WO1996002630A1 - Analytical and preparative methods for purifying phthalyl amidase from xanthobacter agilis - Google Patents

Abstract

An analytical process for purifying phthalyl amidase from Xanthobacter agilis is presented. The process comprises the sequential steps of obtaining a cell free extract, anion exchange chromatography, ammonium sulfate fractionation, hydrophobic interaction chromatography, hydroxylapatite chromatography, and a second anion exchange chromatography. The resulting amidase is about 95 % purity as determined by SDS electrophoresis. A preparative process is also presented which comprises the sequential steps of anion exchange chromatography of the cell free extract followed by hydroxylapatite chromatography.

Description

-ANALYTICALANDPREPARATIVEMETHODSFORPURIFYINGPHTHALYLAMIDASE FROM XANTHOBACTER AGILIS"

Background of the Invention The present invention relates to the discovery of a specific enzyme that has not been previously described, a phthalyl amidase, which readily removes the phthalyl moiety from phthalyl amides. The present invention also relates to an organism isolated from natural sources that produce the enzyme, DNA compounds that encode the enzyme, and methods for producing and using the enzyme. The phthalimido functional group is an important tool in organic synthesis because of the protection it provides against unwanted reactions. However, dephthalylation reactions generally require harsh conditions and often have low yields thereby limiting the situations in which phthalimido protection can be employed.

Removal of a phthalyl protecting group from a phthalyl amide can be accomplished chemically, Kukolja et al., Croatica Chemica Acta 49:779, 1977, but yields are variable especially with substrates that are unstable to harsh reaction conditions.

Certain enzymes have previously been found that could be used to remove benzoyl groups from benzoylated amino acids. Toyoura et al., Chem. Pharm. Bull. 7:789, 1959. These enzymes were specific for benzoyl groups and for the amino acid to which they were attached. Others have also reported enzymes that will hydrolyze phthalate esters. Kurane et al., Agric. Biol. Chem. 44:529, 1980. However, none of these enzymes have been shown to operate on phthalyl amides.

In contrast, the phthalyl amidase enzyme of this invention catalyzes removal of the phthalyl group from a wide variety of phthalyl-containing compounds with improved yields over processes known in the art, exhibits stereochemical selectivity, and eliminates the need for harsh conditions to remove the protecting group. in light of the previously undescribed existence of phthalyl amidase activity, a process for its purification was heretofore unknown.

Summary of the Invention The present invention provides a process for purifying a phthalyl amidase enzyme that may be obtained by a number of means provided by this invention. For example, the phthalyl amidase enzyme may be obtained from Xanthobacter agilis, the organism that produces the natural enzyme; heterologous organisms that have been transformed to express recombinant phthalyl amidase; and in a preferred instance, from the culture broth of recombinant organisms that express phthalyl amidase in secreted soluble form. The present invention provides a process for purifying the phthalyl amidase enzyme which is derived from any of the sources identified. Thus, a process for purifying phthalyl amidase comprises: A) chromatographing a crude cell-free extract of phthalyl amidase, obtained by disruption of phthalyl amidase-containing cells, over an anion exchange resin, eluting bound proteins with a linear KCl gradient of 0-1.5 M, followed by pooling the fractions eluted at between about 1 M and about 1.1 M KCl;

B) fractionating the pooled eluate of step A with ammonium sulfate and solubilizing pellets obtained at an ammonium sulfate concentration of between about 67% and about 97%;

C) chromatographing the solubilized pellets of step B over a hydrophobic interaction resin, eluting bound proteins with a decreasing linear gradient of 2.6-0 M ammonium sulfate, followed by pooling the fractions eluted at between about 0.4 M and 0 M ammonium sulfate and removing the salts contained in the pooled eluate;

D) chromatographing the pooled eluate of step C over hydroxylapatite, eluting bound proteins with a linear gradient of 50-500 mM potassium phosphate, pH 8.0, followed by pooling the fractions eluted at between about 150 and about 190 mM potassium phosphate; and

E) chromatographing the pooled eluate of step D over an anion exchange resin, eluting bound proteins with a linear gradient of 0-1.5 M KCl, followed by pooling the fractions eluted at between about 0.72 M and about 0.8 M KCl; wherein steps A, B, C, D, and E are carried out at a temperature between about 0° C and about 10° C; and wherein each of steps A, B, C, D, and E are carried out in 50 mM potassium phosphate buffer, pH 8.0.

For purposes of this process, Q-Sepharose is a preferred resin for use in step A, Phenyl-Sepharose is a preferred resin for use in step C, and Mono P is a preferred resin for use in step E.

The present invention also provides a preparative scale process for purifying phthalyl amidase, said process comprising: A) chromatographing a crude cell-free extract of phthalyl amidase, obtained by disruption of phthalyl amidase-containing cells, over an anion exchange resin, eluting bound proteins with a linear KCl gradient of

0-1.5 M, followed by concentrating and diafiltering the fractions eluted at between about 1 M and about 1.1 M KCl; and

B) chromatographing the pooled eluate of step A over hydroxylapatite, eluting bound proteins with a linear gradient of 0-500 mM potassium phosphate, pH 8.0, and collecting the fractions eluted at between about 150 and about 190 mM potassium phosphate; wherein steps A and B are carried out at a temperature between about 0° C and about 10° C; and wherein steps A and

B are carried out in 50 mM potassium phosphate buffer, pH 8.0 and the fractions are concentrated and diafiltered in

50 mM potassium phosphate, pH 8.0.

For purposes of the preparative scale process,

Super-Q is a preferred anion exchange resin. In addition, the present invention provides a process for purifying phthalyl amidase comprising chromatographing a cell-free culture broth, obtained by clarifying the culture broth of a cell that secretes soluble phthalyl amidase into the culture medium, over an anion exchange resin, eluting bound proteins with a linear gradient of 0-1.5 M KCl in 50 mM potassium phosphate, pH 8.0 and collecting the fractions eluted at about 0.75 M KCl. For purposes of purifying phthalyl amidase from culture broth, Mono Q, Q-Sepharose, or Super-Q are preferred resins.

Definitions: Coding sequence - the sequence of DNA in the open reading frame of a gene that encodes the amino acid residue sequence of the protein expressed from the gene.

Gene - a segment of DNA that comprises a promoter, translational activating sequence, coding sequence, and 3' regulatory sequences, positioned to drive expression of the gene product.

Promoter - a DNA sequence that directs or initiates the transcription of DNA.

Recombinant DNA vector - any autonomously replicating or integrating DNA agent, including but not limited to plasmids, comprising a promoter and other regulatory sequences positioned to drive expression of a DNA sequence that encodes a polypeptide or RNA. Recombinant DNA sequence - any DNA sequence, excluding the host chromosome from which the DNA is derived, which comprises a DNA sequence that has been isolated, synthesized, or partially synthesized. Restriction fragment - any linear DNA molecule generated by the action of one or more restriction enzymes.

Translation activating sequence - a regulatory DNA sequence that, when transcribed into mRNA, promotes translation of mRNA into protein. All nucleotide and amino acid abbreviations used in this disclosure are those accepted by the united States Patent and Trademark Office as set forth in 37 C.F.R. S1.822(b)(1993).

Brief Description of the Figures The restriction enzyme and function maps presented in the drawings are approximate representations of the recombinant DNA vectors discussed herein. The restriction site information is not exhaustive. There may be more restriction enzymes sites of a given type than are actually shown on the map.

Figure 1 is a restriction enzyme site and function map of plasmid pZPA600. PAorf = phthalyl amidase open reading frame, tsr = gene enabling resistance to thiostrepton.

Figure 2 is a restriction enzyme site and function map of plasmid pZPA400. PA-orf = pthalyl amidase open reading frame. pL97-pro = modified promoter from phage lamda. tet = gene enabling resistance to thiostrepton. cl857 = gene encoding temperature sensitive lamda repressor.

Detailed Description of the Invention The phthalyl amidase of the current invention catalyzes the following type of reaction:

The phthalyl amidase enzyme is characterized by the following: a) Reactivity: said enzyme catalyzes the removal of the phthalyl group from phthalyl amides generating phthalate and an amine; b) Substrate specificity: said enzyme hydrolyzes phthalylated amino acids, peptides, beta- lactams, aromatic and aliphatic amines; substitutions allowed on the phthalyl group include 6-F, 6-NH₂, 3-OH, and a nitrogen in the aromatic ring ortho to the carboxyl group attached to the amine; c) Reactive pH range: 5.5 to 9.0, with optimum pH of 8.0 ± 0.4; d) Reactive temperature range: 10 to 50° C, with optimum temperature of 30° C ± 4° C at pH 8.0; e) Temperature stability: At 200 mM buffer, 80% of enzyme activity retained at 35° C for 48 hours; f) Influence of effectors: Iodoacetate, p- HMB, and Cu⁺⁺ exert inhibitory activity; g) Molecular weight: approximately 49,900 daltons; h) Subunits: one; i) Km: 0.9 mM in 50 mM potassium phosphate buffer, 30° C, pH 8.0, when phthalyl carbacephem is the substrate.

During the course of developing a chiral, shorter, and more efficient synthetic route to loracarbef ([6R-(6A,7B(R) )]-7-[(aminophenylacetyl)amino]-3-chloro-8- oxo-azabicyclo[4,2,0]oct-2-ene-2-carboxylic acid), the Mitsunobu reaction (see e.g. Hughes, D.L. Organic reactions 42:336, 1992; Bose, A.K. et al., Can. J. Chem. 62:2498, 1984) was selected for forming the beta-lactam ring from a chiral linear amino acid ester intermediate. Several reactants with one N-valence protected and a few reactants with both N-valences protected were examined in Mitsunobu reactions. They were either not cyclized or were cyclized in poor yield.

It was discovered that problems in forming the beta-lactam ring via Mitsunobu reactions could be overcome if both valences of the α-nitrogen of the chiral linear amino acid ester intermediate were protected with a phthalimido group. However, no known chemical reaction was available to remove the phthalimido moiety and regenerate free amine in high yield.

Thus, soil samples were examined for microorganisms that could catalyze removal of the phthalamido group from a test substrate (II) that was formed by base cleavage of the phthalimido ring of a bivalently N-protected compound. A culture was identified that demonstrated phthalyl amidase activity that liberated the free amine derivative of the test substrate. Native enzyme was purified and shown to catalyze the following desired reaction:

Phthalyl amidase also has significant value in peptide synthesis applications. Phthalimido amino acid derivatives are very effective reactants for enzymatic coupling of amino acids to form peptides. However, heretofore, methods for removing the phthalimido blocking group from the protected peptide were lacking. The phthalyl amidase of the current invention displays reactivity toward a wide range of substrates and can be used for deblocking phthalimido-protected peptide intermediates.

The isolated phthalyl amidase of this invention demonstrates high specific activity toward phthalylated amides or esters (i.e., having a 1,2 dicarboxylate configuration). Such compounds may have other functional groups on the phthalyl aromatic ring and still serve as substrates for the enzyme. For example, acceptable functional groups include 6-F, 6-NH₂, and 3-OH. Moreover, substrates may include a nitrogen in the aromatic ring ortho to the carboxyl group attached to the amine. Compounds lacking a 2-carboxylate, such as benzoyl, phenylacetate, phenoxyacetate, or their derivatives, are not suitable substrates for this enzyme. The enzyme also exhibits a broad substrate specificity in regard to the amine group attached to the phthalate side chain. For example, phthalylated amino acids and peptides, mono- and bicyclic beta-lactams, aromatic and non-aromatic amines, as well as phthalylated amines attached to heterocycles, are dephthalylated by this enzyme at acceptable catalytic rates. The enzyme also removes the methyl group from mono-methyl phthalate.

The enzyme is stable in the broad range of pH from 6-9, having an optimum stability pH of 8.0 ± 0.4. The enzyme also demonstrates a marked stability dependence on ionic strength. Ionic strength above 20 mM enhances pH and temperature stability of the enzyme. Optimum ionic strength occurs at 200 mM and above.

The enzyme retains good activity in low salt (50 mM) up to 30° C and in high salt (200 mM) up to 40° C. In 200 mM salt, at least 80% of the enzyme activity is retained in temperatures up to 35° C for 48 hours.

Iodoacetic acid (10 mM), p-HMB (1 mM), and Cu⁺⁺ (1 mM) significantly inhibited the enzyme. No organic co- factors, such as ATP, NADPH, or others, stimulated enzyme activity. EDTA, phenanthroline, and metal ions besides Cu"^1""^1" had little or no effect on enzyme activity. The molecular weight of the enzyme is approximately 49,900, as determined by electrospray mass spectrometry, and the molecule consists of one subunit.

The K_m, with phthalamido carbacephem (7- phthalamido-3-chloro-4-carboxy-1-carba-dethioceph-3-em)

(III) as substrate, is 0.9 mM in 50 mM potassium phosphate buffer, pH 8.0, and 30° C. The Vm_ax for this substrate and under these conditions is 7.6 μmol/min/mg.

Phthalyl amidase activity was recovered from a microorganism isolated from soil samples. The organism was characterized by comparison of its fatty acid methyl ester profile with that of known standards, and has been identified as a strain of Xanthobacter agilis .

The organism can be preserved as lyophilized culture and has been deposited with the National Center for Agricultural Utilization Research, 1815 North University Street, Peoria, Illinois 61604-39999, under accession number NRRL B-21115 (date of deposit: 7/16/93). Working cultures are maintained as liquid cultures stored in liquid nitrogen or at temperatures below -78° C.

In order to recover the phthalyl amidase of this invention, Xanthobacter agilis can be cultivated in an aqueous nutrient medium consisting of a source of carbon and nitrogen and mineral salts at an initial pH between 6 and 8 and at 25° to 37° C. A number of agents can be included in the culture medium as inducers of enzyme production, including, for example, phthalate (PAA), phthalyl glycine (PAG), and phthalyl monocyclic beta-lactam (PMBL). The enzyme can be recovered in larger amounts by cultivating Xanthobacter agilis in a known manner in a bioreactor of desired size, for example, with a working volume of 100 liters. Good aerating conditions, and the presence of nutrients in complex form, and a pH between 6 and 8 are important for a successful culture. The cell mass can be separated from the medium and the enzyme purified as shown in Example 4.

It will be recognized by those skilled in the art that phthalyl amidase-producing mutants of the isolated Xanthobacter agilis organism can readily be made by methods known in the art. These mutants are considered within the scope of this invention.

As described, phthalyl amidase, catalyzes the removal of the phthalyl moiety from a wide range of phthalimido-containing compounds. The enzyme actually cleaves the amide bond of a phthalamido substrate, which is formed by the action of mild base on the corresponding phthalimido compound. This conversion proceeds readily under conditions that are suitable for enzyme activity. Thus, the phthalimido-containing compound and the enzyme being concurrently present under conditions that promote enzyme activity result in in situ removal of the phthalyl group. In some chemical reactions involving an amine reactant, the corresponding phthalimido compound is particularly suited to high reaction yields whereas the conversion proceeds poorly with the unprotected amine or with a monovalently protected amine or even when the amine is bivalently protected by an alternative means. Thus, the current invention, which provides an economic source of phthalyl amidase, allows practical synthesis of a variety of amine products via phthalimido-protected amine intermediates.

It will be recognized that the enzyme can also be used in immobilized form to catalyze desired reactions according to procedures known in the art. A specific application of the current invention occurs in a new chiral synthesis of the antibiotic loracarbef. The phthalyl amidase-catalyzed reaction shown above is one step of that synthesis.

Another application occurs in the synthesis of aspartame (N-L-α-aspartyl-L-phenylalanine, 1-methyl ester) as described in Example 16 below.

In both cases phthalic anhydride (or other suitable activated forms of phthalic acid) is used to react with an intermediate containing a key amino group so that a phthalimido moiety is formed for bivalent protection of the amino group. The bivalently protected amine can then be converted efficiently to a desired intermediate. For example, cyclization of a α-phthalimido-β-hydroxy-acid to a beta-lactam, or for example, condensation of an α- phthalimido carboxy-activated amino acid with a carboxy- protected amino acid to form a dipeptide. The phthalimido moiety is hydrolyzed with mild base and the resulting phthalamido moiety is then exposed to phthalyl amidase to catalyze the removal of the phthalyl moiety and release free amine plus phthalic acid.

In addition to identification and isolation of a naturally-occurring phthalyl amidase, the current invention provides isolated DNA compounds that comprise a DNA sequence encoding phthalyl amidase, recombinant DNA vectors encoding phthalyl amidase, host cells transformed with these DNA vectors, and a method for producing recombinant phthalyl amidase. These elements of the current invention provide the opportunity to use phthalyl amidase as a biocatalyst in industrial scale chemical processes.

Phthalyl amidase may be produced by cloning DNA encoding phthalyl amidase into a variety of vectors by means that are well known in the art. A number of suitable vectors may be used, including cosmids, plasmids, bacteriophage, and viruses. One of the principle requirements for such a vector is that it be capable of reproducing itself and transforming a host cell. Preferably, the vector will be a recombinant DNA vector that is capable of driving expression of phthalyl amidase encoded by the DNA compounds of this invention. Typical expression vectors comprise a promoter region, a 5'- untranslated region, a coding sequence, a 3'-untranslated region, an origin of replication, a selective marker, and a transcription termination site.

After the DNA compound encoding phthalyl amidase has been inserted into the vector, the vector may be used to transform a host cell, in general, the host cell may comprise any cellular organism, including a prokaryotic cell or eukaryotic cell, that is capable of being transformed with a vector comprising the DNA of this invention. The techniques of transforming and transfecting cells are well known in the art and may be found in such general references as Maniatis, et al . (1989) or Current Protocols in Molecular Biology (1989).

A particularly preferred method of the current invention generates soluble, extra-cellular enzyme. The method makes use of a DNA compound that comprises SEQ ID NO:l, which enables, when transformed into Streptomyces lividans as part of a self-replicating vector, the host to produce and secrete soluble mature phthalyl amidase in an amount 20-fold in excess of the amount of a cell-bound form of the enzyme produced by Xanthobacter agilis, the bacterium from which the DNA compound was cloned.

SEQ ID N0:1 comprises four functional components: SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:6; and SEQ ID NO:8. SEQ ID NO:3, which includes the promoter-bearing nucleotides 1-135 of SEQ ID NO:l, promotes transcription of the coding sequences. SEQ ID NO:4 (nucleotides 136-261 of SEQ ID NO:l) encodes the signal peptide portion of a proenzyme form of phthalyl amidase (pro-phthalyl amidase (SEQ ID NO:2)). The signal peptide (SEQ ID NO:5), which provides for transport of the proenzyme across the microbial cell wall of Streptomyces lividans, is cleaved from the proenzyme by the cell, thereby enabling extra¬ cellular production of the mature enzyme. SEQ ID NO:6 (nucleotides 262-1620 of SEQ ID NO:l) encodes mature phthalyl amidase (SEQ ID NO:7). SEQ ID NO:8 (nucleotides 1621-3029 of SEQ ID NO:l) is a 3'-untranslated region which assists proper and efficient translation termination of the mRNA that encodes pro-phthalyl amidase.

Moreover, in a more general application of the expression method of the current invention, a wide variety of soluble, extra-cellular, properly-folded, functional proteins may be produced in Streptomyces. The current method comprises propagating Streptomyces lividans that has been transformed with a DNA compound, which encodes the desired enzyme, protein, or peptide, and which includes the transcriptional and translational regulatory elements of the phthalyl amidase gene isolated from the bacterium Xanthobacter agilis. These regulatory elements enable synthesis and secretion of the soluble, properly-folded, functional enzyme, protein, or peptide.

To accomplish the general method, the DNA sequence encoding mature phthalyl amidase (SEQ ID NO:6) may be replaced in SEQ ID NO:l by a heterologous open reading frame from a wide variety of organisms wherein the heterologous open reading frame encodes a mature protein or hormone and introns are absent from those open reading frames, either by nature or by virtue of precise removal from genomic DNA to form cDNA open reading frames. In this arrangement, the regulatory elements of the phthalyl amidase gene continue to function such that proteins and oligopeptides other than phthalyl amidase are produced and secreted from Stre tozπyces transformed with the modified DNA sequence. Thus, substitution of a desired protein- encoding sequence for the coding sequence of mature phthalyl amidase enables economic extra-cellular production of numerous enzymes, peptides, and peptide hormones.

Synthesis of the phthalyl amidase gene and its various elements can be accomplished by recombinant DNA technology. Synthetic genes, the in vitro or in vivo transcription and translation of which will result in the production of the phthalyl amidase enzyme, may be constructed by techniques well known in the art. Owing to the degeneracy of the genetic code, the skilled artisan will recognize that a sizable, yet definite, number of DNA sequences may be constructed, which encode the phthalyl amidase enzyme. All such sequences are provided by the present invention.

A preferred sequence encoding phthalyl amidase is the naturally-occurring phthalyl amidase gene of Xanthobacter agilis, which is SEQ ID NO:l. This preferred gene is available on an 3.2 kb SacI-BamHI restriction fragment of plasmid pZPA600, which can be isolated from Streptomyces lividans TK23/pZPA600 by techniques well known in the art. Streptojπyces lividans TK23/pZPA600 designates Streptomyces lividans strain TK23 which has been transformed with vector pZPA600.

Plasmid pZPA600 was derived by ligating SEQ ID NO:l into Streptomyces vector, pIJ702 (Hopwood, D.A. , et al., Genetic Manipulations of Streptomyces : A Laboratory Manual , The John Innes Foundation, Norwich, England, 1985). The pIJ702 vector contains a pIJlOl Streptomyces replicon and a thiostrepton resistance gene for selection. The ligated material was transformed into Streptomyces lividans TK23 by a standard protoplast fusion technique. After selection on thiostrepton (45 mg/ml), the plasmid designated pZPA600, was isolated and confirmed by restriction analysis. A restriction site and function map of plasmid pZPA600 is found in Figure 1. Streptomyces lividans TK23/pZPA600 is publicly available and on deposit at the National Center for Agricultural Utilization Research, 1815 North University Street, Peoria, Illinois 61604-39999, under accession number NRRL 21290 (date of deposit: June 23, 1994). The Streptomyces lividans TK23 strain has been previously described in Plasmid 12:1936 (1984).

Plasmid pZPA600 allows high level expression of the pro-phthalyl amidase open reading frame and results in secretion of soluble mature phthalyl amidase, which process is especially preferred. -Thus, the invention comprises a process in which Streptomyces lividans TK23/pZPA600 is grown and then separated from its extra-cellular broth so that high concentrations of phthalyl amidase are obtained in that cell-free broth. Other preferred DNA sequences include, for example, SEQ ID NO:6, which encodes mature phthalyl amidase enzyme (SEQ ID NO:7), and SEQ ID NO:9, which encodes the proenzyme form of phthalyl amidase (SEQ ID NO:2). Thus, the present invention also comprises plasmid pZPA400 as a preferred embodiment.

In plasmid pZPA400, the regulatory elements of the native gene were removed and an ATG codon for a methionyl residue was attached to the 5'-terminal nucleotide of the mature phthalyl amidase coding sequence to generate an open reading frame (SEQ ID NO:10) encoding met-phthalyl amidase (SEQ ID NO:11). This sequence was positioned, via a two-cistron configuration, to be driven by a temperature inducible lambda pL promoter. Plasmid pZPA400 also contains the temperature sensitive cI857 repressor gene, a tetracycline resistance gene, and the pBR322-based origin of replication minus the rop region, which controls copy number (Cesareni et al., Proc. Natl. Acad. Sci. 79:6313, 1982). E. coli cells harboring this plasmid ( E. coli DH5α/pZPA400) are induced to produce met- phthalyl amidase (without signal peptide) when the culture temperature is raised from 30° C to 42° C. A restriction site and function map of plasmid pZPA400, which can be isolated from E. coli DH5α/pZPA400 cells by techniques well known in the art, is found in Figure 2.

E. coli DH5α/pZPA400 designates the commercially available E. coli DH5α strain that has been transformed with plasmid pZPA400. E. coli DH5α/pZPA400 cells are publicly available and on deposit at the National Center for Agricultural Utilization Research, 1815 North University Street, Peoria, Illinois 61604-39999, under accession number NRRL B21289 (date of deposit: June 23, 1994).

The phthalyl amidase gene may also be created by synthetic methodology. Such methodology of synthetic gene construction is well known in the art. See Brown et al.

(1979) Methods in Enzymology, Academic Press, N.Y., 68:109. The phthalyl amidase DNA sequence may be generated using a conventional DNA synthesizing apparatus, such as the Applied Biosystems Model 380A of 380B DNA synthesizers (commercially available from Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404.

Synthesis of the phthalyl amidase protein of the present invention may also proceed by solid phase synthesis. The principles of solid phase chemical synthesis of polypeptides are well known in the art and may be found in general texts, such as, Dugas, H. and Penny, C, Bioorganic Chemistry (1981), Springer-Verlag, New York, pp. 54-92. However, recombinant methods are preferred if a high yield is desired. A skilled artisan will recognize that the nucleotide sequences described in the present disclosure may be altered by methods known in the art to produce additional sequences that substantially correspond to the described sequences without changing their functional aspects. These altered sequences are considered to be included in the current invention.

In order that the invention described herein may be more fully understood, the following examples are set forth. It should be understood that the examples are for illustrative purposes only and are not to be construed as limiting the scope of the invention.

EXAMPLE 1 Search for phthalyl amidase producing organisms

240 soil samples (8 to 15 mg of damp dry soil) were individually suspended in 10 ml sterile BL medium (hereinafter defined) containing 100 mg phthalyl monocyclic beta-lactam (PMBL) (I).

BL medium had the following composition:

a₂HPθ₄ 6.0 g

KH₂PO₄ 3.0 g

NaCl 0.5 g

NH₄CI 2.0 g

CaCl₂ 0.1 g

MgS0₄-7H₂0 0.25 g

ZnSθ₄- H₂θ 70 mg

FeCl₃-6H₂0 270 mg

MnS0₄ 80 mg

CUCI2 7.4 mg

CθS0₄-7H₂0 28 mg

H₃B0₃ 3 mg

Yeast Extract 1.0 g

Deionized water 1.0 L

PH 7.0

The cultures were incubated aerobically at 30° C in a rotary shaker at 250 rpm for as long as 2 weeks. Cultures were examined by thin layer chromatography at 7 day intervals for the disappearance of the starting substrate and appearance of the beta-lactam nucleus product. A culture showing the desired catalytic activity was transferred at least two more times under similar conditions of medium and growth. The final culture was diluted with sterile water and plated out on agar plates containing either Trypticase Soy Broth (Difco) or Bac MI medium. Bac MI medium had the following composition:

Peptone 10.0 g Beef Extract 5.0 g

Yeast Extract 2.0 g

NaCl 5.0 g

Deionized water 1.0 L pH 7.0

(Agar plates were prepared by adding 20 g agar per L of medium) .

Individual colonies were picked from the agar and grown in Bac Ml medium containing 10 mg/ml of PMBL for 12 days at 30°C with aeration. Broths were examined for appearance of beta-lactam nucleus and phthalic acid using TLC.

A pure isolated organism that demonstrated rapid hydrolysis of the substrate was then grown in Bac MI medium containing 1 mg/ml phthalate for 48 hours at 30° C with aeration. Cells were centrifuged and then suspended in 50 mM Tris-HCl buffer, pH 8.0, at a ratio of 1 g wet weight cells to 8 ml of buffer. A solution of lysozyme, 2 mg in 1.0 ml 50 mM EDTA, pH 8.2, was added at the ratio of 1 ml lysozyme solution to 8 ml cell suspension. After mixing well and holding at room temperature for 1 hour, the suspension was cooled to 4° C and held overnight. The resultant viscous solution was sonicated only long enough to liquefy the solution. This solution was centrifuged at

10,000 rpm for 15 minutes. The pellet was discarded and the supernatant tested for phthalyl amidase activity.

The cell-free extract was chromatographed on a size exclusion column (1.5 x 100 cm; Sephacryl S-300;

Pharmacia, Piscataway, NJ) at 4° C with an elution buffer consisting of 50 mM potassium phosphate and 150 mM KCl at a flow rate of 0.5 ml/min. The eluant was monitored at a wavelength of 280 run. UV-absorbing fractions were tested for hydrolysis of PMBL by HPLC.

Reference proteins for molecular weight

(daltons) determination were chymotrypsinogen (25,000), ovalbumin (43,000), albumin (67,000), aldolase (158,000), catalase (232,000), ferritin (440,000), and thyroglobulin

(669,000) .

Cell-free extract of the organism subsequently identified as Xanthobacter agilis was determined to contain an enzyme that catalyzed the hydrolysis of PMBL, and which had an approximate weight of 54,000 daltons and a specific activity of 39.7 nmol/min/mg.

EXAMPLE 2 Production of phthalyl amidase from Xaπ i_oj acter agilis

Fermentation of Xanthobacter agilis on a 100 L scale was conducted in 100 L working volume bioreactors, with automatic control for pH (7.9-8.1), temperature (30° C), air flow (1 scfm) , agitation (300 rpm), and back pressure (5 lb) . Dissolved oxygen levels (>50%) were kept constant by small increases in agitation speed. The medium consisted of 1.25% Bacto peptone, 0.3% yeast extract, 0.5% beef extract, 0.5% phthalic acid, 0.5% NaCl, and 0.05% anti-foam. After sterilization, the medium was brought to pH 8.0 with 30% sulfuric acid. The fermenter was inoculated with 1 L of pre-culture which had been incubated at 30° C for 24 hours in the same medium with shaking at

300 rpm. After 48 hours of growth, the fermentation broth was cooled and centrifuged at 17,000 rpm with a flow rate of 1 to 2 L/min to remove the biomass. The cell paste was harvested and stored at -20° C yielding 6.0 g wet cell weight/L.

EXAMPLE 3 Induction of phthalyl amidase

Three compounds at different concentrations were added to aerated cultures of the organism growing at 30° C in Bac MI medium. The compounds tested were phthalate (PAA) , phthalyl glycine (PAG), and PMBL. Cells of Xanthobacter agilis were grown with aeration for 24 hours. This vegetative culture was used to inoculate Bac MI medium (50 ml) containing different concentrations of the compounds to be tested. After 48 hours growth under standard conditions, cells were harvested by centrifugation and wet weight of the cells was determined. Crude cell extracts were prepared by lysozyme treatment of the cells as in Example 1. Suspensions were briefly sonicated to break up the viscous suspension. A cell-free supernatant was obtained by centrifugation of the suspension at 14,000 rpm for 15 minutes.

Enzyme activity in cell-free lysates was determined by monitoring conversion of the chromogenic substrate 4- (2 ' -carboxy-N-benzoyl) amino-2-carboxy- nitrobenzene (II) to 2-nitro-5-amino benzoic acid and phthalic acid, a reaction catalyzed by phthalyl amidase as shown below:

The assay reaction mixture (1 ml) consisted of 0.3 μmol of the chromogenic substrate (II) and 0.001-0.5 μg of enzyme preparation in 50 mM potassium phosphate buffer, pH 8.0 (buffer A) . The enzymatic reaction was conducted at 30° C for 10 minutes and the appearance of product was monitored at 380 nm (or 430 nm) . The amount of substrate hydrolyzed was calculated from a standard curve of the amine product .

As can be seen in Table 1, PAG and PAA increased the wet weight cell mass while PMBL had no effect. However, all three substrates produced a dramatic concentration-dependent increase in the total number of enzyme units recovered. The units of enzyme per gram of wet weight cells also increased with all additions but the increase was most pronounced at high PAA concentrations.

TABLE 1

Total units/ Units/

Inducer Addition Cell weight mg protein g cells

(mg/ml) (g/50 ml) (μmol/min/mg)

Control - 0.29 0.017 0.06

PAG 1 0.33 0.35 1.04

5 0.5 4.4 9.4

PMBL 1 0.35 0.28 0.79

5 0.24 2.0 8.1

PAA 1 0.35 1.4 3.9

2 0.47 4.3 9.3

5 0.7 3.7 5.5

10 0.6 12.0 19.8 EXAMPLE 4 Purification of phthalyl amidase

A. Analytical scale purification of phthalyl amidase Cells of Xanthobacter agilis (200 grams, wet weight) , which contained significant amounts of phthalyl amidase activity, were resuspended to 1800 ml in 50 mM Tris-HCl, pH 8.0, plus 5 mM EDTA. The cells were broken by sonication for 22 minutes at a maximal power below 8° C. DNase (1 μg/ml) and magnesium sulfate (10 mM) were added during the sonication to minimize viscosity and improve cell breakage. After a high-speed centrifugation, the resulting crude extract supernatant served as the source for further enzyme purifica ion. All subsequent purification steps were conducted at 4° C.

The crude extract was loaded onto a Q-Sepharose column (4.4 x 23 cm; Pharmacia), previously equilibrated with buffer A. After washing with buffer A, a linear gradient of 0-1.5 M KCl in buffer A was applied and the phthalyl amidase eluted as a single activity peak between 1 and 1.1 M KCl. Selected fractions containing most of the enzyme activity were pooled as Q-Sepharose eluate.

The Q-Sepharose eluate was subjected to ammonium sulfate fractionation. The majority of the enzyme activity was recovered from 67-77, 77-87 and 87-97% ammonium sulfate pellets. Those pellets were solubilized in buffer A with 0.2 M ammonium sulfate. Ammonium sulfate was added to the 67-97% ammonium sulfate enzyme pool to a final concentration of approximately 2 M. The enzyme pool was loaded onto a Phenyl-Sepharose column (2.6 x 16 cm; Pharmacia), which was previously equilibrated with buffer A plus 2.6 M ammonium sulfate. The phthalyl amidase eluted with a linear gradient decreasing from 2.6 M to 0 M ammonium sulfate in buffer A as a single activity peak between 0 M and 0.5 M ammonium sulfate. Selected fractions containing the majority of the enzyme activity were pooled as Phenyl- Sepharose eluate.

The Phenyl-Sepharose eluate was dialyzed against buffer A and then loaded onto a hydroxylapatite column (1.5 x 90 cm; Clarkson Chemical Company, Williamsport, PA), which was previously equilibrated with buffer A. After washing the column with buffer A, the enzyme eluted with a linear gradient of 50-500 mM potassium phosphate, pH 8.0, as a single activity peak between 150 and 190 mM potassium phosphate. Selected fractions containing most of the enzyme activity were pooled as hydroxylapatite eluate.

After a dilution of the buffer strength from 120 to 50 mM potassium phosphate, the hydroxylapatite eluate was loaded onto a Mono P column (0.5 x 20 cm; Pharmacia), which was previously equilibrated with buffer A. After washing with 3 column volumes of buffer A, a linear gradient of 0-1.5 M KCl in buffer A was applied and the enzyme eluted as a single activity peak between 0.72 and 0.8 M KCl. Those fractions containing the majority of the enzyme activity were pooled as Mono P eluate. The most active enzyme preparation was derived from Mono P FPLC (Fast Protein Liquid Chromatography) .

Table 2 summarizes the results of the purification. Based on sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and Laser Densitometric Scanning, the phthalyl amidase was greater than 95% pure.

The phthalyl amidase activity reported in Table 2 was determined using the chromogenic substrate as in

Example 3. A typical reaction mixture in a total volume of 1 ml contained 0.2 mg of the chromogenic substrate and an aliquot of phthalyl amidase in buffer A. The enzymatic reaction was conducted at 30° C for 10-15 min. Formation of the reaction product was monitored with a spectrophotometer at 430 nm (or 380 nm) and quantitated from a standard curve of the product.

TABLE 2

Step Protein Activity Spec. Act. Purification Yield (mg) (Units) (Units/mg) (fold) (%)

Crude 5475 345 0.063 1 100

Extract

Q Sepharose 230 279 1.214 19 81

Ammonium 145 224 1.547 25 65

Sulfate:

67-97% cut

Phenyl- 63 158 2.505 40 46

Sepharose

Hydroxyl¬ 28 154 5.52 88 45 apatite

Mono P 16.5 119 7.2 119 34

B. Preparative scale purification of phthalyl amidase

Crude extract of Xanthobacter agilis was prepared by adding 1 g of cells (wet weight) and 2 mg lysozyme per 9 ml of 50 mM Tris-HCl buffer, pH 8.0, 1 mM EDTA (600 g cells total) . After 30 minutes at room temperature, DNase (100 U/g of cells) in 10 mM magnesium sulfate was added. The cells were homogenized using a cell homogenizer for 30 minutes at room temperature. After 17 hours of incubation at 8° C, the lysate was centrifuged at 10,000 rpm for 30 minutes.

The crude extract supernatant (4.5 L) was applied to a Super-Q column (7 x 40 cm; TosoHaas, Montgomeryvilie, PA) equilibrated in buffer A. After loading crude extract, the column was washed with 2 column volumes of 50 mM phosphate buffer containing 3.5 M urea, pH 8.0. A second wash (5 L) was used to re-equilibrate the column in buffer A. Phthalyl amidase eluted from the column using a 10 column-volume linear gradient of 0-1.5 M KCl in buffer A. Fractions were collected and assayed for enzyme activity. The active fractions were pooled (1.5 L) , concentrated (250 ml) , and diafiltered with buffer A at 7- 10° C. The concentrated and diafiltered Super-Q mainstream was applied to a hydroxylapatite column (3.2 x 40 cm) equilibrated in buffer A. After washing the column with this buffer, phthalyl amidase was eluted using a linear gradient of 0-500 mM phosphate buffer, pH 8.0. Fractions were assayed according to the chromogenic substrate method (see Example 3) and the active fractions were pooled (1 L) and concentrated (400 ml) .

Table 3 shows the results of this purification. TABLE 3

Step Activity Spec. Act. Purification Yield (Units) (Units/mg) (Fold) (%)

Crude 14,846 0.8 1 100

Extract

Super-Q 6,828 3.0 4 46

Hydroxyl¬ 4,985 9.0 11 34 apatite

EXAMPLE 5 Effect of pH on phthalyl amidase activity

The effect of pH on the reaction rate of the analytical scale purified enzyme was determined using phthalamido carbacephem (III) as substrate.

III

A typical reaction mixture consisted of 1 ml total volume and contained 0.1 mM III, 0.1 μM phthalyl amidase in 50 mM potassium phosphate buffer (pH 5.5-9.0) at 32° C for 20 minutes. The reactions were stopped by the addition of 1 ml methanol. After removal of precipitate by centrifugation, an aliquot of the supernatant fraction (typically 30 μl) was monitored for the beta-lactam nucleus and phthalic acid by HPLC using a Zorbax C8 column (0.46 x 15 cm; MacMod Analytical Inc., Chadds Ford, PA) . The two reaction products were eluted by a mobile phase constructed as continuous mixed gradients from (a) 1% ACN (acetonitrile) /0.2% TFA (trifluoroacetic acid) and (b) 80% ACN/0.2% TFA as follows: 1) 0% (b) , 3 min; 2) 0-50% (b) , 0.5 min; 3) 50-100% (b) , 3 min; 4) 100% (b) , 2.5 min; 5) 100-0 % (b) , 0.1 min; and 6) 0% (b) , 5 min. At a flow rate of 1.5 ml/min, retention times of the beta-lactam nucleus and phthalic acid, as measured at 254 nm, were 2.3 and 7.2 min, respectively.

The results are shown in Table 4. Optimal range for enzyme activity occurred between pH 7.8 and 8.4.

TABLE 4

pH Specific Activity !μmol/min/μmol enzyme)

7.0 125.4 7.2 130.4 7.4 155.2 7.6 172.5 7.8 184.2 8.0 195.3 8.2 201.2 8.4 208.0 8.6 181.7 8.8 185.1 9.0 33.1

EXAMPLE 6 Optimum reaction temperature

Test reactions were carried out similar to Example 5 except that all incubations were performed in 50 mM potassium phosphate buffer at pH 8.2. Solutions of the substrate were pre-incubated for 5 minutes at temperatures between 2 and 60° C. The enzymatic reaction was initiated by the addition of phthalyl amidase and stopped by the addition of 1 ml methanol. Specific activity of the enzyme was determined by monitoring the hydrolysis of III by HPLC as in Example 5.

The maximum reaction rate for the enzyme was reached at 34° C. Little enzyme activity was found below 10° C and above 50° C.

EXAMPLE 7 Optimum salt concentration

Test reactions were carried out similar to

Example 6 except that buffer concentrations ranging from 10 to 200 mM at 32° C were examined. All other conditions and analyses were the same.

As is apparent in Table 5, high salt concentration markedly improved the specific activity of the enzyme. The effect was of a general nature and did not appear to be dependent on specific anions or cations.

TABLE 5

Specific Activity (μmol/mm L/μmol enzyme)

Buffer Cone.

K Phosphate Tris-HCl NH₄ Acetate

(mM)

10 148 73 25

50 300 230 50

100 350 275 75

200 360 300 100

EXAMPLE 8 Stability of phthalyl amidase

A. Effect of ionic strength

The stability of phthalyl amidase at pH values ranging from 6-9 was determined as described in Example 5 at 30° C in both 20 and 200 mM potassium phosphate buffer. In 20 mM buffer, all enzyme activity was lost within 2 hours at any pH of the incubation medium. In 200 mM buffer, the enzyme retained at least 80% of its activity for 100 hours irrespective of the pH of the incubating medium. Twenty mM buffer that was supplemented with 200 mM KCl or NaCl also protected against activity loss, indicating that the enzyme stabilization was primarily dependent on the high ionic strength of the buffer. B. Temperature stability

The phthalyl amidase enzyme was also tested for stability at varying temperatures. The enzyme was incubated at pH 8.2 in the temperature range of 4-50° C for 48 hours in 50 and 200 mM phosphate buffer. In 50 mM buffer, the enzyme retained 90% of its activity for 48 hours when maintained at temperatures below 25° C, while all enzyme activity was lost within 48 hours when the incubation temperature was above 40° C. In 200 mM buffer, 80% of the enzyme activity was retained in temperatures up to 35° C and 30% of the enzyme activity was retained after 48 hours incubation at 40° C.

EXAMPLE 9 Influence of effectors on enzyme activity

The effect of various agents on the enzymatic activity of phthalyl amidase was determined using standard conditions (see Example 5). All agents were tested at 1 mM final concentration unless otherwise indicated.

It can be seen from the data in Table 6 that iodoacetate, p-HMB, and copper ions significantly reduced phthalyl amidase activity. None of the tested compounds stimulated enzyme activity significantly above that of the control.

Table 7 shows the effects of four organic solvents at three concentrations on enzyme catalysis. All four solvents tested significantly decreased enzyme activity at a concentration of 10%. Glycerol caused the least amount of inhibition of the enzyme at the highest concentration tested.

TABLE 6

Effector Agent % of Control Activity

Sulfhvrtrvl aσents p-HMB 65

DTNB 98

NEM 100 lodoacetate, 1 mM 91 lodoacetate, 10 mM 46

Metal chelators

Phenanthroline 104

EDTA 103

Co-factors and reducing aσents

Mercaptoethanol 105

DTT 100

NAD 101

NADH 96

NADP 99

NADPH 99

ATP 96

PLP 106

THF 100

COASH 102

THF + DTT 100

FAD 101

FAD + DTT 100 TABLE 6-continued

Effector Agent % of Control Activity

Metal Cations

NaCl 104

KCl 100

CaCl₂ 89

CoCl 101

CuCl₂ 36

FeCl₂ 102

FeCl₃ 96

MgCl₂ 102

MnCl2 84

NiCl2 94

ZnCl₂ 100

DTT: dithiothreitol p-HMB: para-hydroxy mercuric benzoate

DTNB: dithionitrobenzoate

NEM: N-ethylmaleimide

NAD: nicotinamide adenine nucleotide

NADP: nicotinamide adenine dinucleotide phosphate

NADPH: reduced form of NADP

ATP: adenosine 5 ' -triphosphate

PLP: pyridoxyl-5-phosphate

THF: tetrahydrofolate

FAD: flavin adenine dinucleotide TABLE 7

% Residual enzyme activity

Solvent 1.0% 5.0% 10.0%

Ethanol 99 85 45

DMSO 101 80 71

Glycerol 100 94 85

Methanol 100 90 69

DMSO: dimethyl sulfoxide

EXAMPLE 10 Physical and chemical properties of phthalyl amidase

The molecular weight of the phthalyl amidase was determined to be 49,900 by electrospray mass spectrometry. The enzyme is monomeric with an isoelectric point estimated by isoelectric focusing to be pH 5.5. Chemical hydrolysis and amino acid analysis of the protein by standard methods are shown in Table 8. Repeated attempts to sequence the N- terminus of the purified enzyme failed, indicating that the enzyme was blocked. TABLE 8

Number of residues

Amino Acid in protein

Aspartate/Asparagine 62

Threonine 21

Serine 37

Glutamate/Glutamine 52

Proline 26

Glycine 34

Alanine 50

Cysteine* 2

Valine 23

Methionine 12

Isoleucine 20

Leucine 35

Tyrosine 17

Phenylalanine 13

Histidine 11

Lysine 4

Arginine 20

Tryptophan* 13

* derived from nucleotide sequence of the gene EXAMPLE 11 Substrate specificity of phthalyl amidase

A. Chemical structure requirements for enzyme activity The activity of phthalyl amidase against 25 compounds was determined. The compounds were divided into beta-lactams (Table 9), phthalyl amides (Table 10), and aromatic ring substituted amides (Table 11) . Each reaction mixture (1 ml total volume) contained 2.5 μmol of compound and 0.3 units of enzyme (based on the chromogenic substrate) of the preparative scale purified enzyme, in 50 mM phosphate buffer, pH 8.0. The reactions were conducted at 30° C. Samples of the reaction mixture were taken at various times, and methanol (equal volume) was added to stop the reaction. The samples were examined by HPLC to determine the extent of substrate hydrolysis. The amount of compound hydrolyzed was calculated from a standard curve of the test compound. All substrates were stable in buffer at 30° C and pH 8.0 in the absence of enzyme for 24 hours. As the results in Table 9 indicate, the enzyme recognizes mono- and bicyclic beta-lactam compounds containing a phthalyl group attached to the exocyclic nitrogen. However, the side chain apparently requires a 2- carboxylate group, for example, phthalate, since no hydrolysis is observed in the absence of this functional group.

A wide variety of phthalyl amides are substrates for the enzyme as shown in Table 10. Substrates include phthalylated amino acids, dipeptides, monocyclic and bicyclic beta-lactams, phenyl, benzyl, and aliphatic amines. The enzyme also exhibited esterase activity as demonstrated by its ability to hydrolyze phthalate mono methyl ester (IX) . In this series, compound XIII was the most active compound found.

Using compound XIII as a standard, a variety of aromatic ring substituted compounds were examined for reactivity with the enzyme. Results are shown in Table 11. Aromatic ring substituents at the 6 position of the phthalyl ring such as F and NH₂ were accepted by the enzyme. A hydroxyl group at the 3 position (XXI) of the ring and a nitrogen within the aromatic ring (XX) is also acceptable. Low levels of hydrolysis occur if a tetrazole is substituted for the 2-carboxylate group (XXII) . Moving the carboxylate group to the 3 (XXIV) or 4 (XXIII) position of the aromatic ring completely eliminates hydrolytic activity. Compounds lacking the 2-carboxylate (XXV - XXVIII) are not suitable substrates for the enzyme. These results are consistent with the enzyme being a novel catalyst that removes phthalyl protecting groups from a variety of amines under mild conditions. TABLE 9

Compound Relative

Structure number activity

Compound Relative

R Group number Activity

IX -OCH₃ 207.6

H

X _' ^N__ 40.2

^H n

XI OH 31.8

^H /

XII 9.7

XIV -L-Asp-L-Phe-OMe 118.5

XV -D,L-methionine 220.1

XVI -D,L-leucine 90.2 TABLE 11

Compound Relative

R Group number Activity

XVII 2-COOH 100.0

XVIII 6-F, 2-COOH 159.00

XVIX 6-NH2, 2- -COOH 10.2

XX 2-COOH (with N at 85.9 position 6)

XXI 3-OH, 2-COOH 1.3

XXIII 4-COOH 0

XXIV 3-COOH 0

XXV 2-OH 0

XXVI 3-OH 0

XXVII 3,5- OH 0

XXVIII 2-H 0 B. Kinetic parameters for phthalyl amidase

The kinetic parameters of the enzyme were determined for several representative substrates. Compounds II, XVII, and XVIII were tested using 0.9 μg/ml of enzyme. Compounds III and XI were tested using 5.14 μg/ml of enzyme. Substrate concentrations were between 0 and 25 mM and reaction time was between 2 and 20 minutes, depending on the substrate used. All reactions were run at 32° C and at pH 8.2. The K_m, Vr _x, K_cat, and K_cat/K_m for these substrates are shown in Table 12. K_m is the Michaelis constant for enzyme kinetics, V_m χ is the maximal rate of reaction calculated by the Michaelis-Menten equation, and K_cat is the catalytic constant for an enzyme reaction.

TABLE 12

Substrate

Parameter II III^a XI XVII XVIII^b

K_m (mM) 0.05 0.9 0.14 0.09 0.17

Vmax (μmol/min/mg) 5.95 7.6 0.27 1.41 1.94

K_ca (μmol/sec/μmol) 4.95 6.33 0.22 1.18 1.61

Kc_,t/K_m 99.0 7.0 1.6 13.1 9.5

a - carbacepham nucleus (7-amino-3-chloro-4-carboxy-l- carba-dethioceph-3-em ) (XXXIV) quantitatively monitored as the product of substrate III. b - for the other substrates, phthalic acid was the product monitored during the reaction.

C. Chiral and additional substrate selectivity of phthalyl amidase.

Several additional substrates were tested in a total volume of 1 ml. Reaction mixtures contained 0.009 mg

(0.6 units) of enzyme, 2.5 μmol of substrate, and buffer A.

All reactions were run at 30° C for 2 minutes except for compounds XXX and XXXII, which were run for a longer time period since they were poor substrates for the enzyme.

Reactions were stopped by the addition of methanol, and phthalic acid formation was monitored by HPLC. Results are shown in Table 13. The results show that the enzyme has a marked preference for the D isomer of N-phthalyl-phenylglycine. The L isomer was an extremely poor substrate for the enzyme. Compound XXXI had a relative activity twice that of compound III as a substrate for the enzyme. However, by substituting a sulfonate group for the carboxyl group of the phthalyl moiety, enzyme reactivity is completely lost. Again, these results show the selectivity of this enzyme for N-phthalylated amines and indicate that the enzyme has a chiral preference on the amine side of the substrate.

TABLE 13

Compound Relative

Structure Number Activity

D-isomer

L-isomer

κ

XXXII cς so₃

0 . 0

HN^^ EXAMPLE 12

Preparative scale synthesis of carbacephem nucleus

Phthalimido carbacephem (XXXIII) readily hydrolyzes to phthalamido carbacephem (III) in buffer at pH 8.0. Thus, either compound XXXIII or III can be used to prepare the carbacephem nucleus (XXXIV) . Substrate (4 grams) was added to 20 ml of deionized water and the pH of the solution was adjusted to 8.0 with concentrated ammonium hydroxide. Phthalyl amidase, 80 units as determined using the chromogenic substrate (II), was added to start the reaction. Temperature was maintained at 30° C and the pH maintained at 8.0 by adding 2 N ammonium hydroxide. After 510 minutes under these conditions, HPLC analysis of the samples from the reaction indicated that compound III was 90.0% hydrolyzed and compound XXXIII was 98% hydrolyzed. The pH of the reaction was lowered to 5.0 thereby precipitating the carbacephem nucleus. Isolated yields of the nucleus were between 65% and 77%.

XXXIII XXXIV EXAMPLE 13 Expression of met-phthalyl amidase in Escherichia coli

Several small scale temperature inductions of E. coli DH5α/pZPA400 were carried out to assess the amount of met-phthalyl amidase protein and enzymatic activity generated by E. coli DH5α/pZPA400. Enzymatic activity was observed by incubation of a soluble cell extract with the chromogenic substrate (II) under conditions as described in Example 3. Results are reported in Table 14.

SDS-PAGE gels of the cell extract showed a Coomassie-stained protein band corresponding to approximately 50,000 daltons that increased upon temperature induction. Partial purification of the cell extract by anion exchange chromatography yielded fractions with increased phthalyl amidase specific activity. Phthalyl amidase in these fractions catalyzed cleavage of the phthalyl group from compound III to form compound XXXIV and phthalic acid.

EXAMPLE 14 Expression of pro-phthalyl amidase open reading frame in

Streptomyces lividans

A 5 ml inoculum of Streptomyces l i vidans

TK23/pZPA600 (grown for 48 hours at 30° C, 280 rpm) was added to each of two 2 L shake flasks containing 500 ml Trypticase Soy Broth medium and cultured at 30° C, 280 rpm for 24 hours. Incubations beyond 24 hours were deleterious to production of phthalyl amidase. Cells were removed by centrifugation (4° C, 15 min, 12,000 x g) and phthalyl amidase activity in the cell-free broth was determined with compound III as substrate as in Example 13 (Table 14) . The cell-free broth (800 ml, 0.10 mg/ml) was passed at 1 ml/min through a Mono Q column (10 x 10 mm (8 ml); Pharmacia) . A linear gradient of 0 to 1.5 M KCl in buffer A was passed over the column and 2 ml fractions were collected. Most of the phthalyl amidase activity eluted in fractions 19 and 20 (about 0.75 M KCl) .

A 1 ml aliquot of fraction 19 was concentrated 10-fold via ultrafiltration and analyzed by SDS-PAGE. A major protein band was observed at about 50,000 daltons, which corresponded to the molecular weight observed by electrospray mass spectrometry for purified mature phthalyl amidase obtained from Xanthobacter agilis . It also corresponded closely to the theoretical molecular weight predicted for a protein encoded by SEQ ID NO:6.

TABLE 14

Activity in Activity in

Expressing Plasmid Crude Extract Culture Broth Organism (nmol/min/mg) (nmol/min/L)

XantΛojbacter agilis none 63.0 3465 Escherichia coli PZPA400 0.96 438 Streptomyces

PZPA600 748.8 76,378 lividans

EXAMPLE 15

Use of recombinant phthalyl amidase to remove the phthalyl blocking group from phthalamido carbacephem

Activity was assayed by the addition of phthalyl amidase (30 μl of Mono Q fraction 19 from Example 14, 1.83 μg total protein) to 1.82 μg of compound III in a 1 ml reaction mixture buffered by 200 mM potassium phosphate, pH 8.2. The reaction was carried out at 32° C for 20 minutes and stopped with the addition of 1 ml methanol. After removal of precipitate by centrifugation, an aliquot (30 μl) of the supernatant fraction was monitored by HPLC (254 nm absorbance) for both the carbacephem nucleus (XXXIV) and phthalic acid using a Zorbax C8 column (0.46 x 15 cm; MacMod Analytical Inc.) . The reaction products were eluted by a mobile phase constructed as continuous mixed gradients from (a) 1% acetonitrile/0.2% trifluoroacetic acid and (b) 80% acetonitrile/0.2% trifluoroacetic acid. The above substrate, loracarbef nucleus, and phthalic acid eluted at 11.0, 3.4, and 5.9 minutes, respectively. HPLC peaks were identified and quantitated using data generated by known amounts of authentic compounds. The specific activity of recombinant phthalyl amidase derived from fraction 19 for conversion of substrate was 9.5 μmol/min/mg protein.

EXAMPLE 16

Use of recombinant phthalyl amidase to remove the phthalyl blocking group from phthalimido-aspartame

In the synthesis of aspartame, the bivalent protection of the amino group of L-aspartic acid via a phthalimido moiety gives a superior substrate for a lyase- catalyzed condensation with L-phenylalanine methyl ester. However, an efficient method to convert the phthalimido- protected compound back to the amine was previously lacking. Following the condensation reaction, mild base was used to open the phthalimido moiety to a phthalamido moiety and recombinant phthalyl amidase was then used to catalyze hydrolysis of the latter to aspartame and phthalic acid (see Table 10) . SEQUENCE LISTING

1) GENERAL INFORMATION:

(l) APPLICANT: Evans, Robert R. Kreuzman, Adam J. Vangala, Surya Yeh, Wu-Kuang

(11) TITLE OF INVENTION: METHOD FOR PURIFYING A NOVEL ENZYME: PHTHALYL

AMIDASE

(in) NUMBER OF SEQUENCES: 11

(IV) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Ell Lilly and Company (B) STREET: Lilly Corporate Center

(C) CITY: Indianapolis

(D) STATE: Indiana

(E) COUNTRY: USA

(F) ZIP: 46285

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette, 3.50 inch, 1.0 Mb storage

(B) COMPUTER: Macintosh

(C) OPERATING SYSTEM: Macintosh (D) SOFTWARE: Microsoft Word

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: (C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Cantrell, Paul R.

(B) REGISTRATION NUMBER: 36,470 (C) REFERENCE/DOCKET NUMBER: X9660

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 317-276-3885

(B) TELEFAX: 317-277-1917

(2) INFORMATION FOR SEQ ID NO:l:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3029 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 136. .1617 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

GGATCCTTAG GAATCTAAAC ATTCTGGTTG ACACTCCACA TTTTGAATGT CAGCATTTCG 60 GCCATGGCTG CTATGCAGCC TGTTATTGCA TTTGAAATGG AATAGATCAG CAAACTTATC 120

GGGAGGATGA GTATT ATG ATA ATC AAG GGT AGT GTA CCG GGT AAA GCC GGA 171 Met lie lie Lys Gly Ser Val Pro Gly Lys Ala Gly 1 5 10

GGA AAA CCT CGA GCG ACC ATC TTT CAT AGT TCT ATT GCA ACG CTA CTT 219 Gly Lys Pro Arg Ala Thr lie Phe His Ser Ser lie Ala Thr Leu Leu 15 20 25 TTA ACC ACA GTC TCA CTG TCA GGA GTA GCG CCA GCA TTT GCA CAG GCG 267 Leu Thr Thr Val Ser Leu Ser Gly Val Ala Pro Ala Phe Ala Gin Ala 30 35 40

CCG TCT GTG CAC CAA CAC GTC GCC TTC ACT GAG GAA ATT GGA GAC CTT 315 Pro Ser Val His Gin His Val Ala Phe Thr Glu Glu He Gly Asp Leu 45 50 55 60

CCC GAC GGC TCA AGT TAC ATG ATC CGT GTG CCG GAG AAC TGG AAC GGC 363 Pro Asp Gly Ser Ser Tyr Met He Arg Val Pro Glu Asn Trp Asn Gly 65 70 75

GTG TTA ATT CGC GAC CTA GAC CTT GTC AGC GGC ACC AGC AAT TCT AAC 411 Val Leu He Arg Asp Leu Asp Leu Val Ser Gly Thr Ser Asn Ser Asn 80 85 90

GCC GCA AGG TAC GAA ACC ATG CTG AAA GAA GGT TTT GCC GTT GCT GGC 459 Ala Ala Arg Tyr Glu Thr Met Leu Lys Glu Gly Phe Ala Val Ala Gly 95 100 105 ACG GCG AGG CAT CCC CTT CGG CAA TGG CAA TAT GAC CCC GCT CAC GAG 507 Thr Ala Arg His Pro Leu Arg Gin Trp Gin Tyr Asp Pro Ala His Glu 110 115 120

ATT GAA AAC CTC AAT CAC GTG CTG GAC ACA TTC GAG GAA AAT TAC GGT 555 He Glu Asn Leu Asn His Val Leu Asp Thr Phe Glu Glu Asn Tyr Gly 125 130 135 140

TCA CCT GAA AGA GTT ATC CAG TAC GGT TGC TCG GGT GGG GCA CAC GTG 603 Ser Pro Glu Arg Val He Gin Tyr Gly Cys Ser Gly Gly Ala His Val 145 150 155

TCA CTA GCC GTG GCA GAG GAC TTC TCG GAC CGC GTA GAT GGC TCA GTT 651 Ser Leu Ala Val Ala Glu Asp Phe Ser Asp Arg Val Asp Gly Ser Val 160 165 170

GCT CTA GCT GCT CAT ACT CCT GTC TGG ATA ATG AAT TCT TTC TTG GAC 699 Ala Leu Ala Ala His Thr Pro Val Trp He Met Asn Ser Phe Leu Asp 175 180 185 GGA TGG TTT TCG CTG CAG TCT CTG ATC GGC GAG TAC TAT GTA GAA GCT 747 Gly Trp Phe Ser Leu Gin Ser Leu He Gly Glu Tyr Tyr Val Glu Ala 190 195 200

GGT CAC GGC CCA CTT TCG GAT CTC GCT ATT ACG AAA CTG CCC AAT GAT 795 Gly His Gly Pro Leu Ser Asp Leu Ala He Thr Lys Leu Pro Asn Asp 205 210 215 220

GGT AGC TCT AAT TCG AGC GGT CAT GGA ATG GAA GGA GAT CTT CCT GCC 843

Gly Ser Ser Asn Ser Ser Gly His Gly Met Glu Gly Asp Leu Pro Ala 225 230 235

GCG TGG CGC AAC GCG TTC ACC GCT GCT AAC GCC ACA CCT GAG GGT CGC 891

Ala Trp Arg Asn Ala Phe Thr Ala Ala Asn Ala Thr Pro Glu Gly Arg 240 245 250

GCA CGC ATG GCA CTA GCC TTT GCG CTC GGT CAG TGG TCT CCG TGG TTG 939 Ala Arg Met Ala Leu Ala Phe Ala Leu Gly Gin Trp Ser Pro Trp Leu 255 260 265 GCC GAC AAC ACG CCC CAA CCT GAT CTC GAT GAT CCT GAG GCC ATC GCG 987 Ala Asp Asn Thr Pro Gin Pro Asp Leu Asp Asp Pro Glu Ala He Ala 270 275 280

GAT TCC GTA TAT GAG TCT GCC ATG CGA CTT GCA GGA AGC CCT GGG GGA 1035 Asp Ser Val Tyr Glu Ser Ala Met Arg Leu Ala Gly Ser Pro Gly Gly 285 290 295 300

GAA GCG CGC ATA ATG TTC GAG AAC GCC GCT CGA GGG CAA CAG CTC TCT 1083 Glu Ala Arg He Met Phe Glu Asn Ala Ala Arg Gly Gin Gin Leu Ser 305 310 315

TGG AAC GAC GAC ATC GAC TAT GCG GAT TTC TGG GAG AAC TCA AAC CCA 1131 Trp Asn Asp Asp He Asp Tyr Ala Asp Phe Trp Glu Asn Ser Asn Pro 320 325 330

GCC ATG AAG AGC GCC GTT CAG GAG CTG TAC GAC ACG GCC GGC CTT GAT 1179 Ala Met Lys Ser Ala Val Gin Glu Leu Tyr Asp Thr Ala Gly Leu Asp 335 340 345 CTG CAG TCC GAT ATA GAA ACG GTA AAT TCC CAG CCA CGC ATA GAG GCA 1227 Leu Gin Ser Asp He Glu Thr Val Asn Ser Gin Pro Arg He Glu Ala 350 355 360

TCG CAG TAT GCG CTC GAC TAC TGG AAC ACG CCA GGT CGC AAT GTC ATT 1275 Ser Gin Tyr Ala Leu Asp Tyr Trp Asn Thr Pro Gly Arg Asn Val He 365 370 375 380

GGC GAC CCC GAA GTT CCT GTG CTG CGC CTG CAT ATG ATA GGC GAC TAC 1323 Gly Asp Pro Glu Val Pro Val Leu Arg Leu His Met He Gly Asp Tyr 385 390 395

CAA ATT CCC TAT AGT CTT GTA CAG GGC TAC AGC GAT CTT ATC TCA GAG 1371

Gin He Pro Tyr Ser Leu Val Gin Gly Tyr Ser Asp Leu He Ser Glu 400 405 410

AAC AAC AAT GAT GAC TTG TAC AGA ACT GCT TTT GTG CAA TCC ACT GGA 1419

Asn Asn Asn Asp Asp Leu Tyr Arg Thr Ala Phe Val Gin Ser Thr Gly 415 420 425 CAC TGC AAT TTC ACA GCT GCA GAA AGT TCC GCT GCG ATT GAG GTC ATG 1467 His Cys Asn Phe Thr Ala Ala Glu Ser Ser Ala Ala He Glu Val Met 430 435 440

ATG CAA CGG CTT GAC ACG GGT GAG TGG CCG AGC ACC GAG CCG GAT GAT 1515 Met Gin Arg Leu Asp Thr Gly Glu Trp Pro Ser Thr Glu Pro Asp Asp 445 450 455 460

CTG AAT GCA ATT GCC GAA GCC TCA AAC ACC GGA ACT GAA GCA CGT TTC 1563

Leu Asn Ala He Ala Glu Ala Ser Asn Thr Gly Thr Glu Ala Arg Phe

465 470 475

ATG GCC CTA GAT GGC TGG GAA ATA CCC GAG TAC AAT CGT ACT TGG AAG 1611

Met Ala Leu Asp Gly Trp Glu He Pro Glu Tyr Asn Arg Thr Trp Lys

480 485 490

CCT GAA TAATCACCAT TCTGGAGGCT CACGTTCGCG AAGGGTTGCG GCGAAGAAAA 1667 Pro Glu

CATGCGCCGC AACCTATCCT CCAAACAAGG GCCAGTTCAA CGACGAACAA GCCAGACCGG 1727

CGCAAGCCGC GCTAATCTAA TTCACCGCTC CAACCCGCGA TCTCGCGACC GCCCGCGCTG 1787

CATGTCGAGC TTCTGTTGCT GCGCCCGCTC AAGCGTATAA TCACGCCGGA TAATCGTTTC 1847

CCGCGCTTTG TTCGTGATCC TTGCAACGTC CTTGATGCGA TCGACGTTAC GGGCTGTCTC 1907

TGAAGGCTGT GAGCGTGTGC GATCAAGCGC CTGATCGATA TCGCGATGAT TGCTTGATCC 1967 GAACCGGATC TGCATAGCCC GGGCAATACG TTTGGCTTCA TCAAGCGCCT GTTTGCCATC 2027

AGCCGTCTTT TCGAGCTGAT CGACAAAGCC CGTCCGTGCC TTCGCATCCT TGATCTGATC 2087

GAGCTGCCTG AGCAGGGTTT CGCTGCGAGG TGAGAGGCCA GGAATCTCGA CGCGATCATT 2147

ATTGTCACGC CGCCATTGTT CGGCTTCCTT TTCCTCGGCA AAGCGCCGCG TCCAGGTCTT 2207

CCCCGCCGCG TCCAGATGCG AACTCATCGC CTCGGCCCGC TTGAGGGCAT TTTTTGCGCT 2267 CGGCATTGGC ACCGAACAGG CCGAACTTGC CGCGCAGCTG TTGATTTCTG CTGAGAAGTG 2327

ACCCGGTATT GGAGTGAACC CCTGGGACTG GACCAGCGGG GAAGAAAAGC TGATACGCTC 2387

TGTGGGCCTT GAATGGAGAA GGTCCATGTC ACCAAGAGGT CCCTACCGCC GTCACTCGAT 2447

GCAGTTCAAG CGTAAGCGCC AAGCCTGGCC CGTCTGGTGA TGGCTGCCTT TGAGCGCTAT 2507

CGACACCCCG GAGTTAGTGA TGGGTGTCAT GTTCTATGTC TGCGACTATG CCTGCAGATA 2567 GAAGTTTCCA GTTGATCGAG GCGGTTCCGG ATCGGATGGA GGGCGCTCCG GTTGCGCGGC 2627

GACGCCGGTG GTCGGACGCG TTCAAGGCCG AGATGGTAGC GCGCAGCTTC GAACCTGGAA 2687

CGAATGTGTC GGCACTGGCG CGCGAGATCG GCATCCAGTC CTCGCAGTTG TTCGGCTGGC 2747

GCGCCGAGGC CCTCAAGCGC GGAGAGGTGG AAAGGCGCGA TGTTGATATC GTTGCAACGC 2807

AAGCCTCTCG CTTGGTGAGC GGGACGGTCG AGATCGCGGT CAACGACACG GTGATCCGGG 2867 TCGGCATTGA TATCGGGGAA GACCATTTGC GGCGCGTGAT CCGCGCTGTG CGGTCGGCAT 2927

GATCCCTGCG GGTGTGAAGG TCTATCTGGC CAGCCAGCCG GTAGACTTCA GGAAAGGTCC 2987

AGACGGCCTT GTTGGCCTGG TGCGCGATGC TGGAGCGGAT CC 3029 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 494 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

( i) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met He He Lys Gly Ser Val Pro Gly Lys Ala Gly Gly Lys Pro Arg 1 5 10 15

Ala Thr He Phe His Ser Ser He Ala Thr Leu Leu Leu Thr Thr Val 20 25 30

Ser Leu Ser Gly Val Ala Pro Ala Phe Ala Gin Ala Pro Ser Val His 35 40 45

Gin His Val Ala Phe Thr Glu Glu He Gly Asp Leu Pro Asp Gly Ser

50 55 60 Ser Tyr Met He Arg Val Pro Glu Asn Trp Asn Gly Val Leu He Arg 65 70 75 80

Asp Leu Asp Leu Val Ser Gly Thr Ser Asn Ser Asn Ala Ala Arg Tyr 85 90 95

Glu Thr Met Leu Lys Glu Gly Phe Ala Val Ala Gly Thr Ala Arg His 100 105 110

Pro Leu Arg Gin Trp Gin Tyr Asp Pro Ala His Glu He Glu Asn Leu 115 120 125

Asn His Val Leu Asp Thr Phe Glu Glu Asn Tyr Gly Ser Pro Glu Arg 130 135 140 Val He Gin Tyr Gly Cys Ser Gly Gly Ala His Val Ser Leu Ala Val 145 150 155 160

Ala Glu Asp Phe Ser Asp Arg Val Asp Gly Ser Val Ala Leu Ala Ala 165 170 175

His Thr Pro Val Trp He Met Asn Ser Phe Leu Asp Gly Trp Phe Ser 180 185 190

Leu Gin Ser Leu He Gly Glu Tyr Tyr Val Glu Ala Gly His Gly Pro 195 200 205

Leu Ser Asp Leu Ala He Thr Lys Leu Pro Asn Asp Gly Ser Ser Asn

210 215 220 Ser Ser Gly His Gly Met Glu Gly Asp Leu Pro Ala Ala Trp Arg Asn

225 230 235 240

Ala Phe Thr Ala Ala Asn Ala Thr Pro Glu Gly Arg Ala Arg Met Ala 245 250 255 Leu Ala Phe Ala Leu Gly Gin Trp Ser Pro Trp Leu Ala Asp Asn Thr 260 265 270

Pro Gin Pro Asp Leu Asp Asp Pro Glu Ala He Ala Asp Ser Val Tyr 275 280 285

Glu Ser Ala Met Arg Leu Ala Gly Ser Pro Gly Gly Glu Ala Arg He 290 295 300 Met Phe Glu Asn Ala Ala Arg Gly Gin Gin Leu Ser Trp Asn Asp Asp 305 310 315 320

He Asp Tyr Ala Asp Phe Trp Glu Asn Ser Asn Pro Ala Met Lys Ser 325 330 335

Ala Val Gin Glu Leu Tyr Asp Thr Ala Gly Leu Asp Leu Gin Ser Asp 340 345 350

He Glu Thr Val Asn Ser Gin Pro Arg He Glu Ala Ser Gin Tyr Ala 355 360 365

Leu Asp Tyr Trp Asn Thr Pro Gly Arg Asn Val He Gly Asp Pro Glu

370 375 380 Val Pro Val Leu Arg Leu His Met He Gly Asp Tyr Gin He Pro Tyr 385 390 395 400

Ser Leu Val Gin Gly Tyr Ser Asp Leu He Ser Glu Asn Asn Asn Asp 405 410 415

Asp Leu Tyr Arg Thr Ala Phe Val Gin Ser Thr Gly His Cys Asn Phe 420 425 430

Thr Ala Ala Glu Ser Ser Ala Ala He Glu Val Met Met Gin Arg Leu 435 440 445

Asp Thr Gly Glu Trp Pro Ser Thr Glu Pro Asp Asp Leu Asn Ala He 450 455 460 Ala Glu Ala Ser Asn Thr Gly Thr Glu Ala Arg Phe Met Ala Leu Asp 465 470 475 480

Gly Trp Glu He Pro Glu Tyr Asn Arg Thr Trp Lys Pro Glu 485 490

(2) INFORMATION FOR SEQ ID NO:3

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 135 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 :

GGATCCTTAG GAATCTAAAC ATTCTGGTTG ACACTCCACA TTTTGAATGT CAGCATTTCG 60 GCCATGGCTG CTATGCAGCC TGTTATTGCA TTTGAAATGG AATAGATCAG CAAACTTATC 120 GGGAGGATGA GTATT 135

(2) INFORMATION FOR SEQ ID NO:4:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 126 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..126 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

ATG ATA ATC AAG GGT AGT GTA CCG GGT AAA GCC GGA GGA AAA CCT CGA 48

Met He He Lys Gly Ser Val Pro Gly Lys Ala Gly Gly Lys Pro Arg

1 5 10 15

GCG ACC ATC TTT CAT AGT TCT ATT GCA ACG CTA CTT TTA ACC ACA GTC 96

Ala Thr He Phe His Ser Ser He Ala Thr Leu Leu Leu Thr Thr Val 20 25 30 TCA CTG TCA GGA GTA GCG CCA GCA TTT GCA 126

Ser Leu Ser Gly Val Ala Pro Ala Phe Ala 35 40

(2) INFORMATION FOR SEQ ID NO:5:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 42 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(XI) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Met He He Lys Gly Ser Val Pro Gly Lys Ala Gly Gly Lys Pro Arg 1 5 10 15

Ala Thr He Phe His Ser Ser He Ala Thr Leu Leu Leu Thr Thr Val 20 25 30

Ser Leu Ser Gly Val Ala Pro Ala Phe Ala 35 40

(2) INFORMATION FOR SEQ ID NO:6:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1359 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..1356 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

CAG GCG CCG TCT GTG CAC CAA CAC GTC GCC TTC ACT GAG GAA ATT GGA 48 Gin Ala Pro Ser Val His Gin His Val Ala Phe Thr Glu Glu He Gly 1 5 10 15

GAC CTT CCC GAC GGC TCA AGT TAC ATG ATC CGT GTG CCG GAG AAC TGG 96 Asp Leu Pro Asp Gly Ser Ser Tyr Met He Arg Val Pro Glu Asn Trp 20 25 30 AAC GGC GTG TTA ATT CGC GAC CTA GAC CTT GTC AGC GGC ACC AGC AAT 144 Asn Gly Val Leu He Arg Asp Leu Asp Leu Val Ser Gly Thr Ser Asn 35 40 45

TCT AAC GCC GCA AGG TAC GAA ACC ATG CTG AAA GAA GGT TTT GCC GTT 192 Ser Asn Ala Ala Arg Tyr Glu Thr Met Leu Lys Glu Gly Phe Ala Val 50 55 60

GCT GGC ACG GCG AGG CAT CCC CTT CGG CAA TGG CAA TAT GAC CCC GCT 240 Ala Gly Thr Ala Arg His Pro Leu Arg Gin Trp Gin Tyr Asp Pro Ala 65 70 75 80

CAC GAG ATT GAA AAC CTC AAT CAC GTG CTG GAC ACA TTC GAG GAA AAT 288 His Glu He Glu Asn Leu Asn His Val Leu Asp Thr Phe Glu Glu Asn 85 90 95

TAC GGT TCA CCT GAA AGA GTT ATC CAG TAC GGT TGC TCG GGT GGG GCA 336 Tyr Gly Ser Pro Glu Arg Val He Gin Tyr Gly Cys Ser Gly Gly Ala 100 105 110 CAC GTG TCA CTA GCC GTG GCA GAG GAC TTC TCG GAC CGC GTA GAT GGC 384 His Val Ser Leu Ala Val Ala Glu Asp Phe Ser Asp Arg Val Asp Gly 115 120 125

TCA GTT GCT CTA GCT GCT CAT ACT CCT GTC TGG ATA ATG AAT TCT TTC 432 Ser Val Ala Leu Ala Ala His Thr Pro Val Trp He Met Asn Ser Phe 130 135 140

TTG GAC GGA TGG TTT TCG CTG CAG TCT CTG ATC GGC GAG TAC TAT GTA 480 Leu Asp Gly Trp Phe Ser Leu Gin Ser Leu He Gly Glu Tyr Tyr Val 145 150 155 160

GAA GCT GGT CAC GGC CCA CTT TCG GAT CTC GCT ATT ACG AAA CTG CCC 528 Glu Ala Gly His Gly Pro Leu Ser Asp Leu Ala He Thr Lys Leu Pro 165 170 175

AAT GAT GGT AGC TCT AAT TCG AGC GGT CAT GGA ATG GAA GGA GAT CTT 576 Asn Asp Gly Ser Ser Asn Ser Ser Gly His Gly Met Glu Gly Asp Leu 180 185 190 CCT GCC GCG TGG CGC AAC GCG TTC ACC GCT GCT AAC GCC ACA CCT GAG 624 Pro Ala Ala Trp Arg Asn Ala Phe Thr Ala Ala Asn Ala Thr Pro Glu 195 200 205

GGT CGC GCA CGC ATG GCA CTA GCC TTT GCG CTC GGT CAG TGG TCT CCG 672 Gly Arg Ala Arg Met Ala Leu Ala Phe Ala Leu Gly Gin Trp Ser Pro 210 215 220

TGG TTG GCC GAC AAC ACG CCC CAA CCT GAT CTC GAT GAT CCT GAG GCC 720 Trp Leu Ala Asp Asn Thr Pro Gin Pro Asp Leu Asp Asp Pro Glu Ala 225 230 235 240

ATC GCG GAT TCC GTA TAT GAG TCT GCC ATG CGA CTT GCA GGA AGC CCT 768 He Ala Asp Ser Val Tyr Glu Ser Ala Met Arg Leu Ala Gly Ser Pro 245 250 255

GGG GGA GAA GCG CGC ATA ATG TTC GAG AAC GCC GCT CGA GGG CAA CAG 816 Gly Gly Glu Ala Arg He Met Phe Glu Asn Ala Ala Arg Gly Gin Gin 260 265 270 CTC TCT TGG AAC GAC GAC ATC GAC TAT GCG GAT TTC TGG GAG AAC TCA 864 Leu Ser Trp Asn Asp Asp He Asp Tyr Ala Asp Phe Trp Glu Asn Ser 275 280 285

AAC CCA GCC ATG AAG AGC GCC GTT CAG GAG CTG TAC GAC ACG GCC GGC 912 Asn Pro Ala Met Lys Ser Ala Val Gin Glu Leu Tyr Asp Thr Ala Gly 290 295 300

CTT GAT CTG CAG TCC GAT ATA GAA ACG GTA AAT TCC CAG CCA CGC ATA 960 Leu Asp Leu Gin Ser Asp He Glu Thr Val Asn Ser Gin Pro Arg He 305 310 315 320

GAG GCA TCG CAG TAT GCG CTC GAC TAC TGG AAC ACG CCA GGT CGC AAT 1008

Glu Ala Ser Gin Tyr Ala Leu Asp Tyr Trp Asn Thr Pro Gly Arg Asn

325 330 335

GTC ATT GGC GAC CCC GAA GTT CCT GTG CTG CGC CTG CAT ATG ATA GGC 1056

Val He Gly Asp Pro Glu Val Pro Val Leu Arg Leu His Met He Gly 340 345 350 GAC TAC CAA ATT CCC TAT AGT CTT GTA CAG GGC TAC AGC GAT CTT ATC 1104 Asp Tyr Gin He Pro Tyr Ser Leu Val Gin Gly Tyr Ser Asp Leu He 355 360 365

TCA GAG AAC AAC AAT GAT GAC TTG TAC AGA ACT GCT TTT GTG CAA TCC 1152 Ser Glu Asn Asn Asn Asp Asp Leu Tyr Arg Thr Ala Phe Val Gin Ser 370 375 380

ACT GGA CAC TGC AAT TTC ACA GCT GCA GAA AGT TCC GCT GCG ATT GAG 1200 Thr Gly His Cys Asn Phe Thr Ala Ala Glu Ser Ser Ala Ala He Glu 385 390 395 400

GTC ATG ATG CAA CGG CTT GAC ACG GGT GAG TGG CCG AGC ACC GAG CCG 1248 Val Met Met Gin Arg Leu Asp Thr Gly Glu Trp Pro Ser Thr Glu Pro 405 410 415

GAT GAT CTG AAT GCA ATT GCC GAA GCC TCA AAC ACC GGA ACT GAA GCA 1296 Asp Asp Leu Asn Ala He Ala Glu Ala Ser Asn Thr Gly Thr Glu Ala 420 425 430 CGT TTC ATG GCC CTA GAT GGC TGG GAA ATA CCC GAG TAC AAT CGT ACT 1344 Arg Phe Met Ala Leu Asp Gly Trp Glu He Pro Glu Tyr Asn Arg Thr 435 440 445

TGG AAG CCT GAA TAA 1359 Trp Lys Pro Glu 450

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 452 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7 : Gin Ala Pro Ser Val His Gin His Val Ala Phe Thr Glu Glu He Gly 1 5 10 15

Asp Leu Pro Asp Gly Ser Ser Tyr Met He Arg Val Pro Glu Asn Trp 20 25 30

Asn Gly Val Leu He Arg Asp Leu Asp Leu Val Ser Gly Thr Ser Asn 35 40 45

Ser Asn Ala Ala Arg Tyr Glu Thr Met Leu Lys Glu Gly Phe Ala Val 50 55 60

Ala Gly Thr Ala Arg His Pro Leu Arg Gin Trp Gin Tyr Asp Pro Ala

65 70 75 80 His Glu He Glu Asn Leu Asn His Val Leu Asp Thr Phe Glu Glu Asn

85 90 95

Tyr Gly Ser Pro Glu Arg Val He Gin Tyr Gly Cys Ser Gly Gly Ala

100 105 110

His Val Ser Leu Ala Val Ala Glu Asp Phe Ser Asp Arg Val Asp Gly

115 120 125

Ser Val Ala Leu Ala Ala His Thr Pro Val Trp He Met Asn Ser Phe 130 135 140

Leu Asp Gly Trp Phe Ser Leu Gin Ser Leu He Gly Glu Tyr Tyr Val

145 150 155 160 Glu Ala Gly His Gly Pro Leu Ser Asp Leu Ala He Thr Lys Leu Pro

165 170 175

Asn Asp Gly Ser Ser Asn Ser Ser Gly His Gly Met Glu Gly Asp Leu

180 185 190

Pro Ala Ala Trp Arg Asn Ala Phe Thr Ala Ala Asn Ala Thr Pro Glu

195 200 205

Gly Arg Ala Arg Met Ala Leu Ala Phe Ala Leu Gly Gin Trp Ser Pro 210 215 220 Trp Leu Ala Asp Asn Thr Pro Gin Pro Asp Leu Asp Asp Pro Glu Ala 225 230 235 240

He Ala Asp Ser Val Tyr Glu Ser Ala Met Arg Leu Ala Gly Ser Pro

245 250 255

Gly Gly Glu Ala Arg He Met Phe Glu Asn Ala Ala Arg Gly Gin Gin 260 265 270

Leu Ser Trp Asn Asp Asp He Asp Tyr Ala Asp Phe Trp Glu Asn Ser 275 280 285

Asn Pro Ala Met Lys Ser Ala Val Gin Glu Leu Tyr Asp Thr Ala Gly 290 295 300

Leu Asp Leu Gin Ser Asp He Glu Thr Val Asn Ser Gin Pro Arg He

305 310 315 320 Glu Ala Ser Gin Tyr Ala Leu Asp Tyr Trp Asn Thr Pro Gly Arg Asn

325 330 335

Val He Gly Asp Pro Glu Val Pro Val Leu Arg Leu His Met He Gly 340 345 350

Asp Tyr Gin He Pro Tyr Ser Leu Val Gin Gly Tyr Ser Asp Leu He 355 360 365

Ser Glu Asn Asn Asn Asp Asp Leu Tyr Arg Thr Ala Phe Val Gin Ser 370 375 380

Thr Gly His Cys Asn Phe Thr Ala Ala Glu Ser Ser Ala Ala He Glu 385 390 395 400 Val Met Met Gin Arg Leu Asp Thr Gly Glu Trp Pro Ser Thr Glu Pro

405 410 415

Asp Asp Leu Asn Ala He Ala Glu Ala Ser Asn Thr Gly Thr Glu Ala 420 425 430

Arg Phe Met Ala Leu Asp Gly Trp Glu He Pro Glu Tyr Asn Arg Thr

435 440 445

Trp Lys Pro Glu 450

(2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1409 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: TCACCATTCT GGAGGCTCAC GTTCGCGAAG GGTTGCGGCG AAGAAAACAT GCGCCGCAAC 60 CTATCCTCCA AACAAGGGCC AGTTCAACGA CGAACAAGCC AGACCGGCGC AAGCCGCGCT 120

AATCTAATTC ACCGCTCCAA CCCGCGATCT CGCGACCGCC CGCGCTGCAT GTCGAGCTTC 180

TGTTGCTGCG CCCGCTCAAG CGTATAATCA CGCCGGATAA TCGTTTCCCG CGCTTTGTTC 240

GTGATCCTTG CAACGTCCTT GATGCGATCG ACGTTACGGG CTGTCTCTGA AGGCTGTGAG 300 CGTGTGCGAT CAAGCGCCTG ATCGATATCG CGATGATTGC TTGATCCGAA CCGGATCTGC 360

ATAGCCCGGG CAATACGTTT GGCTTCATCA AGCGCCTGTT TGCCATCAGC CGTCTTTTCG 420

AGCTGATCGA CAAAGCCCGT CCGTGCCTTC GCATCCTTGA TCTGATCGAG CTGCCTGAGC 480

AGGGTTTCGC TGCGAGGTGA GAGGCCAGGA ATCTCGACGC GATCATTATT GTCACGCCGC 540

CATTGTTCGG CTTCCTTTTC CTCGGCAAAG CGCCGCGTCC AGGTCTTCCC CGCCGCGTCC 600 AGATGCGAAC TCATCGCCTC GGCCCGCTTG AGGGCATTTT TTGCGCTCGG CATTGGCACC 660

GAACAGGCCG AACTTGCCGC GCAGCTGTTG ATTTCTGCTG AGAAGTGACC CGGTATTGGA 720

GTGAACCCCT GGGACTGGAC CAGCGGGGAA GAAAAGCTGA TACGCTCTGT GGGCCTTGAA 780

TGGAGAAGGT CCATGTCACC AAGAGGTCCC TACCGCCGTC ACTCGATGCA GTTCAAGCGT 840

AAGCGCCAAG CCTGGCCCGT CTGGTGATGG CTGCCTTTGA GCGCTATCGA CACCCCGGAG 900 TTAGTGATGG GTGTCATGTT CTATGTCTGC GACTATGCCT GCAGATAGAA GTTTCCAGTT 960

GATCGAGGCG GTTCCGGATC GGATGGAGGG CGCTCCGGTT GCGCGGCGAC GCCGGTGGTC 1020

GGACGCGTTC AAGGCCGAGA TGGTAGCGCG CAGCTTCGAA CCTGGAACGA ATGTGTCGGC 1080

ACTGGCGCGC GAGATCGGCA TCCAGTCCTC GCAGTTGTTC GGCTGGCGCG CCGAGGCCCT 1140

CAAGCGCGGA GAGGTGGAAA GGCGCGATGT TGATATCGTT GCAACGCAAG CCTCTCGCTT 1200 GGTGAGCGGG ACGGTCGAGA TCGCGGTCAA CGACACGGTG ATCCGGGTCG GCATTGATAT 1260

CGGGGAAGAC CATTTGCGGC GCGTGATCCG CGCTGTGCGG TCGGCATGAT CCCTGCGGGT 1320

GTGAAGGTCT ATCTGGCCAG CCAGCCGGTA GACTTCAGGA AAGGTCCAGA CGGCCTTGTT 1380

GGCCTGGTGC GCGATGCTGG AGCGGATCC 1409

(2) INFORMATION FOR SEQ ID NO:9 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1485 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(IX) FEATURE: (A) NAME/KEY: CDS ( B ) LOCATION : 1 . . 1482

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: ATG ATA ATC AAG GGT AGT GTA CCG GGT AAA GCC GGA GGA AAA CCT CGA 48 Met He He Lys Gly Ser Val Pro Gly Lys Ala Gly Gly Lys Pro Arg 1 5 10 15

GCG ACC ATC TTT CAT AGT TCT ATT GCA ACG CTA CTT TTA ACC ACA GTC 96 Ala Thr He Phe His Ser Ser He Ala Thr Leu Leu Leu Thr Thr Val

20 25 30

TCA CTG TCA GGA GTA GCG CCA GCA TTT GCA CAG GCG CCG TCT GTG CAC 144 Ser Leu Ser Gly Val Ala Pro Ala Phe Ala Gin Ala Pro Ser Val His 35 40 45

CAA CAC GTC GCC TTC ACT GAG GAA ATT GGA GAC CTT CCC GAC GGC TCA 192

Gin His Val Ala Phe Thr Glu Glu He Gly Asp Leu Pro Asp Gly Ser 50 55 60

AGT TAC ATG ATC CGT GTG CCG GAG AAC TGG AAC GGC GTG TTA ATT CGC 240

Ser Tyr Met He Arg Val Pro Glu Asn Trp Asn Gly Val Leu He Arg 65 70 75 80 GAC CTA GAC CTT GTC AGC GGC ACC AGC AAT TCT AAC GCC GCA AGG TAC 288 Asp Leu Asp Leu Val Ser Gly Thr Ser Asn Ser Asn Ala Ala Arg Tyr 85 90 95

GAA ACC ATG CTG AAA GAA GGT TTT GCC GTT GCT GGC ACG GCG AGG CAT 336 Glu Thr Met Leu Lys Glu Gly Phe Ala Val Ala Gly Thr Ala Arg His 100 105 110

CCC CTT CGG CAA TGG CAA TAT GAC CCC GCT CAC GAG ATT GAA AAC CTC 384 Pro Leu Arg Gin Trp Gin Tyr Asp Pro Ala His Glu He Glu Asn Leu 115 120 125

AAT CAC GTG CTG GAC ACA TTC GAG GAA AAT TAC GGT TCA CCT GAA AGA 432 Asn His Val Leu Asp Thr Phe Glu Glu Asn Tyr Gly Ser Pro Glu Arg 130 135 140

GTT ATC CAG TAC GGT TGC TCG GGT GGG GCA CAC GTG TCA CTA GCC GTG 480 Val He Gin Tyr Gly Cys Ser Gly Gly Ala His Val Ser Leu Ala Val 145 150 155 160 GCA GAG GAC TTC TCG GAC CGC GTA GAT GGC TCA GTT GCT CTA GCT GCT 528 Ala Glu Asp Phe Ser Asp Arg Val Asp Gly Ser Val Ala Leu Ala Ala 165 170 175

CAT ACT CCT GTC TGG ATA ATG AAT TCT TTC TTG GAC GGA TGG TTT TCG 576 His Thr Pro Val Trp He Met Asn Ser Phe Leu Asp Gly Trp Phe Ser 180 185 190

CTG CAG TCT CTG ATC GGC GAG TAC TAT GTA GAA GCT GGT CAC GGC CCA 624 Leu Gin Ser Leu He Gly Glu Tyr Tyr Val Glu Ala Gly His Gly Pro 195 200 205

CTT TCG GAT CTC GCT ATT ACG AAA CTG CCC AAT GAT GGT AGC TCT AAT 672 Leu Ser Asp Leu Ala He Thr Lys Leu Pro Asn Asp Gly Ser Ser Asn 210 215 220 TCG AGC GGT CAT GGA ATG GAA GGA GAT CTT CCT GCC GCG TGG CGC AAC 720 Ser Ser Gly His Gly Met Glu Gly Asp Leu Pro Ala Ala Trp Arg Asn 225 230 235 240 GCG TTC ACC GCT GCT AAC GCC ACA CCT GAG GGT CGC GCA CGC ATG GCA 768 Ala Phe Thr Ala Ala Asn Ala Thr Pro Glu Gly Arg Ala Arg Met Ala 245 250 255

CTA GCC TTT GCG CTC GGT CAG TGG TCT CCG TGG TTG GCC GAC AAC ACG 816 Leu Ala Phe Ala Leu Gly Gin Trp Ser Pro Trp Leu Ala Asp Asn Thr 260 265 270

CCC CAA CCT GAT CTC GAT GAT CCT GAG GCC ATC GCG GAT TCC GTA TAT 864 Pro Gin Pro Asp Leu Asp Asp Pro Glu Ala He Ala Asp Ser Val Tyr 275 280 285

GAG TCT GCC ATG CGA CTT GCA GGA AGC CCT GGG GGA GAA GCG CGC ATA 912

Glu Ser Ala Met Arg Leu Ala Gly Ser Pro Gly Gly Glu Ala Arg He 290 295 300

ATG TTC GAG AAC GCC GCT CGA GGG CAA CAG CTC TCT TGG AAC GAC GAC 960

Met Phe Glu Asn Ala Ala Arg Gly Gin Gin Leu Ser Trp Asn Asp Asp 305 310 315 320 ATC GAC TAT GCG GAT TTC TGG GAG AAC TCA AAC CCA GCC ATG AAG AGC 1008 He Asp Tyr Ala Asp Phe Trp Glu Asn Ser Asn Pro Ala Met Lys Ser 325 330 335

GCC GTT CAG GAG CTG TAC GAC ACG GCC GGC CTT GAT CTG CAG TCC GAT 1056 Ala Val Gin Glu Leu Tyr Asp Thr Ala Gly Leu Asp Leu Gin Ser Asp 340 345 350

ATA GAA ACG GTA AAT TCC CAG CCA CGC ATA GAG GCA TCG CAG TAT GCG 1104 He Glu Thr Val Asn Ser Gin Pro Arg He Glu Ala Ser Gin Tyr Ala 355 360 365

CTC GAC TAC TGG AAC ACG CCA GGT CGC AAT GTC ATT GGC GAC CCC GAA 1152

Leu Asp Tyr Trp Asn Thr Pro Gly Arg Asn Val He Gly Asp Pro Glu 370 375 380

GTT CCT GTG CTG CGC CTG CAT ATG ATA GGC GAC TAC CAA ATT CCC TAT 1200

Val Pro Val Leu Arg Leu His Met He Gly Asp Tyr Gin He Pro Tyr 385 390 395 400 AGT CTT GTA CAG GGC TAC AGC GAT CTT ATC TCA GAG AAC AAC AAT GAT 1248 Ser Leu Val Gin Gly Tyr Ser Asp Leu He Ser Glu Asn Asn Asn Asp 405 410 415

GAC TTG TAC AGA ACT GCT TTT GTG CAA TCC ACT GGA CAC TGC AAT TTC 1296 Asp Leu Tyr Arg Thr Ala Phe Val Gin Ser Thr Gly His Cys Asn Phe 420 425 430

ACA GCT GCA GAA AGT TCC GCT GCG ATT GAG GTC ATG ATG CAA CGG CTT 1344 Thr Ala Ala Glu Ser Ser Ala Ala He Glu Val Met Met Gin Arg Leu 435 440 445

GAC ACG GGT GAG TGG CCG AGC ACC GAG CCG GAT GAT CTG AAT GCA ATT 1392

Asp Thr Gly Glu Trp Pro Ser Thr Glu Pro Asp Asp Leu Asn Ala He 450 455 460 GCC GAA GCC TCA AAC ACC GGA ACT GAA GCA CGT TTC ATG GCC CTA GAT 1440 Ala Glu Ala Ser Asn Thr Gly Thr Glu Ala Arg Phe Met Ala Leu Asp 465 470 475 480 GGC TGG GAA ATA CCC GAG TAC AAT CGT ACT TGG AAG CCT GAA TAA 1485

Gly Trp Glu He Pro Glu Tyr Asn Arg Thr Trp Lys Pro Glu 485 490

(2) INFORMATION FOR SEQ ID NO: 10:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1362 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..1359

(XI) SEQUENCE DESCRIPTION: SEQ ID NO:10:

ATG CAG GCG CCG TCT GTG CAC CAA CAC GTC GCC TTC ACT GAG GAA ATT 48 Met Gin Ala Pro Ser Val His Gin His Val Ala Phe Thr Glu Glu He 1 5 10 15 GGA GAC CTT CCC GAC GGC TCA AGT TAC ATG ATC CGT GTG CCG GAG AAC 96 Gly Asp Leu Pro Asp Gly Ser Ser Tyr Met He Arg Val Pro Glu Asn 20 25 30

TGG AAC GGC GTG TTA ATT CGC GAC CTA GAC CTT GTC AGC GGC ACC AGC 144 Trp Asn Gly Val Leu He Arg Asp Leu Asp Leu Val Ser Gly Thr Ser 35 40 45

AAT TCT AAC GCC GCA AGG TAC GAA ACC ATG CTG AAA GAA GGT TTT GCC 192 Asn Ser Asn Ala Ala Arg Tyr Glu Thr Met Leu Lys Glu Gly Phe Ala 50 55 60

GTT GCT GGC ACG GCG AGG CAT CCC CTT CGG CAA TGG CAA TAT GAC CCC 240 Val Ala Gly Thr Ala Arg His Pro Leu Arg Gin Trp Gin Tyr Asp Pro 65 70 75 80

GCT CAC GAG ATT GAA AAC CTC AAT CAC GTG CTG GAC ACA TTC GAG GAA 288 Ala His Glu He Glu Asn Leu Asn His Val Leu Asp Thr Phe Glu Glu 85 90 95 AAT TAC GGT TCA CCT GAA AGA GTT ATC CAG TAC GGT TGC TCG GGT GGG 336 Asn Tyr Gly Ser Pro Glu Arg Val He Gin Tyr Gly Cys Ser Gly Gly 100 105 110

GCA CAC GTG TCA CTA GCC GTG GCA GAG GAC TTC TCG GAC CGC GTA GAT 384 Ala His Val Ser Leu Ala Val Ala Glu Asp Phe Ser Asp Arg Val Asp 115 120 125

GGC TCA GTT GCT CTA GCT GCT CAT ACT CCT GTC TGG ATA ATG AAT TCT 432 Gly Ser Val Ala Leu Ala Ala His Thr Pro Val Trp He Met Asn Ser 130 135 140 TTC TTG GAC GGA TGG TTT TCG CTG CAG TCT CTG ATC GGC GAG TAC TAT 480

Phe Leu Asp Gly Trp Phe Ser Leu Gin Ser Leu He Gly Glu Tyr Tyr

145 150 155 160

GTA GAA GCT GGT CAC GGC CCA CTT TCG GAT CTC GCT ATT ACG AAA CTG 528

Val Glu Ala Gly His Gly Pro Leu Ser Asp Leu Ala He Thr Lys Leu 165 170 175 CCC AAT GAT GGT AGC TCT AAT TCG AGC GGT CAT GGA ATG GAA GGA GAT 576 Pro Asn Asp Gly Ser Ser Asn Ser Ser Gly His Gly Met Glu Gly Asp 180 185 190

CTT CCT GCC GCG TGG CGC AAC GCG TTC ACC GCT GCT AAC GCC ACA CCT 624 Leu Pro Ala Ala Trp Arg Asn Ala Phe Thr Ala Ala Asn Ala Thr Pro 195 200 205

GAG GGT CGC GCA CGC ATG GCA CTA GCC TTT GCG CTC GGT CAG TGG TCT 672 Glu Gly Arg Ala Arg Met Ala Leu Ala Phe Ala Leu Gly Gin Trp Ser 210 215 220

CCG TGG TTG GCC GAC AAC ACG CCC CAA CCT GAT CTC GAT GAT CCT GAG 720 Pro Trp Leu Ala Asp Asn Thr Pro Gin Pro Asp Leu Asp Asp Pro Glu 225 230 235 240

GCC ATC GCG GAT TCC GTA TAT GAG TCT GCC ATG CGA CTT GCA GGA AGC 768 Ala He Ala Asp Ser Val Tyr Glu Ser Ala Met Arg Leu Ala Gly Ser 245 250 255 CCT GGG GGA GAA GCG CGC ATA ATG TTC GAG AAC GCC GCT CGA GGG CAA 816 Pro Gly Gly Glu Ala Arg He Met Phe Glu Asn Ala Ala Arg Gly Gin 260 265 270

CAG CTC TCT TGG AAC GAC GAC ATC GAC TAT GCG GAT TTC TGG GAG AAC 864 Gin Leu Ser Trp Asn Asp Asp He Asp Tyr Ala Asp Phe Trp Glu Asn 275 280 285

TCA AAC CCA GCC ATG AAG AGC GCC GTT CAG GAG CTG TAC GAC ACG GCC 912 Ser Asn Pro Ala Met Lys Ser Ala Val Gin Glu Leu Tyr Asp Thr Ala 290 295 300

GGC CTT GAT CTG CAG TCC GAT ATA GAA ACG GTA AAT TCC CAG CCA CGC 960 Gly Leu Asp Leu Gin Ser Asp He Glu Thr Val Asn Ser Gin Pro Arg 305 310 315 320

ATA GAG GCA TCG CAG TAT GCG CTC GAC TAC TGG AAC ACG CCA GGT CGC 1008 He Glu Ala Ser Gin Tyr Ala Leu Asp Tyr Trp Asn Thr Pro Gly Arg 325 330 335 AAT GTC ATT GGC GAC CCC GAA GTT CCT GTG CTG CGC CTG CAT ATG ATA 1056 Asn Val He Gly Asp Pro Glu Val Pro Val Leu Arg Leu His Met He 340 345 350

GGC GAC TAC CAA ATT CCC TAT AGT CTT GTA CAG GGC TAC AGC GAT CTT 1104 Gly Asp Tyr Gin He Pro Tyr Ser Leu Val Gin Gly Tyr Ser Asp Leu 355 360 365

ATC TCA GAG AAC AAC AAT GAT GAC TTG TAC AGA ACT GCT TTT GTG CAA 1152 He Ser Glu Asn Asn Asn Asp Asp Leu Tyr Arg Thr Ala Phe Val Gin 370 375 380 TCC ACT GGA CAC TGC AAT TTC ACA GCT GCA GAA AGT TCC GCT GCG ATT 1200

Ser Thr Gly His Cys Asn Phe Thr Ala Ala Glu Ser Ser Ala Ala He 385 390 395 400

GAG GTC ATG ATG CAA CGG CTT GAC ACG GGT GAG TGG CCG AGC ACC GAG 1248

Glu Val Met Met Gin Arg Leu Asp Thr Gly Glu Trp Pro Ser Thr Glu 405 410 415 CCG GAT GAT CTG AAT GCA ATT GCC GAA GCC TCA AAC ACC GGA ACT GAA 1296 Pro Asp Asp Leu Asn Ala He Ala Glu Ala Ser Asn Thr Gly Thr Glu 420 425 430

GCA CGT TTC ATG GCC CTA GAT GGC TGG GAA ATA CCC GAG TAC AAT CGT 1344 Ala Arg Phe Met Ala Leu Asp Gly Trp Glu He Pro Glu Tyr Asn Arg 435 440 445

ACT TGG AAG CCT GAA TAA 1362

Thr Trp Lys Pro Glu 450

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 453 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

Met Gin Ala Pro Ser Val His Gin His Val Ala Phe Thr Glu Glu He 1 5 10 15

Gly Asp Leu Pro Asp Gly Ser Ser Tyr Met He Arg Val Pro Glu Asn 20 25 30

Trp Asn Gly Val Leu He Arg Asp Leu Asp Leu Val Ser Gly Thr Ser 35 40 45

Asn Ser Asn Ala Ala Arg Tyr Glu Thr Met Leu Lys Glu Gly Phe Ala

50 55 ^' 60 Val Ala Gly Thr Ala Arg His Pro Leu Arg Gin Trp Gin Tyr Asp Pro 65 70 75 80

Ala His Glu He Glu Asn Leu Asn His Val Leu Asp Thr Phe Glu Glu 85 90 95

Asn Tyr Gly Ser Pro Glu Arg Val He Gin Tyr Gly Cys Ser Gly Gly 100 105 110

Ala His Val Ser Leu Ala Val Ala Glu Asp Phe Ser Asp Arg Val Asp 115 120 125

Gly Ser Val Ala Leu Ala Ala His Thr Pro Val Trp He Met Asn Ser 130 135 140 Phe Leu Asp Gly Trp Phe Ser Leu Gin Ser Leu He Gly Glu Tyr Tyr 145 150 155 160

Val Glu Ala Gly His Gly Pro Leu Ser Asp Leu Ala He Thr Lys Leu 165 170 175

Pro Asn Asp Gly Ser Ser Asn Ser Ser Gly His Gly Met Glu Gly Asp 180 185 190

Leu Pro Ala Ala Trp Arg Asn Ala Phe Thr Ala Ala Asn Ala Thr Pro 195 200 205

Glu Gly Arg Ala Arg Met Ala Leu Ala Phe Ala Leu Gly Gin Trp Ser 210 215 220 Pro Trp Leu Ala Asp Asn Thr Pro Gin Pro Asp Leu Asp Asp Pro Glu 225 230 235 240

Ala He Ala Asp Ser Val Tyr Glu Ser Ala Met Arg Leu Ala Gly Ser 245 250 255

Pro Gly Gly Glu Ala Arg He Met Phe Glu Asn Ala Ala Arg Gly Gin 260 265 270

Gin Leu Ser Trp Asn Asp Asp He Asp Tyr Ala Asp Phe Trp Glu Asn 275 280 285

Ser Asn Pro Ala Met Lys Ser Ala Val Gin Glu Leu Tyr Asp Thr Ala

290 295 300 Gly Leu Asp Leu Gin Ser Asp He Glu Thr Val Asn Ser Gin Pro Arg

305 310 315 320

He Glu Ala Ser Gin Tyr Ala Leu Asp Tyr Trp Asn Thr Pro Gly Arg 325 330 335

Asn Val He Gly Asp Pro Glu Val Pro Val Leu Arg Leu His Met He

340 345 350

Gly Asp Tyr Gin He Pro Tyr Ser Leu Val Gin Gly Tyr Ser Asp Leu 355 360 365

He Ser Glu Asn Asn Asn Asp Asp Leu Tyr Arg Thr Ala Phe Val Gin

370 375 380 Ser Thr Gly His Cys Asn Phe Thr Ala Ala Glu Ser Ser Ala Ala He

385 390 395 400

Glu Val Met Met Gin Arg Leu Asp Thr Gly Glu Trp Pro Ser Thr Glu 405 410 415

Pro Asp Asp Leu Asn Ala He Ala Glu Ala Ser Asn Thr Gly Thr Glu 420 425 430

Ala Arg Phe Met Ala Leu Asp Gly Trp Glu He Pro Glu Tyr Asn Arg 435 440 445 Thr Trp Lys Pro Glu 450

Claims

We Claim :

1. A process for purifying phthalyl amidase, said process comprising: A) chromatographing a crude cell-free extract of phthalyl amidase, obtained by disruption of phthalyl amidase-containing cells, over an anion exchange resin, eluting bound proteins with a linear KCl gradient of 0-1.5 M, followed by pooling the fractions eluted at between about 1 M and about 1.1 M KCl;

B) fractionating the pooled eluate of step A with ammonium sulfate and solubilizing pellets obtained at an ammonium sulfate concentration of between about 67% and about 97%; C) chromatographing the solubilized pellets of step B over a hydrophobic interaction resin, eluting bound proteins with a decreasing linear gradient of 2.6-0 M ammonium sulfate, followed by pooling the fractions eluted at between about 0.4 M and 0 M ammonium sulfate and removing the salts contained in the pooled eluate;

D) chromatographing the pooled eluate of step C over hydroxylapatite, eluting bound proteins with a linear gradient of 50-500 mM potassium phosphate, pH 8.0, followed by pooling the fractions eluted at between about 150 and about 190 mM potassium phosphate; and

E) chromatographing the pooled eluate of step D over an anion exchange resin, eluting bound proteins with a linear gradient of 0-1.5 M KCl, followed by pooling the fractions eluted at between about 0.72 M and about 0.8 M KCl; wherein steps A, B, C, D, and E are carried out at a temperature between about 0° C and about 20° C; and wherein each of steps A, B, C, D, and E are carried out in 50 mM potassium phosphate buffer, pH 8.0.

2. The prcoess of claim 1 wherein the cell- free extract is obtained from Xanthobacter agilis cells.

3. The process of claim 2 wherein the Xanthobacter agilis cells are Xanthobacter agilis NRRL B- 21115 or phthalyl amidase-producing mutants thereof.

4. The process of claim 1 wherein the anion exchange resin used in step A is Q-Sepharose.

5. The process of claim 1 wherein the hydrophobic interaction resin used in step C is Phenyl- Sepharose.

6. The process of claim 1 wherein the resin used in step E Mono P.

7. A preparative scale process for purifying phthalyl amidase, said process comprising:

A) chromatographing a crude cell-free extract of phthalyl amidase, obtained by disruption of phthalyl amidase-containing cells, over an anion exchange resin, eluting bound proteins with a linear KCl gradient of 0-1.5 M, followed by concentrating and diafiltering the active fractions;

B) chromatographing the pooled eluate of step A over hydroxylapatite, eluting bound proteins with a linear gradient of 0-500 mM potassium phosphate, pH 8.0, and collecting the active fractions; wherein steps A and B are carried out at a temperature between about 0° C and about 20° C; and wherein steps A and B are carried out in 50 mM potassium phosphate buffer, pH 8.0 and the fractions are concentrated and diafiltered in 50 mM potassium phosphate, pH 8.0.

8. The process of claim 7 wherein the resin of step A is Super-Q.

9. The process of claim 7 wherein the resin of step A is washed with 50 mM potassium phosphate containing 3.5 urea, pH 8.0 after the cell-free extract is loaded onto the resin.

10. A process for purifying phthalyl amidase, said process comprising chromatographing a cell-free culture broth, obtained by clarifying the culture medium of a cell that secretes soluble phthalyl amidase into the culture medium, over an anion exchange resin, eluting bound proteins with a linear gradient of 0-1.5 M KCl in 50 mM potassium phosphate, pH 8.0 and collecting the fraction eluted at about 0.75 M KCl.

11. The process of claim 10 wherein the anion exchange resin is Mono Q.

12. The process of claim 10 wherein the cultured cell is Streptomyces 1 ividans/pZP 600 NRRL 21289.