It is known that 7-ACA can be used as the starting product for the semi- synthetic preparation of various cephalosporin antibiotics, and that there exist various processes to prepare 7-ACA on a large scale. Among these known processes the enzymatic processes are preferred from both economic and environmental perspective.

In one of the known production processes, cephalosporin C is decarboxylated by chemical oxidation to form glutaryl-7-ACA, and the so obtained substrate is subsequently processed to form 7-ACA using the enzyme glutaryl amidase. This process has, however, the disadvantage that it involves a high level of environmental pollution, resulting in high costs.

In another known production process (as for example described in W003076640), cephalosporin C is firstly subjected to an oxidative decarboxylation treatment which is carried out enzymatically with the help of D-amino acid oxidase. The glutaryl-7-ACA so obtained is then converted into 7-ACA using the enzyme glutaryl amidase. Although the latter enzymatic process is already much more attractive than the former it still involves several process steps and therefore there is still significant room to develop an improved process for the conversion of cephalosporin C into 7-ACA.

In view of the commercial interest of such an application, many attempts have been made to isolate an amidase that can hydrolyse cephalosporin C to produce 7-ACA. Enzymes from different organisms have been isolated and tested on their capability to hydrolyse cephalosporin C. Examples can be found in patents W00224879, US5229274, EP-A-0283218, EP-A-0322032, and US4774179. Nevertheless, so far the cephalosporin C hydrolysis activity in any of the known amidases is too low for commercial interest.

Also attempts have been made to mutate glutaryl amidase at specific positions in order to increase the cephalosporin C hydrolysis activity. For example, the glutaryl amidase as described in US4774179 was mutated at several positions, but so far no commercially usable variants have been obtained [ref. 1-4]. Further in WO 02/072806 mutations in another glutaryl amidase have been proposed on the basis of structural modelling, without any experimental substantiation [re£ 5]. In yet another approach, mutations were introduced in a random manner, after which variants with an activity towards a desired substrate were selected. As an example of the latter, the glutaryl amidase of Pseudomonas SY-77 was mutated and variants of the amidase with the desired activity were isolated by growth on selective medium. Several mutants, amongst which a variant carrying the mutation Tyrl78 into His, were claimed (US5457032). The latter mutant was shown to have increased hydrolysis activity on adipyl-7-aminodesacetoxycephalosporanic acid (7-ADCA; ref. 6). This compound is, like 7-ACA, an industrially important reagent for the synthesis of semi-synthetic cephalosporin antibiotics [ref. 7]. However, in spite of the fact that this mutant was claimed more than ten years ago, no activity for cephalosporin C was demonstrated so far. It is further observed that mutations at other positions in Pseudomonas diminua cephalosporin acylase were predicted by modelling studies, and claimed to increase the cephalosporin C hydrolysis activity of a glutaryl amidase several fold [ref. 8]. However, no formation of 7-ACA was shown. Only a few variants were predicted and there was clearly no teaching for further improvement by making, for instance, combinations of different mutations.

In order to isolate new mutants of Pseudomonas SY-77 with increased hydrolysis activity towards adipyl-7-ADCA, an unbiased approach using mutagenesis and growth selection on solid medium containing adipyl-leucine was used recently [ref. 6,9]. However, no activity on cephalosporin C was shown.

It has now surprisingly been found that a very attractive single-enzyme process can be developed for the production of 7-ACA from cephalosporin C when use is made of a newly developed class of glutaryl amidases.

Accordingly, one aspect of the present invention relates to an isolated or single mutated glutaryl amidase from Pseudomonas SY-77, or a functional part, derivative or analogue thereof, having an amino acid at position 266 other than asparagine, histidine or serine.

With the term"single mutated"is meant that the glutaryl amidase is derived from a naturally occurring glutaryl amidase that has only been mutated in one position. In other words, Asn266 has been mutated into an amino acid other than asparagine.

A major advantage of this newly developed class of glutaryl amidases is that they enable the direct conversion of cephalosporin C into 7-ACA in a much simpler and cheaper way. Further, these variants can suitably be used to prepare 7-ACA on an industrial scale. In such processes, the glutaryl amidase according to the present invention binds cephalosporin C as a substrate and catalyses the conversion of the cephalosporin C into 7-ACA, whereby the amino acid at position 266 directly or indirectly increases the cephalosporin C hydrolysis activity of said amidase.

Preferably, the present invention relates to an isolated or single mutated glutaryl amidase from Pseudomonas SY-77, or a functional part, derivative or analogue thereof, having a glutamine, methionine or tryptophan at position 266. More preferably such glutaryl amidase has a glutamine or methionine at position 266. Most preferably, such glutaryl amidase has a glutamine at position 266.

Another aspect of the present invention is that the effect of a mutation in position 266 on cephalosporin C hydrolysis activity can be improved by the additional presence of other mutations in or near to the substrate binding pocket. Amino acids that directly or indirectly contribute to substrate binding, or that may otherwise contribute to substrate specificity, are found in

positions: Leul77, Tyrl78, Vall79, His221, Leu222, Phe229, Tyr231, Gln248, Arg255, Phe256, Thr267, Val268, Met271, Gln291, Met347, Tyr351, Thr374, Phe375 and/or Asn442. It cannot be excluded that variants of glutaryl amidase with mutations in these positions have a high cephalosporin C hydrolysis activity even in combination with Asn266.

Accordingly, another attractive aspect of the invention relates to an isolated or multiple mutated glutaryl amidase from Pseudomonas SY-77, or a functional part, derivative or analogue thereof, having an amino acid other than its native amino acid at position Asn266, and having an amino acid other than its native amino acid at one or more of the following positions: Leul77, Tyrl78, Vall79, His221, Leu222, Phe229, Tyr231, Gln248, Arg255, Phe256, Thr267, Val268, Met271, Gln291, Met347, Tyr351, Thr374, Phe375 and/or Asn442.

With the term"multiple mutated"is meant that the glutaryl amidase is derived from a naturally occurring glutaryl amidase that has been mutated in at least two positions. In other words, Asn266 has been mutated into another amino acid, whereas in addition one or more other native amino acids have been mutated into other amino acids.

As mentioned hereinabove, these glutaryl amidases display an improved efficiency in the preparation of 7-ACA from cephalosporin C.

Preferably, the isolated or multiple mutated glutaryl amidase according to the invention has in addition to an amino acid other than asparagine at position 266, one or more mutations at the following positions: Tyrl78, Leu222, Tyr231, Gln248, Arg255, Val268, Tyr351 and/or Phe375.

Most preferably, the isolated or multiple mutated glutaryl amidase comprises in addition to an amino acid other than asparagine at position 266, one or more mutations at the following positions: Tyrl78, Tyr231, Arg255,

Phe375, Tyrl78 + Tyr231, Tyrl78 + Arg255, Tyrl78 + Phe375, Tyr231 + Arg255, Tyr231 + Phe375, Tyr231 + Arg255 + Phe375, Arg255 + Phe375, Tyrl78 + Tyr231 + Arg255, Tyrl78 + Tyr231 + Phe375, Tyrl78 + Arg255 + Phe375, or Tyrl78 + Tyr231 + Arg255 + Phe375.

All amino acid numbering in the present application is with respect to the propeptide chain as shown in fig. 1. The numbering applies to equivalent positions in amino acid sequences of other glutaryl amidases as determined for example by sequence alignment.

In the context of the present invention, a mutation of a position in the glutaryl amidase implies the incorporation of any of the 20 natural amino acids other than the native amino acids at that particular position in the glutaryl amidase (as listed in fig. 1), or the incorporation of a non-natural amino acid at that particular position or a modification of the amino acid at that particular position.

The glutaryl amidase is obtained from a Pseudomonas SY-77, or is a functional part, derivative or analogue thereof. In the context of the present application, derivates or analogues of said Pseudomonas SY-77 glutaryl amidase include: Pseudomonas C427 glutaryl amidase [ref. 10], Pseudomonas sp. 130 glutaryl amidase [ref. 11,12], Pseudomonas GK-16 glutaryl amidase [ref. 13,14], and Pseudomonas diminuta KAC-1 glutaryl amidase [ref. 15,16].

The glutaryl amidase of Pseudomonas SY-77 has, for instance, been described in references [ref. 6,17]. The homologues of Pseudomonas SY-77

glutaryl amidase as used in accordance of the present invention have, for instance, been described in references [ref. 10-16].

The present invention also provides a polynucleotide encoding a variant of a glutaryl amidase as described hereinabove.

The present invention further provides a nucleic acid vector comprising a polynucleotide encoding a variant of a glutaryl amidase as described hereinabove.

Additionally, the present invention provides a host cell comprising a polynucleotide encoding a variant of a glutaryl amidase as described hereinabove.

The present invention also relates to a method for producing any one of the glutaryl amidases according to the present invention, which method comprises culturing the hereinabove-mentioned host cell under suitable conditions which allow for expression of the glutaryl amidase in said host cell.

In the context of the invention, variants of the glutaryl amidase will be produced in host cells by expression of the encoding gene or by production in a cell-free system. Host cells principally can be prokaryotic or eukaryotic cells, but are by preference prokaryotic. More preferably, the host cells are bacterial cells such as Escherichia coli or Bacillus subtilis or Pseudomonas. Examples of such expression systems can be found in ref. 6 and 18. A cell-free system, such as the Rapid Translation System (Roche) can also be used to produce said variants. Alternatively, both subunits of the glutaryl amidase can be co- expressed, as for example described in ref. 11.

By way of an example, recombinant E. coli clones expressing the variant of a glutaryl amidase under control of a strong promoter according to the present invention is typically cultured to a 0.1-10 litre scale. The cells are initially concentrated by centrifugation. Subsequently, they are resuspended in a small volume of a suitable buffer and disrupted. After centrifugation to remove the cell debris, the supernatant is used in a purification strategy to obtain the purified enzyme.

By way of an example, and more suitable for smaller quantities, the cells may be lysed by sonication, followed by a three-step purification using different chromatographic steps as described hereinbelow in Example 1.

Alternatively, and more suitable for an industrial scale, cells may be resuspended in 100 mM phosphate buffer at a pH of 7.2, and disrupted by means of a French press. To remove the cell debris, the suspension is then centrifuged at 10,000 rpm for 15 minutes, and the supernatant is transferred to another tube. The enzyme-containing supernatant solution is then rebuffered in an Amicon ultrafiltration cell or by diafiltration against 0.8-1. 5 M, more preferably in the range of from 1-1.5 M, potassium phosphate buffer at a pH of 7. 8 at 4°C with simultaneous concentration. If the concentrated solution is left to stand in cold conditions, the enzyme crystallises out. The crystals may be enriched by gentle centrifugation and washed with the above- mentioned buffer. In this way large amounts of purified enzyme can be obtained in a simple, swift and cheap manner, without using expensive chromatographic purification steps.

In a preferred embodiment, cephalosporin C hydrolysis is carried out at a pH in the range of from pH 7.5 to 8. 5, more preferably in the range of from 7.5 to 8. 2. In that way, decomposition of cephalosporin C is minimised.

The concentration of cephalosporin C in the above method lies preferably in the range from 20-200 mM, more preferably in the range of from 50 to 120 mM.

In the above method, the variant of the glutaryl amidase is suitably used in a concentration in the range of from 0.25 to 10 mg/ml in the reaction mixture, and preferably in the range of from 1 to 4 mg/ml.

The above method is suitably carried out at a temperature in the range of from 15 to 45°C, preferably in the range of from 25 to 40°C.

The reaction time for cephalosporin C hydrolysis is suitably in the range of from 1 to 40 hour, preferably in the range of from 1 to 20 hour.

The variants of a glutaryl amidase according to the present invention can also be applied carrier-fixed. In this way, a further reduction of costs can be established. Suitable carrier materials that can be used for this purpose have for instance been described in ref. 19. Alternatively, variants of a glutaryl amidase according to the present invention can also be applied as carrier-free cross-linked enzymes as for instance described in Cao et al. [ref. 20].

The variants of a glutaryl amidase according to the present invention can be used to produce 7-ACA directly from cephalosporin C.

Accordingly, the present invention also provides a method for preparing 7-ACA from cephalosporin C, which method comprises reacting cephalosporin C with an isolated, single mutated or multiple mutated glutaryl amidase from Pseudomonas SY-77, or a functional part, derivative or analogue thereof, having an amino acid at position 266 other than asparagine under suitable conditions such that the glutaryl amidase cleaves the cephalosporin C directly to form the 7-aminocephalosporanic acid.

In said method any of the glutaryl amidases according to the present invention can suitably be used.

Preferably, the present invention relates to a method for preparing 7- ACA from cephalosporin C, which method comprises reacting cephalosporin C with a glutaryl amidase under suitable conditions such that the variant of a glutaryl amidase cleaves the cephalosporin C directly to form the 7-ACA, wherein the glutaryl amidase comprises an isolated or multiple mutated glutaryl amidase from Pseudomonas SY-77, or a functional part, derivative or analogue thereof, having an amino acid other than its native amino acid at position Asn266, and having an amino acid other than its native amino acid at one or more of the positions Leul77, Tyrl78, Va1179, His221, Leu222, Phe229, Tyr231, Gln248, Arg255, Phe256, Thr267, Val268, Met271, Gln291, Met347, Tyr351, Thr374, Phe375 and/or Asn442.

Surprisingly, it has also been found that the mutation of Tyrl78 into another amino acid in said Pseudomonas SY-77 glutaryl amidase increases

hydrolysis activity on cephalosporin C (see table 2). This particular mutant has been isolated (ref. 6) but its unexpectedly high activity on cephalosporin C was not known before.

Accordingly, the present invention also relates to a method for preparing 7-aminocephalosporanic acid from cephalosporin C, which method comprises reacting cephalosporin C with an isolated, single or multiple mutated glutaryl amidase from Pseudomonas SY-77, or a functional part, derivative or analogue thereof, having an amino acid other than its native amino acid at position Tyrl78, under suitable conditions such that the glutaryl amidase cleaves the cephalosporin C directly to form the 7-aminocephalosporanic acid.

Preferably, the said glutaryl amidase is a single mutated glutaryl amidase.

More preferably, the amino acid at position Tyrl78 of the said glutaryl amidase is a histidine.

It has further surprisingly been found that some of the single mutants that were isolated have a high hydrolysis activity in respect of the production of adipyl-7-aminodesacetoxycephalosporanic acid (7-ADCA). This compound is, like 7-ACA, an industrially important reagent for the synthesis of semi- synthetic antibiotics.

The present invention therefore also relates to a method for preparing 7- ADCA, which comprises reacting adipyl-7-ADCA with an isolated or single mutated glutaryl amidase from Pseudomonas SY-77, or a functional part, derivative or analogue thereof, having an amino acid other than its native amino acid at position Asn266, under suitable conditions such that the glutaryl amidase cleaves adipyl-7-ADCA to form the 7-ADCA. Preferably, the amino acid at position 266 of said glutaryl amidase is a glutamine, methionine, phenylalanine, tryptophan or tyrosine. More preferably, the amino acid is a methionine or glutamine.

It will be appreciated by the person skilled in the art that the said variants with increased hydrolysis activity to produce 7-ACA or 7-ADCA can

also be used for the synthesis of new cephalosporin antibiotics from these compounds [ref. 21]. Hence, the present invention also relates to a method for preparing 7-ACA based antibiotics comprising incubating 7-ACA in the presence of the glutaryl amidase according to the present invention with alkyl side chains under suitable synthesizing conditions.

The glutaryl amidases according to the present invention consist of two sub-units of different sizes which are formed from a propeptide comprising amino acids through processing steps taking place in the periplasm of the expression organism to form a functional enzyme. The small sub-unit (A chain) comprises 161 amino acids in the functional enzyme, the large sub-unit (B chain) 522 amino acids. The protein sequences of the A and B chains are shown in the sequence listing part of the description respectively. Numbering of residues throughout is with reference to the amino acid sequences.

However, it will be understood that where other glutaryl amidases are used, the numbering applies to the functionally corresponding residues in those sequences that do not have exactly the same numbering as in the sequence listing part. Furthermore, it will be understood by a person skilled in the art that functional parts of different sizes of the said glutaryl amidase or derivative or analogue can be derived without significantly affecting hydrolysis activity.

Mutations in the glutaryl amidase gene sequence may be introduced, for example, using standard site directed mutagenesis techniques, such as those described in ref. 22.

As mentioned earlier, the present invention also provides a polynucleotide encoding a variant of a glutaryl amidase according to the present invention. Such polynucleotide may comprise DNA or RNA. The polynucleotide may include synthetic or modified nucleotides. Various methods to modify oligonucleotides are known in the art. These methods include the use of methylphosphonate and phosphorothioate backbones, with addition of acridine or polylysine chains at the 3'and/or 5'ends of the molecule. The

polynucleotides as described hereinabove may be modified by any method available in the art. It will be appreciated by the skilled person in the art that various different polynucleotides can encode the same polypeptide as a result of the degeneracy of the genetic code.

Another aspect of the invention relates to a method for developing a variant of a cephalosporin amidase with high cephalosporin C hydrolysis activity. In this method use is made of a particular combination of techniques.

Accordingly, the present invention also relates to a method for developing a glutaryl amidase with cephalosporin C hydrolysis activity comprising the steps of : 1) applying a mutagenesis technique in order to create a large library of mutants of a glutaryl amidase according to the present invention that carries a mutation in one or more positions; and subsequently 2) applying a selection procedure comprising growth of proper host cells that express variants of said glutaryl amidase on solid medium containing a selective, aminoadipyl-containing compound and identification of variants with the desired enzymatic activity.

This process enables the identification and preparation of cephalosporin amidases that display a high cephalosporin C hydrolysis activity.

The present invention will now be shown by means of the following Examples, which are non-limiting and have been included for illustration purposes only.

Examples Example 1-Preparation and purification of Pseudomonas SY-77 glutaryl amidase mutants modified at position 266 The megaprimer method [ref. 23] was used in order to introduce the 19 amino acid other than asparagine on position 266. First, the plasmid pMcSY-2 comprising the wild-type (WT) sequence for Pseudomonas SY-77 glutaryl amidase and a primer with a randomised codon 266 was used in a PCR reaction. The PCR fragment was digested with restriction endonucleases BglII and SstII, and cloned into vector pMcSY-2 digested with the same endonucleases by ligation. Plasmid DNA of a total of 96 transformants was sequenced. This resulted in the isolation of mutants carrying 17 different amino acids at this position in a single mutagenesis round. The missing 3 mutants were made by site-directed mutagenesis using specific primers.

Escherichia coli DH10B cells containing the plasmid encoding the (mutant) enzyme were grown in 300 ml 2*YT-medium at 25°C. After 30 hours the cells were harvested by centrifugation. A cell-free extract was made by sonication (10 min, output 4,50% duty cycle on a Sonifier 250, Branson) and centrifugation (30 min at 14000 rpm). This extract was used to purify the enzyme in a 3-step protocol on a Duoflow system (Bio-Rad) using columns from Amersham Pharmacia. The cell free extract was loaded onto a HiTrapQ column and the protein eluted with a linear gradient of 0-1 M NaCl in 50 mM Tris-HCl pH 8. 8. After analysis on SDS-PAGE, the fractions containing the enzyme were loaded onto a HiTrap PhenylSepharose HP column. The protein was eluted with a gradient of 0.7-0 M (NH4) 2SO4. The pooled fractions containing the enzyme were desalted on a HiPrep 26/10 Desalting column, and reloaded onto a HiTrapQ column. After rinsing with 220 mM NaCl, the protein was eluted with 330 mM NaCl. The pooled fractions were stored at-20°C.

Typical yields were 10 mg of more than 90 % pure enzyme per liter of culture.

Example 2-Conversion of cephalosporin C into 7- aminocephalosporanic acid For the determination of activity on cephalosporin C, 70 fig of various SY-77 glutaryl amidase variants and wild-type enzymes were incubated for 5 hours at 37°C with 10 mM cephalosporin C in 300 Fl of phosphate buffer (20M, pH 7.5). Aliquots of 40 Ill were taken from the reaction mixture every hour and transferred to 140 pl of 0.5 M acetate buffer pH 4. 5. 201l1 Of a 1 mg/ml fluorescamine in acetone solution was added, and the absorbance at 380 nm was measured on a spectrophotometer after 1 hour incubation. The product of the deacylation reaction, 7-aminocephalosporanic acid, forms a complex with fluorescamine that absorbs light of 380 nm wavelength [ref. 24].

The mean slope of the conversion of cephalosporin C in the first 4 hours was determined and normalised towards WT.

Example 3-Construction of a library of glutaryl amidase mutants randomised at positions 266 and 375 A pool of vectors harboring glutaryl amidase genes with 20 different codons encoding all 20 naturally occurring amino acids at position 375 was digested with restriction endonucleases NcoI and HindIII, and the small fragment was ligated into the large fragments of a pool of vectors harboring glutaryl amidase genes with 20 different codons encoding all 20 naturally occurring amino acids at position 266 digested with the same restriction endonucleases. This pool of vectors harboring glutaryl amidase genes with altered codons encoding the amino acids at positions 266 and 375 was transformed to E. coli DH10B cells, resulting in a mutant library in which positions 266 and 375 were completely randomized.

Example 4-Construction of a library of glutaryl amidase mutants randomized at positions 178. 231, 255, 266 and 375

A mutant library of glutaryl amidase genes, which are randomized in five codons (encoding amino acids at positions 178,231, 255,266 and 375), was made by the megaprimer method as described by Landt et al [ref. 23]. Primers according to parts of the glutaryl amidase gene, but with randomized codons encoding amino acids at position 178,231, and 255+266 were used to amplify the section containing the codons encoding these 4 positions of the glutaryl amidase gene. The nucleotide sequences of these primers are listed in table 1.

The library was constructed in four steps: Forward primer Backward primer Product Step 1. F1 B1 Pi Step 2. F2 PI P2 Step 3. P2 B2 P3 Step 4. P3 B3 P4 This product P4 was digested with EcoO190I and NcoI, and ligated into the large fragments of a pool of vectors harboring glutaryl amidase genes with 20 different codons encoding all 20 amino acids at position 375 digested with the same restriction endonucleases. This pool of vectors harboring glutaryl amidase genes with altered amino acids at positions 178,231, 255,266 and 375 was transformed to E. coli DH10B cells, resulting in a mutant library in which positions 178,231, 255,266 and 375 were completely randomized.

Example 5-Selection of variants of Pseudomonas SY-77 on hydrolysis activity for aminoadipyl-leucine or other aminoadipyl-amino acids A library as described in example 4 was selected on minimal medium containing aminoadipyl-leucine as sole leucine source. 107 different colonies were starved by incubation in 0.9% NaCl for 2 h at 37°C and plated onto selective minimal medium plates containing 0.1 mg/ml aminoadipyl-leucine as a sole leucine source. Approximately 10000 viable E. coli DH10B cells containing the mutant plasmids were spread onto each plate. Plates were

incubated at 30°C for 6 days. Every day new appearing colonies were marked.

Colonies were selected on the basis of their date of appearance and size. 57 colonies were picked and grown overnight at 30°C. From each culture the plasmid was isolated and the presence of glutaryl amidase was detected using a polyclonal antibody against the glutaryl amidase of Pseudomonas SY-77.

Plasmid were retransformed to E. coli DH1OB cells and streaked again on minimal medium containing adipyl-leucine as sole leucine source to ensure unique colonies were obtained. Single colonies from these second plates should be used to inoculate an overnight culture to determine acylase activity and to isolate plasmid DNA for sequence determination.

Example 6-Some single mutated variants of Pseudomonas SY-77 glutaryl amidase with increased cephalosporin C hydrolysis activity Wild-type (WT) and 15 glutaryl amidases variants mutated at position 266 were produced in E. coli DH10B and purified up to 95% purity. The 4 glutaryl amidases comprising single mutations N266R, N266I, N266K and N266V could not be produced. 20 gag of the mutant and WT enzymes were incubated at 37°C with 10 mM cephalosporin C. The WT enzyme and the variants carrying mutants Asn266 into Gln, Asn266 into His, Asn266 into Met or Asn266 into Trp showed some hydrolysis of the cephalosporin C after 20 hours. These activities were too low to be able to determine Km and kcat. In order to avoid long incubation times that may lead to enzyme destabilisation, the conversion rate of the different mutants was calculated from an assay with more enzyme (70 jig) as given in example 2. The mean slope of the conversion of cephalosporin C in the first 4 hours was determined and normalised towards WT. All the tested variants, carrying mutants Asn266 into Gln, Asn266 into His, Asn266 into Met or Asn266 into Trp, showed an improved conversion of CPC compared to the activity of the wild-type enzyme. Variants carrying mutations Asn266 into Gln or Asn266 into His displayed almost a two-fold

improved conversion of cephalosporin C over wild-type, whereas the variant carrying mutation Asn266 into Met showed an improvement of about 30% (Fig.

2). Hence, this example clearly shows that the variants according to the present inventions are indeed very effective enzymes for the conversion of cephalosporin C into 7-ACA.

Similarly, the variant carrying mutation Tyrl78 into His was shown to be 3- fold more active towards cephalosporin C (Table 2).

Example 7-Some multiple mutated variants of Pseudomonas SY-77 glutaryl amidase with increased cephalosporin C hydrolysis activity The single mutants of the glutaryl amidase from Pseudomonas SY-77 comprising mutations Asn266 into glutamine, Asn266 into histidine or Asn266 into methionine were combined with different other mutations. Some mutants were purified and the hydrolysis activity towards cephalosporin C acylase was determined. The conversion rate of the different mutants was calculated from an assay with 70 gg enzyme. The mean slope of the conversion of cephalosporin C in the first 4 hours was determined and normalised towards WT (see Figure 1). The multiple mutants SY-77N266H+Y178H SY-77N266M+Y178H and SY-77N266Q+Yl78H+F375M appeared to have a higher hydrolysis activity towards cephalosporin C than the wild-type glutaryl amidase (Fig 3).

Example 8-Some single mutated variants of Pseudomonas SY-77 glutaryl amidase carrying a mutation at position Asn266 with increased hydrolysis activity towards adipyl-7-ADCA Wild-type (WT) and 15 glutaryl amidases variants mutated at position 266 were produced in E. coli DH10B and purified up to 95% purity. The 4 glutaryl amidases comprising single mutations N266R, N266I, N266K and N266V could not be produced. The catalytic parameters of the remaining 16 enzymes were determined measuring the initial rate of hydrolysis on a range of

substrate concentrations with a fixed amount of enzyme using the Multiprobe II for all pipetting steps. 140 1li 20 mM phosphate buffer pH 7.5 with substrate (0.06-2 mM glutaryl-7-ACA or 0.2-10 mM adipyl-7-ADCA) was preheated at 37°C. 40, 1 phosphate buffer containing an appropriate amount of purified enzyme was added, starting the reaction. After 10 min incubation at 37°C 40 Ill 2.5 M acetate buffer pH 4.5 was added to stop the reaction. 20 il 1 mg/ml fluorescamine in acetone was added and the A380 was measured after 60 min incubation at room temperature.

Kinetic parameters were obtained by fitting the experimental data from Eadie- Hofstee plots, and the mean and standard deviation of values of at least 4 independent measurements were calculated. The kcat was calculated using the theoretical molecular weight of the mature enzyme, 75.9 kDa. Variants carrying mutations Asn266 into Met, His or Gln displayed a 2-to 15-fold improved catalytic efficiency over wild-type according to the hydrolysis of adipyl-7-ADCA, whereas the variants carrying mutation Asn266 into Phe, Trp, Tyr or Cys were equally or 150% as efficient as WT in the hydrolysis of adipyl- 7-ADCA. Hence, this example clearly shows that the variants according to the present inventions are indeed very effective enzymes for the conversion of adipyl-7-ADCA into 7-ADCA.

Example 9-Some multiple mutated variants of Pseudomonas SY-77 glutaryl amidase selected on adipyl-leucine with increased hydrolysis activity towards adipyl-7-ADCA Two libraries of multiple mutated glutaryl amidase of Pseudomonas SY-77 were constructed. The first one comprised all randomized codons at positions Asn266 and Phe375 as described in example 3. In the second library, the Tyrl78 of all the plasmids of library 1, were replaced by a His. E. coli DH1OB cells containing the mutant plasmids were slowly thawed on ice and washed twice with 0.9% NaCl. They were starved by incubation in 0.9% NaCl for 2 h at 37°C and plated onto selective minimal medium plates containing 0.1 mg/ml

adipyl-leucine as a sole leucine source. Approximately 5000 viable E. coli DH10B cells containing the mutant plasmids were spread onto each plate. Of each library a total of 105 transformants was plated. Plates were incubated at 30°C for at least 10 days. Every day new appearing colonies were marked.

Colonies were selected on the basis of their date of appearance and size, and streaked again on minimal medium containing adipyl-leucine as sole leucine source to ensure unique colonies were obtained. Single colonies from these second plates were used to inoculate an overnight culture to determine acylase activity and to isolate plasmid DNA for sequence determination. Four colonies with unique sequences appeared to have an improved ratio of adipyl-7-ADCA hydrolysis over glutaryl-7-ACA hydrolysis. These mutants were purified and their catalytic parameters on glutaryl-7-ACA and adipyl-7-ADCA were determined as described in example 8. Furthermore, the hydrolysis of cephalosporin C was measured as described in example 2. The hydrolytic properties of these enzymes towards these different substrates are listed in table 2. It is obvious that all measured mutants have a better catalytic efficiency towards adipyl-7-ADCA mostly due to a lowered Km. Furthermore, 3 out of the 4 mutants tested have an improved hydrolysis rate of cephalosporin C. It is not clear if this activity stems from an improved kcat and/or Km. This example shows, however, that variants of the glutaryl amidase with an improved activity towards adipyl-7-ADCA can be isolated using the described selection method.

Example 10-Some rationally designed multiple mutants of Pseudomonas SY-77 glutaryl amidase with increased hydrolysis activity towards adipyl-7-ADCA Combinations were made of mutant Tyrl78His of the a-subunit and six mutants of the 6-sub-unit, i. e. F229L, N266H, N266S, M271V+Q291K+T374S, F375H and F375L. All these mutants show an increased activity towards adipyl-7-ADCA. In the six plasmids encoding the genes with mutations in the

6-subunit Tyrl78 was replaced by His. The mutant enzymes were purified from E. coli DH10B cells that were transformed with these plasmids as described in example 1, and their kinetic parameters towards adipyl-7-ADCA were determined as described in example 8. The hydrolytic properties of the mutant enzymes are listed in table 3. The kcat towards adipyl-7-ADCA of the combination mutants Tyrl78His+Asn266His and Tyrl78His+Phe375Leu are significantly higher that that of both"parent"mutants, clearly showing that combining mutations in the gene for the glutaryl amidase can result in mutant enzymes with more improved kinetic parameters.

Example 11-Glutaryl amidase variants with increased hydrolysis activity towards adipyl-7-ADCA obtained from a library of glutaryl amidase mutants randomized at positions 178, 231, 255, 266 and 375 that was selected on adipyl-leucine A mutant library of glutaryl amidase was constructed as described in example 4. The region encoding the randomized codons was subsequently amplified using the primers 5'-agggggccgaatactggggcccggattacg and 5'- ctcgaagaccgggccatgga. The product was digested, ligated and transformed as described in example 4, resulting in a mutant library sized approximately 6*107 in which positions 178, 231,255, 266 and 375 were randomized.

Subsequent sequencing of 20 randomly picked transformants showed that positions 178,231 and 375 were randomized well, but positions 255 and 266 were randomized to a lesser extent. This mutant library was selected on adipyl-leucine as described in example 9.30 individual colonies were picked from the plates, of which 3 unique mutants showed an increased activity towards adipyl-7-ADCA: mutants Y178F+F375H ; Y178F+F375L; and Y178F+F375M. The three mutant proteins were purified and their catalytic parameters on glutaryl-7-ACA and adipyl-7-ADCA were determined as described in example 8. The hydrolytic properties of these enzymes towards these different substrates are shown in figure 4. It is obvious that all three

double mutants show improved kinetic parameters keat, Km and kcat/Km towards adipyl-7-ADCA when compared to the wild-type enzyme.

Interestingly, the kinetic parameters are also improved when compared to the values for the respective single mutants, which were made and subsequently purified and characterized in the same manner. The best mutant, Y178F+F375H, showed a 36-fold increase of catalytic efficiency when compared to the wild-type enzyme, due to a six-fold increase ofkcat and a six- fold decrease of Km (fig 4). The increase in catalytic efficiency and in kcat of this mutant towards adipyl-7-ADCA was more improved than could be calculated from the respective single mutants (36 versus 6. 3*3. 1=19.4 for catalytic efficiency and 5.8 versus 1. 0*2. 5=2.5 for revealing the unique character of this mutant. In contrast, the combination of mutation Y178H with mutation F375H or F375L, as in double mutants Y178H+F375H and Y178H+F375L, does not improve the kinetic parameters more than could be calculated from the respective single mutants. Obviously, the high activity of mutant Y178F+F375H is highly surprising. As a further surprising result, the single mutant Y178F showed improved kinetic parameters towards glutaryl-7-ACA when compared to the wild-type enzyme. To our knowledge, this is the first mutant of glutaryl amidase to show improved activity towards the recognized preferred substrate glutaryl-7-ACA.

Table 1.

Primers used in the megaprimer method in order to randomize positions 178, 231,255 and 266.

Primer Position Sequence (5'-> 3') randomized F1 178 accgcctgatgaacttcctcnnsgtcgcgtcg F2 atgtgtggaattgtgagcgg Bl 231 gcgcctcgtasnnggtgaagtagtccgtcgtcca B2 255 + 266 ccgttgacggtsnnggtgatgcccatccgctggttgaaggcgaasnngatgaccggc B3 gattggtgacccgcggca Table 2.

The catalytic parameters of on adipyl-leucine selected mutants on glutaryl-7- ACA and adipyl-7-ADCA. The hydrolytic activity towards cephalosporin C is given as the relative activity of the wild type glutaryl amidase. The values of the basic glutaryl amidases of both libraries were also measured.

Sequence at position glutaryl-7-ACA adipyl-7-ADCA ceph. C 178 266 375 kcat Km kcat/Km kcat Km keat/Km rel. act.

His Met Phe 0.9 0.31 2.9 nd nd nd 1.6 His Gln Met 0.7 0.63 1.2 0.7 0.27 2.8 1.3 Tyr Gin Leu 0.7 0.17 4.4 0.4 0.13 2.8 0.8 His His Phe 2.2 0.08 26 1.0 0. 18 6.2 2.6 Tyr Asn Phe 4.6 0.06 83 0.4 0.92 0.5 1 His Asn Phe 5. 0 0.15 40 1.2 0.8 1.5 2.7

Table 3.

The kinetic parameters of rationally designed double mutants of glutaryl amidase towards adipyl-7-ADCA (Triple = Met271Val+Gln291Lys+Thr374Ser). kcat (s-1) Km (mM) kcat/Km (s-i mM-i) WT 0. 38 + 0. 02 0.8 ~ 0. 1 0. 46 ~ 0.05 Tyrl78His 0. 66 ~ 0. 05 0.47 ~ 0. 05 1.40 ~ 0. 08 Tyrl78His+Phe229Leu 0.71 ~ 0. 05 1. 1 ~ 0. 2 0. 7 + 0. 1 Phe299Leu 0.47 ~ 0. 02 0. 52 0. 04 0.91 ~ 0. 04 Tyrl78His+Asn266His 0. 95 ~ 0. 04 0. 19 0. 07 6 2 Asn266His 0.55 ~ 0. 02 0.14 ~ 0. 02 3.9 ~ 0. 3 Tyrl78His+Asn266Ser 0.43 ~ 0. 06 0. 9 ~ 0. 3 0.5 ~ 0. 2 Asn266Ser 0.34 ~ 0. 02 0.42 ~ 0. 04 0. 82 ~ 0. 05 Tyrl78His+Triple 0. 44 ~ 0. 09 0. 8 0. 2 0. 6 ~ 0. 2 Triple 0. 61 ~ 0.04 0.7 ~ 0.1 0.9 ~ 0. 1 Tyrl78His+Phe375His 0. 65 ~ 0. 07 1. 0 0. 2 0.65 ~ 0. 06 Phe375His 1. 16 ~ 0.08 0. 9 0. 2 1.3 ~ 0. 1 Tyrl78His+Phe375Leu 1.0 ~ 0. 1 0.9 ~ 0. 1 1.2 ~ 0. 2 Phe375Leu 0.67 ~ 0. 04 0. 7 ~ 0.1 0. 9 + 0. 1

References 1. Yamada, H. , Ishii, Y. , Noguchi, Y. , Miura, T. , Mori, T. , and Saito, Y.

(1996) Protein engineering of a cephalosporin C acylase. Ann. N. Y. Acad. Sci.

799,74-81.

2. Ishii, Y. , Saito, Y. , Fujimura, T. , Sasaki, H., Noguchi, Y. , Yamada, H. , Niwa, M. , and Shimomura, K. (1995) High-level production, chemical modification and site-directed mutagenesis of a cephalosporin C acylase from Pseudomonas strain N176. Eur. J. Biochem. 230, 773-778.

3. Saito, Y., Fujimura, T., Ishii, Y., Noguchi, Y., Miura, T. , Niwa, M., and Shimomura, K. (1996) Oxidative modification of a cephalosporin C acylase from Pseudomonas strain N176 and site-directed mutagenesis of the gene.

Appl. Environ. Microbiol. 62,2919-2925.

4. Saito, Y. , Ishii, Y. , Fujimura, T. , Sasaki, H., Noguchi, Y. , Yamada, H. , Niwa, M. , and Shimomura, K. (1996) Protein engineering of a cephalosporin C acylase from Pseudomonas strain N176. Ann. N. Y. Acad. Sci.

782, 226-240.

5. Fritz-Wolf, K. Koller, K. P. , Lange, G. , Liesum, A. , Sauber, K., Schreuder, H. , Aretz, W. , and Kabsch, W. (2002) Protein Sci. 11,92-103 6. Sio, C. F. , Riemens, A. M. , van der Laan, J. M. , Verhaert, R. M. D. , and Quax, W. J. (2002) Directed evolution of a glutaryl acylase into an adipyl acylase. Eur. J. Biochem. 269,4495-4504.

7. Elander R. P. (2003) Appl. Microbiol. Biotechnol. 61, 385-392 8. Oh B, Kim M, Yoon J, Chung K, Shin Y, Lee D, Mm Y. (2003) Deacylation activity of cephalosporin acylase to cephalosporin C is improved by changing the side-chain conformations of active-site residues. Biochem Biophys Res Commun. 10; 310 (1) : 19-27.

9. Otten, L. G. , Sio, C. F., Vrielink, J. Cool, R. H. , and Quax, W. J. (2002) J. Biol. Chem. 277 (44), 42121-42127

10. Ishii, Y. , Saito, Y. , Fujimura, T. , Isogai, T. , Kojo, H. , Yamashita, M., Niwa, M. , and Kohsaka, M. (1994) A Novel 7-6- (4-carboxybutanamido)- cephalosporanic acid acylase isolated from Pseudomonas strain C427 and its high-level production in Escherichia coli. Journal of Fermentation and Bioengineering. 77,591-597.

11. Li, Y. , Chen, J. , Jiang, W. , Mao, X. , Zhao, G. , and Wang, E. (1999) In vivo post-translational processing and subunit reconstitution of cephalosporin acylase from Pseudomonas sp. 130. Eur. J. Biochem. 262, 713-719.

12. Li, Y., Jiang, W. , Yang, Y. , Zhao, G. , and Wang, E. (1998) Overproduction and purification of glutaryl 7-amino cephalosporanic acid acylase. Protein Expr. Purif. 12, 233-238.

13. Ichikawa, S., Murai, Y. , Yamamoto, S. , Shibuya, Y. , Fujii, T., Komatsu, K., and Kodaira, R. (1981) The isolation and properties of Pseudomonas mutants with an enhanced productivity of 7-0- (4- carboxybutanamido) -cephalosporanic acid acylase. Agric. Biol. Chem. 45,2225- 2229.

14. Matsuda, A. and Komatsu, K. I. (1985) Molecular cloning and structure of the gene for 7-S- (4-carboxybutanamido) cephalosporanic acid acylase from a Pseudomonas strain. J. Bacteriol. 163,1222-1228.

15. Kim, D. W. , Kang, S. M. , and Yoon, K. H. (1999) Isolation of novel Pseudomonas diminuta KAC-1 strain producing glutaryl 7- aminocephalosporanic acid acylase. J. Microbiol. 37,200-205.

16. Kim, Y. , Yoon, K. , Khang, Y. , Turley, S. , and Hol, W. G. (2000) The 2.0 A crystal structure of cephalosporin acylase. Structure 8, 1059-1068.

17. Shibuya, Y., Matsumoto, K. , and Fujii, T. (1981) Isolation and properties of 7-0- (4-carboxybutanamido) cephalosporanic acid acylase- producing bacteria. Agric. Biol. Chem. 45, 1561-1567.

18. Verhaert et al, 1997, Appl Environ Microbiol. 1997 Sep; 63 (9): 3412-8.

19. Biokatalysatoren und Enzymtechnologie, K. Buchholz and V.

Kasche, VCH Verlag, Weinheim, 1st edition 1997.

20. Cao, L. , Van Langen, L. , and Sheldon, R. A. (2003) Immobilised enzymes: carrier-bound or carrier-free? Curr. Op. Biotechnol. 14,387-394.

21. Bruggink, A. , Roos E. C., de Vroom E., 1998) Organic Process Res Dev. 2,128-133.

22. Ausubel, F. et al, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, 1994-1998 and Supplements.

23. Landt, O., Grunert, H. P. , and Hahn, U. (1990) Gene 1990,96 125- 128.

24. Reyes F, Martinez M. J. , and Soliveri J. (1989) Determination of cephalosporin-C amidohydrolase activity with fluorescamine. J Pharm Pharmacol. 41 (2): 136-7.