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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes

Definitions

• the present invention relates to mutant type Il beta-lactam acylases, to polynucleotides encoding said enzymes and to microorganisms transformed with said polynucleotides as well as to methods of producing the mutant type Il beta lactam.
• the invention furthermore relates to a process for the production of a deacylated beta-lactam compound of interest using the mutant type Il beta lactam acylases of the invention.
• Beta-lactam antibiotics constitute the most important group of antibiotic compounds with a long history of clinical use.
• the prominent ones are the penicillins and cephalosporins.
• Penicillins are naturally produced by various filamentous fungi such as Penicillium (e.g. P. chrysogenum).
• Cephalosporins are naturally produced by various microorganisms such as Acremonium (e.g. A. chrysogenum) and Streptomyces (e.g. Streptomyces clavuligerus).
• the first two steps in the biosynthesis of penicillin in P. chrysogenum aye the condensation of the three amino acids L-5-amino-5-carboxypentanoic acid (L-alphalpha- aminoadipic acid) (A), L-cystein (C) and L-valine (V) into the tripeptide LLD-ACV, followed by cyclization of this tripeptide to form isopenicillin N.
• L-alphalpha- aminoadipic acid) A
• L-cystein C
• L-valine V
• This compound contains the typical beta-lactam structure.
• the third step involves the replacement of the hydrophilic side chain of L-5-amino-5-carboxypentanoic acid by a hydrophobic side chain by the action of the enzyme acy transferase (AT).
• chrysogenum transformants expressing deacetoxycephalosporin C synthase implies the presence of significant amounts of penicillin N, the natural substrate for expandase, in P chrysogenum (Alvi et al , J Antibiot (1995) 48 338-340)
• penicillin N the natural substrate for expandase
• P chrysogenum the D-alpha-am ⁇ no- adipyl side chains of DAOC cannot be easily removed
• Cephalosporins are much more expensive than penicillins
• cephalosporins e g cephalexin
• Cephalosporins are made from penicillins by a number of chemical conversions
• Cephalosporin C by far the most important starting material in this respect, is very soluble in water at any pH, thus implying lengthy and costly isolation processes using cumbersome and expensive column technology
• Cephalosporin C obtained in this way has to be converted into therapeutically used cephalosporins by a number of chemical and enzymatic conversions
• chrysogenum in combination with an adipyl side chain (further referred to as adipyl) as a feedstock, which is a substrate for the acyltransferase enzyme in P. chrysogenum.
• adipyl adipyl side chain
• the side chains of the corresponding 7-ADCA derivatives can be cleaved off either chemically or enzymatically by an acylase enzyme thus yielding 7-ADCA and the corresponding side chain.
• an acylase enzyme thus yielding 7-ADCA and the corresponding side chain.
• Various types of microorganisms have been proposed in the literature as acylase producing strains useful for the deacylation of beta-lactam derivatives obtained by fermentation.
• acylase-producing microorganisms are certain strains of the species Escherichia coli, Kluyvera citrophila, Proteus rettgeri, Pseudomonas sp., Alcaligenes faecalis, Bacillus megaterium, Bacillus sphaericus and Arthrobacter viscosus.
• acylases According to the literature several types of acylases may be envisaged, based on their molecular structure and substrate specificity (Vandamme E. J. Penicillin acylases and beta-lactamases” In: “Microbial Enzymes and Bioconversions” E. H. Rose (Ed.), Economic Microbiology 5 (1980) 467-552, Acad. Press, New York).
• Type-I acylases are specific for Penicillin V. These enzymes are composed of four identical subunits, each having a molecular weight of 35 kDa. A complete nucleotide sequence of the cloned gene from Bacillus sphaericus has been reported (Ollson A. Appl. Environm. Microb. (1976), 203). Type-ll acylases all share a common molecular structure: these enzymes are heterodimers composed of a small alpha-subunit (20-25 kDa) and a large beta-subunit (60-65 kDa).
• Type-ll acylases may be further divided into two groups Type-IIA acylases are very specific for Penicillin G and therefore generally known as penicillin acylases In general, they are not so much specific for the moiety adjacent to the nitrogen atom of the amide group (this might be a cephem group, a penem group, an amino acid, etc ), but the substrate specificity resides in the acyl moiety of the substrate This acyl moiety must be very hydrophobic and is preferably benzyl or (short) alkyl
• substrates that are not hydrolyzed by Type-IIA acylases are those with dicarboxylic acids as acyl moiety succinyl, glutaryl, adipyl and aminoadipyl, the side-chain of CefC
• Type-IIA acylases are the enzymes from Escherichia coli, Kluyvera citrophila, Proteus rettgeri and Alcaligene
• WO2005014821 discloses mutants of the SE83-acylase and EP-A- 1553175 discloses mutants of the N176-acylase, all in order to improve the CEFC- acylase activity. None of the cited references focused on the improvement of the deacylation reaction with other acylated beta-lactam compounds of interest such as adipyl-7-ADCA.
• Figure 1 Multiple alignment of the amino acid sequences of the type Il beta-lactam acylases SE83-acyii from Pseudomonas SE83 (SEQ ID No.1), N 176 from Brevundimonas diminuta N-176 (SEQ ID No. 2) and V22 from Brevundimonas diminuta V22 (SEQ ID No. 3)
• the present invention provides a mutant type Il beta-lactam acylase that is a variant of a model polypeptide with type Il beta-lactam acylase activity whereby the mutant beta-lactam acylase has an at least 1.5-fold improved in vitro beta- lactam acylase activity towards adipyl-7-ADCA in comparison with the model polypeptide with beta-lactam acylase activity.
• the determination of the in vitro beta-lactam acylase activity towards adipyl-7-ADCA is described in detail in the Materials and Methods section.
• the in vitro beta-lactam acylase activity towards adipyl-7-ADCA of the mutant type Il beta-lactam acylase is improved at least 2-fold, more preferably at least 2.5- fold, more preferably at least 3-fold, more preferably at least 4-fold, more preferably at least 5-fold, more preferably at least 6-fold, more preferably at least 7-fold, more preferably at least 8-fold, more preferably at least 9-fold, more preferably at least 10-fold, more preferably at least 11 -fold.
• altered or mutant type Il beta-lactam acylase in the context of the present invention is meant any enzyme having acylase activity, which has not been obtained from a natural source and for which the amino acid sequence differs from the complete amino acid sequences of the natural type Il beta-lactam acylase enzymes.
• the invention also provides a mutant type Il beta-lactam acylase that is a variant of a model polypeptide with type Il beta-lactam acylase activity whereby the mutant type Il beta-lactam acylase has been modified at at least an amino acid position selected from the group consisting of positions 161 , 270, 296, 442 and 589 or from the group consisting of positions 10, 29, 274, 280, 314, 514, 645, 694, 706 and 726 or from the group consisting of 10, 29, 161 , 270, 274, 280, 296, 314, 442, 514, 589, 645, 694, 706 and 726 or from the group consisting of 10, 29, 270, 274, 280, 442, 514, 589, 645, 694 and 726 using the amino acid position numbering of the amino acid sequence of the SE83-acyll acylase of Pseudomonas (SEQ ID NO: 1).
• the present invention provides a mutant type Il beta-lactam acylase that is a variant of a model polypeptide with type Il beta-lactam acylase activity whereby the mutant beta-lactam acylase has an at least 1.5-fold improved in vitro beta- lactam acylase activity towards adipyl-7-ADCA in comparison with the model polypeptide with beta-lactam acylase activity, more preferably at least 2-fold, more preferably at least 2.5-fold, more preferably at least 3-fold, more preferably at least 4-fold, more preferably at least 5-fold, more preferably at least 6-fold, more preferably at least 7-fold, more preferably at least 8-fold, more preferably at least 9-fold, more preferably at least 10-fold, more preferably at least 11-fold and whereby the mutant type Il beta-lactam acylase has been modified at at least an amino acid position selected from group consisting of positions 161 , 270, 296, 442 and 589, or from the group consisting of positions 10, 29,
• the present invention preferably provides a mutant type Il beta-lactam acylase that is a variant of a model polypeptide with type Il beta-lactam acylase activity whereby the mutant type Il beta-lactam acylase has been modified at least at position 10 or at least at position 29, at least at position 161 or at least at position 270 or at least at position 274 or at least at position 280 or at least at position 296 or at least at position 314 or at least at position 442 or at least at position 514 or at least at position 589 or at least at position 645 or at least at position 694 or at least at position 706 or at least at position 726 using the amino acid position numbering of the amino acid sequence of the SE83-acyll acylase of Pseudomonas (SEQ ID NO: 1).
• the invention provides mutant type Il beta-lactam acylases have a single modification at either position 161 or at position 296.
• the present invention also provides a mutant type Il beta-lactam acylase that is a variant of a model polypeptide with type Il beta-lactam acylase activity whereby the mutant type Il beta-lactam acylase has been modified at least at a combination of positions 161+270 or at least at a combination of positions 161+296 or at least at a combination of positions 161+442 or at least at a combination of positions 161+589 or at least at a combination of positions 270+296 or at least at a combination of positions 270+442 or at least at a combination of positions 270+589 or at least at a combination of positions 296+442 or at least at a combination of positions 296+589 or at least at a combination of positions 442+589 or at least at a combination of positions 161+270+296 or at least at a combination of positions 161+270+442 or at least at a combination of positions 161+270+589 or at least at a combination of positions
• the model polypeptide with type Il beta-lactam acylase activity as used in the present invention is selected from the group consisting of a polypeptide with type Il beta- lactam acylase activity, preferably having an amino acid sequence according to SEQ ID NO 1 ( ⁇ e the SE83-acyll acylase of Pseudomonas species SE83) or having an amino acid sequence according to SEQ ID NO 2 ( ⁇ e the N176-acylase of Pseudomonas species N176) or having an amino acid sequence according to SEQ ID NO 3 ( ⁇ e the V22 acylase of Brevundimonas diminuta V22) and polypeptides with type Il beta-lactam acylase activity having an amino acid sequence with a percentage identity with SEQ ID NO 1 of at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95%, or with SEQ ID NO 2 of at least 70%, preferably at least
• the present invention preferably provides mutants of the model type Il beta-lactam acylases selected from the group consisting of the acylase having the amino acid sequence according to SEQ ID NO 1 and the acylase having the amino acid sequence according to SEQ ID NO 2 and the acylase having the amino acid sequence according to SEQ ID NO 3 and polypeptides with type Il beta-lactam acylase activity having an amino acid sequence with a percentage identity with SEQ ID NO 1 of at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95%, or with SEQ ID NO 2 of at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95%, %, or with SEQ ID NO 3 of at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at
• the modification at an amino acid position may comprise a substitution by another amino acid, selected from the group of 20 L-amino acids that occur in Nature - see Table 1
• the modification at an amino acid position may comprise a deletion of the amino acid at said position
• the modification at an amino acid position may comprise a substitution of one or more amino acids at the C-terminal or N-terminal side of said amino acid
• mutant type Il beta-lactam acylase of the invention preferably the mutants of the model type Il beta-lactam acylases selected from the group consisting of the acylase having the amino acid sequence according to SEQ ID NO 1 and the acylase having the amino acid sequence according to SEQ ID NO 2 and the acylase having the ammo acid sequence according to SEQ ID NO 3 may carry one or more of the following modifications
• Substitution of the leucine at position 161 by a smaller and more polar amino acid such threonine, serine, glycine and cystein or a positively charged around pH 9 such as arginine and lysine, preferably by serine or threonine or glycine, most preferably by threonine • Substitution of the histidine at position 274 by an amino acid residue that contains at least a carbon, oxygen or sulphur atom at the gamma position of the side chain and which is smaller in size compared to histidine such as leucine, isoleucine, cystein, threonine, serine, asparagine, valine and proline, preferably by leucine, isoleucine, cystein or threonine, • Substitution of the arginine at position 280 by an amino acid residue that substitutes the positive charge by a negative charge such as aspartic acid and glutamic acid or by an unbranched and uncharged polar side chain such as
• Highly preferred embodiments of the present invention are mutants of the model type Il beta-lactam acylases selected from the group consisting of the acylase having the amino acid sequence according to SEQ ID NO: 1 and the acylase having the amino acid sequence according to SEQ ID NO: 2 and the acylase having the amino acid sequence according to SEQ ID NO:3 and polypeptides with type Il beta-lactam acylase activity having an amino acid sequence with a percentage identity with SEQ ID NO: 1 of at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95%, or with SEQ ID NO: 2 of at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95%, or with SEQ ID NO: 3 of at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least
• More highly preferred embodiments of the present invention are mutants of the model type Il beta-lactam acylases selected from the group consisting of the acylase having the amino acid sequence according to SEQ ID NO 1 and the acylase having the ammo acid sequence according to SEQ ID NO 2 and the acylase having the amino acid sequence according to SEQ ID NO 3 and polypeptides with type Il beta-lactam acylase activity having an amino acid sequence with a percentage identity with SEQ ID NO 1 of at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95%, or with SEQ ID NO 2 of at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95%, %, or with SEQ ID NO 3 of at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least
• the present invention preferably provides mutants of the SE83-acyll acylase of Pseudomonas (SEQ ID NO 1) that have modifications at least at a combination of positions L161+M270 or at least at a combination of positions L161+H296 or at least at a combination of positions L161+E442 or at least at a combination of positions L161+R589 or at least at a combination of positions M270+H296 or at least at a combination of positions M270+E442 or at least at a combination of positions M270+R589 or at least at a combination of positions H296+E442 or at least at a combination of positions H296+R589 or at least at a combination of positions E442+R589 or at least at a combination of positions L161+M270+H296 or at least at a combination of positions L161+M270+E442 or at least at a combination of positions L161+M270+R589 or at least at a
• the invention provides a polynucleotide encoding the mutant type Il beta-lactam acylase of the present invention
• the invention provides also polynucleotides encoding the alpha-subunit of the mutant type Il beta-lactam acylase as well as polynucleotides encoding the beta-subunit of the mutant type Il beta-lactam acylase WO2005/014821 discloses on page 8 and 9 that genes encoding the SE83-group acylases are encoding a polypeptide composed of an ⁇ -subun ⁇ t, a spacer peptide, and a ⁇ -subunit, in that order
• the acylase derived from Pseudomonas sp SE83 is generated in the form of an inactive single chain polypeptide having about 84 kDa in size after undergoing transcription and translation in a host cell After then, two self-digestions occur between the amino acids at position 230 and 231 , and at position 239 and
• the polynucleotide encoding the mutant type Il beta-lactam acylase or the alpha- subunit or the beta-subunit according to the present invention can be any polynucleotide that encodes the proper ammo acid sequence according to the invention
• the polynucleotide of the invention may comprise a coding sequence in which the codon usage for the various amino acids deviates from the codon usage in Pseudomonas
• the codon usage may be adapted to the codon usage of a particular host cell, which will or has been transformed with the DNA fragment encoding the altered type Il beta-lactam acylase
• the invention provides an expression vector or expression cassette comprising the polynucleotide of the invention as defined hereinbefore.
• the invention provides a transformed host cell, transformed with the polynucleotide of the invention or the expression vector or expression cassette of the invention.
• the transformed host cell may be used for the production of the mutant type Il beta-lactam acylase of the invention.
• Host cells for the production of the mutant type Il beta-lactam acylase of the invention are preferably host cells which are known in the art for their efficient protein or enzyme production, either extracellular or intracellular ⁇ , for example microorganisms such as fungi, yeast and bacteria.
• preferred host cells comprise, but are not limited to, the following genera: Aspergillus (e.g. A. niger, A. oryzea), Penicillium (e.g. P. emersonii, P. chrysogenum), Saccharomyces (e.g. S. cerevisiae), Kluyveromyces (e.g. K. lactis), Bacillus (e.g. B. subtilis, B. licheniformis, B.
• Aspergillus e.g. A. niger, A. oryzea
• Penicillium e.g. P. emersonii, P. chrysogenum
• Saccharomyces e.
• the invention provides a process for the production of the mutant type Il beta-lactam acylase of the invention comprising cultivating the transformed host cell according to the invention under conditions conducive to the production of the mutant expandase and, optionally, recovering the mutant expandase.
• the invention provides a process for the production of a deacylated beta-lactam compound of interest comprising the step of deacylating an acylated precursor of the beta-lactam compound of interest using the mutant type Il beta- lactam acylase of the invention.
• Deacylated beta-lactam compounds of interest may be derivates of naturally occurring penicillins or cephalosporins such as 6-APA, 7-ACA, 7- ADCA, 7-ADAC, 7-amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid (e.g. WO2004/106347) and others.
• the deacylated beta-lactam compound of interest is 7-ADCA or 7-ACA, most preferred is 7-ADCA.
• Acylated precursors of the beta- lactam compound of interest may have an acyl belonging to the group of consisting of dicarboxylic acids.
• Preferred acyl groups are succinyl, glutaryl, adipyl, alpha-ketoadipyl and aminoadipyl. More preferred are adipyl and aminoadipyl, highly preferred is adipyl.
• acylated precursors of beta-lactam compound of interest are adipyl-7-ADCA, adipyl-7-ACA, aminoadipyl-7-ADCA and aminoadipyl-7-ACA, the latter known as CEFC; most preferred is adipyl-7-ADCA.
• the process of the invention for the production of a deacylated beta-lactam compound of interest may be carried out in a batchwise mode whereby the mutant type Il beta-lactam acylase is used in a dissolved state in a solution comprising the acylated precursor of the beta-lactam compound of interest
• the mutant type Il beta-lactam acylase is used as an immobilized form
• the mutant type Il beta-lactam acylase may be recovered after completion of the deacylation reaction and be reused for further deacylation reactions
• the cost in use of the mutant type Il beta-lactam acylase can be reduced significantly, thus increasing the economic attractiveness of the deacylation process
• Conditions for the deacylation reaction as well as the immobilization of the enzyme are known in the prior art (e g Kallenberg, A l et al Adv Synth Catal (2005), 347, 905-926)
• the present invention relates to the use of the mutant type Ii beta-lactam acylase of the invention in a process for the production of a deacylated beta- lactam compound of interest which process comprises the step of deacylating an acylated precursor of the beta-lactam compound of interest
• Deacylated beta-lactam compounds of interest may be denvates of naturally occurring penicillins or cephalosporins such as 6- APA, 7-ACA, 7-ADCA, 7-ADAC, 7-am ⁇ no-3-carbamoyloxymethyl-3-cephem-4-carboxyl ⁇ c acid and others
• the deacylated beta-lactam compound of interest is 7-ADCA or 7-ACA, most preferred is 7-ADCA
• Acylated precursors of the beta-lactam compound of interest may have an acyl belonging to the group of consisting of dicarboxylic acids
• Preferred acyl groups are succinyl, glutaryl, adipyl, alpha
• Plasmids with the wt gene or mutant genes were transformed to E coll Top 10 cells (Invitrogen)
• the cells were inoculated in 100 ml flasks using 20 ml 2xTY medium containing 50 ⁇ g/ml zeocine at 37 0 C and 280 rpm
• 50 ⁇ g/ml zeocine and 0,05% arabinose were inoculated with 50 ⁇ l of the culture 1 1000 and grown at 25 0 C and 280 rpm
• the cultures were centrifuged and frozen at -2O 0 C
• the pellets were resuspended in extraction buffer (5OmM Tris/HCL, 0 1 mg/ml DNAsel , 2mg/ml Lysozyme, 1OmM DTT (dithiothreitol), 5mM MgSO4) and incubated After 30 minutes the extract was centrifuged and the supern
• the acylase content was determined using SDS-PAGE gel electrophoresis and analytical HPLC size exclusion chromatography performed on a TSK 3000SWxI column with 0 1 M phosphate buffer pH 7 0 as an eluent Applied chromatographic conditions flow rate 1 0 ml/mm and detection at 280 nm By comparing the observed areas of the acylase peaks one can compare the protein content of different samples The acylase protein content is calculated from the OD280 using a molar extinction coefficient of 154350 (M 1 cm 1 ) In case of additional peaks in the HPLC chromatogram, the E280 value of the sample is corrected for the contribution of the additional peaks
• Ad ⁇ pyl-7ADCA can be prepared from adipic acid and 7-ADCA by enzymatic synthesis as described in WO9848037
• Ad ⁇ pyl-7ADCA can be prepared by chemical synthesis as described by Shibuya et al in Agric Biol Chem , 1981 , 45(7), 1561-1567 starting from adipic anhydride instead of glutaric anhydride
• the assay was carried out at room temperature. The initial substrate concentration was 2% (w/v) adipyl-7ADCA. Initial activity was measured in the presence of 1 5% (w/v) adipic acid.
• Table 5 shows the measured K M values for a number of mutants relative to wild type
• the Michaehs constant K M represents the substrate concentration at which the enzyme operates at 50% of its maximal velocity At substrate concentrations below the K M the enzyme becomes slower, at substrate concentrations above the K M the enzymes operates faster until at high substrate concentration the enzyme becomes fully saturated and operate at maximal velocity At the end of an enzymatic conversion where the substrate gets exhausted a low K M is crucial in order to maintain substantial activity
• the relative K M value for a mutant is ⁇ 1 00, it means that at lower substrate concentrations, e.g. at the end of the conversion, the mutant has an advantage relative to wild type in maintaining a substantial higher activity.
• Relative substrate affinities represented as relative K M values.
• the K M determinations were done using the assay as described earlier.
• the adipyl-7ADCA concentration was varied from 0.5 to 4% Adipyl-7ADCA.
• Immobilization was carried out as described in WO97/04086 using gelatin and chitosan as gelling agents and glutaric dialdehyde as cross-linking agent.
• the performance of the immobilized wild type acylase and mutant acylases was measured by performing a complete hydrolysis of adipyl-7ADCA in a temperature and pH controlled 100 ml reactor. Experiments were performed at 3.2% adipyl-7-ADCA.
• the stability of the immobilized acylases was determined by measuring 20 subsequent conversions of 180 minutes with the same batch of immobilized acylase. The rate between 30 and 50% conversion of each incubation was measured. The residual activity of an immobilized acylase is defined herein as the activity of the 20 th incubation compared to the rate of the first incubation.
• Table 6 summarizes the results. It was observed that in particular under the conditions that shift the thermodynamic equilibrium of the hydrolysis reaction to a more complete conversion (high temperature and high pH) the stability of the mutated acylase is significantly improved compared to the wild type.

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