mannheim) as described by Alkema et al. [Alkema, 1999, Analytical Biochemistry, 275:47-53]. Chemicals
  • HPAAm was from Acros Organics. Penicillin G, amoxillin, 7-ADCA, 6-APA, PGA, and HPGA were provided by DSM Life Sciences (Delft, The Netherlands). NIPAB and NIPGB were synthesized by reacting phenylacetic acid chloride and D-phenylglycine chloride, respectively, with 5-amino-2-nitro-benzoic acid in a water/acteone mixture. PAAm was prepared by adding phenylacetylchloride dropwise to a concentrated ammonia solution, resulting in the formation of a white precipitate, which was filtered off and dried to constant weight.[Alkema, PhD thesis, 2002, Groningen State University)].
  • NIPAB 2-nitro-5-[(phenylacetyl)amino]-benzoic acid
  • NIPGB D-2- nitro-5-[(phenylglycyl)amino]-benzoic acid
  • K values for phenylacetic acid (PAA) and K m values for non-colorimetric substrates were determined by measuring the inhibition on the hydrolysis of NIPGB as described by Alkema et al. [Alkema et al., 1999, Analytical Biochemistry, 275:47-53]..
  • the k cat values were determined separately by monitoring the initial velocities of substrate conversion at substrate concentrations of at least 10 x K m by high-performance liquid chromatography (HPLC) (Alkema et al., 2000, Protein Eng. 13, 857-863).
  • Enzymatic synthesis of ⁇ -lactam antibiotics was carried out by mixing enzyme with solutions of acyl donor [either phenylacetamide (PAAm), D-phenylglycine amide (PGA), p-hydroxyphenylacetamide (HPAAm), or D-p-hydroxyphenylglycine amide (HPGA) or the corresponding esters] and nucleophile (6-APA or 7-ADCA).
  • acyl donor either phenylacetamide (PAAm), D-phenylglycine amide (PGA), p-hydroxyphenylacetamide (HPAAm), or D-p-hydroxyphenylglycine amide (HPGA) or the corresponding esters
  • nucleophile 6-APA or 7-ADCA
  • the initial concentration of acyl donor was 15 mM in all experiments, whereas the concentration of nucleophile varied between 1 and 190 mM. All reactants were monitored in time by HPLC analysis and initial velocities of synthesis and hydrolysis product formation were used to calculate
  • Table 1 Comparison of kinetic constants of S2 with other PenG acylases using NIPAB and PenG as substrates.
  • the purified S2 enzyme preparation was used to study its behavior in the synthesis of different ⁇ -lactam antibiotics.
  • amide derivatives of the respective sidechains were used as acyldonors and 6-APA or 7- ADCA as nucleophiles.
  • two side reactions can occur: (1) hydrolysis of the activated side chain, (2) hydrolysis of the initially formed antibiotic.
  • Qmax maximum in product (antibiotic) accumulation in the course of the reaction (Qmax), which is determined by the enzyme properties.
  • Another enzyme characteristic is the initial ratio between product formation (synthesis) and side reaction 1 (hydrolysis of the activated side-chain), the S/H ini ratio.
  • Figures 2 and 3 show the progress curves for the formation of ampicillin starting from D-phenylglycinamide (D-PGA) and 6-APA by E.coli and S2 acylase, respectively.
  • Figures 4 and 5 show the progress curves for the formation of amoxicillin starting from D-hydroxyphenylglycine amide (D-HPGA) and 6-APA by E.coli and S2 acylase, respectively.
  • the PenG acylase concentration was the same (200nM) for both S2 and E.coli PenG acylases. All reactions were carried out with 15mM acyl donor and 25mM nucleophile.
  • the (S/H) in j ratio for S2 is substantially improved compared to E.coli acylase.
  • a higher accumulation for ampicillin as well as for amoxicillin is observed (higher Qmax).
  • the S2 acylase shows in addition to an improved initial S/H ratio and a higher ampicillin and amoxicillin accumulation also an increased synthesis rate for both products.
  • c0(acyl donor) 15 mM
  • c ⁇ (nucleophile) 25 mM
  • c(acylase) 200 nM.
  • c 0 (acyl donor) 15 mM
  • c 0 (nucleophile) 25 mM
  • c(acylase) 200 nM.
  • D-PGM D-phenylglycine methyl ester
  • D-HPGM D-p- hydroxyphenylglycine methylester

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Abstract

Nouvelle enzyme ayant une activité d'acylase de la pénicilline G, caractérisée (a) par un rapport initial synthèse / hydrolyse (S/Hini) supérieur d'au moins 50 % au S/Hini de l'acylase de la pénicilline G de E. coli et (b) un taux initial de conversion de chaîne latérale activée de plus de 80 % supérieur au taux initial de conversion de chaîne latérale activée par l'acylase de la pénicilline G de E coli. L'amide de D-p-hydroxyphénylglycine (DHPGA) est utilisé en tant que précurseur de chaîne latérale activée et de l'acide 6-amino-pénicillanique est utilisé en tant que nucléophile penam. En raison de ses propriétés uniques, ladite enzyme peut être utilisée pour la production enzymatique d'antibiotiques semi-synthétiques.
PCT/EP2004/006423 2003-06-12 2004-06-10 Acylase de penicilline g WO2004111241A1 (fr)

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EP03076828 2003-06-12

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010054319A2 (fr) * 2008-11-10 2010-05-14 Codexis, Inc. Pénicilline g acylases
WO2019005337A1 (fr) * 2017-06-27 2019-01-03 Codexis, Inc. Pénicilline g acylases

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5457032A (en) * 1990-04-18 1995-10-10 Gist-Brocades Nv Mutated β-lactam acylase genes
WO1996005318A1 (fr) * 1994-08-12 1996-02-22 Gist-Brocades B.V. Genes mutants d'acylases de la penicilline g

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5457032A (en) * 1990-04-18 1995-10-10 Gist-Brocades Nv Mutated β-lactam acylase genes
WO1996005318A1 (fr) * 1994-08-12 1996-02-22 Gist-Brocades B.V. Genes mutants d'acylases de la penicilline g

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ALKEMA ET AL.: "The role of hydrophobic active-site residues in substrate specificity and acyl transfer activity of penicillin acylase", EUR. J. BIOCHEM., vol. 269, 2002, pages 2093 - 2100, XP002264819 *
DATABASE EM_PRO [online] EMBL; 24 April 2003 (2003-04-24), ZHU ET AL.: "Achromobacter xylosoxidans subsp. xylosoxidans penicillin G acylase gene, complet cds.", XP002264821, retrieved from EBI Database accession no. AF490005 *
DATABASE SWALL [online] 1 June 2003 (2003-06-01), ZHU ET AL.: "Penicillin G acylase", XP002264820, retrieved from EBI Database accession no. Q83YY8 *
SCHUMACHER G ET AL: "PENICILLIN ACYLASE FROM E. COLI: UNIQUE GENE-PROTEIN RELATION", NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 14, no. 14, 1986, pages 5713 - 5727, XP001097402, ISSN: 0305-1048 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010054319A2 (fr) * 2008-11-10 2010-05-14 Codexis, Inc. Pénicilline g acylases
WO2010054319A3 (fr) * 2008-11-10 2010-08-19 Codexis, Inc. Pénicilline g acylases
US8247192B2 (en) 2008-11-10 2012-08-21 Codexis, Inc. Penicillin-G acylases
US8569013B2 (en) 2008-11-10 2013-10-29 Codexis, Inc. Penicillin-G acylases
WO2019005337A1 (fr) * 2017-06-27 2019-01-03 Codexis, Inc. Pénicilline g acylases
US11643642B2 (en) 2017-06-27 2023-05-09 Codexis, Inc. Penicillin-g acylases

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