WO2002086111A2 - Recombinant alpha-amino ester hydrolases and uses thereof - Google Patents

Recombinant alpha-amino ester hydrolases and uses thereof Download PDF

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WO2002086111A2
WO2002086111A2 PCT/EP2002/004536 EP0204536W WO02086111A2 WO 2002086111 A2 WO2002086111 A2 WO 2002086111A2 EP 0204536 W EP0204536 W EP 0204536W WO 02086111 A2 WO02086111 A2 WO 02086111A2
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seq
sequence
alpha
amino acid
polynucleotide
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PCT/EP2002/004536
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WO2002086111A3 (en
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Van Der Jan Metske Laan
Jolanda Jannie Polderman-Tijmes
Thomas Reinier Maxim Barends
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Dsm Ip Assets B.V.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01043—Alpha-amino-acid esterase (3.1.1.43)

Definitions

  • the present invention relates to recombinant alpha-amino ester hydrolases and uses thereof.
  • the present invention also relates to isolated nucleic acids encoding said recombinant alpha-amino ester hydrolases or functional equivalents thereof.
  • Cells containing heterologous nucleic acids encoding alpha-amino ester hydrolases or vectors containing nucleic acids encoding alpha-amino ester hydrolases are also part of the invention.
  • mutants of the wild type sequence with an improved synthesis over hydrolysis ratio are part of the invention.
  • Description of the related art ⁇ -Lactam antibiotic compounds constitute the most important group of antibiotic compounds, with a long history of clinical use.
  • ⁇ - lactam antibiotic compounds are the penicillins and cephalosporins.
  • ⁇ - lactam antibiotic compounds, or, in short, ⁇ -lactam antibiotics consist of a nucleus, the so-called amino ⁇ -lactam nucleus, which is linked through its primary amino group to the so-called side chain via a linear amide bond.
  • ⁇ -lactam antibiotics are prepared by semi-synthetic methods. These semi-synthetic ⁇ -lactam antibiotics are obtained by modifying an N- substituted ⁇ -lactam product by one or more chemical and/or enzymatic reactions. Typically, one of the reactions involved in the modification of the N-substituted ⁇ -lactam is acylation of the amino ⁇ -lactam nucleus, which has been obtained from the N- substituted ⁇ -lactam.
  • ⁇ -lactam antibiotics have been prepared in chemical procedures. However, such chemical methods have a number of grave disadvantages. They comprise many complex reactions, wherein by-products are formed which give rise to effluent and purification problems.
  • the semi-synthetic routes mostly start from fermentation products such as isopenicillin N, penicillin G, penicillin V and cephalosporin C, which products are enzymatically converted to an amino ⁇ -lactam nucleus, for instance in a manner as has been disclosed in Bioprocess. Technol., 16, 67-88 (1993) by K. Matsumoto, in Process Biochemistry, 146-154 (1989) by J.G. Shewale & H. Sivaraman, in Biotechnology of Industrial Antibiotics (Ed. E.J. Vandamme) Marcel Dekker, New York (1984) by T.A. Savidge, or in Process Biochemistry International, June, 97-103 (1990) by J.G.
  • a D-(-)-phenylglycine side chain may be attached to a 6-aminopenicillanic acid (6-APA) nucleus, in a reaction with a suitable derivative of said D-(- phenylglycine, to yield ampicillin, or to a 7-aminodesacetoxycephalosporanic acid (7-ADCA) nucleus to yield cephalexin.
  • 6-APA 6-aminopenicillanic acid
  • 7-ADCA 7-aminodesacetoxycephalosporanic acid
  • a disadvantage of the known methods is that the coupling reaction starts from an amino ⁇ -lactam nucleus, which has been isolated prior to the coupling reaction. During the isolation of the amino ⁇ -lactam nucleus, which is usually performed by crystallization, up to about 10% of the theoretical yield is lost. Due to the amphoteric nature of the ⁇ -lactam nucleus, it dissolves readily in an aqueous environment at any pH value. As a result a great part of the production of the ⁇ -lactam nucleus is lost in the mother-liquor resulting from the crystallization.
  • a process for preparing a ⁇ -lactam antibiotic wherein the antibiotic is prepared from a mixture in which a N-substituted ⁇ -lactam a dicarboxylate acylase and a penicillin acylase and a desired side chain precursor.
  • the process includes the in-situ preparation of a ⁇ -lactam nucleus which is formed when the N-subtituted ⁇ -lactam is liberated enzymatically from its side chain.
  • the N-substituted ⁇ -lactam starting material is limited to a ⁇ -lactam compounds containing a succinyl, glutaryl or an adipyl or analogous thereof as the N-substituent.
  • Acetobacter turbidans ATCC 9325, X. citri IFO 3835 and A. pasteurianus ATCC 6033 were first described in 1972 by Takahashi etal as organisms able to synthesise cephalosporins. Since only ⁇ -amino acid derivatives act as a substrate and due to the preference for esters over amides, the enzyme involved was named alpha-amino acid ester or ⁇ -amino-acid ester hydrolase (abbreviated as AEH or ⁇ -AEH) (Takahashi, et al., Biochem. J., (1974) 137; 497-503).
  • AEH or ⁇ -AEH alpha-amino acid ester hydrolase
  • AEH (E.C. 3.1.1.43) activity is defined as the ability of an enzyme to catalyse the following general reaction scheme: ⁇ -amino acid ester + Nucleophile- ⁇ -amino acid acyl derivative + Alcohol
  • AEH activity has been described for several organisms.
  • the enzymes are able to catalyse the transfer of the alpha-amino-acyl group from alpha- amino acid esters to amine nucleophiles, which is more commonly referred to as synthesis or transferase reaction, where the nucleophile may belong to the group of 7- aminocephem or 6-amino penams.
  • the enzymes may transfer the acyl group to water which is commonly referred to as the hydrolysis reaction. Presumably an acyl-enzyme intermediate is involved in this transfer reaction
  • The-AEHs show biocatalytic properties for the enzymatic synthesis of the semi-synthetic ⁇ -lactam antibiotics using esters as the acyl side chain precursors. Due to the preference for esters, higher product accumulation can be reached in a synthesis reaction compared to the penicillin G acylase. (Takahashi, et al., Biochem. J., (1974) 137; 497-503, Ryu and Ryu, Enzyme Microb.
  • a turbidans is described as a heterodimer, ⁇ 2 ⁇ 2 with subunits of 70 en 72 kD (Ryu and Ryu, Enzyme Microb. Technol., (1987) 9; 339-344),
  • X. citri is shown to be a homotetramer, ⁇ 4 , with subunits of 72 kD (Kato, et al., Agric. Biol. Chem., (1980) 44; 1069-1074).
  • P. melanogenum is described as a homodimer, ⁇ _>, with subunits of 72 kD as well (Kim and Byun, Biochim. Biophys. Acta, (1990) 1040; 12-18)).
  • citri involved a pBR322 chromosomal library which was screened for growth on D-alanyl-L-leucine. This method is based on the auxotrophic complementation of the E. coli leuB mutant using a minimal medium with D-alanyl-L- leucine as sole source of L-leucine. One clone capable to grow on this medium was found. However this clone displayed no hydrolysis activity with ampicillin. Sequence analysis and homology searches showed that the cloned gene encoded a proline iminopeptidase.
  • the invention relates to recombinantly produced alpha-amino ester hydrolases.
  • the nucleic acid sequences provided herein may be used in any art-known method to transform a micro-organism and subsequently the transformed micro-organism may be used to produce an alpha-amino ester hydrolase.
  • the nucleic acid sequences provided herein may also be used to readily find homologous sequences in other organisms that encode variant alpha-amino ester hydrolases.
  • the nucleic acid sequences provided herein may also be used to identify homologues sequences with unknown function as being an ⁇ -AEH. These homologous sequences may then in turn be used to transform other micro-organisms to express the homologous alpha-amino ester hydrolase of said other organism.
  • the invention also relates to recombinant vectors comprising a nucleic acid sequence accordinging to the invention as well as a method of manufacturing any of the above nucleic acids.
  • the invention also relates to recombinantly produced AEH, in particular an isolated polypeptide selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10. Variants, homologues and fragments of these polypeptides are also part of the invention.
  • nucleic acids encoding mutant AEHs with improved synthesis over hydrolysis ratios are part of the invention. Detailed description of the invention
  • the invention provides isolated nucleic acids preferably encoding functionally active AEH.
  • the cloning of the gene encoding the ⁇ -AEH allows us to optimise expression and purification by placing the gene in front of Myc-epitope and a 6xHis-tag under control of an araBAD promoter. In this way the expression was improved by a factor 10.
  • the construct is easy to cultivate to high optical densities and on a relatively cheap and simple medium (Luria Broth). This is in contrast to the medium described in the literature for A. pasteurianus.A. turbidans and X.
  • citri which contains thioglycollate, glycerol and glucose (Takahashi, et al., J Am. Chem. Soc, (1972) 94; 4035-7). Additionaly, higher optical density can be reached, especially in comparison to A pasteurianus.
  • placing a His-tag directly C-terminal without the Myc-epitope in front of it, resulted in inactive protein under several induction conditions. This indicates that the/Wyc-epitope (15 amino acids) probably functions as a linker, rendering a certain distance necessary for the attachment of the 6xHis tag without losing the ⁇ -AEH activity.
  • the purification (up to 80% pure) is now a one-step procedure with use of Ni-agarose. In general this purity is sufficient for the majority of applications, reducing the intensive purification procedures described in the literature from 5 to a single step procedure. When required further purification can be done by an additional gel filtration step.
  • the ae 7-gene provides a tool to attribute a function to other proteins with yet unknown function.
  • the gene of Zymomonas mobilis ZM4 which was described as a putative glutaryl acylase was in fact an AEH.
  • the sequence of the Z. mobilis gene and gene product is shown in SEQ ID NO: 4 and SEQ ID NO: 9 respectively. Cloning of this gene and initial synthesise experiments indicated that this enzyme is able to synthesis cefalexine from phenylglycine methyl ester and 7-ADCA.
  • ⁇ -AEHs having a higher activity for esters than amide have now been further optimised to reduce the unwanted hydrolysis of the antibiotic during synthesis.
  • the invention provides an isolated polynucleotide encoding a functional alpha- amino ester hydrolase with a S/H ini ratio higher than 1.9, preferably higher than 2, 2.5, 3.0, 4.5, 5, 8, 10 and most preferably higher than 15. It also provides an isolated polynucleotide encoding a functional alpha-amino ester hydrolase with a Q max (mM) ratio higher than 8, preferably higher than 10, 12 and most preferably higher than 15. Also provided is an isolated polynucleotide encoding a functional alpha-amino ester hydrolase with a S/H max ratio higher than 1.2, preferably higher than 2, 2.5, 3.0, 4.5, 5, 8, 10 and most preferably higher than 15.
  • the cloning of the ⁇ -AEH of the plant pathogen X. citri has an additional advantage since it allows purification of its ⁇ -AEH from a non-pathogenic host .
  • gene and “recombinant gene” refer to nucleic acid molecules which may be isolated from chromosomal DNA, which include an open reading frame encoding a protein having alpha-amino ester hydrolase activity.
  • a gene may include coding sequences, non-coding sequences, introns and regulatory sequences.
  • a gene refers to an isolated nucleic acid molecule as defined herein.
  • a nucleic acid molecule of the present invention such as a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1 , SEQ ID NO: 2 , SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO: 5 or functional equivalents thereof, can be isolated using standard molecular biology techniques using the sequence information provided herein.
  • nucleic acid molecules according to the invention can be isolated using standard hybridization and cloning techniques from a variety of organisms (e. g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
  • nucleic acid molecule encompassing all or a portion of SEQ ID NO:1 , SEQ ID NO: 2 , SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO: 5 can be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence information provided herein.
  • PCR polymerase chain reaction
  • a nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid thus amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to or hybridisable to nucleotide sequences according to the invention can be prepared by standard synthetic techniques, e. g., using an automated DNA synthesizer.
  • an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO:1 , SEQ ID NO: 2 , SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO: 5.
  • the sequence of SEQ ID NO: 1 corresponds to the coding region of the A. turbidans alpha-amino ester hydrolase cDNA.
  • SEQ ID NO: 2 is an AEH gene from A. pasteurianus
  • SEQ ID NO:3 is an AEH gene from X. citri
  • SEQ ID NO:4 is an AEH gene from Z. mobilis
  • SEQ ID NO: 5 is an AEH gene form X fastidiosa.
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NO: 1 , SEQ ID NO: 2 , SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 or functional equivalents thereof.
  • a nucleic acid molecule which is complementary to another nucleotide sequence is one which is sufficiently complementary to the other nucleotide sequence such that it can hybridize to the other nucleotide sequence thereby forming a stable duplex.
  • One aspect of the invention pertains to isolated nucleic acid molecules that encode a polypeptide of the invention or a functional equivalent thereof such as a biologically active fragment or domain, as well as nucleic acid molecules sufficient for use as hybridisation probes to identify nucleic acid molecules encoding a polypeptide of the invention and fragments of such nucleic acid molecules suitable for use as PCR primers for the amplification or mutation of nucleic acid molecules.
  • an "isolated polynucleotide” or “isolated nucleic acid” is a DNA or RNA that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived.
  • an isolated nucleic acid includes some or all of the 5' non-coding (e.g., promotor) sequences that are immediately contiguous to the coding sequence.
  • the term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding an additional polypeptide that is substantially free of cellular material, viral material, or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicafs (when chemically synthesized).
  • an "isolated nucleic acid fragment” is a nucleic acid fragment that is not naturally occurring as a fragment and would not be found in the natural state.
  • the terms "polynucleotide” or “nucleic acid molecule” are intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
  • the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • the nucleic acid may be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.
  • oligonucleotide analogs or derivatives e.g., inosine or phosphorothioate nucleotides.
  • Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.
  • Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to an alpha-amino ester hydrolases nucleic acid molecule, e.g., the coding strand of an alpha-amino ester hydrolases nucleic acid molecule. Also included within the scope of the invention are the complement strands of the nucleic acid molecules described herein.
  • sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases.
  • the specific sequences disclosed herein can be readily used to isolate the complete gene from other micro-organisms or other strains or the starins used herein which in turn can easily be subjected to further sequence analyses thereby identifying sequencing errors.
  • all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors.
  • Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule.
  • the actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art.
  • a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
  • the person skilled in the art is capable of identifying such erroneously identified bases and knows how to correct for such errors.
  • a nucleic acid molecule according to the invention may comprise only a portion or a fragment of the nucleic acid sequence shown in SEQ ID NO:1 , SEQ ID NO: 2 , SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, for example a fragment which can be used as a probe or primer or a fragment encoding a portion of an alpha-amino ester hydrolase protein.
  • the nucleotide sequence determined from the cloning of the alpha- amino acid hydrolase genes and cDNAs allows for the generation of probes and primers designed for use in identifying and/or cloning other alpha-amino acid hydrolase family members, as well as alpha-amino acid hydrolase homologues from other species.
  • the probe/primer typically comprises substantially purified oligonucleotide which typically comprises a region of nucleotide sequence that hybridizes preferably under highly stringent conditions to at least about 12 or 15, preferably about 18 or 20, preferably about 22 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 or more consecutive nucleotides of a nucleotide sequence shown in SEQ ID NO:1 , SEQ ID NO: 2 , SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 or functional equivalents thereof.
  • Probes based on the alpha-amino acid hydrolase nucleotide sequences can be used to detect alpha-amino acid hydrolase sequences encoding the same or homologous proteins for instance in other organisms.
  • the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor.
  • the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor.
  • Such probes can also be used as part of a diagnostic test kit for identifying cells that express an alpha-amino acid hydrolase protein. Identity & homology
  • the terms “homology” or “percent identity” are used interchangeably herein.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • % identity number of identical positions/total number of positions (i.e. overlapping positions) x 100).
  • the two sequences are the same length.
  • the skilled person will be aware of the fact that several different computer programms are available to determine the homology between two sequences. For instance, a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.qcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.
  • the percent identity between two nucleotide sequences may be determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1 , 2, 3, 4, 5, or 6.
  • the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W.
  • nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403 — 10.
  • Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • Hybridizing is intended to describe conditions for hybridization and washing under which nucleotide sequences at least about 40%, 50%, at least about 60%, at least about 70%, more preferably at least about 80%, even more preferably at least about 85% to 90%, more preferably at least 95% homologous to each other typically remain hybridized to each other.
  • a preferred, non-limiting example of such hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45 °C, followed by one or more washes in 1 X SSC, 0.1 % SDS at 50 °C, preferably at 55 °C, preferably at 60 °C and even more preferably at 65 °C.
  • Highly stringent conditions include, for example, hybridizing at 68°C in 5x SSC/5x Denhardt's solution/l.0% SDS and washing in 0.2x SSC/0.1% SDS at room temperature. Alternatively washing may be performed at 42°C.
  • a polynucleotide which hybridizes only to a poly A sequence such as the 3' terminal poly(A) tract of mRNAs), or to a complementary stretch of T (or U) resides, would not be included in a polynucleotide of the invention used to specifically hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule contain a poly (A) stretch or the complement thereof (e.g., practically any double-standed cDNA clone).
  • DNA libraries constructed from other organisms e.g. belonging to the proteobacteria can be screened.
  • strains of proteobacteria can be screened for homologous alpha- amino acid hydrolase polynucleotides by Northern blot analysis.
  • DNA libraries can be constructed from the appropriate strain, utilizing standard techniques well known to those of skill in the art.
  • a total genomic DNA library or cDNA library of a eukaryotic organism can be screened using a probe hybridisable to an alpha-amino acid hydrolase polynucleotide according to the invention.
  • Homologous gene sequences can be isolated, for example, by performing PCR using two degenerate oligonucleotide primer pools designed on the basis of nucleotide sequences as taught herein.
  • the template for the reaction can be cDNA obtained by reverse transcription of mRNA prepared from eukaryotic strains known or suspected to express a polynucleotide according to the invention.
  • the PCR product can be subcloned and sequenced to ensure that the amplified sequences represent the sequences of a new alpha-amino acid hydrolase nucleic acid sequence, or a functional equivalent thereof.
  • the PCR fragment can then be used to isolate a full length cDNA clone by a variety of known methods.
  • the amplified fragment can be labeled and used to screen a bacteriophage or cosmid cDNA library.
  • the labeled fragment can be used to screen a genomic library.
  • RNA can be isolated, following standard procedures, from an appropriate cellular or tissue source.
  • a reverse transcription reaction can be performed on the RNA using an oligonucleotide primer specific for the most 5' end of the amplified fragment for the priming of first strand synthesis.
  • the resulting RNA/DNA hybrid can then be "tailed" (e.g., with guanines) using a standard terminal transferase reaction, the hybrid can be digested with RNase H, and second strand synthesis can then be primed (e.g., with a poly-C primer).
  • cDNA sequences upstream of the amplified fragment can easily be isolated.
  • vectors preferably expression vectors, containing a nucleic acid encoding an alpha-amino acid hydrolase protein or a functional equivalent thereof.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector is another type of vector, wherein additional DNA segments can be ligated into the viral genome.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors
  • certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors”.
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and "vector” can be used interchangeably herein as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • viral vectors e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vector includes one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.
  • "operatively linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signal). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in a certain host cell (e.g. tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, encoded by nucleic acids as described herein (e.g. alpha-amino acid hydrolase proteins, mutant forms of alpha- amino acid hydrolase proteins, fragments, variants or functional equivalents thereof, fusion proteins, etc.).
  • nucleic acids as described herein (e.g. alpha-amino acid hydrolase proteins, mutant forms of alpha- amino acid hydrolase proteins, fragments, variants or functional equivalents thereof, fusion proteins, etc.).
  • the recombinant expression vectors of the invention can be designed for expression of alpha-amino acid hydrolase proteins in prokaryotic or eukaryotic cells.
  • alpha-amino acid hydrolase proteins can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells, fungal cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Expression vectors useful in the present invention include chromosomal-, episomal- and virus-derived vectors e.g., vectors derived from bacterial plasmids, bacteriophage, yeast episome, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • the DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E.
  • promoters are preferred that are capable of directing a high expression level of recombinant proteins. Such promoters are known in the art.
  • the expression constructs may contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the mature transcripts expressed by the constructs will include a translation initiating AUG at the beginning and a termination codon appropriately positioned at the end of the polypeptide to be translated.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of artfecognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-percipitation, DEAE-dextran-mediated transfection, transduction, infection, lipofection, cationic lipidmediated transfection or electroporation.
  • Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2 d ,ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Davis et al., Basic Methods in Molecular Biology (1986) and other laboratory manuals.
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methatrexate.
  • Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding an alpha-amino acid hydrolase protein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g. cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • Fusion vectors add a number of amino acids to a protein encoded therein, e.g. to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three purposes: 1 ) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • Such enzymes, and their cognate recognation sequences include Factor Xa, thrombin and enterokinase.
  • the expression vectors will preferably contain selectable markers.
  • Such markers include dihydrofolate reductase or neomycin resistance for eukarotic cell culture and tetracyline or ampicilling resistance for culturing in E. co//and other bacteria.
  • Representative examples of appropriate host include bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS and Bowes melanoma; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
  • vectors preferred for use in bacteria are pQE70, pQE60 and PQE- ⁇ , available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16A, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
  • preferred eukaryotic vectors are PWLNEO, pSV2CAT, pOG44, pZT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
  • bacterial promotors for use in the present invention include £. coli lacl and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR, PL promoters and the trp promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus ("RSV”), and metallothionein promoters, such as the mouse metallothionein-l promoter.
  • RSV Rous sarcoma virus
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type.
  • enhancers include the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovims early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • secretation signal may be incorporated into the expressed polypeptide.
  • the signals may be endogenous to the polypeptide or they may be heterologous signals.
  • the polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals but also additional heterologous functional regions.
  • a region of additional amino acids, particularly charged amino acids may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification or during subsequent handling and storage.
  • peptide moieties may be added to the polypeptide to facilitate purification.
  • Polypeptides according to the invention provides an isolated polypeptide having the amino acid sequence according to SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:9 or SEQ ID NO: 10, or an amino acid sequence obtainable by expressing the polynucleotides of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4 or SEQ ID NO: 5 in an appropriate host. Also, a peptide or polypeptide comprising a functional equivalent of the above polypeptides is comprised within the present invention. The above polypeptides are collectively comprised in the term "polypeptides according to the invention"
  • peptide and oligopeptide are considered synonymous (as is commonly recognized) and each term can be used interchangeably as the context requires to indicate a chain of at least two amino acids coupled by peptidyl linkages.
  • polypeptide is used herein for chains containing more than seven amino acid residues. All oligopeptide and polypeptide formulas or sequences herein are written from left to right and in the direction from amino terminus to carboxy terminus. The one-letter code of amino acids used herein is commonly known in the art and can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2" d ,ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989)
  • isolated polypeptide or protein is intended a polypeptide or protein removed from its native environment.
  • recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the invention as are native or recombinant polypeptides which have been substantially purified by any suitable technique such as, for example, the single-step purification method disclosed in Smith and Johnson, Gene 67:31-40 (1988).
  • the alpha-amino acid hydrolase according to the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.
  • HPLC high performance liquid chromatography
  • Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host- mediated processes.
  • a protein according to the invention may be a precursor protein containing a leader sequence, a hybrid protein, a protein obtained as a pro sequence or pre-pro sequence, or any other type of immature form.
  • the invention also features biologically active fragments of the polypeptides according to the invention.
  • Biologically active fragments of a polypeptide of the invention include polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the alpha-amino acid hydrolase protein (e.g., the amino acid sequences of SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:9 or SEQ ID NO: 10), which include fewer amino acids than the full length protein, and exhibit at least one biological activity of the corresponding full-length protein.
  • biologically active fragments comprise a domain or motif with at least one activity of the alpha-amino acid hydrolase protein.
  • a biologically active fragment of a protein of the invention can be a polypeptide. which is, for example, 10, 25, 50, 100 or more amino acids in length.
  • other biologically active portions, in which other regions of the protein are deleted can be prepared by recombinant techniques and evaluated for one or more of the biological activities of the native form cf a polypeptide of the invention.
  • the invention also features nucleic acid fragments which encode the above biologically active fragments of the alpha-amino acid hydrolase protein. Fusion proteins
  • proteins of the present invention or functional equivalents thereof can be operatively linked to a non-alpha-amino acid hydrolase polypeptide (e.g., heterologous amino acid sequences) to form fusion proteins.
  • a non-alpha-amino acid hydrolase polypeptide e.g., heterologous amino acid sequences
  • an alpha-amino acid hydrolase "chimeric protein” or “fusion protein” comprises an alpha-amino acid hydrolase polypeptide operatively linked to a non-alpha-amino acid hydrolase polypeptide.
  • alpha-amino acid hydrolase polypeptide refers to a polypeptide having an amino acid sequence corresponding to alpha-amino acid hydrolase
  • a non-alpha-amino acid hydrolase polypeptide refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the alpha-amino acid hydrolase protein, e.g., a protein which is different from the alpha-amino acid hydrolase protein and which is derived from the same or a different organism.
  • the alpha-amino acid hydrolase polypeptide can correspond to all or a portion of an alpha-amino acid hydrolase protein.
  • an alpha- amino acid hydrolase fusion protein comprises at least one biologically active fragment of an alpha-amino acid hydrolase protein.
  • an alpha- amino acid hydrolase fusion protein comprises at least two biologically active portions of an alpha-amino acid hydrolase protein.
  • the term "operatively linked" is intended to indicate that the alpha-amino acid hydrolase polypeptide and the non-alpha-amino acid hydrolase polypeptide are fused in-frame to each other.
  • the non-alpha-amino acid hydrolase polypeptide can be fused to the N- terminus or C-terminus of the alpha-amino acid hydrolase polypeptide or the non- alpha-amino hydrolase polypeptide can be inserted at a suitable position in the alpha- amino acid hydrolase polypeptide.
  • the fusion protein is a GST-alpha-amino acid hydrolase fusion protein in which the alpha-amino acid hydrolase sequences are fused to the C-terminus of the GST sequences.
  • Such fusion proteins can facilitate the purification of recombinant alpha-amino acid hydrolase.
  • the fusion protein is an alpha-amino acid hydrolase protein containing a heterologous signal sequence at its N-terminus.
  • expression and/or secretion of alpha-amino acid hydrolase can be increased through use of a hetereologous signal sequence.
  • the gp67 secretory sequence of the baculovirus envelope protein can be used as a heterologous signal sequence (Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, 1992).
  • Other examples of eukaryotic heterologous signal sequences include the secretory sequences of melittin and human placental alkaline phosphatase (Stratagene; La Jolla, California).
  • useful prokarytic heterologous signal sequences include the phoA secretory signal (Sambrook et al., supra) and the protein A secretory signal (Pharmacia Biotech; Piscataway, New Jersey).
  • a signal sequence can be used to facilitate secretion and isolation of a protein or polypeptide of the invention.
  • Signal sequences are typically characterized by a core of hydrophobic amino acids which are generally cleaved from the mature protein during secretion in one or more cleavage events.
  • Such signal peptides contain processing sites that allow cleavage of the signal sequence from the mature proteins as they pass through the secretory pathway.
  • the signal sequence directs secretion of the protein, such as from a eukaryotic host into which the expression vector is transformed, and the signal sequence is subsequently or concurrently cleaved.
  • the protein can then be readily purified from the extracellular medium by art recognized methods.
  • the signal sequence can be linked to the protein of interest using a sequence which facilitates purification, such as with a GST domain.
  • the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide, which facilitates purification of the fused polypeptide.
  • the marker sequence is a hexa- histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described in Gentz et al, Proc. Natl. Acad. Sci.
  • hexa-histidine provides for convenient purificaton of the fusion protein.
  • the HA tag is another peptide useful for purification which corresponds to an epitope derived of influenza hemaglutinin protein, which has been described by Wilson et al., Cell 37:767 (1984), for instance.
  • an alpha-amino acid hydrolase chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
  • anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • many expression vectors are commercially available that already encode a fusion moiety (e.g, a GST polypeptide).
  • An alpha-amino acid hydrolase-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the alpha- amino acid hydrolase protein.
  • Functional equivalents are used interchangeably herein.
  • Functional equivalents of alpha-amino acid hydrolase DNA are isolated DNA fragments that encode a polypeptide that exhibits a particular function of the alpha-amino acid hydrolase as defined herein.
  • a functional equivalent of an alpha- amino acid hydrolase polypeptide according to the invention is a polypeptide that exhibits at least one function of an alpha-amino ester hydrolase as defined herein.
  • Functional protein or polypeptide equivalents may contain only conservative substitutions of one or more amino acids of SEQ ID NO: 2 or substitutions, insertions or deletions of non-essential amino acids.
  • a non-essential amino acid is a residue that can be altered in SEQ ID NO: 2 without substantially altering the biological function.
  • Amino acids conserved among the alpha-amino acid hydrolase proteins according to the present invention and related enzymes are not likely to be amenable to alteration.
  • substitution is intended to mean that a substitution in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • These families are known in the art and include amino acids with basic side chains (e.g. lysine, arginine and hystidine), acidic side chains (e.g.
  • aspartic acid glutamic acid
  • uncharged polar side chains e.g., glycine, asparagines, glutamine, serine, threonine, tyrosine, cysteine
  • non-polar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine tryptophan, histidine
  • nucleic acid equivalents may typically contain silent mutations or mutations that do not alter the biological function of encoded polypeptide. Accordingly, the invention provides nucleic acid molecules encoding alpha-amino acid hydrolase proteins that contain changes in amino acid residues that are not essential for a particular biological activity. Such alpha-amino acid hydrolase proteins differ in amino acid sequence from SEQ ID NO: 2 yet retain at least one biological activity.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises a substantially homologous amino acid sequence of at least about more than 40%, 50%, 60%, 65%, 70%, 75%, 80%, 857o, 90%, 95%, 96%, 97%, 98%, 99% or more homologous to the amino acid sequence shown in SEQ ID NO: 2.
  • An isolated nucleic acid molecule encoding an alpha-amino acid hydrolase protein homologous to the protein according to SEQ ID NO: 2 can be created by introducing one or more nucleotide substitutions, additions or deletions into the coding nucleotide sequences according to SEQ ID NO: 1 such that one or more amino acid substitutions, deletions or insertions are introduced into the encoded protein.
  • Such mutations may be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • orthologues of the alpha- amino acid hydrolase proteins also encompasses orthologues of the alpha- amino acid hydrolase proteins from other organisms than the ones described described herein.
  • Orthologues of the alpha-amino acid hydrolase protein are proteins that can be isolated from other strains or species and possess a similar or identical biological activity. Such orthologues can readily be identified as comprising an amino acid sequence that is substantially homologous to SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:9 or SEQ ID NO: 10.
  • substantially homologous refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., with similar side chain) amino acids or nucleotides to a second amino acid or nucleotide sequence such that the first and the second amino acid or nucleotide sequences have a common domain.
  • amino acid or nucleotide sequences which contain a common domain having about 40%, 50%, 60%, preferably 65%, more preferably 70%, even more preferably 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity or more are defined herein as substantially homologous.
  • nucleic acids encoding other alpha-amino acid hydrolase family members which thus have a nucleotide sequence that differs from SEQ ID NO:1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 are within the scope of the invention.
  • Nucleic acid molecules corresponding to variants (e.g. natural allelic variants) and homologues of the alpha-amino acid hydrolase DNA of the invention can be isolated based on their homology to the alpha-amino acid hydrolase nucleic acids disclosed herein using the cDNAs disclosed herein or a suitable fragment thereof, as a hybridisation probe according to standard hybridisation techniques preferably under highly stringent hybridisation conditions.
  • allelic variants of the alpha-amino acid hydrolase sequence the skilled person will recognise that changes can be introduced by mutation into the nucleotide sequences of SEQ ID NO:1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 thereby leading to changes in the amino acid sequence of the alpha-amino acid hydrolase protein without substantially altering the biological activity of the alpha-amino acid hydrolase protein.
  • improved alpha-amino acid hydrolase proteins are provided.
  • Improved alpha-amino acid hydrolase proteins are proteins wherein at least one biological activity is improved.
  • Such proteins may be obtained by randomly introducing mutations along all or part of the alpha-amino acid hydrolase coding sequence, such as by saturation mutagenesis, and the resulting mutants can be expressed recombinantly and screened for biological activity.
  • the art provides for standard assays for measuring the enzymatic activity of alphaamino ester hydrolases and thus improved proteins may easily be selected.
  • Improved mutants may also be obtained by targeting the mutagenesis to certain regions or just to certain residues which are important for modulation of catalytic functionality.
  • the data provided herein allow the skilled person to modify the enzyme at DNA level to produce mutant ⁇ -AEHs with different, even more desired, properties.
  • the property that the ⁇ -AEHs have a higher activity for esters than amide has been optimised to further reduce the unwanted hydrolysis of the antibiotic during synthesis.
  • the alpha-amino acid hydrolase protein has an amino acid sequence according to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.
  • the alpha-amino acid hydrolase polypeptide is substantially homologous to the amino acid sequence according to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 and retains at least one biological activity of a polypeptide according to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10, yet differs in amino acid sequence due to natural variation or mutagenesis as described above.
  • the alpha-amino acid hydrolase protein has an amino acid sequence encoded by an isolated nucleic acid fragment capable of hybridising to a nucleic acid according to SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, preferably under highly stringent hybridisation conditions.
  • the alpha-amino acid hydrolase protein is a protein which comprises an amino acid sequence at least about 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous to the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 and retains at least one functional activity of the polypeptide according to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.
  • Functional equivalents of a protein according to the invention can also be identified e.g. by screening combinatorial libraries of mutants, e.g. truncation mutants, of the protein of the invention for alpha-amino ester hydrolase activity.
  • a variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level.
  • a variegated library of variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential protein sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display).
  • libraries of fragments of the coding sequence of a polypeptide of the invention can be used to generate a variegated population of polypeptides for screening a subsequent selection of variants.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment cf the coding sequence of interest with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which encodes N-terminal and internal fragments of various sizes of the protein of interest.
  • REM Recursive ensemble mutagenesis
  • DNA sequence polymorphisms that may lead to changes in the amino acid sequence of the alpha-amino acid hydrolase protein may exist within a given population. Such genetic polymorphisms may exist in cells from different populations or within a population due to natural allelic variation. Allelic variants may also include functional equivalents.
  • Fragments of a polynucleotide according to the invention may also comprise polynucleotides not encoding functional polypeptides. Such polynucleotides may function as probes or primers for a PCR reaction. Such polynucleotides may also be useful when it is desired to abolish the functional activity of an alpha-amino acid hydrolase in a particular organism (knock-out mutants).
  • Nucleic acids according to the invention irrespective of whether they encode functional or non-functional polypeptides, can be used as hybridization probes or polymerase chain reaction (PCR) primers.
  • Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having an alpha-amino acid hydrolase activity include, inter alia, (1) isolating the gene encoding the alpha-amino acid hydrolase protein, or allelic variants thereof from a cDNA library e.g. from other organisms than A. niger; (2) in situ hybridization (e.g.
  • FISH FISH to metaphase chromosomal spreads to provide precise chromosomal location of the alpha-amino acid hydrolase gene as described in Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988); (3) Northern blot analysis for detecting expression of alpha-amino acid hydrolase mRNA in specific tissues and/or cells and 4) probes and primers that can be used as a diagnostic tool to analyse the presence of a nucleic acid hybridisable to the alpha-amino acid hydrolase probe in a given biological (e.g. tissue) sample.
  • a nucleic acid hybridisable to the alpha-amino acid hydrolase probe in a given biological (e.g. tissue) sample.
  • Also encompassed by the invention is a method of obtaining a functional equivalent of an alpha-amino acid hydrolase gene or cDNA.
  • a method entails obtaining a labelled probe that includes an isolated nucleic acid which encodes all or a portion of the sequence according to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 or a variant thereof; screening a nucleic acid fragment library with the labelled probe under conditions that allow hybridisation of the probe to nucleic acid fragments in the library, thereby forming nucleic acid duplexes, and preparing a full-length gene sequence from the nucleic acid fragments in any labelled duplex to obtain a gene related to the alpha-amino acid hydrolase gene.
  • an alpha-amino acid hydrolase nucleic acid of the invention is at least 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homologous to a nucleic acid sequence shown in SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 or the complement thereof.
  • an alpha-amino acid hydrolase polypeptide of the invention is at least 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homologous to the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.
  • the invention features cells, e.g., transformed host cells or recombinant host cells that contain a nucleic acid encompassed by the invention.
  • a "transformed cell” or “recombinant cell” is a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a nucleic acid according to the invention.
  • the host cell may over-express the polypeptide, and techniques for engineering over-expression are well known.
  • the host may thus have two or more copies of the encoding polynucleotide (and the vector may thus have two or more copies accordingly).
  • Both prokaryotic and eukaryotic cells are included, e.g., bacteria, fungi, yeast, and the like, especially preferred are cells from Escherichia coli, Pichia pastoris, Kluyveromyces lactis, Aspergillus niger, Lactococcus lactis, and Bacillus subtilis. Bacteria from the genus Bacillus are very suitable as heterologous hosts because of their capability to secrete proteins into the culture medium.
  • yeast host cells for the expression of the DNA sequence encoding the polypeptide is of the general. Saccharomyces, Kluyveromyces, Hansenula, Pichia, Yarrowia, and Schizosaccharc- myces. More preferably a yeast host cell is selected from the group consisting of the species Saccharomyces cerevisiae, Kluyveromyces lactis (also known as Kluyveromyces marxianus var. lactis), Hansenula polymorpha, Pichia pastoris, Yarrowia lipolytica.and Schizosaccharomyces pombe.
  • Preferred filamentous fungal host cells are selected from the group consisting of the genera Aspergillus, Trichoderma, Fusarium, Disporotrichum, Penicillium,
  • a filamentous fungal host cell may belong to the species Aspergillus oyzae, Aspergillus sojae, Aspergillus nidulans, or to a species from the Aspergillus niger group.
  • Aspergillus niger include but are not limited to Aspergillus niger, Aspergillus awamori, Aspergillus tubingensis, Aspergillus aculeatus, Aspergillus foetidus, Aspergillus nidulans, Aspergillus japonicus, Aspergillus oryzae and Aspergillus ficuum, and further consisting of the species Trichoderma reesei, Fusarium graminearum, Penicillium chrysogenum, Acremonium alabamense, Neurospora crassa, Myceliophtora thermophilum, Sporotrichum cellulophilum, Disporotrichum dimorphosporum and Thielavia terrestris.
  • Examples of expression hosts within the scope of the present invention are gram negative bacteria such E.coli and Pseudomonas species; garm positive bacteria such as Bacillus species, e.g. Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens; fungi such as Aspergillus species and Trichoderma species; Pseudomonas species; and yeasts such as Kluyveromyces species, e.g.
  • Kluyveronmyces lactis and Saccharomyces species e.g. Saccharomyces cerevisiae.
  • a host cell can be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in a specific, desired fashion. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may facilitate optimal functioning of the protein.
  • Various host cells have characteristic and specific mechanisms for post- translational processing and modification of proteins and gene products.
  • Appropriate cell lines or host systems familiar to those of skill in the art of molecular biology and/or microbiology can be chosen to ensure the desired and correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used.
  • Such host cells are well known in the art.
  • Host cells also include, but are not limited to, mammalian cell lines such as CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and choroid plexus cell lines.
  • mammalian cell lines such as CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and choroid plexus cell lines.
  • the polypeptides according to the invention can be produced by a stably-transfected cell line.
  • a number of vectors suitable for stable transfection of mammalian cells are available to the public, methods for constructing such cell lines are also publicly known, e.g., in Ausubel et al. (supra). Culture of host cells and recombinant production
  • polypeptides of the invention can be effected by culturing of microbial expression hosts, which have been transformed with one or more polynucleotides of the present invention, in a conventional nutrient fermentation medium.
  • the recombinant host cells according to the invention may be cultured using procedures known in the art. For each combination of a promoter and a host cell culture conditions are available which allow for production of expression of the DNA sequence encoding the polypeptide of the invention. After reaching the desired cell density or titre of the polypeptide the culture is stopped and the polypeptide is recovered using known procedures. In this way the optimal combination of promoter and host for expression of the polypeptide of the invention may be established.
  • the fermentation medium may comprise a known culture medium containing a carbon source (e.g. glucose, maltose, molasses, etc), a nitrogen source (e.g. ammonium sulphate, ammonium nitrate, ammonium clored etc.), inorganic nutrient sources (e.g. phosphate, magnesium, maltodextrin, or xylogalacturonan) may be included.
  • a carbon source e.g. glucose, maltose, molasses, etc
  • a nitrogen source e.g. ammonium sulphate, ammonium nitrate, ammonium clored etc.
  • inorganic nutrient sources e.g. phosphate, magnesium, maltodextrin, or xylogalacturonan
  • the selection of the right medium may be based on choice of expression host or based on regulatory requirements of the expression. Such media are known to those skilled in the art.
  • the medium may contain additional components favouring the transformed expression hosts over other potentially contaminating microorganisms.
  • the proper control of the fermentation with respect to aeration and feed supply in combination with certain promoter / host combination can steer the expression of the polypeptide of the invention during the fermentation. E.g. by such control growth of the cells and expression of the desired protein can be uncoupled to certain extent, which is useful in cases where overexpression hinders the normal growth of the micro- organism.
  • the fermentation can be performed over a period of 0.5-30 days. It may be a batch, continuous or fed batch process, suitably at a temperature in the range of between 0 and 45°C and for example at a pH between 2 and 10.
  • Preferred fermentation conditions are a temperature in the range of 20 and 37°C and/or a pH between 3 and 9. The appropriate conditions are usually selected based on choice of expression host and the protein expressed. In certain systems the fermentative production of enzymes may benefit from the addition of protease inhibitors which significantly reduce losses due to proteolysis.
  • the cells may be killed. This is usually carried out by heat treatment or pH treatment. The use of additives such as for example detergents make such treatments more effective. Subsequently the cells can be removed from the fermentation broth by means procedures including, but not limited to centrifugation, filtration, extraction, spray-drying or precipitation. In those cases in which the polypeptide of the invention is not excreted, the producing cells have to be disrupted to enable purification of the protease.
  • the collected cell mass is best ground with an abrasive, milled with beads, ultrasonicated or subjected to a French press or a Manton-Gaulin homogeniser and then filtered or centrifuged
  • filtration aids and flocculants may improve the results of centrifugation and filtration steps, in particular after grinding of the cells.
  • Use of reversible protease inhibitors may increase the yields during recovery. Washing and concentration steps are commonly performed by ultrafiltration. For a further purification large scale chromatography modules may be applied as is known in the art.
  • the final product of the recovery may be used as such but may also be formulated in order to prolong shelf-life or to adapt the product better to application conditions.
  • the product may be in a liquid form or it my be part of a dry formulation.
  • the polypeptide of the invention is combined with suitable (solid or liquid) carriers or diluents including buffers to produce a composition .
  • suitable (solid or liquid) carriers or diluents including buffers to produce a composition .
  • the invention provides in a further aspect a composition comprising a polypeptide of the invention. This may be in a form suitable for packaging, transport and/or storage preferably where biological activity is retained.
  • Compositions include paste, liquid, emulsion, powder, flake, granulate, pellet or other extrudate forms.
  • the composition may further comprise additional ingredients such as one or more enzymes or proteins, preservatives, stabilisers.
  • the polypeptide may be attached to or mixed with a carrier, e.g. immobilized on a solid carrier.
  • the enzymes may also be encapsulated e.g. by entrapment into gel matrices such as polyacrylamide, alginte, sol-gel matrices, plastic materials.
  • chemical modification may be used to modify performance.
  • chemical cross-linking may be used to stabilize enzymes in certain applications Aggregates of the enzyme may be cross-linked and used as such in bioconversions.
  • the enzymes may be crystallized and used as crystals which may be further stabilised by cross-linking.
  • the enzyme according to the invention is able to hydrolyse a broad substrate range.
  • the amino group on the C ⁇ -atom is essential.
  • esters and amides can be hydrolysed resulting in an ⁇ -amino acid and the corresponding alcohol or amine, the enzyme shows a strong preference for esters.
  • the enzyme is able to transfer the ⁇ -amino acyl group to a number of amino nucleophiles, a property that can be used for the synthesis of semi-synthetic antibiotics.
  • an enzyme that is defined as an ⁇ -amino acid ester hydrolase catalyses the transfer of an ⁇ -amino-acid acyl group from a precursor to an amine nucleophile (the acceptor).
  • suitable precursors are phenylglycine methylester (PGM) and hydroxyphenylglycine methyl ester (HPGM).
  • PGM phenylglycine methylester
  • HPGM hydroxyphenylglycine methyl ester
  • An ⁇ -amino acid ester hydrolase shows a high preference to hydrolyse the phenylglycine methyl ester (PGM) compared to the corresponding phenylacetic acid methyl ester (PAM) that lacks the ⁇ -amino group.
  • the preference or specificity towards a certain substrate is clearly dependant both on the binding affinity for the substrate(s) and on the velocity at which the substrate(s) are converted. Therefore the preference of an enzyme for a certain substrate with respect to another substrate is indicated by the ratio of the kcat/Km value of the one substrate over that of the other substrate.
  • the kcat/Km ratio is better known as the specificity constant (A.Fersht ,1977, Enzyme Structure and Mechanism, W.H. Freeman and Company). The higher the kcat/Km ratio, the more preferred the substrate is.
  • kcat represents the turn-over rate and Km represents an apparent dissociation constant.
  • the kcat/Km ratio has a unit of a second order rate constant and is expressed as 1/(concentration * time).
  • Assays that may be performed in order to recognize an enzyme as being an AEH are described in Example 1. Legends to the figures Figure 1. Ampicillin production at pH 7.2 and pH 6.2.
  • the ampicillin productions by the cell-free extracts of X. maltophilia, X. citri, B. megaterium, Achromobacter, A. turbidans, A. pasteurianus and E. coli were measured at pH 7.2 and pH 6.2.
  • X. citri, A. turbidans and A. pasteurianus were found to produce significant amounts at both pH-values.
  • CFE cell free extracts
  • Figure 2 A representative tree of the relative distance between the homologous proteins to the ⁇ -amino acid ester hydrolase of A turbidans ATCC 9325 as found by BLAST search. The distance can be read as number of nucleotide substitutions per site. In the circle the proteins which share 60% or more identity are situated. The tree was constructed using Clustal W and TreeView . EXAMPLES
  • an enzyme as being an AEH the following assay may be employed. Incubate an appropriate amount of enzyme with 30 mM of a nucleus and 15 mM acyl donor in 50 mM sodium phosphate buffer (pH 6.2) at 30°C. Samples are taken as function of incubation time and quenched. The products of the acyl transfer to the nucleus (antibiotic) and water were subsequently detected by HPLC.
  • Suitable acyl donors comprise phenylglycine esters (e.g. the methyl ester PGM) or p- hydroxyphenylglycine esters (e.g. the methyl ester HPGM). Alternatively, the corresponding amides can be used but the rate of acylation will be considerably lower.
  • the acyl donors carry a primary amino group at the alpha carbon. Hardly any transfer is possible using the corresponding phenyl acetic amide/ester.
  • the nucleus can be either 6-aminopenicillanic acid (6-APA) or 7-aminodesacetoxycephalosporanic acid (7- ADCA) forming ampicillin or cephalexin, respectively when using PGM as the acyl donor, amoxycillin and cephadroxyl respectively when using HPGM as the acyldonor.
  • Other amino-acid esters or amino acid ester derivatives such as for example dihydrophenylglycine or dihydroxyphenylglycine esters may be used as acyl donor.
  • Suitable nuclei may include 7-aminocephem compounds such as 7-amino-cephalosporanic acid (7-ACA), 7-amino-cephem-3- chloor-4-carboxylic acid (7-ACCA), 7-amino-3-[(Z)-1-propenyl)-ceph-3-em-4-carboxylic acid (exomethylene 7ADCA), 7-amino-3-((1,2,3-triazol-5-yl)-thiomethyl)- cephalosporanic acid (7-TACA) and 7-amino-cephem-3-methoxy-4-carboxylic acid.
  • suitable nuclei are not limited to ⁇ -lactams, but may also comprise other compounds containing primary amino groups such as amino acids.
  • a typical example is the synthesis of cephalexin by an ⁇ -AEH that was carried out as follows. Enzyme incubations were done at 30°C and contained 30 mM 7- ADCA and 15 mM D-PGM in 50 mM sodium-phosphate buffer pH 6.2. Before analysis the samples were quenched and diluted 50-fold by the addition of HPLC eluent. The synthesis and hydrolysis experiments were followed by reverse-phase HPLC using a Chrompack C 18 column with Jasco PU-980 pumps and a Jasco MD-910 detector set at 214 nm.
  • cephalexin synthesis unit is defined as the amount of enzyme needed to produce one ⁇ mol of cephalexin per min under the indicated conditions.
  • the initial synthesis over hydrolysis ratio (usually called S/H-ratio) is determined by dividing the initial slopes of the formation of the antibiotic (synthesis) by the formation of hydrolysis product (hydrolysis, corrected for background hydrolysis).
  • S/H Qmax is the maximal concentration antibiotic divided by the concentration hydrolysis product (side chain) at Q max .
  • An ⁇ -amino acid ester hydrolase shows a high preference to hydrolyse the phenylglycine methyl ester (PGM) compared to the corresponding phenylacetic acid methyl ester (PAM) that lacks the ⁇ -amino group.
  • An assay to establish this preference is the following. Incubate an appropriate amount of enzyme with a mixture of PGM and PAM, in 50 mM phosphate buffer pH 6.2, and follow the hydrolysis of PGM and PAM respectively by the formation of the hydrolysis product phenyl glycine (PG) or phenylaceticacid (PAA) by HPLC analysis.
  • the ratio of the initial velocities of formation of PG and PAA reflect the ratio of the specificity constants according to the relationship:
  • v a /v b ⁇ (kcat/Km) a [A] ⁇ / ⁇ (kcat/Km) ⁇ ] ⁇
  • [A] and [B] represent the substrate concentration of the competing substrates PGM and PAM, respectively.
  • substrate inhibition has been observed for some ⁇ -AEHs, it is recommended to use for the determination of activity a range of substrate concentrations, for example 1 , 5, 10, 25, 50mM. Determination of the activity should be done using the initial values (less than or equal to 10% hydrolysis of the substrate), this to prevent significant effects of product inhibition.
  • the enzyme was incubated with varying concentrations in the range of 0 to 25 mM for cephalexin, ampicillin, HPGM, and cefadroxil, or 0 to 50 mM for D-PGM and NIPGB, or 0 to 10 mM for amoxicillin. Reactions were done at 30°C in 50 mM phosphate buffer, pH 6.2. The calculations involved nonlinear regression fitting (Scientist, Micromath) using Michaelis- Menten and substrate inhibition kinetics, and the calculated kinetic parameters are given with their standard deviations. The hydrolysis of PGA was measured at 5 and 50 mM and the kcat/Km was calculated from the initial linear slope of the Michaelis- Menten curve. Hydrolysis of glutaryl 7-ACA and adipoyl 7-ADCA was measured at 5 and 25 mM.
  • Example 2 Selection of suitable organisms for cloning of AEH From the literature, 12 organisms were selected that have been described to be able to hydrolyse and synthesise ampicillin (Table 3).
  • Acetobacter turbidans ( ) 2 ( ⁇ ) 2 (Takahashi, et al., Biochem. J.,
  • Kluyvera cryocrescens ⁇ (Shimizu, et al., Agr. Biol. Chem.,
  • Acetobacter - (Takahashi, et al., J. Am. Chem. pasteurianus Soc, (1972) 94; 4035-7)
  • Achromobacter NRRL - (Fujii, et al., Process Biochem.,
  • a pasteurianus was selected for further molecular and enzymological studies.
  • D-PG-L-Leucine can be converted by ⁇ -AEH releasing the leucine necessary for growth. Using these screenings methods no positive clone could be detected. This might have been due to a bad expression, a too small library, or the acylase gene is not situated on the chromosome but on one of the 5 plasmids which are present in A pasteurianus.
  • coli HB101 according to the alkaline lysis method and purified by ultracentrifugation using a CsCI gradient (Sambrook, Fritsch and Maniatus, 1989, Molecular Cloning: a laboratory manual, 2 nd edition).
  • the chromosomal DNA of A turbidans was partially digested with Sat 3A to yield fragments with an average size of 30-50 kb. These fragments were ligated in the cosmid pLAFR3 (Tc r ) which had been completely digested with BamHl and dephosphorylated with alkaline phosphatase.
  • In vitro packaging and infection of E. coli HB101 was carried out according to the recommendations of the manufacturer (Roche).
  • AEHs from the different organisms were purified. Pure AEH was thereafter used to determine the N-terminal or internal (after digestion) amino acid sequences, which were used to make a genetic probe.
  • AEHAf A turbidans
  • the retained protein eluted in a linear gradient of 0-1 M KCI (30 ml/min) at 0.2 M.
  • Activity containing fractions were pooled and (NH 4 ) 2 SO was added to a final concentration of 1.5 M, after which the pool was loaded on a hydrophobic interaction column (Resource Phenyl, 2.6 by 7.5 cm, Amersham Pharmacia) pre-equilibrated with 1.5 M (NH 4 ) 2 SO 4 , 50 mM Na- phosphate buffer, pH 6.2.
  • AEHAt eluted at 0.8-0.68 M (NH 4 ) 2 SO 4 in a decreasing linear gradient from 1.5 M to 0 M (NH 4 ) 2 SO 4 in 50 mM Na-phosphate buffer (pH 6.2) at 5 ml/min.
  • Fractions that contained AEHA ⁇ were pooled and concentrated by ultrafiltration (YM30, Amicon bioseparations, Millipore, Bedford, USA) and loaded on a Superdex 200 HR 10/30 column (24-ml bed volume, Amersham Pharmacia).
  • AEHA ⁇ was eluted at 1 ml/min in 50 mM Na-phosphate buffer (pH 6.2), 0.15 M NaCl.
  • cexU is the amount of enzyme needed to produce one ⁇ mol cephalexin per min at 30°C from 30 mM 7-ADCA and 15 mM PGM at pH 6.2 (50 mM Na-phosphate buffer).
  • the membrane was washed with 2 x SSC, 0.1 % SDS (10 x SSC is 1.5 M NaCl with 0.15 M Na-citrate) at room temperature and with 0.5 x SSC, 0.1 % SDS for 15 min at 68°C.
  • the DIG-labeled DNA was visualized using alkaline phosphatase and a chemiluminescence substrate,
  • AEHXc The ⁇ -AEH of X. citri
  • AEHXc was purified from a 10 litre culture.
  • AEHXc was purified by ion exchange, hydrophobic interaction and gelfiltration as described for the A. turbidans.
  • the retained protein was eluted from CM sepharose in a linear gradient of 0-1 M KCI at 0.45 M KCI.
  • AEHXc eluted from Resource Phenyl in a decreasing linear gradient from 1.5 M to 0 M (NHt) 2 SO 4 around 0.36 M (NH 4 ) 2 SO 4 .
  • the enzyme was purified to SDS homogeneity by gelfiltration (Sephacryl S300, 1.6 by 65 cm, Amersham Pharmacia Biotech ltd., Hertfordshire, United Kingdom). Results are summarized in Table 6.
  • Fragment 1 A A G L E Q L P W W H K Possible codons 4 4 4 6 2 2 6 4 1 1 2 2
  • YTTRTGCCACCANGGNARYTGYTC-3' (Y is T or C; R is A or G; N is any base) were designed and used to amplify part of the aehXc gene by PCR from chromosomal DNA.
  • the PCR-product was isolated from gel (Qiaquick kit from Qiagen, GmbH, Hilden, Germany), cloned and sequenced.
  • a gene probe for the AEHXc gene was made using matching primers based on the DNA sequence of the PCR fragment.
  • the forward primer was 5'-ACCGATGCCTGGGACACC-3'(upstream of pF) and the reverse primer was 5'-CAGGCCTGCGGCCTTGGC-3'(downstream of pR).
  • These primers were used to amplify a 317-bp fragment (ProbeXc) with Taq polymerase using the PCR DIG probe synthesis mix from Roche.
  • Example 4 Expression and purification of cloned AEHs Cloning of aehAt into an expression host.
  • the vector pETAT (aehAt cloned in pET9) was constructed.
  • the aehAt gene was cloned in the A/del- and BamHl site of pET9 using a forward primer based on the N-terminal sequence including the leader sequence in which an Asnl site is incorporated, 5' CCGC-
  • CGCCGATTAATGGTGGGACAGATTACCCTTT-3' (Asnl site underlined, start codon in bold) and a reverse primer in which a BamHl site was incorporated (underlined), 5'- ACCCATAC-TGGATCCTTACTGTTTCACAACCGGGAG-3'.
  • the gene was also cloned without the N-terminal leader sequence, where the leader sequence was replaced by a methionine, using 5'-GGTCGCGCATTAATGGCTCCGGCAGCG-GATGC-3' (Asnl site underlined, start codon in bold) as a primer.
  • the first 5 N-terminal amino acid residues of the recombinant enzyme were determined and found to be 40-AAPXAD, which is in agreement with the predicted cleavage site.
  • the determined N-terminus had a high degree of heterogeneity, indicative of varying N-termini within the give N-terminal sequence, it indicates that the signal sequence is processed in a similar way as in A turbidans. Therefore, it can be concluded that the leader sequence is needed for the production of active enzyme and is processed properly in E. coli Isolation of AEHAt from E. coli.
  • the recombinant AEHA was purified from E. coli BL21(DE3)pLysS (Cm R ) cells carrying the pETAT (Km R ) construct.
  • the cells were harvested from two 2.5 I cultures by centrifugation and the crude extract was prepared as described above.
  • the extract was loaded on a DEAE Sepharose column (5 by 13 cm column, Amersham Pharmacia) pre-equilibrated with 50 mM Na-phosphate buffer, pH 6.2.
  • the AEHA activity was eluted from the column in the non-binding fraction in the equilibration buffer at 30 ml/min.
  • CM-HAP ceramic hydroxy apetite column, 2.6 by 11 cm column, Amersham Pharmacia
  • AEHAt activity was eluted from the column at 275 mM Na-phosphate in a linear gradient of 50 to 500 mM Na-phosphate (pH 6.2) at 10 ml/min.
  • the AEHAt was purified further to SDS-PAGE homogeneity by hydrophobic interaction and gelfiltration chromatography as described above.
  • the aehAtgene was cloned in pBAD/Myc-HisA (pBADAT), coupling both yc-epitope and the 6xHis-tag C-terminally to the protein.
  • pBADAT pBAD/Myc-HisA
  • the Ncol restriction site was first removed from the gene cloned in pAT (3).
  • the forward primer (aehAhisf), 5'- CGCGCCACACCATGGTGGGACAGATTA-3' (start codon in bold), was based on the N-terminal sequence including the signal sequence and an ⁇ /col site (underlined) was introduced.
  • the reverse primer (aehAhisr), 5'-
  • Protein purification - Wild type AEH was expressed in E. coli TOP10 from the pBAD/Myc-HisA derived constructs.
  • E. coli TOP10 To obtain soluble protein two 2.5 liter cultures supplemented with l-arabinose (0.01% w/v) were inoculated with a 1 ml overnight culture grown at 30°C and incubated for 64 h at 14°C. Induced cells were harvested from the cultures by centrifugation at 5000 g and suspended in 50 mM Na- phosphate buffer pH 6.2. All further steps were carried out at 4°C. The cytoplasmic content was released by sonification and the remaining cell debris was removed by centrifugation at 13.000 g for 40 min.
  • the supernatant was added to 1 ml Ni-agarose (Qiagen) equilibrated with wash buffer (25 mM imidazole, 500 mM NaCl, 50 mM Na- phosphate, pH 7.4). After mixing by inversion for 90 min at 4°C the bed was allowed to form (20 x 8 mm in a polyprep chromatography column (Biorad Laboratories, Hercules, CA, USA)). The unbound protein was washed from the column with 30 column volumes of wash buffer.
  • wash buffer 25 mM imidazole, 500 mM NaCl, 50 mM Na- phosphate, pH 7.4
  • the bound protein eluted from the column at 100 mM imidazole in a stepwise gradient from 50 to 200 mM imidazole, 150 mM NaCl, 50 mM Na-phosphate, pH 7.4, in 20 column volumes.
  • the protein was brought to 50 mM Na-phosphate buffer pH 6.2 with use of an Econpac gelfiltration column (Biorad). All purification steps were monitored by SDS-PAGE and enzymatic activity was measured with NIPGB.
  • the protein concentrations were measured using the Bradford method with bovine serum albumin as the standard.
  • Cephalexin synthesis To check whether the properties of AEH had changed upon addition of the myc- epitope and His-tag the kinetic parameters of the purified enzyme for cephalexin were measured and compared to untagged recombinant protein.
  • the K M values of both proteins appeared to be nearly identical, 0.45 and 0.34 mM, respectively.
  • the k cat of the fusion protein is somewhat lower than for the untagged recombinant protein, 274 and 347 s "1 respectively, but the values are in the same order of magnitude. This indicates the proper folding of the recombinant protein occurred and shows that there is no dramatic influence of the added amino acids.
  • Percent identity was calculated using the pairwise blast option at NCBI on the world wide web.
  • TTTA ⁇ CTCA4GC7TrTATGGGATAACCGGCAA-3' (H/ndlll site is shown in italics and the stop codon in bold). From this construct the gene was amplified with the primers pfz and przhis, S'-TTTATTCTCA GCTTTGGGATAACCGGCAA-S' (H/ndlll site is shown in italics).
  • Enzyme incubations were done at 30°C and contained 30 mM 7-ADCA and 15 mM D-PGM in 50 mM sodium-phosphate buffer pH 6.2.
  • An initial S/H-ratio of 1.9 ⁇ 0.7 and a maximum product accumulation of 5.5 ⁇ 1.5 mM cefalexin with a S/H Qmax of 0.5 ⁇ 0.2 were measured at pH 6.2 (50 mM Na-phosphate).
  • NIPAB 2-Nitro-5-phenylacetylbenzoic acid; - : no activity; + : activity observed
  • Example 6 Sequence comparison of aeh with other related sequences
  • the deduced amino acid sequence of A turbidans and X c/fr/ was used for homology searches to find related enzymes.
  • the ⁇ -AEHs showed homology with several proteins, most of which originated from genome sequencing projects.
  • the most closely related protein, 28-29% identity, for which the activity is described, is the intracellular cocaine esterase from the gram-positive strain Rhodococcus sp. strain MB1 (Bresler, et al., Appl. Environ. Microbiol., (2000) 66; 904-908). This enzyme hydrolyses the ester bond in cocaine resulting in benzoate and ecgonine methyl ester.
  • laterosporus unlike other known glutaryl 7-ACA acylases (Kumar, et al., Hind. Antibiotics Bull., (1993) 35; 112-125), is composed of a single polypeptide with molecular size of 70 kD, which corresponds to the size of the subunits found for ⁇ - AEHs from A turbidans and X citri .
  • the subunit composition of the native glutaryl-7- aminocephalosporanic acid acylase has not been described. Cloning of this enzyme in Bacillus subtilis resulted in an extracellular localisation (Aramori, et al., J. Bacteriol., (1991) 173; 7848-55).
  • PSI-Blast (Altschul, et al., Nucleic Acids Res., (1997) 25; 3389-3402) indicated low identity (average 14%) to seven X-prolyl dipeptidyl aminopeptidases from Lactococcus lactis and Lactobacillus strains. The relation of cocaine esterase with these enzymes has been reported as well (Bresler, et al., Appl. Environ. Microbiol., (2000) 66; 904-908). The X-prolyl dipeptidyl aminopeptidases belong to the peptidase_S15 family as defined by the Pfam database (Bateman, et al., Nucleic Acids Research., (2000) 28; 263-266).
  • the ⁇ -AEH of A.turbidans (2.4 ⁇ M, 144 kDa) was inactivated by incubation with p-NPGB (1 mM, 1% DMF) for 15 min at 30°C. Control experiments involved incubation under the same conditions of solely enzyme and enzyme with 1% DMF. The inactivated enzyme was diluted 76 times in 15 mM NIPGB, 50 mM Na-phosphate, pH 6.2. The reactivation was monitored by measuring the hydrolysis of NIPGB at 30°C and 405 nm. The observed inhibition by p-NPGB indicated the importance of a serine residue for the activity of the enzyme.
  • p-NPGB p-nitrophenol
  • DMF dimethylformamide
  • ACN acetonitrile
  • the molar extinction coefficient of p-NP at pH 7 was determined as 9200 M-1 cm-1.
  • the pre-steady state kinetics of p-NPGB conversion was determined by stopped-flow using an Applied Photophysics SX17MV stopped-flow instrument. A stock of p-NPGB (100 mM) was made in DMF, the final concentration of DMF in reaction mixture was 2% or lower. All pre-steady state reactions were performed in 50 mM 4-morpholinepropanesulfonic acid buffer at pH 7, with 1 mM p-NPGB. The enzyme concentration used was 1.36 and 0.66 ⁇ M (a2; 144 kDa).
  • the slow conversion of the acyl enzyme intermediate during reaction of p- NPGB enabled us to covalently label the enzyme.
  • the enzyme was incubated with p- NPGB in a 1 :35 ratio in 50 mM Na-phosphate buffer, pH 6.2, with 0.5 % dimethylformamide for 15 min at 30°C.
  • the excess p-NPGB was removed by dialysis against 70% formic acid.
  • the enzyme solution was dialyzed against 70% formic acid with b-mercaptoethanol (2 mM). After removing the b- mercaptoethanol by dialysis against solely 70% formic acid the labeled protein was treated with a 100-fold molar excess of CNBr over the Met content.
  • the reaction was allowed to proceed for 24 h at room temperature under N2 in the dark and was stopped by the addition of 10 volumes water and freeze-dried.
  • the generated peptides were separated by reversed phase HPLC using a nucleosil-5 C18 column (4.6 by 300 mm, Alltech) at 1 ml/min in a linear gradient from 0 to 67% acetonitrile with 0.1 % trifluoroacetic acid.
  • the peptide profile was monitored at 280 nm.
  • the control involved the same conditions as described above except no p-NPGB was added.
  • Electrospray mass spectrometry was performed on an API3000 mass spectrometer (Applied Biosystems/MDS-SCIEX, Toronto, Canada), a triple quadrupole mass spectrometer supplied with an atmospheric pressure ionization source and ionspray interface. The spectra were scanned in the range between m/z 400 and 1600. MS/MS product ion spectra were recorded on the same instrument by selectively introducing the m/z 1229.5 (singly charged unlabeled peptide) and m/z 695.9 (doubly charged labeled peptide) precursor ions from the first quadrupole into the collision cell (second quadrupole). The collision gas was nitrogen with 30 eV collision energy.
  • the product ions resulting from the collision were scanned over a range of m/z 10 to 1395 with a step size of 0.1 amu and a dwell time of 2 ms.
  • the elution pattern of the peptide mixture of the labeled AEHAt showed a few different peaks compared to the control. These peaks were individually collected and analyzed by ES/MS.
  • the only peptide with a different mass when it was isolated from the labeled and unlabeled protein was identified as peptide 202-TGSSYEGFTWM-213 (1228.6 Da). Upon labeling the same peptide fragment had a mass of 1390 Da.
  • Example 8 Identification of the catalytic triad of ⁇ -AEH by site directed mutagenesis.
  • Site directed mutagenesis of AEHA was performed on pBADAT using QuickChange site directed mutagenesis kit of Stratagene (La Jolla, USA) according to the procedure recommended by the manufacturer. When possible, a restriction site was introduced in the mutational primers (Table 1 ).
  • the PCR reaction mixture was directly used to transform chemically competent E. co// ' Top10 cells (Invitrogen, Leek, The Netherlands). For isolation of vector the cultires were grown overnight on LB medium at 30°C.
  • the mutant proteins were purified as described for the wild-type enzyme using the Ni-agarose (Example 4).
  • Oligonucleotides used in site-directed mutagenesis of AEHAt onlythe sense primers are shown. Introduced restriction sites are underlined, sequence differences with wild type are shown in bold.
  • the data was converted to mean residue ellipticity (qMRE, deg.cm2.dmol-2). From the CD spectra the percentage of secondary structure elements was calculated using CD spectra deconvolution (CDNN version 2.1 , available on the world wide web). These values were standardized to 100% total structure elements.
  • AEH is a serine hydrolase and contains a classical catalytic triad of Ser205, Asp338 and His370.
  • ⁇ - AEH's can be aligned with X.citri too, subsequently allowing for modelling the 3- dimensional structure.
  • the procedure is generally known as homology modelling.
  • Typical computer programs which allow for generating such models are Insight, DISCOVER, HOMOLOGY and MODELER (Accelrys Ltd. 230/250 The Quorum, Barnwell Road.Cambridge CB5 8RE, UK).
  • X.citri ⁇ -AEH Ser174 corresponds to Ser205 in A. turbidans, which was identified previously as the catalytic serine (see alignment in table 14).
  • the 3- dimensional model of X.citri is defined in its atomic coordinates. Such coordinates can be used by proper software to display a 3-dimensional model.
  • a suitable sofware package is Insight & Discover (Accelrys Ltd).
  • Relevant atomic coordinates that describe the environment of the catalytic serine 174 have been set out in table 15 Examination of the structure around the catalytic serine for a catalytic base increases the nucleophilicity of the Ser174 by accepting the hydroxyl proton reveals that the N ⁇ of His340 is within hydrogen bonding distance of the hydroxyl group.
  • N5 of this His340 forms a hydrogen bond with the carboxyl group of Asp307, which tends to increase the basicity of the histidine. So, the structure of X.citri reveals a catalytic triad consisting of Ser174, His340 and Asp307.
  • Table 15 Relevant atomic coordinates that describe the environment of the catalytic serine 174
  • H H H -H H -1 > > > > > > > > > > > > > > O O I I I I I I I I I TJ TJ TJ TJ TJ TJ TJ TJ TJ TJ TJ TJ TJ TJ TJ TJ TJ o r- r- r- TJ TJ TJ TJ O O o O o O o o - ⁇ - ⁇ - ⁇
  • CD1 TRP 209 45,125 76,87 1 ,311
  • Ol _OT 00 00 cn OT en CO CO 44 co ro J* 0 co 0 -vl 00 co OT -vl OT 44 CO 44
  • the 3-dimensional model of the X.citri active site allows for the calculation of the solvent accessible surface around the catalytic Ser174.
  • the accessible surface reveals a binding pocket in which for example a substrate such as ampicillin can be fitted.
  • the coordinates for the ampicillin molecule, which has been fitted into the active site of ⁇ -AEH are shown in table 15.
  • the model reveals some features which are essential for the specificity of ⁇ -AEH.
  • the carboxyl groups of Asp 208, Glu309 and Asp310 form a cation binding pocket which binds positively charged substituents at the substrate C ⁇ position. In ampicillin the positively charge ⁇ -amino group of the acyl side chain is bound in this pocket.
  • sequence homology should be at least 60% with one of the given ⁇ -AEH's.
  • sequence should contain the following pattern typical for the catalytical serine: Gxx[G/A]xxGxSYx[G/A] where x indicates any amino acid at that position, [/] indicates a number of possibilities e.g.
  • [G/A] means Glycine or Alanine.
  • sequence should contain the following pattern which reveals the catalytic histidine: GPWxH.
  • Gxx[G/A]xxGxSYx[G/A] and GPWxH the preferred ⁇ AEH should contain the following two sequence patterns: [W/Y/F]xGDDW and [W/Y/F]DxEDxx[G/A] in the same order as given here, starting from the N-terminus.
  • Example 11 Oxyanion hole as a tool to modify S/H ratio
  • the 3-dimensional structure of X.citri allows for the calculation of the accessible surface around the catalytic Ser174. The accessible surface reveals a clear binding pocket in which for example ampicillin can be fitted.
  • the coordinates for the ampicillin molecule fitted in active site of ⁇ -AEH are shown in table 15. Hydrolysis of the substrate goes through formation of a so-called acyl enzyme. During formation as well as degradation of the acyl-enzyme a negative charge develops on the carbonyl oxygen, which is commonly stabilized by hydrogen bond donating groups on the protein. Such a binding spot fitted to stabilize the negatively charged oxygen is usually called the oxyanion hole.
  • the most likely candidates for the hydrogen donors in AEHX ⁇ are the backbone amide of Tyr175 and the side chain hydroxyl group of Tyr82.
  • Table 16 Kinetic parameters of PGM hydrolysis for mutants of AEHAt.
  • Both AEH ⁇ f Ser156 and AEHAt His610 do not interact with any of the residues forming the oxyanion hole.
  • the closest residue forming the oxyanion hole with respect to AEHAf Ser156 is AEHAf Tyr206 at 13 Angstroms.
  • the closest residue to AEH ⁇ f His 610 is AEHAt Tyr112 at 30 Angstroms.
  • a mutation was made at position A.turbidans AEH ⁇ f Arg85 corresponding to AEHXf Arg55. Distance of this residue to the active site is about 15 Angstroms. The kinetic properties did not differ significantly from the wild type enzyme.
  • the effective synthesis over hydrolysis ratio is a delicate balance of the specificity of the ⁇ -AEH for the acyl side chain donor, the nucleophile and the water and ultimately also the final acylation product.
  • the specificity of the acyl side chain of the precursor may be tuned by modification of the binding site for precursor acyl site chain. In the table below the interactions with the phenylglycine side chain of ampicillin have been mapped. Substitution of amino acids at the mapped positions not only improve the specificity for phenylglycine even further, but it also improves or shifts the specificity towards different side chains, e.g. p-hydroxylphenylglycine which is found in amoxycillin and cephadroxyl.
  • Table 19 Positions in ⁇ -AEH which allows for optimising S/H ratio's with different acyl side chain donors.
  • the acyl intermediate has formed the competition between water (hydrolysis) and the nucleus (the acceptor) will be dependant very much on how well the acceptor can be positioned towards a nucleophilic attack of the acyl intermediate with respect to water.
  • the orientation of the nucleophile with respect to the transition state can be improved by mutagenesis at the positions given below.
  • Table 20 Positions in ⁇ -AEH which allows for optimising S/H ratio's with different nucleophiles.

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Abstract

The present invention relates to recombinant alpha-amino ester hydrolases (AEH) and uses thereof. The present invention also relates to isolated nucleic acids encoding said recombinant alpha-amino ester hydrolases or functional equivalents thereof. Cells containing heterologous nucleic acids encoding alpha-amino ester hydrolases or vectors containing nucleic acids encoding alpha-amino ester hydrolases are also part of the invention. In particular the invention provides cloned AEH genes isolated from A. turbidans, A. pasteurianus, X citri, Z. mobilis and X. fastidiosa. The invention also provides mutant AEH genes expressing AEHs from A. turbidans with improved synthesis to hydrolysis ratios.

Description

RECOMBINANT ALPHA-AMINO ESTER HYDROLASES AND USES THEREOF
Field of the invention The present invention relates to recombinant alpha-amino ester hydrolases and uses thereof. The present invention also relates to isolated nucleic acids encoding said recombinant alpha-amino ester hydrolases or functional equivalents thereof. Cells containing heterologous nucleic acids encoding alpha-amino ester hydrolases or vectors containing nucleic acids encoding alpha-amino ester hydrolases are also part of the invention. In particular, mutants of the wild type sequence with an improved synthesis over hydrolysis ratio are part of the invention. Description of the related art β-Lactam antibiotic compounds constitute the most important group of antibiotic compounds, with a long history of clinical use. Among this group, the prominentβ- lactam antibiotic compounds are the penicillins and cephalosporins. In general, β- lactam antibiotic compounds, or, in short, β-lactam antibiotics consist of a nucleus, the so-called amino β-lactam nucleus, which is linked through its primary amino group to the so-called side chain via a linear amide bond.
Presently, most of the β-lactam antibiotics used are prepared by semi-synthetic methods. These semi-synthetic β-lactam antibiotics are obtained by modifying an N- substituted β-lactam product by one or more chemical and/or enzymatic reactions. Typically, one of the reactions involved in the modification of the N-substitutedβ-lactam is acylation of the amino β-lactam nucleus, which has been obtained from the N- substituted β-lactam. Conventionally, β-lactam antibiotics have been prepared in chemical procedures. However, such chemical methods have a number of grave disadvantages. They comprise many complex reactions, wherein by-products are formed which give rise to effluent and purification problems. Also, because many steps have to be performed in order to obtain the final antibiotic product, the overall yield is quite low. Recently, there has been a lot of interest for so-called enzymatic semi-synthetic routes to β-lactam antibiotics. These routes involve enzymatically-catalyzed processes and lack many of the disadvantages of the conventional synthetic methods for preparing β-lactam antibiotics. The enzymatically-catalyzed reactions are highly selective, thus the production of many by-products, and the effluent and purification problems, which result therefrom, are reduced or avoided. Furthermore, enzymatic processes can be performed in aqueous environment.
The semi-synthetic routes mostly start from fermentation products such as isopenicillin N, penicillin G, penicillin V and cephalosporin C, which products are enzymatically converted to an amino β-lactam nucleus, for instance in a manner as has been disclosed in Bioprocess. Technol., 16, 67-88 (1993) by K. Matsumoto, in Process Biochemistry, 146-154 (1989) by J.G. Shewale & H. Sivaraman, in Biotechnology of Industrial Antibiotics (Ed. E.J. Vandamme) Marcel Dekker, New York (1984) by T.A. Savidge, or in Process Biochemistry International, June, 97-103 (1990) by J.G.
Shewale et al. The obtained amino β-lactam nucleus is subsequently converted to the desired β-lactam antibiotic by coupling the nucleus to a suitable side chain, as has been described in inter alia German patent application No. DE 2163792, Dutch patent No. NL 158847 B, German patent No. DE 2621618 C, European patent No. EP339751 B and International patent applications No. WO 9201061 and WO 9312250. Enzymatic acylation of an amino β-lactam nucleus with a side chain precursor which is a carbonyl- activated derivative (i.e. amide or alkyl ester) of a side chain acid has also been described in Biochem. J. (1969) 747-756 by M. Cole and in Ann. N.Y. Acad. Sci. 99- 105 (1996) by V. Kasche et al, Industrial transformations of penicillins and cephalopsporins, J.Verweij & E de Vroom, 1993, Reel Trav Chim Pays Bas 112, 66-81 ; Penicillin Acylase in Indiustrial Production of β-lactam antibiotics, A.Bruggink, E.C.Roos, E de Vroom, Organic Progress Research & Development, 1998, vol 2, 2, 128-133; Innovations in Cephalosporins and Penicillin Production: Painting the Antibiotics Industry Green, E.J.A.X van de Sandt & E de Vroom, Chimica Oggi 2000, 18, 72-75; E.de Vroom, The central role of penicillin acylase in antibiotics production, Chimica Oggi / Chemsitry today 1999, Nov/Dec pages 65-68.
By making different combinations of side chains and amino β-lactam nuclei, a variety of penicillin and cephalosporin antibiotics may be obtained. For example, a D-(-)-phenylglycine side chain may be attached to a 6-aminopenicillanic acid (6-APA) nucleus, in a reaction with a suitable derivative of said D-(- phenylglycine, to yield ampicillin, or to a 7-aminodesacetoxycephalosporanic acid (7-ADCA) nucleus to yield cephalexin.
A disadvantage of the known methods is that the coupling reaction starts from an amino β-lactam nucleus, which has been isolated prior to the coupling reaction. During the isolation of the amino β-lactam nucleus, which is usually performed by crystallization, up to about 10% of the theoretical yield is lost. Due to the amphoteric nature of the β-lactam nucleus, it dissolves readily in an aqueous environment at any pH value. As a result a great part of the production of the β-lactam nucleus is lost in the mother-liquor resulting from the crystallization.
In the International patent application WO 98/48038, a process for preparing a β-lactam antibiotic is described wherein the antibiotic is prepared from a mixture in which a N-substituted β-lactam a dicarboxylate acylase and a penicillin acylase and a desired side chain precursor. The process includes the in-situ preparation of a β-lactam nucleus which is formed when the N-subtituted β-lactam is liberated enzymatically from its side chain. In WO 98/48038, however, the N-substitutedβ-lactam starting material is limited to a β-lactam compounds containing a succinyl, glutaryl or an adipyl or analogous thereof as the N-substituent.
In search for microorganisms applicable in the biocatalytic production of semisynthetic antibiotics, Acetobacter turbidans ATCC 9325, X. citri IFO 3835 and A. pasteurianus ATCC 6033 were first described in 1972 by Takahashi etal as organisms able to synthesise cephalosporins. Since only α-amino acid derivatives act as a substrate and due to the preference for esters over amides, the enzyme involved was named alpha-amino acid ester or α-amino-acid ester hydrolase (abbreviated as AEH or α-AEH) (Takahashi, et al., Biochem. J., (1974) 137; 497-503).
AEH (E.C. 3.1.1.43) activity is defined as the ability of an enzyme to catalyse the following general reaction scheme: α-amino acid ester + Nucleophile- α-amino acid acyl derivative + Alcohol
AEH activity has been described for several organisms. In particular the enzymes are able to catalyse the transfer of the alpha-amino-acyl group from alpha- amino acid esters to amine nucleophiles, which is more commonly referred to as synthesis or transferase reaction, where the nucleophile may belong to the group of 7- aminocephem or 6-amino penams. In addition the enzymes may transfer the acyl group to water which is commonly referred to as the hydrolysis reaction. Presumably an acyl-enzyme intermediate is involved in this transfer reaction
(Takahashi, et al., Biochem. J., (1974) 137; 497-503, Blinkovsky and Markaryan, Enzyme Microb. Technol., (1993) 15; 965-973). The-AEHs show biocatalytic properties for the enzymatic synthesis of the semi-synthetic β-lactam antibiotics using esters as the acyl side chain precursors. Due to the preference for esters, higher product accumulation can be reached in a synthesis reaction compared to the penicillin G acylase. (Takahashi, et al., Biochem. J., (1974) 137; 497-503, Ryu and Ryu, Enzyme Microb. Technol., (1988) 10; 239-245, Hernandez-Jύstiz, et al., Enz. Microbiol. Tech., (1999) 25; 336-343). Moreover, the stabilised AEH of A turbidans showed high specificity toward the D-form of phenylglycine methyl ester (PGM, the activated acyl donor). This enables an ampicillin synthesis starting from a racemic mixture of acyl donors, which is not feasible with the E. coli penicillin acylase (Femandez-Lafuente, et al., J. Mol. Catal., (2001) 11; 633-638). In contrast to penicillin acylase from E. colj ti was claimed that the α-amino acid ester hydrolases are able to accept charged substrates (Blinkovsky and Markaryan, Enzyme Microb. Technol., (1993) 15; 965-973). The lower pH-optimum of the α-amino acid ester hydrolases, pH 6 compared to optimum pH of 7.5-8 for penicillin G acylases (Kutzbach and Rauenbusch, Hoppe Seylers Z. Physiol. Chem., (1974) 355; 45-53), is also interesting for biocatalytic applications. The α-AEHs are not inhibited by phenylacetic acid (Blinkovsky and Markaryan, Enzyme Microb. Technol., (1993) 15; 965-973), a side product from the hydrolysis of penicillin G. This makes it possible to use a reaction mixture containing 6-aminopenicillanic acid and phenylacetic acid resulting from the cleavage of penicillin G by K. citrophila or E. coli acylase as proposed for the α-AEH of Pseudomonas melanogenum (Okachi and Nara, Agr. Biol. Chem., (1973) 37; 2979-2804). The structural characterisation of the α-AEHs is limited to the determination of the subunit size. The α-AEHs from Acetobacter turbidans ATCC9325 (Takahashi, et al., Biochem. J., (1974) 137; 497-503, Ryu and Ryu, Enzyme Microb. Technol., (1987) 9; 339-344), from Xanthomonas citri IFO 3835 (Kato, et al., Agric. Biol. Chem., (1980) 44; 1069-1074) and from Pseudomonas melanogenum IFO 12020 (Kim and Byun, Biochim. Biophys. Acta, (1990) 1040; 12-18) have been purified. All three enzymes have similar subunit sizes. However, there is some dissimilarity in the native subunit composition; A turbidans is described as a heterodimer,α2β2 with subunits of 70 en 72 kD (Ryu and Ryu, Enzyme Microb. Technol., (1987) 9; 339-344), X. citri is shown to be a homotetramer, α4, with subunits of 72 kD (Kato, et al., Agric. Biol. Chem., (1980) 44; 1069-1074). P. melanogenum is described as a homodimer, α_>, with subunits of 72 kD as well (Kim and Byun, Biochim. Biophys. Acta, (1990) 1040; 12-18)).
The fermentation of AEH producing organisms is quite cumbersome and the subsequent recovery of the α-AEH's is difficult because the expression levels of the desired AEH's are very low with respect to total protein (Takahashi, et al., Biochem. J., (1974) 137; 497-503, Ryu and Ryu, Enzyme Microb. Technol., (1987) 9; 339-344, Kato, et al., Agric. Biol. Chem., (1980) 44; 1069-1074, Kim and Byun, Biochim. Biophys. Acta, (1990) 1040; 12-18). The recovery and isolation procedures are rather elaborate and comprehend 4-5 steps, which makes it also a time-consuming procedure. The different values reported for the specific activity do suggest that further purification might be required in some cases.
For the above reasons, a strong desire exists in the art to clone and recombinantly express AEHs in overexpressing organisms. Despite of several attempts this goal has not yet been achieved. Illustrative examples are the unsuccessful cloning attempts of AEH from both A turbidans and X. citri gene (Alonso and Garcia,
Microbiology, (1996) 142; 2951-2957, Nam and Ryu, Kor. J. Appl. Microbiol. Bioeng., (1988) 16; 363-368). For A turbidans a polyclonal antibody against purified α-AEH (Ryu and Ryu, Enzyme Microb. Technol., (1987) 9; 339-344) was used for immunochemical detection of a λgtl 1 genomic library. Two positive clones were found which produced a protein of 105 and 180 kD. However, these clones appeared to be false-positives because they did not encode a protein with α-AEH activity. The attempt to clone the α-AEH of X. citri involved a pBR322 chromosomal library which was screened for growth on D-alanyl-L-leucine. This method is based on the auxotrophic complementation of the E. coli leuB mutant using a minimal medium with D-alanyl-L- leucine as sole source of L-leucine. One clone capable to grow on this medium was found. However this clone displayed no hydrolysis activity with ampicillin. Sequence analysis and homology searches showed that the cloned gene encoded a proline iminopeptidase.
Accordingly, it is an object of the present invention to provide recombinant alpha-amino ester hydrolases. It is also an object of the present invention to provide recombinant alpha-amino ester hydrolases with an improved synthesis/hydrolysis ratio. Summary of the invention
The invention relates to recombinantly produced alpha-amino ester hydrolases. The nucleic acid sequences provided herein may be used in any art-known method to transform a micro-organism and subsequently the transformed micro-organism may be used to produce an alpha-amino ester hydrolase. The nucleic acid sequences provided herein may also be used to readily find homologous sequences in other organisms that encode variant alpha-amino ester hydrolases. In addition the nucleic acid sequences provided herein may also be used to identify homologues sequences with unknown function as being an α-AEH. These homologous sequences may then in turn be used to transform other micro-organisms to express the homologous alpha-amino ester hydrolase of said other organism.
The invention also relates to recombinant vectors comprising a nucleic acid sequence acording to the invention as well as a method of manufacturing any of the above nucleic acids.
The invention also relates to recombinantly produced AEH, in particular an isolated polypeptide selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10. Variants, homologues and fragments of these polypeptides are also part of the invention.
In particular, nucleic acids encoding mutant AEHs with improved synthesis over hydrolysis ratios are part of the invention. Detailed description of the invention
In a first embodiment, the invention provides isolated nucleic acids preferably encoding functionally active AEH. The cloning of the gene encoding theα-AEH (aeh gene) allows us to optimise expression and purification by placing the gene in front of Myc-epitope and a 6xHis-tag under control of an araBAD promoter. In this way the expression was improved by a factor 10. The construct is easy to cultivate to high optical densities and on a relatively cheap and simple medium (Luria Broth). This is in contrast to the medium described in the literature for A. pasteurianus.A. turbidans and X. citri which contains thioglycollate, glycerol and glucose (Takahashi, et al., J Am. Chem. Soc, (1972) 94; 4035-7). Additionaly, higher optical density can be reached, especially in comparison to A pasteurianus. Using the pET28 vector, placing a His-tag directly C-terminal (without the Myc-epitope in front of it), resulted in inactive protein under several induction conditions. This indicates that the/Wyc-epitope (15 amino acids) probably functions as a linker, rendering a certain distance necessary for the attachment of the 6xHis tag without losing theα-AEH activity. The purification (up to 80% pure) is now a one-step procedure with use of Ni-agarose. In general this purity is sufficient for the majority of applications, reducing the intensive purification procedures described in the literature from 5 to a single step procedure. When required further purification can be done by an additional gel filtration step.
The ae 7-gene provides a tool to attribute a function to other proteins with yet unknown function. In this way we discovered that the gene of Zymomonas mobilis ZM4 which was described as a putative glutaryl acylase was in fact an AEH. The sequence of the Z. mobilis gene and gene product is shown in SEQ ID NO: 4 and SEQ ID NO: 9 respectively. Cloning of this gene and initial synthesise experiments indicated that this enzyme is able to synthesis cefalexine from phenylglycine methyl ester and 7-ADCA.
Furthermore, the cloning allows us to modify the enzyme at DNA level to produce mutant α-AEHs with different, even more desired, properties. As is demonstrated herein, α-AEHs having a higher activity for esters than amide have now been further optimised to reduce the unwanted hydrolysis of the antibiotic during synthesis.
The invention provides an isolated polynucleotide encoding a functional alpha- amino ester hydrolase with a S/H ini ratio higher than 1.9, preferably higher than 2, 2.5, 3.0, 4.5, 5, 8, 10 and most preferably higher than 15. It also provides an isolated polynucleotide encoding a functional alpha-amino ester hydrolase with a Q max (mM) ratio higher than 8, preferably higher than 10, 12 and most preferably higher than 15. Also provided is an isolated polynucleotide encoding a functional alpha-amino ester hydrolase with a S/H max ratio higher than 1.2, preferably higher than 2, 2.5, 3.0, 4.5, 5, 8, 10 and most preferably higher than 15.
The cloning of the α-AEH of the plant pathogen X. citri has an additional advantage since it allows purification of its α-AEH from a non-pathogenic host .
As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules which may be isolated from chromosomal DNA, which include an open reading frame encoding a protein having alpha-amino ester hydrolase activity. A gene may include coding sequences, non-coding sequences, introns and regulatory sequences. Moreover, a gene refers to an isolated nucleic acid molecule as defined herein.
A nucleic acid molecule of the present invention, such as a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1 , SEQ ID NO: 2 , SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO: 5 or functional equivalents thereof, can be isolated using standard molecular biology techniques using the sequence information provided herein. For example, using all or portion of the nucleic acid sequence of SEQ ID NO: 1 as a hybridization probe, nucleic acid molecules according to the invention can be isolated using standard hybridization and cloning techniques from a variety of organisms (e. g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
Moreover, a nucleic acid molecule encompassing all or a portion of SEQ ID NO:1 , SEQ ID NO: 2 , SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO: 5 can be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence information provided herein.
A nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid thus amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
Furthermore, oligonucleotides corresponding to or hybridisable to nucleotide sequences according to the invention can be prepared by standard synthetic techniques, e. g., using an automated DNA synthesizer.
In a preferred embodiment, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO:1 , SEQ ID NO: 2 , SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO: 5. The sequence of SEQ ID NO: 1 corresponds to the coding region of the A. turbidans alpha-amino ester hydrolase cDNA.,SEQ ID NO: 2 is an AEH gene from A. pasteurianus, SEQ ID NO:3 is an AEH gene from X. citri, SEQ ID NO:4 is an AEH gene from Z. mobilis and SEQ ID NO: 5 is an AEH gene form X fastidiosa. These cDNAs comprise sequences encoding the alpha-amino ester hydrolases according to SEQ ID NO:6, SEQ ID NO: 7 , SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 respectively. In another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NO: 1 , SEQ ID NO: 2 , SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 or functional equivalents thereof.
A nucleic acid molecule which is complementary to another nucleotide sequence is one which is sufficiently complementary to the other nucleotide sequence such that it can hybridize to the other nucleotide sequence thereby forming a stable duplex.
One aspect of the invention pertains to isolated nucleic acid molecules that encode a polypeptide of the invention or a functional equivalent thereof such as a biologically active fragment or domain, as well as nucleic acid molecules sufficient for use as hybridisation probes to identify nucleic acid molecules encoding a polypeptide of the invention and fragments of such nucleic acid molecules suitable for use as PCR primers for the amplification or mutation of nucleic acid molecules.
An "isolated polynucleotide" or "isolated nucleic acid" is a DNA or RNA that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. Thus, in one embodiment, an isolated nucleic acid includes some or all of the 5' non-coding (e.g., promotor) sequences that are immediately contiguous to the coding sequence. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding an additional polypeptide that is substantially free of cellular material, viral material, or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicafs (when chemically synthesized). Moreover, an "isolated nucleic acid fragment" is a nucleic acid fragment that is not naturally occurring as a fragment and would not be found in the natural state. As used herein, the terms "polynucleotide" or "nucleic acid molecule" are intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. The nucleic acid may be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.
Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to an alpha-amino ester hydrolases nucleic acid molecule, e.g., the coding strand of an alpha-amino ester hydrolases nucleic acid molecule. Also included within the scope of the invention are the complement strands of the nucleic acid molecules described herein.
Sequencing errors
The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The specific sequences disclosed herein can be readily used to isolate the complete gene from other micro-organisms or other strains or the starins used herein which in turn can easily be subjected to further sequence analyses thereby identifying sequencing errors. Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion. The person skilled in the art is capable of identifying such erroneously identified bases and knows how to correct for such errors.
Nucleic acid fragments, probes and primers
A nucleic acid molecule according to the invention may comprise only a portion or a fragment of the nucleic acid sequence shown in SEQ ID NO:1 , SEQ ID NO: 2 , SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, for example a fragment which can be used as a probe or primer or a fragment encoding a portion of an alpha-amino ester hydrolase protein. The nucleotide sequence determined from the cloning of the alpha- amino acid hydrolase genes and cDNAs allows for the generation of probes and primers designed for use in identifying and/or cloning other alpha-amino acid hydrolase family members, as well as alpha-amino acid hydrolase homologues from other species. The probe/primer typically comprises substantially purified oligonucleotide which typically comprises a region of nucleotide sequence that hybridizes preferably under highly stringent conditions to at least about 12 or 15, preferably about 18 or 20, preferably about 22 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 or more consecutive nucleotides of a nucleotide sequence shown in SEQ ID NO:1 , SEQ ID NO: 2 , SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 or functional equivalents thereof.
Probes based on the alpha-amino acid hydrolase nucleotide sequences can be used to detect alpha-amino acid hydrolase sequences encoding the same or homologous proteins for instance in other organisms. In preferred embodiments, the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. Such probes can also be used as part of a diagnostic test kit for identifying cells that express an alpha-amino acid hydrolase protein. Identity & homology
The terms "homology" or "percent identity" are used interchangeably herein. For the purpose of this invention, it is defined here that in order to determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions/total number of positions (i.e. overlapping positions) x 100). Preferably, the two sequences are the same length.
The skilled person will be aware of the fact that several different computer programms are available to determine the homology between two sequences. For instance, a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.qcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.
As an alternative, the percent identity between two nucleotide sequences may be determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1 , 2, 3, 4, 5, or 6. Alternatively, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989) which has been incorporated into the ALIGN program (version 2.0) (available at http://vega/igh.cnrs.fr/bin/align- quess.cgi), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403 — 10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to alpha-amino acid hydrolase nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to alpha-amino acid hydrolase protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
Hybridization As used herein, the term "hybridizing" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least about 40%, 50%, at least about 60%, at least about 70%, more preferably at least about 80%, even more preferably at least about 85% to 90%, more preferably at least 95% homologous to each other typically remain hybridized to each other. A preferred, non-limiting example of such hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45 °C, followed by one or more washes in 1 X SSC, 0.1 % SDS at 50 °C, preferably at 55 °C, preferably at 60 °C and even more preferably at 65 °C.
Highly stringent conditions include, for example, hybridizing at 68°C in 5x SSC/5x Denhardt's solution/l.0% SDS and washing in 0.2x SSC/0.1% SDS at room temperature. Alternatively washing may be performed at 42°C.
The skilled artisan will know which conditions to apply for stringent and highly stringent hybridisation conditions. Additional guidance regarding such conditions is readily available in the art, for example, in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; and Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology, (John Wiley & Sons, N.Y.).
Of course, a polynucleotide which hybridizes only to a poly A sequence (such as the 3' terminal poly(A) tract of mRNAs), or to a complementary stretch of T (or U) resides, would not be included in a polynucleotide of the invention used to specifically hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule contain a poly (A) stretch or the complement thereof (e.g., practically any double-standed cDNA clone).
Obtaining full length DNA from other organisms
In a typical approach, DNA libraries constructed from other organisms, e.g. belonging to the proteobacteria can be screened.
For example, strains of proteobacteria can be screened for homologous alpha- amino acid hydrolase polynucleotides by Northern blot analysis. Upon detection of transcripts or genomic polynucleotides homologous to polynucleotides according to the invention, DNA libraries can be constructed from the appropriate strain, utilizing standard techniques well known to those of skill in the art. Alternatively, a total genomic DNA library or cDNA library of a eukaryotic organism can be screened using a probe hybridisable to an alpha-amino acid hydrolase polynucleotide according to the invention.
Homologous gene sequences can be isolated, for example, by performing PCR using two degenerate oligonucleotide primer pools designed on the basis of nucleotide sequences as taught herein.
The template for the reaction can be cDNA obtained by reverse transcription of mRNA prepared from eukaryotic strains known or suspected to express a polynucleotide according to the invention. The PCR product can be subcloned and sequenced to ensure that the amplified sequences represent the sequences of a new alpha-amino acid hydrolase nucleic acid sequence, or a functional equivalent thereof.
The PCR fragment can then be used to isolate a full length cDNA clone by a variety of known methods. For example, the amplified fragment can be labeled and used to screen a bacteriophage or cosmid cDNA library. Alternatively, the labeled fragment can be used to screen a genomic library.
PCR technology also can be used to isolate full length cDNA sequences from other organisms. For example, RNA can be isolated, following standard procedures, from an appropriate cellular or tissue source. A reverse transcription reaction can be performed on the RNA using an oligonucleotide primer specific for the most 5' end of the amplified fragment for the priming of first strand synthesis. The resulting RNA/DNA hybrid can then be "tailed" (e.g., with guanines) using a standard terminal transferase reaction, the hybrid can be digested with RNase H, and second strand synthesis can then be primed (e.g., with a poly-C primer). Thus, cDNA sequences upstream of the amplified fragment can easily be isolated. For a review of useful cloning strategies, see e.g..Sambrook et al., supra; and Ausubel et al., supra.
Vectors
Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding an alpha-amino acid hydrolase protein or a functional equivalent thereof. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. The terms "plasmid" and "vector" can be used interchangeably herein as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vector includes one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operatively linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signal). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in a certain host cell (e.g. tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, encoded by nucleic acids as described herein (e.g. alpha-amino acid hydrolase proteins, mutant forms of alpha- amino acid hydrolase proteins, fragments, variants or functional equivalents thereof, fusion proteins, etc.).
The recombinant expression vectors of the invention can be designed for expression of alpha-amino acid hydrolase proteins in prokaryotic or eukaryotic cells. For example, alpha-amino acid hydrolase proteins can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells, fungal cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression vectors useful in the present invention include chromosomal-, episomal- and virus-derived vectors e.g., vectors derived from bacterial plasmids, bacteriophage, yeast episome, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. co//lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled person. In a specific embodiment, promoters are preferred that are capable of directing a high expression level of recombinant proteins. Such promoters are known in the art. The expression constructs may contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will include a translation initiating AUG at the beginning and a termination codon appropriately positioned at the end of the polypeptide to be translated.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of artfecognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-percipitation, DEAE-dextran-mediated transfection, transduction, infection, lipofection, cationic lipidmediated transfection or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2d,ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Davis et al., Basic Methods in Molecular Biology (1986) and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In σder to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methatrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding an alpha-amino acid hydrolase protein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g. cells that have incorporated the selectable marker gene will survive, while the other cells die).
Expression of proteins in prokaryotes is often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, e.g. to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1 ) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognation sequences, include Factor Xa, thrombin and enterokinase. As indicated, the expression vectors will preferably contain selectable markers.
Such markers include dihydrofolate reductase or neomycin resistance for eukarotic cell culture and tetracyline or ampicilling resistance for culturing in E. co//and other bacteria. Representative examples of appropriate host include bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS and Bowes melanoma; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
Among vectors preferred for use in bacteria are pQE70, pQE60 and PQE-Θ, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16A, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Among preferred eukaryotic vectors are PWLNEO, pSV2CAT, pOG44, pZT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan. Among known bacterial promotors for use in the present invention include £. coli lacl and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR, PL promoters and the trp promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus ("RSV"), and metallothionein promoters, such as the mouse metallothionein-l promoter.
Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovims early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretation signal may be incorporated into the expressed polypeptide. The signals may be endogenous to the polypeptide or they may be heterologous signals.
The polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals but also additional heterologous functional regions. Thus, for instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification.
Polypeptides according to the invention The invention provides an isolated polypeptide having the amino acid sequence according to SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:9 or SEQ ID NO: 10, or an amino acid sequence obtainable by expressing the polynucleotides of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4 or SEQ ID NO: 5 in an appropriate host. Also, a peptide or polypeptide comprising a functional equivalent of the above polypeptides is comprised within the present invention. The above polypeptides are collectively comprised in the term "polypeptides according to the invention"
The terms "peptide" and "oligopeptide" are considered synonymous (as is commonly recognized) and each term can be used interchangeably as the context requires to indicate a chain of at least two amino acids coupled by peptidyl linkages. The word "polypeptide" is used herein for chains containing more than seven amino acid residues. All oligopeptide and polypeptide formulas or sequences herein are written from left to right and in the direction from amino terminus to carboxy terminus. The one-letter code of amino acids used herein is commonly known in the art and can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2"d,ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989)
By "isolated" polypeptide or protein is intended a polypeptide or protein removed from its native environment. For example, recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the invention as are native or recombinant polypeptides which have been substantially purified by any suitable technique such as, for example, the single-step purification method disclosed in Smith and Johnson, Gene 67:31-40 (1988).
The alpha-amino acid hydrolase according to the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification.
Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host- mediated processes.
Moreover, a protein according to the invention may be a precursor protein containing a leader sequence, a hybrid protein, a protein obtained as a pro sequence or pre-pro sequence, or any other type of immature form.
Protein fragments
The invention also features biologically active fragments of the polypeptides according to the invention. Biologically active fragments of a polypeptide of the invention include polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the alpha-amino acid hydrolase protein (e.g., the amino acid sequences of SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:9 or SEQ ID NO: 10), which include fewer amino acids than the full length protein, and exhibit at least one biological activity of the corresponding full-length protein. Typically, biologically active fragments comprise a domain or motif with at least one activity of the alpha-amino acid hydrolase protein. A biologically active fragment of a protein of the invention can be a polypeptide. which is, for example, 10, 25, 50, 100 or more amino acids in length. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the biological activities of the native form cf a polypeptide of the invention.
The invention also features nucleic acid fragments which encode the above biologically active fragments of the alpha-amino acid hydrolase protein. Fusion proteins
The proteins of the present invention or functional equivalents thereof, e.g., biologically active portions thereof, can be operatively linked to a non-alpha-amino acid hydrolase polypeptide (e.g., heterologous amino acid sequences) to form fusion proteins. As used herein, an alpha-amino acid hydrolase "chimeric protein" or "fusion protein" comprises an alpha-amino acid hydrolase polypeptide operatively linked to a non-alpha-amino acid hydrolase polypeptide. An"alpha-amino acid hydrolase polypeptide" refers to a polypeptide having an amino acid sequence corresponding to alpha-amino acid hydrolase, whereas a "non-alpha-amino acid hydrolase polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the alpha-amino acid hydrolase protein, e.g., a protein which is different from the alpha-amino acid hydrolase protein and which is derived from the same or a different organism. Within an alpha-amino acid hydrolase fusion protein the alpha-amino acid hydrolase polypeptide can correspond to all or a portion of an alpha-amino acid hydrolase protein. In a preferred embodiment, an alpha- amino acid hydrolase fusion protein comprises at least one biologically active fragment of an alpha-amino acid hydrolase protein. In another preferred embodiment, an alpha- amino acid hydrolase fusion protein comprises at least two biologically active portions of an alpha-amino acid hydrolase protein. Within the fusion protein, the term "operatively linked" is intended to indicate that the alpha-amino acid hydrolase polypeptide and the non-alpha-amino acid hydrolase polypeptide are fused in-frame to each other. The non-alpha-amino acid hydrolase polypeptide can be fused to the N- terminus or C-terminus of the alpha-amino acid hydrolase polypeptide or the non- alpha-amino hydrolase polypeptide can be inserted at a suitable position in the alpha- amino acid hydrolase polypeptide.
For example, in one embodiment, the fusion protein is a GST-alpha-amino acid hydrolase fusion protein in which the alpha-amino acid hydrolase sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant alpha-amino acid hydrolase. In another embodiment, the fusion protein is an alpha-amino acid hydrolase protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian and Yeast host cells), expression and/or secretion of alpha-amino acid hydrolase can be increased through use of a hetereologous signal sequence.
In another example, the gp67 secretory sequence of the baculovirus envelope protein can be used as a heterologous signal sequence (Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, 1992). Other examples of eukaryotic heterologous signal sequences include the secretory sequences of melittin and human placental alkaline phosphatase (Stratagene; La Jolla, California). In yet another example, useful prokarytic heterologous signal sequences include the phoA secretory signal (Sambrook et al., supra) and the protein A secretory signal (Pharmacia Biotech; Piscataway, New Jersey).
A signal sequence can be used to facilitate secretion and isolation of a protein or polypeptide of the invention. Signal sequences are typically characterized by a core of hydrophobic amino acids which are generally cleaved from the mature protein during secretion in one or more cleavage events. Such signal peptides contain processing sites that allow cleavage of the signal sequence from the mature proteins as they pass through the secretory pathway. The signal sequence directs secretion of the protein, such as from a eukaryotic host into which the expression vector is transformed, and the signal sequence is subsequently or concurrently cleaved. The protein can then be readily purified from the extracellular medium by art recognized methods. Alternatively, the signal sequence can be linked to the protein of interest using a sequence which facilitates purification, such as with a GST domain. Thus, for instance, the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide, which facilitates purification of the fused polypeptide. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa- histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described in Gentz et al, Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purificaton of the fusion protein. The HA tag is another peptide useful for purification which corresponds to an epitope derived of influenza hemaglutinin protein, which has been described by Wilson et al., Cell 37:767 (1984), for instance.
Preferably, an alpha-amino acid hydrolase chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g, a GST polypeptide). An alpha-amino acid hydrolase-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the alpha- amino acid hydrolase protein.
Functional equivalents The terms "functional equivalents" and "functional variants" are used interchangeably herein. Functional equivalents of alpha-amino acid hydrolase DNA are isolated DNA fragments that encode a polypeptide that exhibits a particular function of the alpha-amino acid hydrolase as defined herein. A functional equivalent of an alpha- amino acid hydrolase polypeptide according to the invention is a polypeptide that exhibits at least one function of an alpha-amino ester hydrolase as defined herein. Functional protein or polypeptide equivalents may contain only conservative substitutions of one or more amino acids of SEQ ID NO: 2 or substitutions, insertions or deletions of non-essential amino acids. Accordingly, a non-essential amino acid is a residue that can be altered in SEQ ID NO: 2 without substantially altering the biological function. Amino acids conserved among the alpha-amino acid hydrolase proteins according to the present invention and related enzymes are not likely to be amenable to alteration.
The term "conservative substitution" is intended to mean that a substitution in which the amino acid residue is replaced with an amino acid residue having a similar side chain. These families are known in the art and include amino acids with basic side chains (e.g. lysine, arginine and hystidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagines, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine).
Functional nucleic acid equivalents may typically contain silent mutations or mutations that do not alter the biological function of encoded polypeptide. Accordingly, the invention provides nucleic acid molecules encoding alpha-amino acid hydrolase proteins that contain changes in amino acid residues that are not essential for a particular biological activity. Such alpha-amino acid hydrolase proteins differ in amino acid sequence from SEQ ID NO: 2 yet retain at least one biological activity. In one embodiment the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises a substantially homologous amino acid sequence of at least about more than 40%, 50%, 60%, 65%, 70%, 75%, 80%, 857o, 90%, 95%, 96%, 97%, 98%, 99% or more homologous to the amino acid sequence shown in SEQ ID NO: 2.
For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie, J.U. et al., Science 247:1306-1310 (1990) wherein the authors indicate that there are two main approaches for studying the tolerance of an amino acid sequence to change. The first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection. The second approach uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene and selects or screens to identify sequences that maintain functionality. As the authors state, these studies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which changes are likely to be permissive at a certain position of the protein. For example, most buried amino acid residues require non-polar side chains, whereas few features of surface side chains are generally conserved. Other such phenotypically silent substitutions are described in Bowie et al, supra, and the references cited therein. An isolated nucleic acid molecule encoding an alpha-amino acid hydrolase protein homologous to the protein according to SEQ ID NO: 2 can be created by introducing one or more nucleotide substitutions, additions or deletions into the coding nucleotide sequences according to SEQ ID NO: 1 such that one or more amino acid substitutions, deletions or insertions are introduced into the encoded protein. Such mutations may be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
The term "functional equivalents" also encompasses orthologues of the alpha- amino acid hydrolase proteins from other organisms than the ones described described herein. Orthologues of the alpha-amino acid hydrolase protein are proteins that can be isolated from other strains or species and possess a similar or identical biological activity. Such orthologues can readily be identified as comprising an amino acid sequence that is substantially homologous to SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:9 or SEQ ID NO: 10. As defined herein, the term "substantially homologous" refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., with similar side chain) amino acids or nucleotides to a second amino acid or nucleotide sequence such that the first and the second amino acid or nucleotide sequences have a common domain. For example, amino acid or nucleotide sequences which contain a common domain having about 40%, 50%, 60%, preferably 65%, more preferably 70%, even more preferably 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity or more are defined herein as substantially homologous.
Also, nucleic acids encoding other alpha-amino acid hydrolase family members, which thus have a nucleotide sequence that differs from SEQ ID NO:1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 are within the scope of the invention. Nucleic acid molecules corresponding to variants (e.g. natural allelic variants) and homologues of the alpha-amino acid hydrolase DNA of the invention can be isolated based on their homology to the alpha-amino acid hydrolase nucleic acids disclosed herein using the cDNAs disclosed herein or a suitable fragment thereof, as a hybridisation probe according to standard hybridisation techniques preferably under highly stringent hybridisation conditions.
In addition to naturally occurring allelic variants of the alpha-amino acid hydrolase sequence, the skilled person will recognise that changes can be introduced by mutation into the nucleotide sequences of SEQ ID NO:1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 thereby leading to changes in the amino acid sequence of the alpha-amino acid hydrolase protein without substantially altering the biological activity of the alpha-amino acid hydrolase protein.
In another aspect of the invention, improved alpha-amino acid hydrolase proteins are provided. Improved alpha-amino acid hydrolase proteins are proteins wherein at least one biological activity is improved. Such proteins may be obtained by randomly introducing mutations along all or part of the alpha-amino acid hydrolase coding sequence, such as by saturation mutagenesis, and the resulting mutants can be expressed recombinantly and screened for biological activity. For instance, the art provides for standard assays for measuring the enzymatic activity of alphaamino ester hydrolases and thus improved proteins may easily be selected. Improved mutants may also be obtained by targeting the mutagenesis to certain regions or just to certain residues which are important for modulation of catalytic functionality.
Furthermore, the data provided herein allow the skilled person to modify the enzyme at DNA level to produce mutantα-AEHs with different, even more desired, properties. For example, the property that the α-AEHs have a higher activity for esters than amide has been optimised to further reduce the unwanted hydrolysis of the antibiotic during synthesis.
An extended search for homologous proteins revealed homology of a certain region of α-AEH to the X-prolyl peptidases. These enzymes are serine proteases with the active-site serine located in the consensus sequence GxSYxG, where X is a non- conserved amino acid (Chich, et al., FEBS, (1992) 314; 139-142). Theα-AEH shows conservation of this motif and its direct surroundings, suggesting that the enzyme according to the invention is be a serine hydrolase.
In a preferred embodiment the alpha-amino acid hydrolase protein has an amino acid sequence according to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10. In another embodiment, the alpha-amino acid hydrolase polypeptide is substantially homologous to the amino acid sequence according to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 and retains at least one biological activity of a polypeptide according to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10, yet differs in amino acid sequence due to natural variation or mutagenesis as described above.
In a further preferred embodiment, the alpha-amino acid hydrolase protein has an amino acid sequence encoded by an isolated nucleic acid fragment capable of hybridising to a nucleic acid according to SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, preferably under highly stringent hybridisation conditions.
Accordingly, the alpha-amino acid hydrolase protein is a protein which comprises an amino acid sequence at least about 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous to the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 and retains at least one functional activity of the polypeptide according to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.
Functional equivalents of a protein according to the invention can also be identified e.g. by screening combinatorial libraries of mutants, e.g. truncation mutants, of the protein of the invention for alpha-amino ester hydrolase activity. In one embodiment, a variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level. A variegated library of variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential protein sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display). There are a variety of methods that can be used to produce libraries of potential variants of the polypeptides of the invention from a degenerate oligonucleotide sequence. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al.(1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11 :477) Methods for gene shuffling are known in the art, as for instance described in Stemmer, Nature, (1994) 370; 389-391.
In addition, libraries of fragments of the coding sequence of a polypeptide of the invention can be used to generate a variegated population of polypeptides for screening a subsequent selection of variants. For example, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment cf the coding sequence of interest with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal and internal fragments of various sizes of the protein of interest.
Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations of truncation, and for screening cDNA libraries for gene products having a selected property. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify variants of a protein of the invention (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327- 331 ).
In addition to the alpha-amino acid hydrolase gene sequence shown in SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 it will be apparent for the person skilled in the art that DNA sequence polymorphisms that may lead to changes in the amino acid sequence of the alpha-amino acid hydrolase protein may exist within a given population. Such genetic polymorphisms may exist in cells from different populations or within a population due to natural allelic variation. Allelic variants may also include functional equivalents.
Fragments of a polynucleotide according to the invention may also comprise polynucleotides not encoding functional polypeptides. Such polynucleotides may function as probes or primers for a PCR reaction. Such polynucleotides may also be useful when it is desired to abolish the functional activity of an alpha-amino acid hydrolase in a particular organism (knock-out mutants).
Nucleic acids according to the invention irrespective of whether they encode functional or non-functional polypeptides, can be used as hybridization probes or polymerase chain reaction (PCR) primers. Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having an alpha-amino acid hydrolase activity include, inter alia, (1) isolating the gene encoding the alpha-amino acid hydrolase protein, or allelic variants thereof from a cDNA library e.g. from other organisms than A. niger; (2) in situ hybridization (e.g. FISH) to metaphase chromosomal spreads to provide precise chromosomal location of the alpha-amino acid hydrolase gene as described in Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988); (3) Northern blot analysis for detecting expression of alpha-amino acid hydrolase mRNA in specific tissues and/or cells and 4) probes and primers that can be used as a diagnostic tool to analyse the presence of a nucleic acid hybridisable to the alpha-amino acid hydrolase probe in a given biological (e.g. tissue) sample.
Also encompassed by the invention is a method of obtaining a functional equivalent of an alpha-amino acid hydrolase gene or cDNA. Such a method entails obtaining a labelled probe that includes an isolated nucleic acid which encodes all or a portion of the sequence according to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 or a variant thereof; screening a nucleic acid fragment library with the labelled probe under conditions that allow hybridisation of the probe to nucleic acid fragments in the library, thereby forming nucleic acid duplexes, and preparing a full-length gene sequence from the nucleic acid fragments in any labelled duplex to obtain a gene related to the alpha-amino acid hydrolase gene.
In one embodiment, an alpha-amino acid hydrolase nucleic acid of the invention is at least 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homologous to a nucleic acid sequence shown in SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 or the complement thereof.
In another preferred embodiment an alpha-amino acid hydrolase polypeptide of the invention is at least 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homologous to the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.
Host cells
In another embodiment, the invention features cells, e.g., transformed host cells or recombinant host cells that contain a nucleic acid encompassed by the invention. A "transformed cell" or "recombinant cell" is a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a nucleic acid according to the invention.
The host cell may over-express the polypeptide, and techniques for engineering over-expression are well known. The host may thus have two or more copies of the encoding polynucleotide (and the vector may thus have two or more copies accordingly). Both prokaryotic and eukaryotic cells are included, e.g., bacteria, fungi, yeast, and the like, especially preferred are cells from Escherichia coli, Pichia pastoris, Kluyveromyces lactis, Aspergillus niger, Lactococcus lactis, and Bacillus subtilis. Bacteria from the genus Bacillus are very suitable as heterologous hosts because of their capability to secrete proteins into the culture medium. Other bacteria suitable as hosts are those from the genera Streptomyces and Pseudomonas. A preferred yeast host cell for the expression of the DNA sequence encoding the polypeptide is of the general. Saccharomyces, Kluyveromyces, Hansenula, Pichia, Yarrowia, and Schizosaccharc- myces. More preferably a yeast host cell is selected from the group consisting of the species Saccharomyces cerevisiae, Kluyveromyces lactis (also known as Kluyveromyces marxianus var. lactis), Hansenula polymorpha, Pichia pastoris, Yarrowia lipolytica.and Schizosaccharomyces pombe.
Preferred filamentous fungal host cells are selected from the group consisting of the genera Aspergillus, Trichoderma, Fusarium, Disporotrichum, Penicillium,
Acremonium, Neurospora, Thermoascus, Myceliophtora, Sporotrichum, Thielavia, and Talaromyces. A filamentous fungal host cell may belong to the species Aspergillus oyzae, Aspergillus sojae, Aspergillus nidulans, or to a species from the Aspergillus niger group. These include but are not limited to Aspergillus niger, Aspergillus awamori, Aspergillus tubingensis, Aspergillus aculeatus, Aspergillus foetidus, Aspergillus nidulans, Aspergillus japonicus, Aspergillus oryzae and Aspergillus ficuum, and further consisting of the species Trichoderma reesei, Fusarium graminearum, Penicillium chrysogenum, Acremonium alabamense, Neurospora crassa, Myceliophtora thermophilum, Sporotrichum cellulophilum, Disporotrichum dimorphosporum and Thielavia terrestris.
Examples of expression hosts within the scope of the present invention are gram negative bacteria such E.coli and Pseudomonas species; garm positive bacteria such as Bacillus species, e.g. Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens; fungi such as Aspergillus species and Trichoderma species; Pseudomonas species; and yeasts such as Kluyveromyces species, e.g.
Kluyveronmyces lactis and Saccharomyces species, e.g. Saccharomyces cerevisiae.
A host cell can be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in a specific, desired fashion. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may facilitate optimal functioning of the protein.
Various host cells have characteristic and specific mechanisms for post- translational processing and modification of proteins and gene products. Appropriate cell lines or host systems familiar to those of skill in the art of molecular biology and/or microbiology can be chosen to ensure the desired and correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such host cells are well known in the art.
Host cells also include, but are not limited to, mammalian cell lines such as CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and choroid plexus cell lines. If desired, the polypeptides according to the invention can be produced by a stably-transfected cell line. A number of vectors suitable for stable transfection of mammalian cells are available to the public, methods for constructing such cell lines are also publicly known, e.g., in Ausubel et al. (supra). Culture of host cells and recombinant production
The production of polypeptides of the invention can be effected by culturing of microbial expression hosts, which have been transformed with one or more polynucleotides of the present invention, in a conventional nutrient fermentation medium.
The recombinant host cells according to the invention may be cultured using procedures known in the art. For each combination of a promoter and a host cell culture conditions are available which allow for production of expression of the DNA sequence encoding the polypeptide of the invention. After reaching the desired cell density or titre of the polypeptide the culture is stopped and the polypeptide is recovered using known procedures. In this way the optimal combination of promoter and host for expression of the polypeptide of the invention may be established.
The fermentation medium may comprise a known culture medium containing a carbon source (e.g. glucose, maltose, molasses, etc), a nitrogen source (e.g. ammonium sulphate, ammonium nitrate, ammonium clored etc.), inorganic nutrient sources (e.g. phosphate, magnesium, maltodextrin, or xylogalacturonan) may be included.
The selection of the right medium may be based on choice of expression host or based on regulatory requirements of the expression. Such media are known to those skilled in the art. The medium may contain additional components favouring the transformed expression hosts over other potentially contaminating microorganisms. The proper control of the fermentation with respect to aeration and feed supply in combination with certain promoter / host combination can steer the expression of the polypeptide of the invention during the fermentation. E.g. by such control growth of the cells and expression of the desired protein can be uncoupled to certain extent, which is useful in cases where overexpression hinders the normal growth of the micro- organism.
The fermentation can be performed over a period of 0.5-30 days. It may be a batch, continuous or fed batch process, suitably at a temperature in the range of between 0 and 45°C and for example at a pH between 2 and 10. Preferred fermentation conditions are a temperature in the range of 20 and 37°C and/or a pH between 3 and 9. The appropriate conditions are usually selected based on choice of expression host and the protein expressed. In certain systems the fermentative production of enzymes may benefit from the addition of protease inhibitors which significantly reduce losses due to proteolysis.
After fermentation, if necessary, the cells may be killed. This is usually carried out by heat treatment or pH treatment. The use of additives such as for example detergents make such treatments more effective. Subsequently the cells can be removed from the fermentation broth by means procedures including, but not limited to centrifugation, filtration, extraction, spray-drying or precipitation. In those cases in which the polypeptide of the invention is not excreted, the producing cells have to be disrupted to enable purification of the protease. In such cases the collected cell mass is best ground with an abrasive, milled with beads, ultrasonicated or subjected to a French press or a Manton-Gaulin homogeniser and then filtered or centrifuged The use of filtration aids and flocculants may improve the results of centrifugation and filtration steps, in particular after grinding of the cells. Use of reversible protease inhibitors may increase the yields during recovery. Washing and concentration steps are commonly performed by ultrafiltration. For a further purification large scale chromatography modules may be applied as is known in the art. The final product of the recovery may be used as such but may also be formulated in order to prolong shelf-life or to adapt the product better to application conditions. The product may be in a liquid form or it my be part of a dry formulation. Coveniently the polypeptide of the invention is combined with suitable (solid or liquid) carriers or diluents including buffers to produce a composition . Thus the invention provides in a further aspect a composition comprising a polypeptide of the invention. This may be in a form suitable for packaging, transport and/or storage preferably where biological activity is retained. Compositions include paste, liquid, emulsion, powder, flake, granulate, pellet or other extrudate forms. The composition may further comprise additional ingredients such as one or more enzymes or proteins, preservatives, stabilisers.
The polypeptide may be attached to or mixed with a carrier, e.g. immobilized on a solid carrier. In addition the enzymes may also be encapsulated e.g. by entrapment into gel matrices such as polyacrylamide, alginte, sol-gel matrices, plastic materials. Also chemical modification may be used to modify performance. In particular chemical cross-linking may be used to stabilize enzymes in certain applications Aggregates of the enzyme may be cross-linked and used as such in bioconversions. As an alternative the enzymes may be crystallized and used as crystals which may be further stabilised by cross-linking.
The enzyme according to the invention is able to hydrolyse a broad substrate range. However, the amino group on the Cα-atom is essential. Although both esters and amides can be hydrolysed resulting in an α-amino acid and the corresponding alcohol or amine, the enzyme shows a strong preference for esters. In addition to the hydrolysis reaction the enzyme is able to transfer theα-amino acyl group to a number of amino nucleophiles, a property that can be used for the synthesis of semi-synthetic antibiotics. As used herein, an enzyme that is defined as an α-amino acid ester hydrolase catalyses the transfer of an α-amino-acid acyl group from a precursor to an amine nucleophile (the acceptor). Examples of suitable precursors are phenylglycine methylester (PGM) and hydroxyphenylglycine methyl ester (HPGM). Anα-amino acid ester hydrolase shows a high preference to hydrolyse the phenylglycine methyl ester (PGM) compared to the corresponding phenylacetic acid methyl ester (PAM) that lacks the α-amino group. The preference or specificity towards a certain substrate is clearly dependant both on the binding affinity for the substrate(s) and on the velocity at which the substrate(s) are converted. Therefore the preference of an enzyme for a certain substrate with respect to another substrate is indicated by the ratio of the kcat/Km value of the one substrate over that of the other substrate. The kcat/Km ratio is better known as the specificity constant (A.Fersht ,1977, Enzyme Structure and Mechanism, W.H. Freeman and Company). The higher the kcat/Km ratio, the more preferred the substrate is. In the specificity constant kcat represents the turn-over rate and Km represents an apparent dissociation constant. The kcat/Km ratio has a unit of a second order rate constant and is expressed as 1/(concentration*time). Assays that may be performed in order to recognize an enzyme as being an AEH are described in Example 1. Legends to the figures Figure 1. Ampicillin production at pH 7.2 and pH 6.2.
The ampicillin productions by the cell-free extracts of X. maltophilia, X. citri, B. megaterium, Achromobacter, A. turbidans, A. pasteurianus and E. coli were measured at pH 7.2 and pH 6.2. X. citri, A. turbidans and A. pasteurianus were found to produce significant amounts at both pH-values.
Synthesis was performed using cell free extracts (CFE) (with exception of E. coli from which a partially purified sample was used). The substrate concentrations were 20 mM 6-APA and 20 mM PGM in 83 mM phosphate buffer. Used volumes of CFE's corresponds to the following amount of protein, for X. citri (closed circle) 2,6 mg, X.maltophilia (open square): 2 mg, B. megaterium (triangle): 3 mg, Achromobacter (closed square) 0,6 mg, A. pasteurianus (plus): 0,48 mg, A. turbidans (open circle): 1 ,26 mg and for E. coli (X): 0.4 mg (0,7 NIPAB units).
Figure 2. A representative tree of the relative distance between the homologous proteins to the α-amino acid ester hydrolase of A turbidans ATCC 9325 as found by BLAST search. The distance can be read as number of nucleotide substitutions per site. In the circle the proteins which share 60% or more identity are situated. The tree was constructed using Clustal W and TreeView . EXAMPLES
Example 1 : AEH Assays
To identify an enzyme as being an AEH the following assay may be employed. Incubate an appropriate amount of enzyme with 30 mM of a nucleus and 15 mM acyl donor in 50 mM sodium phosphate buffer (pH 6.2) at 30°C. Samples are taken as function of incubation time and quenched. The products of the acyl transfer to the nucleus (antibiotic) and water were subsequently detected by HPLC. Suitable acyl donors comprise phenylglycine esters (e.g. the methyl ester PGM) or p- hydroxyphenylglycine esters (e.g. the methyl ester HPGM). Alternatively, the corresponding amides can be used but the rate of acylation will be considerably lower. The acyl donors carry a primary amino group at the alpha carbon. Hardly any transfer is possible using the corresponding phenyl acetic amide/ester. The nucleus can be either 6-aminopenicillanic acid (6-APA) or 7-aminodesacetoxycephalosporanic acid (7- ADCA) forming ampicillin or cephalexin, respectively when using PGM as the acyl donor, amoxycillin and cephadroxyl respectively when using HPGM as the acyldonor. Other amino-acid esters or amino acid ester derivatives such as for example dihydrophenylglycine or dihydroxyphenylglycine esters may be used as acyl donor. Alternative penicillins or cephalosporins can be prepared by contacting the suitable acyl ester with the appropriate nucleus. Suitable nuclei may include 7-aminocephem compounds such as 7-amino-cephalosporanic acid (7-ACA), 7-amino-cephem-3- chloor-4-carboxylic acid (7-ACCA), 7-amino-3-[(Z)-1-propenyl)-ceph-3-em-4-carboxylic acid (exomethylene 7ADCA), 7-amino-3-((1,2,3-triazol-5-yl)-thiomethyl)- cephalosporanic acid (7-TACA) and 7-amino-cephem-3-methoxy-4-carboxylic acid. However, suitable nuclei are not limited to β-lactams, but may also comprise other compounds containing primary amino groups such as amino acids.
A typical example is the synthesis of cephalexin by an α-AEH that was carried out as follows. Enzyme incubations were done at 30°C and contained 30 mM 7- ADCA and 15 mM D-PGM in 50 mM sodium-phosphate buffer pH 6.2. Before analysis the samples were quenched and diluted 50-fold by the addition of HPLC eluent. The synthesis and hydrolysis experiments were followed by reverse-phase HPLC using a Chrompack C18 column with Jasco PU-980 pumps and a Jasco MD-910 detector set at 214 nm. All compounds were eluted isocratically using a solution containing 340 mg/l SDS, 5 mM phosphate, 30% acetonitril, which was adjusted to pH 3.0 with phosphoric acid. One cephalexin synthesis unit (cexU) is defined as the amount of enzyme needed to produce one μmol of cephalexin per min under the indicated conditions.
The initial synthesis over hydrolysis ratio (usually called S/H-ratio) is determined by dividing the initial slopes of the formation of the antibiotic (synthesis) by the formation of hydrolysis product (hydrolysis, corrected for background hydrolysis). The S/HQmax is the maximal concentration antibiotic divided by the concentration hydrolysis product (side chain) at Qmax.
An α-amino acid ester hydrolase shows a high preference to hydrolyse the phenylglycine methyl ester (PGM) compared to the corresponding phenylacetic acid methyl ester (PAM) that lacks the α-amino group. An assay to establish this preference is the following. Incubate an appropriate amount of enzyme with a mixture of PGM and PAM, in 50 mM phosphate buffer pH 6.2, and follow the hydrolysis of PGM and PAM respectively by the formation of the hydrolysis product phenyl glycine (PG) or phenylaceticacid (PAA) by HPLC analysis. The ratio of the initial velocities of formation of PG and PAA reflect the ratio of the specificity constants according to the relationship:
va/vb ={(kcat/Km)a[A]} / {(kcat/Km)^]}
where [A] and [B] represent the substrate concentration of the competing substrates PGM and PAM, respectively.
Since substrate inhibition has been observed for some α-AEHs, it is recommended to use for the determination of activity a range of substrate concentrations, for example 1 , 5, 10, 25, 50mM. Determination of the activity should be done using the initial values (less than or equal to 10% hydrolysis of the substrate), this to prevent significant effects of product inhibition.
Table 1 : Some kinetic parameters A.turbidans α-AEH
Substrate8 Km kcat ^^ vn
(mM) (s-1) (s'1.mW1) NIPGB 1.1 ± 0.5 0.4 ± 0.07 0.4 ± 0.2
D-PGM 7 ± 2 1035 ± 123 148 ± 46
D-PGA >13 >43 3.3
HPGM 11 ± 3 263 ± 30 24 ± 7
Table 2 : Some kinetic parameters X.citri α-AEH
Substrate mM) IζtW) kcat/ *
NIPGB 0.07 0.4
PGA n.d. 1.6a
PGM 90 1860 21
HPGM > 40 > 520 < 13
PAM - 0
Penicillin G - 0
Activity of AEH was routinely assayed at 30°C by following the hydrolysis of 15 mMD- 2-Nitro-5-[(phenylglycyl) amino] benzoic acid (NIPGB) in a spectrophotometer at 405 nm in 50 mM phosphate buffer, pH 6.2. The initial rates (_10% conversion) of hydrolysis of all the substrates were determined by measuring product formation by HPLC except for NIPGB, which was monitored as described above. The enzyme was incubated with varying concentrations in the range of 0 to 25 mM for cephalexin, ampicillin, HPGM, and cefadroxil, or 0 to 50 mM for D-PGM and NIPGB, or 0 to 10 mM for amoxicillin. Reactions were done at 30°C in 50 mM phosphate buffer, pH 6.2. The calculations involved nonlinear regression fitting (Scientist, Micromath) using Michaelis- Menten and substrate inhibition kinetics, and the calculated kinetic parameters are given with their standard deviations. The hydrolysis of PGA was measured at 5 and 50 mM and the kcat/Km was calculated from the initial linear slope of the Michaelis- Menten curve. Hydrolysis of glutaryl 7-ACA and adipoyl 7-ADCA was measured at 5 and 25 mM.
Example 2 Selection of suitable organisms for cloning of AEH From the literature, 12 organisms were selected that have been described to be able to hydrolyse and synthesise ampicillin (Table 3).
Table 3. The selected organisms, for each the subunit composition of the α-AEH is indicated.
Organism Subunits Reference
Xanthomonas citri (α)4 (Kato, et al., Agric. Biol. Chem.,
IFO 3835 72 kDa (1980) 44, 1069-1074)
Acetobacter turbidans ( )2(β)2 (Takahashi, et al., Biochem. J.,
ATCC 9325 70/72 kDa (1974) 137; 497-503)
Pseudomonas ( )2 (Kim and Byun, Biochim. Biophys. melanogenum 70 kDa Acta, (1990) 1040; 12-18)
IFO 12020
Proteus rettgeri αβ (Daumy, et al., J. Bacteriol.,
ATCC 9250 24/62 kDa (1985) 163; 925-932)
Kluyvera cryocrescens αβ (Shimizu, et al., Agr. Biol. Chem.,
ATCC 21285 24/62 kDa (1975) 39; 1655-1661)
Pseudomonas diminuta αβ (Aramori, et al., J. Ferment.
N176 26/58 kDa Bioen., (1991) 72; 232-243)
Pseudomonas sp. αβ (Matsuda, et al., J. Bacteriol.,
SE83 25/58 kDa (1987) 169; 5815-5820)
FERM BP817
Bacillus sphaericus (α)4 (Olsson, et al., Appl. Environ.
ATCC 14577 35 kDa Microbiol., (1985) 49; 1084-1089)
Bacillus megaterium αβ (Chiang and Bennette, J.
ATCC 14945 24/62 kDa Bacteriol. 93, (1967) 302-308)
Arthrobacter viscosus αβ (Konstantinovic, et al., Gene,
DSM 20159 24/62 kDa (1994) 143; 79-83)
Acetobacter - (Takahashi, et al., J. Am. Chem. pasteurianus Soc, (1972) 94; 4035-7)
ATCC 6033
Achromobacter NRRL - (Fujii, et al., Process Biochem.,
B-5393 (1976) 11 : 21-24) Organisms were tested for their ability to hydrolyse NIPGB and synthesise ampicillin at two pH values (pH 7.2 and 6.2 (Fig. 1)).
X. citri, A. turbidans and A pasteurianus were interesting due to their relatively high ampicillin production, indicative of the expression level in the wild type organism, and their ability to produce significant amounts at both pH-values with comparable S/H- ratio's to E. coli. To compare these three organisms the ampicillin production and hydrolysis per mg of protein was determined (Table 4).
Table 4: S/H ratios of AEH enzymes from different organisms
Organism acyl Synthesis (1) Hydrolysis (2) S/H Ratio (3) donor
A pasteurianus PGM 4,4 0,4 11
A. turbidans PGM 2,2 0,4 5,5
X. citri PGM 1 ,7 0,7 2,4
E. coli PGA 1 ,1 1 ,2 0,9
(1) Synthesis: μmol ampicillin production at pH 6.2 after 60 min/mg protein
(2) Hydrolysis: μmol ampicillin hydrolysis at pH 6.2 after 60 min/mg protein
(3) ) S/H Ratio: synthesis/hydrolysis
Based on the low ampicillin hydrolysis-activity and the high ampicillin production per mg of protein, A pasteurianus was selected for further molecular and enzymological studies.
Example 3 Cloning of AEH
To clone the gene encoding the α-AEH it was decided to make a cosmid library of the total DNA of A pasteurianus ATCC 6033. We used the broad host range cosmid pLAFR3 and E. coli HB101 (led) as a host. The library contained 690 clones and with an average insert size of 20 kb the library was for 97% complete. Therefore, it was decided to screen the library for colony activity on D-2-Nitro-5-[(phenylglycyl) amino] benzoic acid (NIPGB, Syncom, Groningen, The Netherlands) and for growth in the absence of leucine but in the presence of D-PG-L-Leucine. D-PG-L-Leucine can be converted by α-AEH releasing the leucine necessary for growth. Using these screenings methods no positive clone could be detected. This might have been due to a bad expression, a too small library, or the acylase gene is not situated on the chromosome but on one of the 5 plasmids which are present in A pasteurianus.
As an alternative, and to circumvent some of the problems described above, we decided to make a cosmid library of A turbidans ATCC 9325. Genomic DNA was isolated as described by Poelarends et al. (Appl. Environ. Microbiol 1998, 64:2931-2936). An incubation of 30 min at 37°C with proteinase K (0.10 mg/ml) after the first hour of incubation with SDS was added to the procedure. DNA of the cosmid pLAFR3 used for the construction of the gene library was isolated from E. coli HB101 according to the alkaline lysis method and purified by ultracentrifugation using a CsCI gradient (Sambrook, Fritsch and Maniatus, 1989, Molecular Cloning: a laboratory manual, 2nd edition). The chromosomal DNA of A turbidans was partially digested with Sat 3A to yield fragments with an average size of 30-50 kb. These fragments were ligated in the cosmid pLAFR3 (Tcr) which had been completely digested with BamHl and dephosphorylated with alkaline phosphatase. In vitro packaging and infection of E. coli HB101 was carried out according to the recommendations of the manufacturer (Roche). Recombinant clones were stored at -80°C in microtiter plates. A cosmid library which was complete for 99.9% was obtained and screened with both activity assays. No active clone was found. This might be due to a low expression resulting from the low copy number of pl_AFR3.
It was decided to make also a genomic library of .c/ In order to avoid the low copy number of pLAFR3 , in making the genomic library of X. citrit e cosmid pWE15 (Stratagene) was used, which is assumed to have a higher copy number. A library with 99.9% completeness was obtained. The library was screened for activity on NIPGB and for the ability to synthesise cefalexine. No positive clones were found.
To check for cephalexine synthesis an HPLC method was developed in which 50 colonies were resuspended from plate and incubated with a PGM and 7ADCA mixture. In time samples were taken and analysed by HPLC. We were able to see one positive clone per 50 clones. This method was found to be the most sensitive method. Therefore, this method was used to screen the genomic libraries of X. citri A. pasteurianus and A turbidans again. Unfortunately, no positive clone was obtained in either of the libraries. Since a cosmid with a higher copy number did not result in finding a positive clone, we concluded that the expression or processing was the bottle neck. This may be overcome by expression in a other host. Therefore, the libraries in pLAFR3 of A pasteurianus and A turbidans were conjugated via triparental mating to Pseudomonas US2. The resulting clones were tested for activity on NIPGB and for the ability to synthesise cephalexine. Again, no positive clones were detected. By using a genetic probe the expression or processing would not interfere with the cloning of the gene. In order to obtain such a probe, new attempts to purify the α-AEHs to homogeneity were started.
Purification of AEHs
To screen the library with a genetic probe the AEHs from the different organisms were purified. Pure AEH was thereafter used to determine the N-terminal or internal (after digestion) amino acid sequences, which were used to make a genetic probe.
A. turbidans ATCC 9325
For purification of α-AEH 10 liters of A turbidans (AEHAf) was grown. Cells of A. turbidans were harvested in the stationary phase by continuous centrifugation at 6,000 x g, washed twice with 10 mM potassium phosphate buffer (pH 6.2) and resuspended in this buffer. All further steps were carried out at 4°C. A cell extract was made by sonification and cell debris was removed by centrifugation at 13,000 x g for 40 min. To the supernatant DNase and RNase (final concentration 6 mg/l each) were added in the presence of 5 mM MgSO4 The solution was incubated for 3 h under mild stirring and centrifuged at 50,000 rpm in a type 50 Ti rotor (Beckman) for 60 min and then applied to a CM sepharose fast flow column (5 by 15 cm column, Amersham Pharmacia Biotech Ltd., Hertfordshire, United Kingdom) pre- equilibrated with 10 mM K2HPO4-KH2PO4, pH 6.2. Prior to elution the non-binding proteins were washed from the column with equilibration buffer. The retained protein eluted in a linear gradient of 0-1 M KCI (30 ml/min) at 0.2 M. Activity containing fractions were pooled and (NH4)2SO was added to a final concentration of 1.5 M, after which the pool was loaded on a hydrophobic interaction column (Resource Phenyl, 2.6 by 7.5 cm, Amersham Pharmacia) pre-equilibrated with 1.5 M (NH4)2SO4, 50 mM Na- phosphate buffer, pH 6.2. After washing with the equilibration buffer the AEHAt eluted at 0.8-0.68 M (NH4)2SO4 in a decreasing linear gradient from 1.5 M to 0 M (NH4)2SO4in 50 mM Na-phosphate buffer (pH 6.2) at 5 ml/min. Fractions that contained AEHA^were pooled and concentrated by ultrafiltration (YM30, Amicon bioseparations, Millipore, Bedford, USA) and loaded on a Superdex 200 HR 10/30 column (24-ml bed volume, Amersham Pharmacia). AEHA^was eluted at 1 ml/min in 50 mM Na-phosphate buffer (pH 6.2), 0.15 M NaCl. Finally very little protein was obtained (Table 5a) but it was enough for the purification and the determination of the N-terminus (Eurosequence BN., Groningen). The determined Ν-terminal sequence is shown in Table 5b. Based on the first 12 amino acids, and adding a starting methionine, a degenerated oligonucleotide primer (pΝTd; 5'-
ATGGCΝCCΝGCΝGCΝGAYGCΝGCΝCARGCΝCAYGA-3' (Y=T/C; R=A/G; N=any)) was designed. From total DNA of A turbidans, digested with Hind\\\ and ligated to the corresponding cassettes of the LA PCR in vitro cloning kit (TaKaRa Biomedicals, Takara Shuzo Co., Ltd., Otsu, Shiga, Japan), a PCR product of 2.6 kb was obtained using the primers pNTd and C1. Sequence analysis of the 2.6 kb fragment indicated that the fragment contained the correct gene since downstream of the primer (pNTd) the sequence corresponding to the remaining 10 amino acids of the determined N- terminus was confirmed.
Table 5a. Purification of α-amino acid ester hydrolase from A turbidans ATCC 9325
Purification step Total Total Specific Purification Recovery protein activity activity3 (fold) (%)
(mg) (cexU) (cexU/mg)
Cell free extract 461 599 1.3 1 100
CM-sepharose 31 477 15.4 12 80
Hydrophobic 0.37 23 62.2 47 3.8 interaction
Gel filtration 0.011 7.4 673 518 1.2
One cexU is the amount of enzyme needed to produce one μmol cephalexin per min at 30°C from 30 mM 7-ADCA and 15 mM PGM at pH 6.2 (50 mM Na-phosphate buffer).
Figure imgf000041_0001
To ensure the completeness of the gene and to be able to study the surroundings of the genomic DNA library of A turbidans was screened with a DIG labelled probe (NTaeh) based on part of the gene found on the 2.6 kb PCR product. Therefore matching primers were designed based on the DNA sequence of the fragment amplified by the LA PCR in vitro cloning kit (TaKaRa). The forward primer, 5'- CCGCTAAGCGTGCAGACCGGCAGC-3' (upstream of pNTd), and the reverse primer, 5'-CATGCATACCGTGCCAGAACG-3', were used to amplify a 696 bp fragment with Taq polymerase using the PCR DIG probe synthesis mix from Boehringer (NTaeh). Colony hybridization was essentially carried out as described by Van Hylckama Vlieg et al. (J. Bacteriol. 2000, 182:1956-1963) using an AEH specific probe. An incubation of the membrane with proteinase K for 30 min at room temperature after fixation of the DNA was included in the procedure. After hybridization at 68°C the membrane was washed with 2 x SSC, 0.1 % SDS (10 x SSC is 1.5 M NaCl with 0.15 M Na-citrate) at room temperature and with 0.5 x SSC, 0.1 % SDS for 15 min at 68°C. The DIG-labeled DNA was visualized using alkaline phosphatase and a chemiluminescence substrate,
CPSD (C18H20CIO7PNa2; Roche) following the recommendations of the manufacturer.
Out of the 1248 colonies screened, two hybridised with the probe. From one of these clones the cosmid, pLATC3 was isolated and its insert sequenced. Since in the literature the α-AEH of A turbidans is described as a heterotetramer with subunits of 70 and 72 kD (Takahashi, et al., Biochem. J., (1974) 137; 497-503, Ryu and Ryu,
Enzyme Microb. Technol., (1987) 9; 339-344), we sequenced the gene coding for the 70 kD subunit including 3 kb up- and downstream, looking for a gene coding for the subunit of 72 kD. No other subunit was found, concluding that theα-AEH is encoded by one gene (aehAt) A. pasteurianus ATCC 6033
The purification of the α-AEH of A pasteurianus starting (AEHAp) from a 2.500-liter culture was very troublesome. The expression was very low, and the growth of A pasteurianus was very bad, resulting in large volumes with low activity. From this material we were not able to purify theα-AEH. Using the DIG-labelled probe of A.turbidans 620 clones of the pl_AFR3 cosmid library were screened and three positive clones were found. The sequence appeared very homologous to the gene found in A turbidans X. citri IFO 3835
The α-AEH of X. citri (AEHXc) was purified from a 10 litre culture. AEHXc was purified by ion exchange, hydrophobic interaction and gelfiltration as described for the A. turbidans. The retained protein was eluted from CM sepharose in a linear gradient of 0-1 M KCI at 0.45 M KCI. AEHXc eluted from Resource Phenyl in a decreasing linear gradient from 1.5 M to 0 M (NHt)2SO4 around 0.36 M (NH4)2SO4. Finally, the enzyme was purified to SDS homogeneity by gelfiltration (Sephacryl S300, 1.6 by 65 cm, Amersham Pharmacia Biotech ltd., Hertfordshire, United Kingdom). Results are summarized in Table 6.
Table 6: Purification of α-amino acid ester hydrolase from X. citri IFO 3835
Figure imgf000043_0001
Since the N-terminus was blocked, the protein was digested with trypsin. For the internal sequence analysis of AEHXc, approximately 15 μg of protein was sliced from a SDS-PAGE gel and subjected to digestion by trypsin (Eurosequence BV, Groningen, The Netherlands). To allow primer design with a low degeneracy, those peptides containing a tryptophane (high absorbance at 297 nm) were selected for sequencing and their amino acid sequence (Table 7) determined by automated Edman degradation (Model 477A, Applied Biosystems).
Table 7: Internal sequences of theα-AEH from X. citri.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Fragment 1 A A G L E Q L P W W H K Possible codons 4 4 4 6 2 2 6 4 1 1 2 2
Fragment 2 G P L N P S E V D H A T D A X D T I D X Possible codons 4 4 6 2 4 6 2 4 2 2 4 4 2 4 - 2 4 3 2 - X : tryptophane or cysteine
Based on internal amino acid sequences two primers pF, 5'- AAYCCNAGYGARGTNGAYCAYGC -3' and pR, 5'-
YTTRTGCCACCANGGNARYTGYTC-3' (Y is T or C; R is A or G; N is any base) were designed and used to amplify part of the aehXc gene by PCR from chromosomal DNA. The PCR-product was isolated from gel (Qiaquick kit from Qiagen, GmbH, Hilden, Germany), cloned and sequenced. A gene probe for the AEHXc gene was made using matching primers based on the DNA sequence of the PCR fragment. The forward primer was 5'-ACCGATGCCTGGGACACC-3'(upstream of pF) and the reverse primer was 5'-CAGGCCTGCGGCCTTGGC-3'(downstream of pR). These primers were used to amplify a 317-bp fragment (ProbeXc) with Taq polymerase using the PCR DIG probe synthesis mix from Roche.
Example 4 Expression and purification of cloned AEHs Cloning of aehAt into an expression host.
The gene encoding the AEH from A turbidans (aehAt) encoded for a precursor protein with a molecular weight of 74,060 D, corresponding to a polypeptide of 667 amino acids. The determined N-terminal amino acid sequence was found at position 41-62, indicating that the first 40 amino acids of the gene were processed during maturation in A turbidans. It is therefore likely that the ORF encodes a precursor of α-AEH that undergoes processing to yield active enzyme.
For expression of the aehAt gene in E. coli the vector pETAT (aehAt cloned in pET9) was constructed. The aehAt gene was cloned in the A/del- and BamHl site of pET9 using a forward primer based on the N-terminal sequence including the leader sequence in which an Asnl site is incorporated, 5' CCGC-
CGCCGATTAATGGTGGGACAGATTACCCTTT-3'(Asnl site underlined, start codon in bold) and a reverse primer in which a BamHl site was incorporated (underlined), 5'- ACCCATAC-TGGATCCTTACTGTTTCACAACCGGGAG-3'. The gene was also cloned without the N-terminal leader sequence, where the leader sequence was replaced by a methionine, using 5'-GGTCGCGCATTAATGGCTCCGGCAGCG-GATGC-3' (Asnl site underlined, start codon in bold) as a primer. After denaturation of the DNA (pLAFR3 (aehA)) the amplifications were established in 30 cycles of 30 s at 94°C, 1 min at 58°C and 1.5 min at 72°C. Products were digested with Asnl and BamHl and ligated into pET9 cut with Ndel and BamHl. The ligation mixture was used to transform CaCI2- competent E. coli BL21 (DE3)pLysS. The constructs were confirmed by sequence analysis. For cloning in the NdeMHindW site of pEC the gene was amplified with the forward primers as described above and the following reverse primer; 5'- CATACTGGCAAGCTJTTA-CTGTTTCACAAC-CGGGAGCAG-3'(H/'ncfl 11 site underlined, stop codon in bold). Cloning of the aeh gene in E. coli without the leader sequence, starting with M-41-APAAD, resulted in inactive clones, using two different expression systems (pET9, pEC). Therefore it was concluded that the leader sequence is necessary for the production of active enzyme in £. coli. To determine the way of processing in E. coli the first 5 N-terminal amino acid residues of the recombinant enzyme were determined and found to be 40-AAPXAD, which is in agreement with the predicted cleavage site. Although the determined N-terminus had a high degree of heterogeneity, indicative of varying N-termini within the give N-terminal sequence, it indicates that the signal sequence is processed in a similar way as in A turbidans. Therefore, it can be concluded that the leader sequence is needed for the production of active enzyme and is processed properly in E. coli Isolation of AEHAt from E. coli.
The recombinant AEHA was purified from E. coli BL21(DE3)pLysS (CmR) cells carrying the pETAT (KmR) construct. The cells were harvested from two 2.5 I cultures by centrifugation and the crude extract was prepared as described above. The extract was loaded on a DEAE Sepharose column (5 by 13 cm column, Amersham Pharmacia) pre-equilibrated with 50 mM Na-phosphate buffer, pH 6.2. The AEHA activity was eluted from the column in the non-binding fraction in the equilibration buffer at 30 ml/min. The activity was then applied to a CM-HAP (ceramic hydroxy apetite column, 2.6 by 11 cm column, Amersham Pharmacia) which was equilibrated with 50 mM Na-phosphate, pH 6.2. After washing with the equilibration buffer the AEHAt activity was eluted from the column at 275 mM Na-phosphate in a linear gradient of 50 to 500 mM Na-phosphate (pH 6.2) at 10 ml/min. The AEHAt was purified further to SDS-PAGE homogeneity by hydrophobic interaction and gelfiltration chromatography as described above.
Table 8. Purification of α-amino acid ester hydrolase from E. coli BL21 (DE3)pLysS(pETAT).
Purification step Total Total Specific Purification Recovery protein activity activity3 (fold) (%)
(mg) (cexU) (cexU/mg)
Cell free extract 1174 2348 2 1 100
DEAE 60 2050 34 17 87
CM-Hap 5.8 1109 191 96 47
Hydrophobic 1.0 633 605 303 27 interaction
Gelfiltration 0.18 153 859 430 7
Cephalexin synthesis activity.
To achieve a higher expression level and an easier purification of AEHAt that what was obtained with the pETAT construct the aehAtgene was cloned in pBAD/Myc-HisA (pBADAT), coupling both yc-epitope and the 6xHis-tag C-terminally to the protein. To clone aehAt in the Ncol and Hindlll site of pBAD/Myc-HisA, resulting in pBADAT, the Ncol restriction site was first removed from the gene cloned in pAT (3). This was accomplished by PCR using the sense primer 5'- GAACTGCCTGTGTCTATGGATATTTTCCGGGGC-3\ its compatible reverse complement primer and the QuickChange site directed mutagenesis kit of Stratagene (La Jolla, USA) resulting in pATdelNco. From this construct the gene encoding AEH was amplified by PCR using two mutagenic primers to allow cloning in theΛ/col and Hind\\\ site of pBAD/ZWyc-HisA. The forward primer (aehAhisf), 5'- CGCGCCACACCATGGTGGGACAGATTA-3' (start codon in bold), was based on the N-terminal sequence including the signal sequence and an Λ/col site (underlined) was introduced. The reverse primer (aehAhisr), 5'-
CATACTGGCAAGCTTCTGTTTCACAACCGGGAG-3' (Hind\\\ site is underlined), lacked the stop codon to allow the C-terminal attachment of the myc-epitope and the polyhistidine region of pBAD/Myc-HisA.
Protein purification - Wild type AEH was expressed in E. coli TOP10 from the pBAD/Myc-HisA derived constructs. To obtain soluble protein two 2.5 liter cultures supplemented with l-arabinose (0.01% w/v) were inoculated with a 1 ml overnight culture grown at 30°C and incubated for 64 h at 14°C. Induced cells were harvested from the cultures by centrifugation at 5000 g and suspended in 50 mM Na- phosphate buffer pH 6.2. All further steps were carried out at 4°C. The cytoplasmic content was released by sonification and the remaining cell debris was removed by centrifugation at 13.000 g for 40 min. The supernatant was added to 1 ml Ni-agarose (Qiagen) equilibrated with wash buffer (25 mM imidazole, 500 mM NaCl, 50 mM Na- phosphate, pH 7.4). After mixing by inversion for 90 min at 4°C the bed was allowed to form (20 x 8 mm in a polyprep chromatography column (Biorad Laboratories, Hercules, CA, USA)). The unbound protein was washed from the column with 30 column volumes of wash buffer. The bound protein eluted from the column at 100 mM imidazole in a stepwise gradient from 50 to 200 mM imidazole, 150 mM NaCl, 50 mM Na-phosphate, pH 7.4, in 20 column volumes. The protein was brought to 50 mM Na-phosphate buffer pH 6.2 with use of an Econpac gelfiltration column (Biorad). All purification steps were monitored by SDS-PAGE and enzymatic activity was measured with NIPGB. The protein concentrations were measured using the Bradford method with bovine serum albumin as the standard.
The use of the arabinose promoter in the pBADAT plasmid resulted in an overproduction of 5 fold (1% of the total protein in a cell free extract) compared to the expression in the wild-type A turbidans strain and pETAT system. Furthermore, with the resulting construct, the number of necessary purification steps were reduced from 4 to 2 by use of a Ni2+-agarose column (Table..).
Table 9 Purification of AEH-6xHis from E. coli.
Purification step Total Total Total Specific Purification Recovery volume protein activity3 activity3 (fold) (%)
(ml) (mg) (cexU) (cexU/mg)
Cell free extract 72 312 5551 17.8 1 100
Ni2+-agarose 4.8 2.7 3587 1329 75 65
Desalting 8.0 2 3344 1672 94 60
Cephalexin synthesis To check whether the properties of AEH had changed upon addition of the myc- epitope and His-tag the kinetic parameters of the purified enzyme for cephalexin were measured and compared to untagged recombinant protein. The KM values of both proteins appeared to be nearly identical, 0.45 and 0.34 mM, respectively. The kcatof the fusion protein is somewhat lower than for the untagged recombinant protein, 274 and 347 s"1 respectively, but the values are in the same order of magnitude. This indicates the proper folding of the recombinant protein occurred and shows that there is no dramatic influence of the added amino acids.
Example 5 Identification of new α-AEH by In Silico screening
Comparing the sequences of the three α-AEH sequences from X citri, A. pasteurianus and A. turbidans we found that the enzymes have at least 60% identity
Table 10. Percentage identity.
Identity (%) X. A past. A Z. Rhodo B. X. fast. citri turb. mobil. late.
X. citri 100 60 60 61 28 28 77
A. pasteurianus 100 99 61 28 26 60
A. turbidans 100 61 29 26 60
Z. mobilis 100 29 29 60
Rhodococcus sp. 100 34 29
B. laterosporus 100 28
X. fastidiosa 100
Percent identity was calculated using the pairwise blast option at NCBI on the world wide web.
Surprisingly, two more sequences from the database showed extensive homology with the cloned sequences. Zymomonas mobilis ZM4 and Xylella fastidiosa appeared to encode enzymes with more than 60% homology to the cloned sequences. In order to verify whether these enzymes which were so far annotated as hypothetical proteins glutaryl-7-ACA-acylase can now be classified as genuine AEHs, we decided to clone the gene from Z. mobilis
Cloning of aehZ into an expression host. The gene encoding the AEH from Zymomonas mobilis ATCC31821 (aehZ) was cloned in the Nde\ and H/ndlll site of pEC resulting in pZM. Therefore, the gene was amplified from the freeze-dried cells supplied by ATCC with the forward primer, pfz, 5'- CAGGGAGGGC/ TArGTCTCGATTAAAGCTC-3' (Λ/cfel site is shown in italics and start codon in bold) and the reverse primer, prz, 5'-
TTTAπCTCA4GC7TrTATGGGATAACCGGCAA-3' (H/ndlll site is shown in italics and the stop codon in bold). From this construct the gene was amplified with the primers pfz and przhis, S'-TTTATTCTCA GCTTTGGGATAACCGGCAA-S' (H/ndlll site is shown in italics). After restriction the gene was cloned in the Λ/cfel and H/ndlll sites of a modified pBAD/Myc-HisA in which the Λ/col site was changed into a Λ/cfel site and the tree Λ/cfel sites were removed by site directed mutagenesis (Kamerbeek, to be published) resulting in pBADZm. The constructs were confirmed by sequence analysis. Some kinetic properties of Z. mobilis α-AEH are shown in table 11.
Enzyme incubations were done at 30°C and contained 30 mM 7-ADCA and 15 mM D-PGM in 50 mM sodium-phosphate buffer pH 6.2. An initial S/H-ratio of 1.9 ± 0.7 and a maximum product accumulation of 5.5 ± 1.5 mM cefalexin with a S/HQmax of 0.5 ± 0.2 were measured at pH 6.2 (50 mM Na-phosphate).
Table 11 : Characterisation of cloned AEH of Zymomonas mobilis which was expressed in E.coli
Figure imgf000049_0001
NIPAB : 2-Nitro-5-phenylacetylbenzoic acid; - : no activity; + : activity observed
This example shows that the cloning of the aen-gene provides a tool to attribute a function to other proteins with yet unknown function. In this way it was discovered that the gene ol Zymomonas mobilisZM4 which was described as a putative glutaryl acylase was in fact an AEH. The sequence of the Z. mobilis gene and gene product is shown in SEQ ID NO: 4 and SEQ ID NO: 9 respectively. Cloning of this gene and initial synthesis experiments indicated that this enzyme is able to synthesis cefalexine from phenylglycine methyl ester and 7-ADCA. Z. mobilis and X. fastidiosis appeared to encode enzymes with more than 60% homology to the cloned sequences. Within this homology range we have shown that enzymes exhibit α-AEH activity. These enzymes were so far annotated as hypothetical proteins and can now be classified as genuine AEHs.
Other members, likely to belong to this class of β-lactam antibiotic acylases, considering their subunit sizes and substrate range, are the AEHs from Pseudomonas melanogenum (α2, 72 kDa, (Kim and Byun, Biochim. Biophys. Acta, (1990) 1040;12- 18) and Xanthomonas rubrilineans (at, 70-72 kDa) (Krest'ianova, et al., Biokhimiia (Russian), (1990) 55; 2226-2238 ).
PCR with the primers pF and pR on whole cells of X rubrilineans and Achromobacter B-402-2 NRRL B-5393, another organism with AEH activity (Fujii, et al., Process Biochem., (1976) 11 ;21-24), showed a product of 0.4 kb as was found for X citri indicating that it is very likely that these organisms have homologues of α-AEH.
Example 6 Sequence comparison of aeh with other related sequences The deduced amino acid sequence of A turbidans and X c/fr/was used for homology searches to find related enzymes. The α-AEHs showed homology with several proteins, most of which originated from genome sequencing projects. The most closely related protein, 28-29% identity, for which the activity is described, is the intracellular cocaine esterase from the gram-positive strain Rhodococcus sp. strain MB1 (Bresler, et al., Appl. Environ. Microbiol., (2000) 66; 904-908). This enzyme hydrolyses the ester bond in cocaine resulting in benzoate and ecgonine methyl ester. No synthesis or hydrolysis of β-lactam antibiotics has been described for this enzyme. The next most related studied protein, 26-29 % identity, is the enzyme glutaryl-7- aminocephalosporanic acid acylase of Bacillus laterosporus. This enzyme is able to hydrolyse glutaryl-7-aminocephalosporanic acid to 7-aminocephalosphoranic acid, an important reaction in the production of 7-ACA from cephalosporin C, through cleavage of an acyl linkage. Synthesis or hydrolysis of β-lactam antibiotics with an α-amino acid group has not been studied. Glutaryl-7-aminocephalosporanic acid acylase from B. laterosporus, unlike other known glutaryl 7-ACA acylases (Kumar, et al., Hind. Antibiotics Bull., (1993) 35; 112-125), is composed of a single polypeptide with molecular size of 70 kD, which corresponds to the size of the subunits found forα- AEHs from A turbidans and X citri . The subunit composition of the native glutaryl-7- aminocephalosporanic acid acylase has not been described. Cloning of this enzyme in Bacillus subtilis resulted in an extracellular localisation (Aramori, et al., J. Bacteriol., (1991) 173; 7848-55). Since no homology was found with any other known penicillin acylase we conclude that α-AEHs, probably together with the glutaryl 7-ACA acylase from B. laterosporus, discloses a new class of β-lactam acylases. A graphic representation of the relation of α-AEHs among each other and with the enzymes described above is shown in fig. 2. An extended search for homologous proteins using the position-specific iterated
Blast program, PSI-Blast (Altschul, et al., Nucleic Acids Res., (1997) 25; 3389-3402) indicated low identity (average 14%) to seven X-prolyl dipeptidyl aminopeptidases from Lactococcus lactis and Lactobacillus strains. The relation of cocaine esterase with these enzymes has been reported as well (Bresler, et al., Appl. Environ. Microbiol., (2000) 66; 904-908). The X-prolyl dipeptidyl aminopeptidases belong to the peptidase_S15 family as defined by the Pfam database (Bateman, et al., Nucleic Acids Research., (2000) 28; 263-266). These enzymes are serine proteases with the active- site serine located in the consensus sequence GxSYxG, where X is a non-conserved amino acid (Chich, et al., FEBS, (1992) 314; 139-142). The α-AEHs shows conservation of this motif and its direct surroundings, suggesting that the enzyme according to the invention is a serine hydrolase.
Example 7 Identification of the active site serine of AEHAf by labeling by p-NPGB
The α-AEH of A.turbidans (2.4 μM, 144 kDa) was inactivated by incubation with p-NPGB (1 mM, 1% DMF) for 15 min at 30°C. Control experiments involved incubation under the same conditions of solely enzyme and enzyme with 1% DMF. The inactivated enzyme was diluted 76 times in 15 mM NIPGB, 50 mM Na-phosphate, pH 6.2. The reactivation was monitored by measuring the hydrolysis of NIPGB at 30°C and 405 nm. The observed inhibition by p-NPGB indicated the importance of a serine residue for the activity of the enzyme.
To test whether p-NPGB is a substrate, AEHA was incubated with p-NPGB and the the hydrolysis of p-NPGB was followed at concentrations varying from 0.1 to 1 mM with 1.5 μM enzyme. The release of p-nitrophenol (p-NP) was measured at 405 nm at 30°C using a spectrophotometer (Lambda Bio 10 and software package UV WinLab, Perkin Elmer, Norwalk, U.S.A.). A stock solution of p-NPGB (10 mM) was made in dimethylformamide (DMF) and acetonitrile (ACN) in a 1 :4 ratio. The steady state reactions were done in 50 mM Na-phosphate buffer, pH 7.0. The molar extinction coefficient of p-NP at pH 7 was determined as 9200 M-1 cm-1. The pre-steady state kinetics of p-NPGB conversion was determined by stopped-flow using an Applied Photophysics SX17MV stopped-flow instrument. A stock of p-NPGB (100 mM) was made in DMF, the final concentration of DMF in reaction mixture was 2% or lower. All pre-steady state reactions were performed in 50 mM 4-morpholinepropanesulfonic acid buffer at pH 7, with 1 mM p-NPGB. The enzyme concentration used was 1.36 and 0.66 μM (a2; 144 kDa). Progress curves were fit to obtain the amplitude and the first order rate constant for the burst phase and the velocity of the steady-state reaction, using the program Scientist. The data showed hydrolysis of p-NPGB in a biphasic time course consisting of an initial "burst" of p-NP followed by a second phase corresponding to the steady state rate of p-NPGB hydrolysis. Therefore, hydrolysis of p-NPGB must proceed via an acyl-enzyme intermediate. The formation of the intermediate is faster than its hydrolysis, resulting in an accumulation of the acyl-enzyme indicated by the burst of p- NP, which is in agreement with what is expected for an active site covalent inhibitor. Subsequently the acyl-enzyme complex is slowly hydrolyzed.
The slow conversion of the acyl enzyme intermediate during reaction of p- NPGB enabled us to covalently label the enzyme. The enzyme was incubated with p- NPGB in a 1 :35 ratio in 50 mM Na-phosphate buffer, pH 6.2, with 0.5 % dimethylformamide for 15 min at 30°C. The excess p-NPGB was removed by dialysis against 70% formic acid. To reduce any disulfide bridges the enzyme solution was dialyzed against 70% formic acid with b-mercaptoethanol (2 mM). After removing the b- mercaptoethanol by dialysis against solely 70% formic acid the labeled protein was treated with a 100-fold molar excess of CNBr over the Met content. The reaction was allowed to proceed for 24 h at room temperature under N2 in the dark and was stopped by the addition of 10 volumes water and freeze-dried. The generated peptides were separated by reversed phase HPLC using a nucleosil-5 C18 column (4.6 by 300 mm, Alltech) at 1 ml/min in a linear gradient from 0 to 67% acetonitrile with 0.1 % trifluoroacetic acid. The peptide profile was monitored at 280 nm. The control involved the same conditions as described above except no p-NPGB was added. The peaks that were different from the control experiment were collected and rechromatographed on the same column in a linear gradient from 0-67 % acetonitrile in 0.1% ammoniumacetate, pH 5.0. The individual peaks were collected, concentrated and injected directly into the mass spectrometer.
Electrospray mass spectrometry (ES/MS) was performed on an API3000 mass spectrometer (Applied Biosystems/MDS-SCIEX, Toronto, Canada), a triple quadrupole mass spectrometer supplied with an atmospheric pressure ionization source and ionspray interface. The spectra were scanned in the range between m/z 400 and 1600. MS/MS product ion spectra were recorded on the same instrument by selectively introducing the m/z 1229.5 (singly charged unlabeled peptide) and m/z 695.9 (doubly charged labeled peptide) precursor ions from the first quadrupole into the collision cell (second quadrupole). The collision gas was nitrogen with 30 eV collision energy. The product ions resulting from the collision were scanned over a range of m/z 10 to 1395 with a step size of 0.1 amu and a dwell time of 2 ms. The elution pattern of the peptide mixture of the labeled AEHAt showed a few different peaks compared to the control. These peaks were individually collected and analyzed by ES/MS. The only peptide with a different mass when it was isolated from the labeled and unlabeled protein was identified as peptide 202-TGSSYEGFTWM-213 (1228.6 Da). Upon labeling the same peptide fragment had a mass of 1390 Da. This mass (1390 Da) is in agreement with the fragment of 1228.6 Da plus the guanidino benzoate label (161 Da). To determine which Ser (204 or 205) of the labeled peptide 202-TGSSYEGFTWM-213 was modified by p-NPGB, the labeled peptide was analyzed by ES/MS/MS using product ion scan to obtain the significant fragments. The product ion scan of the (M + 2H)2+ ion at m/z 695.5 of the labeled peptide showed an increase in the masses with 161 Da of the b+-fragments starting at b4,compared to the unlabeled protein. The same increase in the mass was found for the detectable fragments y9 + (hsl-Ser205) to y11+ (hsl-Gly203) of the labeled peptide compared to wild-type. These fragments are in agreement with the label positioned on Ser205, and exclude labeling at position 204.
Example 8 Identification of the catalytic triad of α-AEH by site directed mutagenesis. Site directed mutagenesis of AEHA was performed on pBADAT using QuickChange site directed mutagenesis kit of Stratagene (La Jolla, USA) according to the procedure recommended by the manufacturer. When possible, a restriction site was introduced in the mutational primers (Table 1 ). The PCR reaction mixture was directly used to transform chemically competent E. co//'Top10 cells (Invitrogen, Leek, The Netherlands). For isolation of vector the cultires were grown overnight on LB medium at 30°C. The mutant proteins were purified as described for the wild-type enzyme using the Ni-agarose (Example 4).
Table 12
Synthetic oligonucleotides
Oligonucleotides used in site-directed mutagenesis of AEHAt, onlythe sense primers are shown. Introduced restriction sites are underlined, sequence differences with wild type are shown in bold.
Restri
Amino acid
Oligonucleotide sequence 5' → 3' ction substitution site
C GAG GTT ATG GTA CCC ATG GCG GAC GGC GTG AAG CTG Rsa\ Arg85Ala GTT ATG GTA CCC ATG CGG GCC GGC GTG AAG CTG Rsa\ Asp86Ala GC GGG AAA TAT GGC GCT CAG GGC GAT TAT G HaeW Ser156Ala GGT ATG ACA GGG TCG GCC TAT GAG GGC TTT ACT Aspl Ser205Ala GGT ATG ACA GGG TCG TGC TAT GAG GGC TTT ACT G Aspl Ser205Cys G GGT ATG ACA GGG TCG TCC TTT GAG GGC TTT ACT G Aspl Tyr206Phe G ACG CGT ACC CCC GCT AAC GCC AAA GGC CGG Mlu\ Tyr112Ala GAA CAG GGC TTG TGG GCT CAG GAA GAT ATG TG - Asp338Ala G ATG GGC CCA TGG CGG GCT AGT GGG GTG AAC Λ/col His370Ala G TTT GTA GAG GGC GGC GCT ATC CGC GTG TTT CAG - Tyr143Ala CA GAA TCC CGC CCG GCT GTG GTG ACA TAT GAA AC Λ/cfel Asp509Ala
C CAT GTG TTT GCA AAA GGG GCT CGG ATT ATG GTG CAG - His610Ala G GGT ATG ACA GGG TCG TCC GCT GAG GGC TTT ACT G Aspl Tyr206Ala GGT ATG ACA GGG TCG TCC AAT GAG GGC TTT ACT GTT G Aspl Tyr206Asn G GGT ATG ACA GGG TCG TCC TGG GAG GGC TTT ACT G Aspl Tyr206Phe GGT ATG ACA GGG TCG TCC AAT GAG GGC TTT ACT GTT G Aspl Tyr206Trp
The effects of the inactivating mutations on the secondary structure were evaluated with circular dichroism. Far-UV CD spectra from 250 to 190 nm were recorded on an AVIV circular dichroism spectrometer model 62A DS (AVIV associates, Lakewood, NJ, USA) at 25°C using a quartz cuvette with a path length of 0.1 cm. The concentration of wild-type and mutant enzymes was 0.2 mg/ml in 50 mM Na-phosphate buffer, pH 6.2. Per sample three separate spectra were collected and averaged using a step interval of 0.5 nm/min and an averaging time of 5 s. The phosphate buffer was used as a blank and subtracted form each recording. The data was converted to mean residue ellipticity (qMRE, deg.cm2.dmol-2). From the CD spectra the percentage of secondary structure elements was calculated using CD spectra deconvolution (CDNN version 2.1 , available on the world wide web). These values were standardized to 100% total structure elements.
Slight variations in expression were observed for the mutants, but they were similar to wild-type AEH according to their behavior in the standard purification procedure and observed molecular weights on SDS-PAGE.
Table 13.
Kinetic parameters of cephalexin hydrolysis for mutants of AEHAt.
KM Acat Relative activity
Enzyme , / KM
(mM) (s-1) (%)
AEH-His 0.45 ± 0.06 274 ± 7 609 100
S205A - 0.03 - < 0.01
D338A 0.35 ± 0.09 0.201 ± 0.004 0.6 0.07
H370A 0.43 ± 0.06 0.190 ± 0.002 0.4 0.07
Replacement of the Ser205, Asp338 or His370 by an alanine reduced the activity drastically. These radical changes were not observed for the other purified mutants. The effects of the inactivating mutations on the secondary structure were evaluated with circular dichroism. The spectra were superimposable and the calculated percentages of the secondary structure elements were essentially the same as calculated from the wild-type data. This indicates that the inactivation caused by the mutations of Ser205, Asp338 or His370 did not result from drastic changes in the secondary structure of the enzyme. The KMfor cephalexin was in the same order of magnitude for all mutants, except for the Tyr206 mutant, of which the KM increased significantly. The unchanged K values for the inactive Asp338Ala and His370Ala mutants indicate that these residues have a large influence on either the formation or the hydrolysis of the acyl enzyme intermediate, whichever is rate determining.
Based on the kinetic characterization of the mutants we conclude that AEH is a serine hydrolase and contains a classical catalytic triad of Ser205, Asp338 and His370.
Example 9 Identification of the active site of α-AEH.
Purified X.citri α-AEH was crystallised from a solution containing 5mg/ml protein in 0.1 M cacodylate buffer pH=6.5 using 12-14% polyethyleneglycol (PEG8000) as a precipitant. Phasing was carried out using a highly redundant three-wavelength Multiple Anomalous Dispersion (MAD) X-ray diffraction dataset out to 2.3 Angstrom. The data were collected from X.citri α-AEH crystals in which the methionine had been substituted for Selenomethionine. Initially 62 selenium sites were found using SnB("Howell, P.L., Blessing, R.H., Smith, G.D. and Weeks, CM. (2000), Acta
Crystallographica D56, 604-617" & "Smith, G.D., Nagar, B., Rini, J.M., Hauptmann, H.A. and Blessing, R.H. (1998) Acta Crystallographica D54, 799-804"). Further refinement was carried out using the programs SOLVE and RESOLVE (Terwilliger, T.C. and Berendzen, J. (1999) Acta Crystallographica D55, 849-861" resp. "Terwilliger T.C. (1999) Acta Crystallographica D55, 1863-1871 ). Ultimately further diffraction data were added to 1.8 Angstroms allowing for extending the phases and autobuilding with wARP (Perrakis, A., Morris, R.M. and Lamzin, V.S. (1999) Nature Structural Biology 6, 458-463). Final refinement was done with RefmacS. The final three dimensional structure of X.citri α-AEH reveals a tetrameric assembly of four identically folded α- AEH peptide chains.
The alignment of the sequences of the five α-AEH sequences from X citri, A. pasteurianus, A. turbidans , Z. mobilis and X. fastidiosa is shown in table 14. The alignment indicates that the α-AEH's of A. pasteurianus, A. turbidans , Z. mobilis and X. fastidiosa have at least 60% identity with theα-AEH of X.citri. Based on such an extensive homology it is allowed to conclude that the 3-dimensional structure A. pasteurianus, A. turbidans , Z. mobilis and X. fastidiosa will be very similar to the 3- dimensional structure of X.citri which has been determined above. Therefore it is allowed to speak of corresponding residues in the different α-AEH's. Corresponding amino acid positions does not only mean that amino acids are at the same position in the sequence alignment for α-AEH as given in alignment 14 but it also indicates that the amino acids will be more or less identically positioned in 3-dimensional space. It means that the coordinates of the X.citri 3-dimensional model and an alignment as given in the alignment of table 14 will allow a person skilled in the art for generating 3- dimensional models for the other α-AEH's. In fact this modelling approach is not limited to the presently known α-AEH's as shown in alignment table 14. Newly discovered α- AEH's can be aligned with X.citri too, subsequently allowing for modelling the 3- dimensional structure. The procedure is generally known as homology modelling. Typical computer programs which allow for generating such models are Insight, DISCOVER, HOMOLOGY and MODELER (Accelrys Ltd. 230/250 The Quorum, Barnwell Road.Cambridge CB5 8RE, UK).
Table 14 Alignment of α-AEH sequences from X citri, X. fastidiosa, Z. mobilis ,A. pasteurianus, and A. turbidans.
Xc 1 MRRLATCLLA TAIAAASGSA
Xf 1 RRFIA ALFFILPLAA
Zm 1 —MSRLKLSV NLSAMKKYLM RGSWASLTS ILAIPALSSA Ap 1 MVGQITLSKQ KSVLQKKSLW ASVALSGVLL AATLPVAQAA APAADAAQAH
At 1 MVGQITLSKQ KSVLQKKSLW ASVALSGVLL AATLPVAQAA APAADAAQAH
Xc 21 WAQTSPMTPD ITGKPFVAAD ASNDYIKREV MIPMRDGVKL HTVIVLPKGA Xf 17 IAQTAPMTPD ITGRKFIVPT ERNDYIKREA MIPMRDGTKL HTVIIIPKKA
Zm 39 PDKTIWPENG DIAMHFSAPT AHYDYEKREV MIPMRDGVKL HTVIWPKNG
Ap 51 DPLSVQTGSD IPASVHMPTD QQRDYIKREV MVAMRDGVKL YTVIVIPKNA
At 51 DPLSVQTGSD IPASVHMPTD QQRDYIKREV MVPMRDGVKL YTVIVIPKNA
Xc 71 KNAPIVLTRT PYDASGRTER LA-SPHMKDL LSAGDDVFVE GGYIRVFQDV
Xf 67 QHAPMLLTRT PYNANERSER LL-SPHMKNL LPQGDDVFAT GDYIRVFQDV
Zm 89 RNLPILLTRT PYDASSRTSR SD-SPSMLAT LPEGDEVFVR DGYIRVFQDV
Ap 101 RNAPILLTRT PYNAKGRANR VPNALTMREV LPQGDDVFVE GGYIRVFQDI At 101 RNAPILLTRT PYNAKGRANR VPNALTMREV LPQGDDVFVE GGYIRVFQDI
XC 120 RGKYGSEGDY VMTRPLRGPL NPSEVDHATD AWDTIDWLVK NVSESNGKVG
Xf 116 RGKYGSEGDY VVTRPLRGVL NLTNIDHATD AWDTIDWLVK NIKESNGNVG Zm 138 RGKYGSEGVY WTRPPVGDL NPTKVDHTTD AWDTIEWLTK HIPESNGRVG Ap 151 RGKYGSQGDY VMTRPPHGPL NPTKTDETTD AWDTVDWLVH NVPESNGRVG At 151 RGKYGSQGDY VMTRPPHGPL NPTKTDETTD AWDTVDWLVH NVPESNGRVG
#* Xc 170 MIGSSYEGFT VVMALTNPHP ALKVAVPESP MIDGWMGDDW FNYGAFRQVN Xf 166 MIGSSYEGFT VVMALTDPHP ALKVAAPESP MIDGWMGDDW FNYGAFRQVN Zm 188 MIGSSYEGFT VVAALLNPHP ALKVAAPESP MVDGWMGDDW FHYGAFRQAA Ap 201 MTGSSYEGFT VVMALLDPHP ALKVAAPESP MVDGWMGDDW FHYGAFRQGA At 201 MTGSSYEGFT VVMALLDPHP ALKVAAPESP MVDGWMGDDW FHYGAFRQGA
Xc 220 FDYFTGQLSK RGKGAGIARQ GHDDYSNFLQ AGSAGDFAKA AGLEQLPWWH
Xf 216 FDYFSGQMTN RGKGLSIARQ GYDDYSNFLQ AGSAGNYAKA AGLEQLPWWH
Zm 238 FDYFLRQMTA KGTGAPPVHG AYDDYKAFLE TGSAGNWAKK EGIDQIPWWQ Ap 251 FDYFVSQMTA RGGGNDIPRR DADDYTNFLK AGSAGSFATQ AGLDQYPFWQ
At 251 FDYFVSQMTA RGGGNDIPRR DADDYTNFLK AGSAGSFATQ AGLDQYPFWQ
#
Xc 270 KLTEHAAYDA FWQEQALDKV MARTPLKVPT MWLQGLWDQE DMWGAIHSYA Xf 266 KLTEHPAYDS FWQEQALDKV MARTPLKVPT MWLQGLWDQE DMWGAIHSYA
Zm 288 RLSIHPAYDK FWQGQALDQL VAAHPSHVPT MWIQGLWDQE DMWGAIHSFE
Ap 301 RMHAHPAYDA FWQGQALDKI LAQRKPTVPM LWEQGLWDQE DMWGAIHAWQ
At 301 RMHAHPAYDA FWQGQALDKI LAQRKPTVPM LWEQGLWDQE DMWGAIHAWQ
#
Xc 320 AMEPRDKRNT LNYLVMGPWR HSQVNYDGSA LGALNFEGDT ARQFRHDVLR
Xf 316 AMEPRDVHND KNYLVMGPWR HSQVNYDGSN LGTLKFDGNT ALQFRHDVLK
Zm 338 SLKNAG-HID TNYLVMGPWR HSQVNYNGSS LGALHWDGDT ALQFRRDTLL
Ap 351 ALKDAD-VKA PNTLVMGPWR HSGVNYNGST LGPLEFEGDT AHQYRRDVFR
At 351 ALKDAD-VKA PNTLVMGPWR HSGVNYNGST LGPLEFEGDT AHQYRRDVFR
XC 370 PFFDQYLVDG APKADTPPVF IYNTGENHWD RLKAWPRSCD KGCAATSKPL Xf 366 PFFDQYLIDG ASKADTPPVL IYNTGENHWD RMQHWPRSCE RGCEYTSKPL
Zm 387 PFFNRYLKDK QPAEETPKAL IYNTGENHWD KLNDWQTDKE K QLTPL
Ap 400 PFFDEYLKPG SASVHLPDAI IYNTGDQKWD YYRSWPSVCE SNCTGGLTPL
At 400 PFFDEYLKPG SASVHLPDAI IYNTGDQKWD YYRSWPSVCE SNCTGGLTPL
Xc 420 YLQAGGKLSF QPPVAGQAGF EEYVSDPAKP VPFVPRPVDF ADRAMWTTWL
Xf 416 YLNAGGTLSF QTSQRKQNDY DEYISDPANP VPFMPRPINF KDNAMWTTWL
Zm 433 YLQAQSALSF TKPTSGNAPS DQYISDPKKP VPYLPTPITF ADTTRWKQWL
Ap 450 YLADRHGLSF THPAADG—A DSYVSDPAHP VPFISRPFAF AQSSRWKPWL At 450 YLADGHGLSF THPAADG—A DSYVSDPAHP VPFISRPFAF AQSSRWKPWL
Xc 470 VHDQRFVDGR PDVLTFVTEP LTEPLQIAGA PDVHLQASTS GSDSDWVVKL
Xf 466 VQDQRFVDNR PDVLTYLSEP LTTPLQIAGV PRINLHASTS GTDSDWVVKL Zm 483 IEDQRFAASR PDVLTYETPV LDHPEKLRGA PFANLLAATT GSDVDWVVKL
Ap 498 VQDQREAESR PDVVTYETEV LDEPVRVXGV PVADLFAATS GTDSDWVVKL
At 498 VQDQREAESR PDVVTYETEV LDEPVRVSGV PVADLFAATS GTDSDWVVKL
Xc 520 IDVYPEEMAS NPKMGGYELP VSLAIFRGRY RESFSTPKPL TSNQPLAFQF
Xf 516 IDVYPDEIAS DPKMGGNELA ISLGIFRGRY RTSFQYPTPM TPNQPLLYRF
Zm 533 IDVYPDEIPS DPKMGGYQLA ISMDIFRGRY RNSFEKPSPV PAGKVQQYRF
Ap 548 IDVQPAMTPD DPKMGGYELP VSMDIFRGRY RKDFAKPEAL QPDATLHYHF
At 548 IDVQPAMTPD DPKMGGYELP VSMDIFRGRY RKDFAKPEAL QPDATLHYHF Xc 570 GLPTANHTFQ PGHRVMVQVQ SSLFPLYDRN PQTYVPNIFF AKPGDYQKAT
Xf 566 DLPNVNHTFL TGHRIMVQVQ SSLFPLYDRN PQRYVPNIFF AKPDDYIKAT
Zm 583 RLPVVDHVFL PGHRIMVQIQ SSLFPLYDRN PQRYVENIMF AKPADYAATV Ap 598 TLPAVNHVFA KGHRIMVQIQ SSWFPLYDRN PQKFVPNIFD AKPVDYTVAT
At 598 TLPAVNHVFA KGHRIMVQIQ SSWFPLYDRN PQKFVPNIFD AKPADYTVAT
Xc 620 QRVYVSPEQP SYISLPVR— 637 Xf 616 QRIWHTPRQP SFIELPVVNP DMLTQRTHWN ISSSHKTTLP TPSLSDWL 663
Zm 633 ETVMHSPDQA SSVELPVIP- 666
Ap 648 QSIHHGGKEA TSILLPVVKQ 667
At 648 QSIHHGGKEA TSILLPVVKQ 667
In X.citri α-AEH Ser174 corresponds to Ser205 in A. turbidans, which was identified previously as the catalytic serine (see alignment in table 14). The 3- dimensional model of X.citri is defined in its atomic coordinates. Such coordinates can be used by proper software to display a 3-dimensional model. A suitable sofware package is Insight & Discover (Accelrys Ltd). Relevant atomic coordinates that describe the environment of the catalytic serine 174 have been set out in table 15 Examination of the structure around the catalytic serine for a catalytic base increases the nucleophilicity of the Ser174 by accepting the hydroxyl proton reveals that the Nεof His340 is within hydrogen bonding distance of the hydroxyl group. In addition N5 of this His340 forms a hydrogen bond with the carboxyl group of Asp307, which tends to increase the basicity of the histidine. So, the structure of X.citri reveals a catalytic triad consisting of Ser174, His340 and Asp307.
Table 15: Relevant atomic coordinates that describe the environment of the catalytic serine 174
ATOι\ 1 RES NR X Y Z
N THR 78 53,318 97,394 4,304
CA THR 78 52,47 96,207 4,328
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ATOM RES NR X Y Z
OD1 ASP 208 44,211 80,759 7,377
OD2 ASP 208 46,041 80,8 6,202
C ASP 208 43,636 76,811 5,944
0 ASP 208 42,593 76,626 6,537
N TRP 209 43,872 76,308 4,729
CA TRP 209 42,852 75,596 3,927
CB TRP 209 43,143 75,603 2,406
CG TRP 209 43,827 76,87 1 ,834
CD1 TRP 209 45,125 76,87 1 ,311
NE1 TRP 209 45,574 78,143 0,945
CE2 TRP 209 44,524 78,963 1 ,273
CD2 TRP 209 43,433 78,209 1 ,81
CE3 TRP 209 42,2 78,838 2,077
CZ3 TRP 209 42,064 80,18 1,757
CH2 TRP 209 43,127 80,913 1 ,212
CZ2 TRP 209 44,352 80,319 0,957
C TRP 209 42,57 74,162 4,448
0 TRP 209 41 ,405 73,764 4,566
N PHE 210 43,632 73,412 4,767
CA PHE 210 43,545 72,084 5,378
CB PHE 210 43,849 70,925 4,381
CG PHE 210 43,099 70,962 3,061
CD1 PHE 210 42,132 70,012 2,781
CE1 PHE 210 41,485 69,976 1,567
CZ PHE 210 41 ,795 70,904 0,608
CE2 PHE 210 42,779 71 ,833 0,853
CD2 PHE 210 43,442 71 ,848 2,069
C PHE 210 44,654 72,008 6,429
0 PHE 210 45,596 72,804 6,418
N ASN 211 44,557 71 ,024 7,31
CA ASN 211 45,643 70,594 8,185
CB ASN 211 45,329 70,842 9,682
CG ASN 211 45,586 72,276 10,134
OD1 ASN 211 45,629 72,529 11 ,331
ND2 ASN 211 45,75 73,214 9,187 o O o o O z O O z z o z O O O O z O O O 0 O O O O 0 O z O O O O z O O m σ CD CD > I X N m σ CD CD > O m N CD CO > CD > to ro to m g 0
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ATOM RES NR X Y Z
CE3 TRP 267 42,118 79,591 -4,327
CZ3 TRP 267 43,425 79,244 -3,971
CH2 TRP 267 43,731 78,805 -2,675
CZ2 TRP 267 42,76 78,708 -1 ,712
C TRP 267 38,166 77,836 -5,13
0 TRP 267 38,887 76,915 -4,787
N TRP 268 36,846 77,782 -5,005
CA TRP 268 36,208 76,615 -4,444
CB TRP 268 34,729 76,871 -4,232
CG TRP 268 34,048 75,648 -3,757
CD1 TRP 268 33,092 74,965 -4,391
NE1 TRP 268 32,735 73,86 -3,659
CE2 TRP 268 33,488 73,825 -2,514
CD2 TRP 268 34,34 74,925 -2,554
CE3 TRP 268 35,214 75,128 -1 ,493
CZ3 TRP 268 35,212 74,233 -0,445
CH2 TRP 268 34,369 73,135 -0,443
CZ2 TRP 268 33,496 72,906 -1 ,469
C TRP 268 36,432 75,36 -5,33
0 TRP 268 36,612 74,269 -4,815
N LEU 271 39,974 74,19 -4,747
CA LEU 271 40,074 73,378 -3,533
CB LEU 271 39,273 74 -2,395
CG LEU 271 39,889 75,207 -1 ,728
CD1 LEU 271 38,894 75,841 -0,763
CD2 LEU 271 41 ,113 74,814 -1 ,024
C LEU 271 39,581 71,959 -3,758
0 LEU 271 40,252 70,987 -3,377
N TRP 281 49,437 72,105 -4,654
CA TRP 281 49,843 72,505 -3,297
CB TRP 281 49,148 71 ,633 -2,261
CG TRP 281 47,704 71 ,838 -2,289
CD1 TRP 281 46,774 70,953 -2,68
NE1 TRP 281 45,521 71 ,507 -2,611
CE2 TRP 281 45,635 72,792 -2,163 o O z O O O 0 0 O O 2 O O O 0 O O 0 z O 0 2 0 0 O O 0 z O O O O O 0 0
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Example 10 Identification sequence patterns which identify preferred α-AEH's
The 3-dimensional model of the X.citri active site allows for the calculation of the solvent accessible surface around the catalytic Ser174. The accessible surface reveals a binding pocket in which for example a substrate such as ampicillin can be fitted. The coordinates for the ampicillin molecule, which has been fitted into the active site of α-AEH are shown in table 15. Surprisingly the model reveals some features which are essential for the specificity of α-AEH. The carboxyl groups of Asp 208, Glu309 and Asp310 form a cation binding pocket which binds positively charged substituents at the substrate Cα position. In ampicillin the positively charge α-amino group of the acyl side chain is bound in this pocket. Given a number of other typical features observed for the family of proteins that comprises the α-AEH's, we can now set out the following sequence criteria to identify the useful α-AEH enzymes within this much larger family. After alignment of the amino acid sequence of a potential α-AEH with the theα-AEH's of X.citri, A.turbidans, A.pasteurianus, A.fastidiosa, Z.mobilis the sequence homology should be at least 60% with one of the given α-AEH's. The sequence should contain the following pattern typical for the catalytical serine: Gxx[G/A]xxGxSYx[G/A] where x indicates any amino acid at that position, [/] indicates a number of possibilities e.g. [G/A] means Glycine or Alanine. In addition the sequence should contain the following pattern which reveals the catalytic histidine: GPWxH. In between the sequence patterns Gxx[G/A]xxGxSYx[G/A] and GPWxH the preferred αAEH should contain the following two sequence patterns: [W/Y/F]xGDDW and [W/Y/F]DxEDxx[G/A] in the same order as given here, starting from the N-terminus. Example 11 Oxyanion hole as a tool to modify S/H ratio The 3-dimensional structure of X.citri allows for the calculation of the accessible surface around the catalytic Ser174. The accessible surface reveals a clear binding pocket in which for example ampicillin can be fitted. The coordinates for the ampicillin molecule fitted in active site of α-AEH are shown in table 15. Hydrolysis of the substrate goes through formation of a so-called acyl enzyme. During formation as well as degradation of the acyl-enzyme a negative charge develops on the carbonyl oxygen, which is commonly stabilized by hydrogen bond donating groups on the protein. Such a binding spot fitted to stabilize the negatively charged oxygen is usually called the oxyanion hole. The most likely candidates for the hydrogen donors in AEHXϊ are the backbone amide of Tyr175 and the side chain hydroxyl group of Tyr82. In α- AEHAt the replacement of the corresponding Tyr112 by an alanine led to an inactive protein, confirming the importance of the hydroxyl group of Tyr82 in AEHXf, respectively Tyr112 in AEHAt, for the catalytic mechanism 2-AEH's.
So drastic modifications such as the removal of the hydrogen donating group lead to an inactive enzyme. However small perturbation of the oxyanion hole lead to subtle changes in for example the ratio synthesis over hydrolysis. Such small perturbation may be introduced by substitutions in the close environment of the groups forming the oxyanion hole. In particular amino acids that interact with the amino acids that form the oxyanion hole are preferred for this substitution. As in the case of AEHXt Tyr175 only the amide of the peptide bond is involved in formation of the oxyanion hole and not the amino acid side chain itself. Therefore also AEHXf Tyr175 itself is a good candidate for substitution in order to evoke a perturbation of the oxyanion hole. The corresponding oxyanion hole in A.turbidans is formed by residues AEHAt Tyr112 and AEHAt Tyr206.
Site directed mutagenesis of A.turbidans AEH>4t Tyr206 was performed as described before in example 8. The mutants were expressed, purified and characterised with respect to their kinetic properties. Nucleotide and sequence data of these mutants are provided in SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 , SEQ ID NO: 15, SEQ ID NO: 15, SEQ ID NO: 16 , SEQ ID NO: 17 and SEQ ID NO: 18. Results are shown in table 16
Table 16: Kinetic parameters of PGM hydrolysis for mutants of AEHAt.
Enzyme PGM
KM (mM) kca, (s-1) /cca(/KM (s-1.mM'1)
AEHΛπHis 1 ± 0.1 1067 ± 56 1067
Y206A 5.3 ± 0.9 655 ± 39 124
Y206F 14 ± 3 2333 ± 198 167
Y206N 6.3 ± 0.8 154 ± 7 24
Y206W 21 ± 4 388 ± 31 18
The mutation of the AEHAt Tyr206 to an alanine resulted in mutants with a better S/H-ratio and a higher Qmax (Table 17). In addition, other amino acids were placed on this position. An improvement in catalytic properties for the synthesis of cephalexin was also observed for AEHAt Y206N mutant. This is a very interesting position for saturated site directed mutagenesis. In addition mutations were made in A.turbidans AEHAt at positions Serl 56 and His 610, cooresponding to AEHXf Ser 125 and AEHXf His582 respectively. Both residues were replaced by alanine. The mutations did not show any effect on the synthesis over hydrolysis ratio. Both AEHΛf Ser156 and AEHAt His610 do not interact with any of the residues forming the oxyanion hole. The closest residue forming the oxyanion hole with respect to AEHAf Ser156 is AEHAf Tyr206 at 13 Angstroms. The closest residue to AEHΛf His 610 is AEHAt Tyr112 at 30 Angstroms. In additions also a mutation was made at position A.turbidans AEHΛf Arg85 corresponding to AEHXf Arg55. Distance of this residue to the active site is about 15 Angstroms. The kinetic properties did not differ significantly from the wild type enzyme.
Table 17
Kinetic parameters of cephalexin hydrolysis and synthesis of AEHAt mutants on position 206.
Enzyme Cephalexin hydrolysis Cephalexin synthesis
KM (mM) /cca, (s-1) S/Hjni Qmax (mM) /Hmax
AtHis 0.45 ± 0.06 274 ± 7 1.9 ± 0.3 8 ± 2 1.2 ± 0.1
Y206A 4.0 ± 0.2 120 ± 2 8 ± 2 10.7 ± 0.9 2.2 ± 0.1
Y206F 2.2 ± 0.3 77 ± 3 1.4 ± 0.6 8.9 ± 0.1 1.2 ± 0.03
Y206N 11 ± 2 31 ± 3 3.5 ± 0.4 11.2 ± 0.1 2.6 ± 0.06
Y206W 5.6 ± 0.9 14.8 ± 0.9 2.1 ± 0.1 5.5 + 0.1
Based on the above principle of perturbing the oxyanion binding site, instead of replacing the AEHXf Tyr175 side chain one may also change the position of the AEHXf Tyr175 side chain by mutation of neighbouring residues. Therefor the X.citri model was examined for residue which influence the orientation of the Tyr175 side chain. The same was done for AEHXf Tyr82 which is the second residue that contributes to the formation of the oxyanion binding hole. The results are shown in the table 18 below. Table 18 : Positions in α-AEH which influence S/H ratio
Figure imgf000093_0001
In α-AEH substitution of the residues at positions corresponding to Arg79, Thr80, Pro81 , Arg87Nal119, Lys122 Tyr129, Met131 , Thr132, Phe178, His146,Glu176,Thr179, Tyr222, Gln226 in X.citri will modify the ratio synthesis over hydrolysis. The substitution, which will give the most optimal result for a certain precursor side chain and an acceptor nucleophile combination can easily determined by subjecting the positions mentioned above to site-saturated mutagenesis and selecting for the best performing mutant.
The effective synthesis over hydrolysis ratio is a delicate balance of the specificity of the α-AEH for the acyl side chain donor, the nucleophile and the water and ultimately also the final acylation product. The specificity of the acyl side chain of the precursor may be tuned by modification of the binding site for precursor acyl site chain. In the table below the interactions with the phenylglycine side chain of ampicillin have been mapped. Substitution of amino acids at the mapped positions not only improve the specificity for phenylglycine even further, but it also improves or shifts the specificity towards different side chains, e.g. p-hydroxylphenylglycine which is found in amoxycillin and cephadroxyl.
Table 19: Positions in α-AEH which allows for optimising S/H ratio's with different acyl side chain donors.
Figure imgf000094_0001
In α-AEH substitution of the residues at positions corresponding to
Met200, Asp208, Trp209, Val218, Arg216, Asn219, Tyr222, Phe223, Trp267, Trp281 , Leu271 , Glu309, Asp310, Trp465 in X.citri will change the preference for a certain acyl- sidechain. As a result a much better synthesis over hydrolysis ratio with such a side chain will be obtained. Substitution at given positions allows for improving efficiency the synthesis of semi-synthetic β-lactam antibiotics with side chain precursers which are poor substrates with the corresponding wild type α-AEH's
Once the acyl intermediate has formed the competition between water (hydrolysis) and the nucleus (the acceptor) will be dependant very much on how well the acceptor can be positioned towards a nucleophilic attack of the acyl intermediate with respect to water. The orientation of the nucleophile with respect to the transition state can be improved by mutagenesis at the positions given below.
Table 20: Positions in α-AEH which allows for optimising S/H ratio's with different nucleophiles.
Figure imgf000095_0001
In α-AEH substitution of the residues at positions corresponding to Arg87, Serl 73, Gln308, Glu309, Arg339, Gln342, Asn344, Leu350, Ser341 , Tyr345, Arg462, Thr466, Thr467 in X.citri will affect specificity for the nucleophile in the transfer of acyl side chains from a precursor to that nucleophile.

Claims

1. An isolated polynucleotide having at least 40% sequence identity to a polynucleotide selected from the group consisting of SEQ ID NO:1 , SEQ ID NO: 2 , SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO: 5 and fragments and variants thereof, provided the sequence is not 100% identical with a sequence according to SEQ ID NO:4 or SEQ ID NO: 5
2. An isolated polynucleotide according to claim 1 encoding a functional alpha- amino ester hydrolase.
3. An isolated polynucleotide according to claims 1 or 2 encoding a functional alpha-amino ester hydrolase with a higher synthesis to hydrolysis ratio than the wild type
4. An isolated polynucleotide according to any of claims 1 to 3 encoding a functional alpha-amino ester hydrolase with a S/H ini ratio higher than 1.9, preferably higher than 2, 2.5, 3.0, 4.5, 5, 8, 10 and most preferably higher than
15.
5. An isolated polynucleotide according to any of claims 1 to 4 encoding a functional alpha-amino ester hydrolase with a Q max (mM) ratio higher than 8, preferably higher than 10, 12 and most preferably higher than 15.
6. An isolated polynucleotide according to any of claims 1 to 5 encoding a functional alpha-amino ester hydrolase with a S/H max ratio higher than 1.2, preferably higher than 2, 2.5, 3.0, 4.5, 5, 8, 10 and most preferably higher than 15.
7. An isolated polynucleotide according to any of claims 3 to 6 comprising a sequence selected from the group consisting of SEQ ID NO: 11 , SEQ ID NO:
12 , SEQ ID NO: 13 and SEQ ID NO: 14.
8. An isolated polynucleotide encoding a functional alpha-amino ester hydrolase hybridisable to a polynucleotide according to any of claims 1 to 7.
9. An isolated polynucleotide according to claim 8 hybridisable under high stringency conditions to a polynucleotide according to claim 1 to 7
10. An isolated polynucleotide according to claims 1 to 9 obtainable from a microorganism.
11. An isolated polynucleotide according to claim 10 obtainable from a microorganism selected from the group of Proteobacteria
12. An isolated polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7 SEQ ID NO: 8, SEQ ID NO: 15 , SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO: 18 or fragments or functional equivalents thereof with the provision that said isolated polynucleotide does not encode a sequence according to SEQ ID NO: 9 or SEQ ID NO: 10
13. An isolated polynucleotide according to claim 12 encoding a functional alpha- amino ester hydrolase.
14. An isolated polynucleotide selected from the group consisting of SEQ ID NO:1 , SEQ ID NO: 2 and SEQ ID NO:3.
15. A vector comprising a polynucleotide sequence selected from the group consisting of a polynucleotide according to claims 1 to 14 and a polynucleotide according to SEQ ID NO: 4 and SEQ ID NO: 5.
16. A vector according to claim 15 wherein said polynucleotide sequence is operatively linked with regulatory sequences suitable for expression of said polynucleotide sequence in a suitable host cell.
17. A vector according to claim 16 wherein said suitable host cell is selected from the group consisting of Escherichia coli, Pichia pasteuris, Aspergillus nidulans, Lactococcus lactis, and Bacillus subtilis
18. A method for manufacturing a polynucleotide according to claims 1 to14 or a vector according to claims 15 to 17 comprising the steps of culturing a host cell transformed with said polynucleotide or said vector and isolating said polynucleotide or said vector from said host cell.
19. Recombinantly produced alpha-amino ester hydrolase .
20. An isolated polypeptide having an amino acid sequence that is at least 40% identical with a sequence selected from the group consisting of SEQ ID NO:6,
SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 or functional equivalents thereof, with the proviso that said amino acid sequence is not 100% identical with a sequence according to SEQ ID NO:9 or SEQ ID NO: 10.
21. An isolated polypeptide obtainable by expressing a polynucleotide according to claims 1 to 14 or a vector according to claims 15 to 17 in an appropriate host cell.
22. Recombinant nucleic acid encoding an enzyme comprising at least one functional domain of an alpha-amino ester hydrolase
23. A method for manufacturing a polypeptide according to claims 19 to 21 comprising the steps of transforming a suitable host cell with an isolated polynucleotide according to claims 1 to 14 or a vector according to claims 15 to 17, culturing said cell under conditions allowing expression of said polynucleotide and optionally purifying the encoded polypeptide from said cell or culture medium.
24. A recombinant host cell comprising a polynucleotide according to claims 1 to 14 or a vector according to claims 15 to 17.
25. A recombinant host cell expressing a polypeptide according to claims 19 to 21.
26. A recombinant host cell comprising a polynucleotide encoding a functionally active alpha-amino ester hydrolase.
27. Use of a polynucleotide selected from the group consisting of a polynucleotide according to claims 1 to 14 or a vector according to claims 15 to 17 or a polynucleotide according to SEQ ID NO: 4 or SEQ ID NO: 5 for the production of an AEH.
28. Use of a polypeptide according to claims 19 to 21 or according to SEQ ID NO: 9 or SEQ ID NO: 10 in the production of β-lactam antibiotics.
29. Purified antibodies reactive with a polypeptide according to claims 19 to 21.
30. Fusion protein comprising a polypeptide sequence according to claims 19 to 21.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005119244A1 (en) * 2004-06-03 2005-12-15 Phylogica Limited Peptide modulators of cellular phenotype and bi-nucleic acid fragment library
US7803765B2 (en) 1999-05-05 2010-09-28 Phylogica Limited Methods of constructing biodiverse gene fragment libraries and biological modulators isolated therefrom
RU2502797C1 (en) * 2012-09-18 2013-12-27 Федеральное государственное унитарное предприятие "Государственный научно-исследовательский институт генетики и селекции промышленных микроорганизмов" (ФГУП "ГосНИИгенетика") STRAIN Escherichia coli - PRODUCER OF HYDROLASE OF ALPHA-AMINO ACIDS FROM Xanthomonas rubrilineans, AND METHOD FOR MICROBIOLOGICAL SYNTHESIS OF HYDROLASE OF ALPHA-AMINO ACIDS ESTERS BASED ON ABOVE SAID STRAIN
WO2016114479A1 (en) * 2015-01-16 2016-07-21 아미코젠주식회사 Modified α -amino acid ester hydrolase with increased amoxicillin productivity

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL [Online] EBI; Zymomomas mobilis fosmid clone 43D2, 11 May 1999 (1999-05-11) LEE H.J., KANG. H.S.: "Sequence analysis of 43D2 fosmid clone of Zymomomas mobilis ZM4" Database accession no. AF124757 XP002224300 *
FERNANDEZ-LAFUENTE ROBERTO ET AL: "Biotransformations catalyzed by multimeric enzymes: Stabilization of tetrameric ampicillin acylase permits the optimization of ampicillin synthesis under dissociation conditions." BIOMACROMOLECULES, vol. 2, no. 1, 1 April 2001 (2001-04-01), pages 95-104, XP002224298 Spring, 2001 ISSN: 1525-7797 *
POLDERMAN-TIJMES JOLANDA J ET AL: "Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding an alpha-amino acid ester hydrolase from Acetobacter turbidans." APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 68, no. 1, January 2002 (2002-01), pages 211-218, XP001121414 January, 2002 ISSN: 0099-2240 *
POLDERMAN-TIJMES JOLANDA J ET AL: "Identification of the catalytic residues of alpha-amino acid ester hydrolase from Acetobacter turbidans by labeling and site-directed mutagenesis." JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 277, no. 32, 9 August 2002 (2002-08-09), pages 28474-28482, XP002224299 August 9, 2002 ISSN: 0021-9258 *
SIMPSON A J ET AL: "The genome sequence of the plant pathogen Xylella fastidiosa. The Xylella fastidiosa Consortium of the Organization for Nucleotide Sequencing and Analysis." NATURE. ENGLAND 13 JUL 2000, vol. 406, no. 6792, 13 July 2000 (2000-07-13), pages 151-157, XP002224297 ISSN: 0028-0836 -& DATABASE EMBL [Online] EBI; Xylella fastidiosa 9a5c, section 86 of 229, 18 July 2000 (2000-07-18) SIMPSON A.J.G. ET AL.: "The genome sequence of the plant pathogen Xylella fastidiosa." Database accession no. AE003940 XP002224359 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7803765B2 (en) 1999-05-05 2010-09-28 Phylogica Limited Methods of constructing biodiverse gene fragment libraries and biological modulators isolated therefrom
WO2005119244A1 (en) * 2004-06-03 2005-12-15 Phylogica Limited Peptide modulators of cellular phenotype and bi-nucleic acid fragment library
RU2502797C1 (en) * 2012-09-18 2013-12-27 Федеральное государственное унитарное предприятие "Государственный научно-исследовательский институт генетики и селекции промышленных микроорганизмов" (ФГУП "ГосНИИгенетика") STRAIN Escherichia coli - PRODUCER OF HYDROLASE OF ALPHA-AMINO ACIDS FROM Xanthomonas rubrilineans, AND METHOD FOR MICROBIOLOGICAL SYNTHESIS OF HYDROLASE OF ALPHA-AMINO ACIDS ESTERS BASED ON ABOVE SAID STRAIN
WO2016114479A1 (en) * 2015-01-16 2016-07-21 아미코젠주식회사 Modified α -amino acid ester hydrolase with increased amoxicillin productivity

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