EP1974029A2

EP1974029A2 - Mutant acylases

Info

Publication number: EP1974029A2
Application number: EP06841245A
Authority: EP
Inventors: Jan Metske Laan Van Der; Richard Kerkman; Willem Bijleveld; Bianca Gielesen
Original assignee: DSM IP Assets BV
Current assignee: Centrient Pharmaceuticals Netherlands BV
Priority date: 2005-12-28
Filing date: 2006-12-28
Publication date: 2008-10-01
Also published as: ES2571865T3; EP2851423B1; WO2007073947A2; CN101351549A; KR20080084986A; BRPI0621278A2; WO2007073947A3; EP2851423A1; CN101351549B; HK1128305A1

Abstract

The present invention describes 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.

Description

MUTANT ACYLASES

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. Among this group, 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).

As a result of classical strain improvement techniques, the production levels of the antibiotics in P. chrysogenum and A. chrysogenum have increased remarkably over the past decades. With the increasing knowledge of the biosynthetic pathways leading to penicillins and cephalosporins and the advent of recombinant DNA technology, new tools for the improvement of production strains have become available.

Most enzymes involved in beta-lactam biosynthesis have been identified and their corresponding genes have been cloned as can be found in lngolia and Queener, Med Res Rev (1989) 9:245-264 (biosynthesis route and enzymes) and Aharonowitz, Cohen, and Martin, Ann Rev Microbiol (1992) 46:461-495 (gene cloning).

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. 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). In EP-A-0448180 it has been described that the enzymatic exchange reaction mediated by AT takes place inside a cellular organelle, the microbody. The observation that substantial quantities of deacetoxycephalosporin C (DAOC) can be formed by non- precursed P. chrysogenum transformants expressing deacetoxycephalosporin C synthase (EC 1.14.20.1 - DAOCS, further indicated herein as expandase) 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) However, the D-alpha-amιno- adipyl side chains of DAOC cannot be easily removed

Cephalosporins are much more expensive than penicillins One reason is that some cephalosporins (e g cephalexin) are made from penicillins by a number of chemical conversions Another reason is that, so far, only cephalosporins with a D- alpha-amino-adipyl side chain could be fermented 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

The methods currently favoured in industry to prepare the intermediate 7-amιno- deacetoxycephaloporanic acid (7-ADCA) involve complex chemical steps leading to the expansion and deπvatization of penicillin G One of the necessary chemical steps to produce 7-ADCA involves the expansion of the 5-membered penicillin ring structure to a 6-membered cephalosporin ring structure (see for instance US 4,003,894) This complex chemical processing is both expensive and noxious to the environment Consequently, there is a great desire to replace such chemical processes with enzymatic reactions such as enzymatic catalysis, preferably during fermentation A key to the replacement of the chemical expansion process by a biological process is the central enzyme in the cephalosporin biosynthetic pathway, expandase The expandase enzyme from the bacterium Streptomyces clavuligerus (S clavuligerus) was found to carry out, in some cases, penicillin ring expansions When introduced into P chrysogenum, it can convert the penicillin ring structure into the cephalosporin ring structure, as described in Cantwell et al , Proc R Soc Lond B (1992) 248 283-289 Since the expandase enzyme catalyses the expansion of the 5-membered thiazolidine ring of penicillin N to the 6- membered dihydrothiazine ring of DAOC, this enzyme would be of course a logical candidate to replace the ring expansion steps of the chemical process Unfortunately, the enzyme works on the penicillin N intermediate of the cephalosporin biosynthetic pathway, but not or very inefficiently on the readily available inexpensive penicillins as produced by P chrysogenum, like penicillin V or penicillin G Penicillin N is commercially not available and even when expanded, its D-alpha-amino-adipyl side chain cannot be easily removed by penicillin acylases It has been reported that the expandase enzyme is capable of expanding penicillins with particular side chains to the corresponding 7-ADCA derivative. This feature of the expandase has been exploited in the technology as disclosed in WO93/05158, WO95/04148 and WO95/04149. In these disclosures the conventional chemical in vitro conversion of penicillin G to 7-ADCA has been replaced by the in vivo conversion of certain 6-aminopenicillanic acid (6-APA) derivatives in recombinant Penicillium chrysogenum strains transformed with an expandase gene. More particularly, WO93/05158 teaches the in vivo use of the expandase enzyme in P. 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. This leads to the formation of adipyl-6-APA, which is converted by an expandase enzyme introduced into the P. chrysogenum strain to yield adipyl-7-ADCA which is excreted by the fungal cells into the surrounding medium.

In a subsequent step, 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. 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. Examples of such 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.

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). With respect to the substrate specificity, 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 Examples of 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 Examples of Type-IIA acylases are the enzymes from Escherichia coli, Kluyvera citrophila, Proteus rettgeri and Alcaligenes faecalis Type-IIB acylases have been reported to be capable of hydrolyzing cephalosporins (including the desacetoxy-denvative) with succinyl, glutaryl, adipyl and α- ketoadipyl as an acyl moiety and even CefC to a very limited degree The group of Type-IIB acylases again can be divided into two groups on the basis of amino acid sequence homology These subgroups will be defined here as the SY77-group and SE83-group and are named after the acylase from Pseudomonas SY77 and Pseudomonas SE83-acyll respectively

Matsuda et al (J Bacterid (1985), 163, 1222 have cloned and sequenced the gene encoding the SY77-acylase and demonstrated that the enzyme was active towards glutaryl-7ACA but much less toward succιnyl-7ACA and adιpyl-7ACA The 3-dιmensιonal structure for the SY77-precursor is known (J Biol Chem (2002), 277, 2823)

Later, Matsuda et al (J Bacteriol (1987), 169, 5815 and J Bacteriol (1987), 169, 5821) cloned and sequenced the gene encoding the SE83-acyll acylase and demonstrated that this enzyme was active towards (in decreasing order) glutaryl-7ACA, adιpyl-7ACA, succιnyl-7ACA and CEFC (Cephalosporin C) All studies related to SE83 focused on the capacity of the enzyme to hydrolyse derivatives of 7-ACA, in particular to hydrolyse CEFC

In WO91 /16435 it has been shown that the amino acid homology between SY77 and SE83-acyll is very low approximately 25% for the alpha-subunits and 28% for the beta-subunits of the acylases WO9512680 discloses another SE83-group acylase from Brevundimonas diminuta, named N176, which is approximately 94% homologous to SE83-acyll and which was tested for its CEFC-acylase activity A third member of the SE83-group is V22 from Brevundimonas diminuta V22 The ammo acid sequences of these three acylases are disclosed by Aramori et al in Journal of Fermentation and Bioengineering 72, 232-243 (1991). Table 1 shows the full length sequence identity matrix of the amino acid sequences of the various Type-IIB acylases of the SE83-group.

Table 1.

Several attempts have been made to increase the CEFC-acylase activity of a few existing acylases: 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. Therefore, there is still an urgent need for an acylase with an improved deacylating activity towards adipyl-7ADCA and which can be used advantageously in a process for the production of 7-ADCA from adipyl-7-ADCA produced for instance by fermentation of a transformed Penicillium strain.

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)

Figure 2: Conversion of adipyl-7ADCA by immobilized acylase at pH=8.8 and 30⁰C

(Figure 2a) and at pH=9.5 and 40⁰C (Figure 2b). Peudomonas SE83 ACYii wild type immobilized acylase (solid line) and mutant L161T immobilized acylase (dashed line). The rate (ml KOH/min on the Y-axis) is plotted as a function of the percentage conversion (% on the X-axis) .

In a first aspect, 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. More preferably 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.

With "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).

More preferably, 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, 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 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). In one embodiment 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 161+296+589 or at least at a combination of positions 296+442+589 or at least at a combination of positions 161+296+442 or at least at a combination of positions 161 +296+589 or at least at a combination of positions 296+442+589 or at least at any combination of 4 positions selected from the groups consisting of 161 , 270, 296, 442 and 589 or at least at the combination of positions 161 , 270, 296, 442 and 589 and whereby the mutant type Il beta- lactam acylases may have modifications at other amino acid positions in addition to those positions and all possible combinations thereof as described before using the amino acid position numbering of the ammo acid sequence of the SE83-acyll acylase of Pseudomonas (SEQ ID NO 1)

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 (ι e the SE83-acyll acylase of Pseudomonas species SE83) or having an amino acid sequence according to SEQ ID NO 2 (ι e the N176-acylase of Pseudomonas species N176) or having an amino acid sequence according to SEQ ID NO 3 (ι 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 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 least 95% More preferred as model polypeptide with type Il beta-lactam acylase activity as used in the present invention is a polypeptide with type Il beta-lactam acylase activity, having the amino acid sequence according to SEQ ID NO 1 or having the amino acid sequence according to SEQ ID NO 2 or having the amino acid sequence according to SEQ ID NO 3 Most preferred as model polypeptide with type Il beta-lactam acylase activity is the SE83-acyll acylase of Pseudomonas (SEQ ID NO 1)

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 least 95% and whereby the mutants have modifications 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 In one embodiment the invention provides mutant type Il beta-lactam acylases have a single modification at either position 161 or at position 296 using the amino acid position numbering of the amino acid sequence of the SE83- acyll acylase of Pseudomonas (SEQ ID NO 1)

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 Alternatively, the modification at an amino acid position may comprise a deletion of the amino acid at said position Furthermore, 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

Table 1

The 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 glutamate (SEQ ID NO 1) or alanine (SEQ ID NO 2 and SEQ ID NO 3) at position 10 by a positively charged amino acid residue such as lysine or arginine or a small amino acid residue with conformational preference for α-helιx formation such as alanine, preferably by lysine

• Substitution of the serine at position 29 by an amino acid with an aromatic (like) side chain such as phenylalanine, tyrosine, tryptophane and histidine or a larger uncharged polar or positively charged side chain such as asparagine, glutamine, arginine and lysine, preferably by asparagine or phenylalanine,

• 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 glutamine, asparagine and serine, preferably glutamine and asparagine, most preferably glutamine • Substitution of the histidine at position 296 by a charged or polar amino acid or an amino acid residue which is able to replace the in the model acylase existing hydrogen bonding to the N-delta or N-epsilon atoms of the histidine residue such as by asparagine and glutamine, preferably by a glutamine

• Substitution of the isoleucine at position 314 by a smaller amino acid residue with β-branching such as valine or by a medium size polar side chain such as glutamine, asparagine serine and threonine, preferably by valine or glutamine

• Substitution of the glutamic acid at position 442 by an amino acts residue with no or a small hydrophobic side chain such as glycine, alanine, leucine, valine and isoleucine, preferably glycine • Substitution of the proline at position 514 by an amino acid residue with a more polar and/or more flexible side chain which is able to contribute to additional hydrogen bonding such as glutamine, asparagine, threonine, serine, cystein, aspartic acid and glutamic acid, preferably glutamine • Substitution of the arginine at position 589 by an amino acid residue that can maintain a positive charge in a certain environment such as histidine and lysine or by aromatic side chains able to form hydrogen bonds such as tyrosine and tryptophane or by amino acid residues which are able to replace the in the model acylase existing hydrogen bonding to the N-delta or N-epsilon atoms of the histidine residue such as by asparagine and glutamine, preferably by histidine.

• Substitution of the alanine at position 645 by a small amino acid residue with an increased preference for β-strand formation such as threonine, valine, serine, cystein and leucine, preferably by threonine

• Substitution of the asparagine at position 694 by an amino acid residue with a side chain smaller than asparagine such as alanine, threonine, serine, cystein, valine and glycine, preferably by a threonine.

• Substitution of the tyrosine at position 706 by an amino acid residue with no or a side chain smaller than leucine, such as glycine, alanine, valine, serine, cystein, threonine and proline, preferably a glycine • Substitution of the valine at 726 by an amino acid residue with a larger hydrophobic side chain such as isoleucine, leucine and methionine, preferably an isoleucine.

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 90%, most preferably at least 95% and which carry one of the following modifications H296Q, L161G, L161S or L161T

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 90%, most preferably at least 95% and which carry a modification at position Most preferred are mutant acylases having modifications at the following combinations of 2 positions [161 +10], [161+29] or [161+694] or [161+726] or [161+274] or [161+706] or [161+442] or [161 +589] or [161+314] or have modifications at the following combinations of 3 positions [161+29+274], [161 +29+706], [161+29+514], [161+274+589] or [161+274+706] or at the following combinations of 4 positions [161+29+274+726], [161+274+280+314], or the following combinations at 5 positions [161+29+274+314+694], [161+274+280+514+726] or the following combinations of 6 positions [161+29+280+314+645+726]

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 combination of positions L161+H296+R589 or at least at a combination of positions H296+E442+R589 or at least at a combination of positions L161+H296+E442 or at least at a combination of positions L161+H296+R589 or at least at a combination of positions H296+E442+R589 or at least at any combination of 4 positions selected from the groups consisting of L161 , M270, H296, E442 and R589 or at least at the combination of positions L161 , M270, H296, E442 and R589 and whereby the mutant type Il beta-lactam acylases may have modifications at other amino acid positions in addition to those positions and all possible combinations thereof as described before Highly preferred embodiments of the present inventions are the Pseudomonas SE83-Acyll mutants summarized in Tables 2-5 of the Examples

In a second aspect, 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 α-subunιt, a spacer peptide, and a β-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 240 in the amino acid sequence of SEQ ID NO 1 , which results in removing the spacer peptide consisting of 9 amino acids, and separating into a 25 kDa α-subunιt and a 58 kDa β- subunit One α-subunιt is bound to one β-subunit through hydrophobic interactions, to form an about 83 kDa heterodimer having acylase activity As is generally known, the first codon (ATG) encoding the N-terminal methionine is needed for translation initiation during protein synthesis in a prokaryote The methionine is removed after the translation

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 Alternatively, 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 For example, 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 In a third aspect, the invention provides an expression vector or expression cassette comprising the polynucleotide of the invention as defined hereinbefore.

In a fourth aspect, 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. Examples of 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. amyloliquefaciens). Escherichia, (E. coli), Streptomyces (e.g. S. clavuligerus), Pseudomonas. In a fifth aspect, 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.

In a sixth aspect, 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. Preferably, 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. Preferred 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

More preferably, the mutant type Il beta-lactam acylase is used as an immobilized form The advantage thereof is that the mutant type Il beta-lactam acylase may be recovered after completion of the deacylation reaction and be reused for further deacylation reactions In this way, 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)

In a seventh aspect, 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-amιno-3-carbamoyloxymethyl-3-cephem-4-carboxylιc acid and others Preferably, 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-ketoadipyland aminoadipyl More preferred are adipyl and aminoadipyl, highly preferred is adipyl The most preferred acylated precursors of the beta-lactam compound of interest are adιpyl-7-ADCA, adιpyl-7- ACA, adιpyl-7-amιno-3-carbamoyloxymethyl-3-cephem-4-carboxylιc acid, alpha- ketoadιpyl-7-ADCA, alpha-ketoadιpyl-7-ACA, amιnoadιpyl-7-ADCA, amιnoadιpyl-7-ACA, the latter known as CEFC 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 More preferably, the mutant type Il beta-lactam acylase is used in an immobilized form

MATERIALS AND METHODS Preparation of acylase

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 μg/ml zeocine at 37⁰C and 280 rpm After 24 hours flasks with 100 ml 2xTY medium, 50 μg/ml zeocine and 0,05% arabinose were inoculated with 50 μl of the culture 1 1000 and grown at 25⁰C and 280 rpm The cultures were centrifuged and frozen at -2O⁰C In order to prepare a cell free extract, 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 supernatant containing the acylase activity was used for activity measurements

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

Purification

Cell pellets from 100 ml cultures were resuspended with 1 ml of 20 mM Tris pH 8 After 9x10 seconds of sonification (Soniprep 150 I-BU03) with an amplitude of 10μ on ice (15 second breaks) the cell suspension was centrifuged for 5 minutes at 14000 rpm and 4°C in micro-centrifuge tubes After the supernatant was brought to pH of 5 3-5 4 with 0 1 M

HCI, it was centrifuged to remove the precipitate Subsequently the supernatant was titrated back to pH=8 with NaOH About 100-400μl was applied to a 1 ml MonoQ column, which was equilibrated with 20 mM Tris pH8 containing 10% NaCI Buffer A (20 mM Tris pH8) and buffer B (20 mM Tris pH8 + 1 M NaCI) were mixed as followed during elution minutes 0-1 10%B/90%A, 1-5 20%B/80%A, 5-9 40%B/60%A, 9-12 60%B/40%A,

12-15 100%B The peak fractions containing acylase activity were collected and applied to a gel filtration column, TSKGeI 3000SWxI, which was equilibrated with 10OmM sodium phosphate buffer pH7 Peak fractions were collected and stored for further use

Reagents

Adιpyl-7ADCA can be prepared from adipic acid and 7-ADCA by enzymatic synthesis as described in WO9848037 In addition Adιpyl-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

An 8% (w/v) stock solution of adιpyl-7ADCA substrate was prepared in the appropriate buffer and adjusted to the desired pH with 4 N NaOH The colour reagent 4-(dιmethylamιno)-benzaldehyde (p-DMBA) was freshly prepared by dissolving 200 mg in 100ml citric acid (315 5 g citric acid monohydrate dissolved in 1 liter ethanol)

Activity measurements in the range pH=8 0 to pH=10 0 were carried out in 0 2 M CHES buffer (2-(N-Cyclohexylamιno)ethane sulfonic acid) adjusted to the desired pH with 4N HCI or 4N NaOH as needed

Measurement of acylase activity

180 μl of the appropriate buffer was mixed with 200 μl of the substrate stock solution in the corresponding buffer and 20 μl enzyme solution and incubated during 20 minutes at the desired temperature, usually room temperature unless stated otherwise Adding 600 μl colour reagent stopped the reaction After 10 minutes at room temperature the absorbance was measured at 415 nm The blanc measurements were done by adding colour solution to the assay before the enzyme is added The acylase activity is calculated as the increase in optical density (OD) per minute (delta OD/min) In order to calculate absolute activities a 7-ADCA calibration line in the range 0 1 to 1 g 7-ADCA per liter was used

K_M determinations and pH curves

K_M determinations were carried out using the assay as described However the adιpyl-7-ADCA concentration was varied from 0 5 to 4% (w/v) adιpyl-7ADCA

EXAMPLES

Example 1

Acylase activity of SE83 ACYM mutants

The acylase activity of the mutants with adιpyl-7-ADCA as a substrate was determined at pH=8 5 and pH=9 5 Results are shown in Table 2 Table 2 Relative activities of wild type and mutant acylases on adιpyl-7ADCA setting the activity of wild type at pH=8 5 to 1 In parentheses the activities relative to wild type activity at pH=9 5 The assay was carried out at room temperature Initial rates were measured in the presence of 4% (w/v) adιpyl-7ADCA

At pH=8 5 as well as pH=9 5 the activity of the mutants is significantly higher compared to the wild type acylase In addition, when comparing the activity at pH=9 5 with the activity at pH=8 5 it becomes clear that the activity of the mutant acylase at pH 9 5 is relatively higher compared to wild type The pH activity profile of the mutants has been shifted to higher pH, making these mutants in particular suitable for use at elevated pH This is of particular importance, because the yield of the conversion will increase at higher pH due to shifting the thermodynamic equilibrium further towards completion of the hydrolysis reaction

Since during the process of conversion of adιpyl-7ADCA into 7-ADCA and adipic acid, the concentration of the latter will increase, product inhibition could reduce the improvements of the mutants measured under conditions of initial rate Therefore, the activity of wild type and mutant acylases was measured in the presence of 1 5% (w/v) adipic acid Table 3 shows that under these conditions the activity of the mutants is significantly higher compared to the wild type acylase at pH=8 5 as well as pH=9 5 The pH activity profile for these mutants has not been shifted to higher pH

The acylase activity of wild type and mutant acylases using adιpyl-7-ADCA as a substrate was determined at pH=8 6, pH=9 1 and pH=9 5 in the presence of 1 5% (w/v) adipic acid Results are shown in Table 4

Table 4 shows that at pH=8 6, pH=9 1 as well as pH=9 5 the activity of the mutants is significantly higher compared to the wild type acylase When comparing the activity improvement at pH=9.1 with the activity improvement at pH=8.6 (columns with ratio of activities) it becomes clear that the activity of the mutant acylases at pH 9.1 is relatively more improved compared to wild type. The pH activity profile of the mutants has been shifted to higher pH, making these mutants in particular suitable for use at elevated pH. When comparing pH=9.5 with pH=8.6 the activity of most mutant acylases is still more improved at pH=9.5 than at pH=8.5 compared to wild type.

Table 3: Relative activities of wild type and mutant acylases on adipyl-7ADCA setting the activity of wild type at pH=8.5 to 1 In parentheses the activities relative to wild type activity at pH=9.5. The assay was carried out at room temperature. The initial substrate concentration was 4% (w/v) adipyl-7ADCA. Initial activity was measured in the presence of 1.5% (w/v) adipic acid.

Table 4: Relative activities of wild type and mutant acylases on adipyl-7ADCA setting the activity of wild type at pH=8.6 to 1. In parentheses the activities relative to wild type activity at pH=9.1 and pH=9.5 respectively. 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.

Example 2

Substrate affinity indicate by K_M measurement of SE83 ACYii mutants

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 In case 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.

Table 5: 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.

Example 3

Acylase activity of immobilized SE83 ACYii mutants

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. Immobilized enzyme was dosed in such a way that at least 90% conversion could be obtained within 120 minutes under the desired conditions. Conversions were carried out at pH=8.8 and 30⁰C and at pH=9.5 at 40⁰C. The same amount (in weight) of wild type and mutant acylase was used for the conversions. During the reaction the pH was kept constant by the addition of a solution of 1 M KOH. The activity of the immobilized acylase is expressed as ml KOH per min. In Figures 2a and 2b the rate, expressed as ml KOH per min, as a function of the conversion is shown.

The average of 6 runs was taken at pH=8.8 and 30⁰C. The data of the first 30% conversion was not included because the system was not completely stabilized, giving rise to a large scattering of the data. At pH=9.5 and 40⁰C the average of two runs was taken. Therefore, the variation is higher. However, the calculated slope gives a good indication of the activity. Figures 2a and 2b show that the activity of the mutant acylase is significantly higher during the whole conversion.

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.

As a consequence of this higher hydrolytic activity and higher stability of the mutated acylase, the productivity per gram of the mutated acylase enzyme was increased considerably.

Table 6: Residual activity after 20 conversions at the conditions indicated.

Reaction Condition Acylase Residual Activity (%) wild type 103% pH 8.8; 30 ⁰C

L161T 106% wild type 98% pH 9.5; 30 ⁰C

L161 T 102% wild type 88% pH 8.8; 40 ⁰C

L161T 99% wild type 66% pH 9.5; 40 ⁰C

L161T 76%

Claims

1. 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.

2. 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 10, 29, 161 , 270, 274, 280, 296, 314, 442, 514, 589, 645, 694, 706 and 726 using the amino acid position numbering of the amino acid sequence of the SE83-acyll acylase of Pseudomonas (SEQ ID NO: 1).

3. A mutant type Il beta-lactam acylase according to claim 1 whereby the mutant type Il beta-lactam acylase has been modified at at least an amino acid position selected from group consisting of positions 10, 29, 161 , 270, 274, 280, 296, 314, 442, 514, 589, 645, 694, 706 and 726 using the amino acid position numbering of the amino acid sequence of the SE83-acyll acylase of Pseudomonas (SEQ ID NO: 1).

4. A mutant type Il beta-lactam acylase according to any of the previous claims whereby the model polypeptide with type Il beta-lactam acylase activity is selected from the group consisting of the SE83-acyll acylase of Pseudomonas (SEQ ID NO: 1), N176 (SEQ ID NO:2) from Brevundimonas diminuta and the V22 acylase (SEQ ID NO:3) from Brevundimonas diminutia 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% or with SEQ ID NO: 2 of at least 70% or with SEQ ID NO: 3 of at least 70%.

5. A mutant type Il beta-lactam acylase according to claim 4 whereby the model peptide is the SE83-acyll acylase of Pseudomonas (SEQ ID NO: 1).

6 A mutant type Il beta-lactam acylase according any of the previous claims whereby the mutant type Il beta-lactam acylase has been modified at position 161 preferably in combination with at least one other modification at a position selected from the group consisting of 10, 29, 270, 274, 280, 296, 314, 442, 514, 589, 645, 694, 706 and 726

7 A polynucleotide encoding the mutant type Il beta-lactam acylase of anyone of the preceding claims

8 An expression vector or cassette comprising the polynucleotide of claim 7

9 A host cell transformed with the polynucleotide of claim 7 or the vector or cassette of claim 8

10 A method of producing the mutant type Il beta-lactam acylase of claims 1-6 comprising cultivating a host cell according to claim 9 under conditions conducive to the production of the mutant type Il beta-lactam acylase and recovering the polypeptide

11 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 claims 1-6

12 The process according to claim 11 wherein the deacylated beta-lactam compound of interest is 6-APA, 7-ACA, 7-ADCA, 7-ADAC or 7-amιno-3-carbamoyloxymethyl-3- cephem-4-carboxylιc acid

13 The process according to any of claims 11 or 12 wherein the acyl group of the acylated precursor of the beta-lactam compound of interest belongs to the group of dicarboxylic acids, preferably succinyl, glutaryl, adipyl, alpha-ketoadipyl and aminoadipyl, more preferably, adipyl

14 The process according to any of claims 11-13 wherein the mutant type Il beta- lactam acylase is used in an immobilized form

15. Use of the mutant type Il beta-lactam acylase of claims 1-6 for the deacylation of an acylated precursor of a beta-lactam compound of interest.

16. Use according to claim 15, wherein the beta-lactam compound of interest is 7- ADCA or 7-ACA.

17. Use according to claim 15 or 16, wherein the acylated precursor of the beta-lactam compound of interest belongs to the group of dicarboxylic acids, preferably succinyl, glutaryl, adipyl, alpha-ketoadipyl and aminoadipyl, more preferably, adipyl.

18. Use according to any of 15-17 wherein the mutant type Il beta-lactam acylase is used in an immobilized form.