# Patent

## Title
Production of streptavidin-like polypeptides

## Abstract

The invention provides a gene encoding penicillin G acylase, the enzyme encoded by said gene and a method for the production of said enzyme by incorporating said gene in a host and bringing the same to expression. The gene is preferably obtained from a strain of the microorganism Alcaligenes faecalis.

## Background
This invention relates to a gene encoding penicillin G acylase, to penicillin G acylase encoded by this gene and to a method for the production of this enzyme.

Penicillin G acylase (benzylpenicillin amidohydrolase, also named penicillin amidase; EC 3.5.1.11) is an enzyme used commercially to hydrolyse penicillin G or 3-desacetoxycephalosporin G to phenylacetic acid and 6-aminopenicillanic acid (6-APA) or 7-aminodesacetoxycephalosporanic acid (7-ADCA), respectively, the most important intermediates for the industrial production of semi-synthetic penicillins and cephalosporins. This enzyme also catalyses the reverse reaction, viz. the N-acylation of 6-APA and 7-ADCA with organic esters to generate their corresponding N-acetylated penicillin and 3-cephem compounds, respectively. See the reviews of Vandamme, E.J., In: Microbial Enzymes and Bioconversions, E.H. Rose (Ed.), Economic Microbiology 5, 467-552 (1980); and P.B. Mahajan, Appl. Biochem. Biotechnol. 1, 83-86 (1982).

Various types of microorganisms have been proposed in the literature as Penicillin G acylase producing strains useful for the deacylation of penicillin G and 3-desacetoxycephalosporin G. Examples of such acylase producing microorganisms are certain strains of the species Escherichia coli, Kluyvera citrophila and Proteus rettgeri. It is to be noted that some penicillin G acylase activity has been described in the whole cell fraction of Alcaligenes faecalis (C.A. Claveridge et al., Nature 4733, 237-238 (1960)). However, no enzyme or some enzymes responsible for this activity from A faecalis have been described up to now.

The use of recombinant DNA methods has enabled an increase of the production levels of commercially used penicillin G acylases (Mayer et al., Adv. Biotechnol. 1, 83-86 (1982)) and has enlarged the insight into the processing of these enzymes (Schumacher et al., Nucleic Acids Res. 14, 5713-5727 (1986)). The penicillin G acylase of E. coli was found to be produced as a large precursor protein, which was further processed into the periplasmic mature protein constituting a small (.alpha.) and a large (.beta.) subunit. Cloning and sequencing of the Kluyvera citrophila acylase gene has revealed a close harmony with the E. coli acylase gene (J.L. Barbero et al., Gene 49, 69-80 (1986)). Also for the Proteus rettgeri penicillin G acylase gene a small and a large subunit has been described (G.O. Daumy et al., Gene 49, 69-80 (1986); Spanish patent application No. 8602933).

In one aspect of the invention a gene is provided encoding penicillin G acylase which has essentially the structure given in FIG. 1. The gene is preferably obtained from A. faecalis.

In another aspect of the invention a vector is provided comprising said gene. Also provided is a host system which comprises one or more copies of said gene.

The invention further provides said penicillin G acylase gene under the control of a regulon comprising transcription and/or translation regulating sequences where said regulating sequences are replaced by other transcription and/or translation regulating sequences. These latter regulating sequences may be obtained from the same or another organism.

The present invention further provides a vector comprising this penicillin G acylase gene, manipulated with respect to the regulating sequences as indicated above, and a host comprising said vector. The penicillin G acylase enzyme, resulting from the expression of said gene has a surprisingly good stability and a high specific activity.

In still another aspect of the invention said penicillin G acylase enzyme is provided in isolated form. When used in large scale acylation or deacylation processes it is preferably used in immobilized form.

The present invention further provides a method for producing penicillin G acylase by fermentation of a transformed host encoding said enzyme, and recovery of the penicillin G acylase in isolated form.

These and other embodiments will be outlined below in more detail.

FIG. 1: Physical map of the A. faecalis penicillin G acylase gene region. The insert of pAF1 has been depicted.

FIG. 2: Structure of the plasmid pMcTNde.

FIG. 3: Structure of the plasmid pMCTAF1A.

FIG. 4: Structure of the tac promoter.

FIG. 5: Structure of the trp promoter.

FIG. 6: Structure of the plasmid pKTAFA.

FIG. 7: Structure of the p78 promoter.

FIG. 8: Structure of the pf3 promoter.

SEQ ID NO: 1: Nucleotide sequence of the penicillin G acylase gene of A. faecalis and derived sequence of amino acids of the new pac enzyme

SEQ ID NO: 2: Nucleotide sequence of the tac promoter

SEQ ID NO: 3: Nucleotide sequence of the trp promoter

SEQ ID NO: 4: Nucleotide sequence of the p78 promoter

SEQ ID NO: 5: Nucleotide sequence of the pf3 promoter

The new Alcaligenes faecalis penicillin G acylase (pac) enzyme can be isolated in a manner known per se: for example, an A. faecalis strain is grown in a suitable culture medium, for instance consisting of yeast extract in a buffered solution, particularly in a phosphate buffer at a pH of about 6-8, particularly about 7, optionally in the presence of the inducer KPA. Any A. faecalis strain can be used. The enzyme is then purified by a method known per se, preferably in two steps, the first one with for instance an esterified cellulose and subsequently the application of for instance gel chromatography, particularly applying hydroxyapatite.

The purified enzyme can be subjected to amino acid sequence analysis of peptide fragments, preferably the NH.sub.2 terminus of each of the subunits. From the determined amino acid sequence a DNA probe to detect the gene sequence may be derived. It was established for the first time that the penicillin hydrolyzing activity of A. faecalis resides in the purified penicillin acylase, which turns out to be a heterodimer of two subunits of 26 kD and 59 kD in size.

The A. faecalis pac gene can be identified from chromosomal DNA in a manner known per se. The gene is essentially of a structure as given in FIG. 1. It is understood that all homologous genes which can hybridize with the sequence depicted in FIG. 1 and which encode an enzyme of essentially the same structure are comprised in this invention. The following equation, which has been derived from analyzing the influence of different factors on hybrid stability: Tm=81+16.6 (log10 Ci)+0.4 (% G+C)-600/n-1.5% mismatch (Ausubel et al., supra) where

Ci=ionic strength (M)

G+C=base composition,

can be used to determine which level of homology can be detected using DNA-DNA hybridisation techniques.

Therefore the term "essentially of a structure" is intended to embrace sequences which can include conservative mutations, where the sequence encodes the same amino acid, but which may differ by up to 35% in the DNA sequence according to the above equation, more typically by up to 10%.

A homology comparison of the amino acid sequence of the new A. faecalis pac enzyme with the published amino acid sequence of E. coli penicillin G acylase (Schumacher et al., supra) revealed an overall homology of only 43%. Furthermore, the homology to the known amino acid sequence of the Kluyvera citrophila pac enzyme (Barbero et al., supra) is also only 44%.

The A. faecalis pac gene can be expressed in E. coli under the guidance of its own promoter and/or of the inducible tac or trp promoter (see FIGS. 4 and 5). The results of the penicillin G acylase production are surprisingly good. Using this pac gene with the tac promoter the production is as high as that of the original A. faecalis strain, whereas in the latter case potassium phenylacetate needs to be used as an inducer. The E. coli strain with the A. faecalis pac gene under the control of the trp promoter shows --without any induction--a penicillin G acylase production which is as much as five times higher than that of the strains mentioned above. In both strains the dependence on induction using a potassium phenylacetate inducer, which is expensive, can now be avoided.

In order to obtain a production strain with only homologous DNA, transformation into A. faecalis itself was developed. Successful DNA transformation into A. faecalis can be suitably achieved by two different methods. First of all, conjugational transfer of broad-host range plasmids from E. coli to A. faecalis can be obtained. Secondly, electroporation of A. faecalis with plasmids based on replicon RSF 1010 can be used (BIORAD-gene pulser).

Upon cloning of the A. faecalis gene different transcription sequences can be used. Firstly, the A. faecalis pac gene can be cloned in A. faecalis under the control of its own promoter. The production of penicillin G acylase is highly improved compared with the A. faecalis strain without extra pac gene(s) and scarcely dependent on potassium phenylacetate induction. Secondly, the use of the E. coli trp promoter, independently of any inducer, leads to a similarly high amount of penicillin G acylase being obtained. Thirdly, by the application of two expression elements derived from Pseudomonas aeruoinosa phage pf3, promoters pf3 and p78, (Luiten, PhD thesis, Nijmegen (1986)) the production of penicillin G acylase is somewhat lower than that achieved with an A. faecalis strain with extra pac gene(s) under the control of its own or E. coli tro promoter, but is still improved compared to the A. faecalis strain without extra pac gene(s) and is much less dependent on potassium phenylacetate induction.

The prior art does not teach the use of A. faecalis to produce penicillin G acylase in isolated form. Also neither the improved properties of the new A. faecalis penicillin G acylase enzyme related to that of E. coli nor the use of recombinant DNA methods to increase and facilitate the production of penicillin G acylase from A. faecalis have been suggested in the prior art up to now.

Penicillin G acylase, produced with the aid of the A. faecalis pac gene, cloned particularly in a gram-negative microorganism, preferably in an Alcaligenes or Escherichia microorganism, more preferably in A. faecalis, is produced in surprisingly large amounts. Furthermore, the stability and especially the specific activity of the penicillin G acylase gene is much greater than that of the penicillin acylases known sofar.

A purified preparation of A. faecalis is also provided, showing a higher specific activity of this enzyme on the preferred substrate penicillin G than any of the presently known penicillin acylases. Furthermore the purified preparation is used to determine the thermostability of A. faecalis penicillin G acylase in comparison to E. coli penicillin acylase. The stability of A. faecalis penicillin G acylase is significantly better than that of E. coli penicillin G acylase, allowing a prolonged use under industrial conditions.

Preferably, in industrial processes penicillin G acylase, and also that provided by the present invention, is used in immobilized form. The carrier on which it is immobilized typically comprises gelatin optionally crosslinked with chitosan, aluminum oxide, silicium oxide, ion exchange resins or acrylate beads, such as, for instance, Eupergit.COPYRGT..

The following examples will further illustrate the present invention.

General cloning techniques were performed as described by Maniatis et al. (1982 and 1989, Molecular Cloning, Cold Spring Harbor Laboratory) or Ausubel et al. (1987, Current Protocols in Molecular Biology, John Wiley and Sons Inc., New York) or B. Perbal (1988, A Practical Guide to Molecular Cloning, 2nd edition, John Wiley and Sons Inc., New York). These handbooks describe in detail the protocols for construction and propagation of rDNA molecules, the procedures for making gene libraries and the protocols for mutating DNA in a site directed or random fashion. Enzymes used for DNA manipulations were purchased from and used according to instructions from commercial suppliers. Plasmids and E. coli cloning hosts were obtained from public culture collections as the Phabagen Collection (Utrecht).

Selective media for phenylacetyl L-leucine (fal) were prepared as described (Garcia et al., ibid). Minimal plates are as follows: M63 minimal agar, 2 g/l glucose, 1 mg/l thiamine, 10 mg/l L-proline and the appropriate antibiotic (50 .mu.g/ml chloramphenicol (cap) or 25 .mu.g/ml ampicillin (amp)). Transformants of E. coli HB101 (Leu.sup.-) growing exclusively in the presence of the acyl L-leucine are considered to harbor an acylase gene. .multidot.7H.sub.2 O, 10 g/l KH.sub.2 PO.sub.4, 3.5 g/l Na(NH.sub.4) HPO.sub.4, 1.6 g/l sodium citrate. casaminoacids 4 g/l, basildon 0.25 g/l (antifoam 86-103Basildon Chemical Corporation), pH 7.0. HPO.sub.4.2H.sub.2 O 4.5 g/l, KH.sub.2 PO.sub.4 3.4 g/l, pH 7.0 (in case of induction: potassium phenylacetic acid (KPA) 1.0 g/l)

Phenylacetyl leucine was purchased from LGSS, Transferbureau Nijmegen.

The A. faecalis strain ATCC 19018 (also deposited as NCTC 415) was used as a donor strain for the A. faecalis pac gene, and as a host for recombinant plasmids.

E. coli strains JM101, WK6 and HB101 (Phabagen, Utrecht) were used as hosts for recombinant plasmids.

Strain A. faecalis ATCC 19018 was grown in a AF medium. Cells were harvested by centrifugation and resuspended in the following buffer: Tris 0.1 M pH 8.0; EDTA 0.2 mM; lysozyme 0.02 mg/ml and incubated for 2 hours at 30.degree. C. Cell debris was removed by centrifugation.

The penicillin G acylase (pac) was purified in two steps. The first one was obtained with carboxymethyl cellulose (CM-52 Whatman). The second one consisted in the elimination of the remaining contaminating proteins through a hydroxyapatite gel chromatography (Biogel HTP from Biorad). The resulting pure pac was shown to be composed of two nonidentical subunits. The small (.alpha.) and large (.beta.) subunit were subjected to NH.sub.2 terminal amino acid analysis. The result was as follows: --Q--X--Q--X--V--E--V--M--X--T --S--N--L--W--S--T--X--P--E--X--V

Chromosomal DNA of A. faecalis (ATCC 19018) was isolated and partially digested with Sau3A. Fractions ranging from 4 kb to 7 kb were purified and ligated into vector pACY184, which was digested with BamHI. DNA was transformed into E. coli HB101 and plated onto fal-plates (see methods). Two positive clones pAFI and pAF2 could be identified. These clones were also tested with positive result in the Serratia marcescens overlay technique (Meevootisom, V. et al., Appl. Microbiol. Biotechnol. 25, 372-378 (1987)). The 6.4 kb insert of the pAF1 plasmid is shown in FIG. 1. The localization of the gene was determined with the aid of an oligonucleotide designed on the NH2 terminal sequence of the .beta.-subunit of A. faecalis pac.

The following oligonucleotide was used as a hybridisation probe on the pAF1 insert (SEQ ID NO: 8):

AGC AAC CTG TGG AGC A/C C/G C TGC CCG GAG TGC GT

From the position of the hybridising signal on the restriction map the orientation of the A. faecalis pac gene was determined (FIG. 1).

The 3.9 kb Sau3A-NdeI subclone of the 6.4 kb insert, was shown to give pac activity, whereas the 3.1 kb Sau3A-Sph1 fragment was inactive (FIG. 1). The DNA sequence of the 3.9 kb insert was determined by deoxy sequencing of suitable fragments in pTZ18R and pTZ19R (Pharmacia). The encoding DNA sequence and the derived amino acid sequence for A. faecalis pac is shown in SEQ ID NO: 1. From the derived amino acid sequence it can be concluded that A. faecalis pac is encoded as a large single polypeptide chain which undergoes processing into two different subunits named .alpha. and .beta.. At the 5' site of the precursor a typical signal sequence responsible for the translocation of the enzyme into the periplasm is shown.

Plasmid pAF1 was digested with SalI and a 4.8 kb fragment was purified. The fragment was ligated into SalI linearized vector pMCT-Nde. The latter vector was constructed from plasmid pMc5-8 (EP-A-0351029) by insertion of a fragment containing the tac promoter followed by a RBS site and a NdeI cloning site (FIG. 2). The resulting plasmid pMcTAFIA (FIG. 3) obtained after transformation into E. coli HB101 expresses pac under guidance of its own promoter and/or of the inducible tac promoter (De Boer et al., Proc. Natl. Acad. Sci., 80, 21 (1983)).

In order to further improve the expression level of pac two strong E. coli promoters were cloned in a precise fusion to the acylase startcodon. To do this an NdeI site was constructed at the ATG startcodon using oligonucleotide site directed mutagenesis (Stanssens et al., 1989) of plasmid pMcTAF1A resulting in plasmid pMcTAF1ANde. This plasmid was digested with NdeI and recircularized resulting in the correct positioning of the tac promoter in front of the acylase gene (plasmid pMcAFtac). In order to insert another promoter plasmid pMcTAF1Nde was digested with EcoRI and NdeI and the large fragment was purified on agarose gel. The tryptophan promoter fragments were inserted into this purified EcoRI-NdeI fragment of pMcTAF1ANde using 6 synthetic oligonucleotides.

The DNA sequences of these promoters are depicted in SEQ ID NO: 2 and 3, respectively, and the structures of these promoters in FIGS. 4 and 5, respectively.

These promoter constructs were transformed into E. coli HB101 and tested for expression of pac. Table 1 shows the results as compared to the expression level of A. faecalis  ATCC 19018. Induction of the tac promoter with isopropylthio-.beta.-galactoside (IPTG) and the trp promoter with indol acrylic acid (IAA) was also tested. ______________________________________ Expression of pac in E. coli Strain         KPA    IAA     IPTG  PAC units* ______________________________________ A. faecalis ATCC 19018 -      -       -     0.1 A. faecalis ATCC 19018 +      -       -     1 pMcAFtac       -      -       -     1 pMcAFtac       -      -       +     17 pMcAFtrp       -      -       -     4 pMcAFtrp       -      +       -     5 ______________________________________ *relative units with A. faecalis Atcc 19018 in the medium with KPA standarized as 1.0

E. coli HB101 containing various plasmids was grown in 4XLBC for 24 hours. A. faecalis was grown in AF medium for 24 hours.

In order to enable stable transfer of qenetic information into A. faecalis a DNA transformation system and a stable cloning vector had to be researched. Surprisingly it was found that triparental mating of plasmid pKT248 (Bagdasarian et al., Gene 16, 237-247 (1981)) into A. faecalis was possible by applying a technique as described (Friedman et al., Gene 18, 289-296 (1982)) with the following modifications: Natl. Acad. Sci. 76, 1648 (1979)) is mixed with E. coli HB101 (pKT248) and A. faecalis recipient strain on 2xTY agar plates. take place. minimal E* medium including citrate, spore elements and the selective antibiotics step (50 .mu.g/ml) and cap (25 .mu.g/ml). Incubation at 30.degree. C. overnight is performed. Due to auxotrophic markers the E. coli strains are counterselected for. The A. faecalis colonies are subsequently spread on 2xTY plates containing 300 .mu.1/ml streptomycine.

Subcloning of the A. faecalis pac gene was done into the unique SalI site of plasmid pKT248. The TthIII-HpaI fragment of plasmid pAFI was isolated and filled in with Klenov polymerase. The SalI site of linearized pKT248 was also made blunt end and plasmid pKTAFA (FIG. 6) was obtained after transformation into E. coli HB101 and selection on fal plates. After isolation this plasmid was transferred into A. faecalis using the triparental mating technique as described above. The strain obtained was grown in shake flasks with A, faecalis growth medium and compound to the original strain. As can be seen in Table 2 the production of pac in the strain with pKTAFA is highly improved. Furthermore it can be seen that even in absence of the inducer KPA a high production can be obtained. This allows the omission of the expensive inducer KPA from industrial fermentation media.

In order to test heterologous promoters in front of the pac gene the EcoRI-SalI fragment of pMcAFtrp, pMcAFpf3 and pMcAFp78, respectively, were subcloned into EcoRI, SalI linearized vector pJRD215 (Davison et al., Gene 51, 275-280 (1987)). All three promoter constructions were obtained in E. coli HB101 as pJRDAFtrp, pJRDAFpf3 and pJRDAFp78 and subsequently transferred into A. faecalis ATCC 19018. Expression of pac from these plasmids was tested in the presence or absence of KPA (Table 2). ______________________________________ Production of pac in A. faecalis transformants Strain     KPA         IAA    PAC units* ______________________________________ pKT248     -           -      0.1 pKT248     +           -      1 pKTAFA     -           -      18 pKTAFA     +           -      22 pJRDAFtrp  -           -      18 pJRDAFtrp  -           +      19 pJRDAFpf3  -           -      5 pJRDAFpf3  +           -      8 pJRDAFp78  -           -      3 pJRDAFp78  +           -      5 ______________________________________ *relative units with A. faecalis ATCC 19018 in the medium with KPA standarized as 1.0

All plasmids were transferred via triparental mating to A. faecalis ATCC 19018. The promoters p78 and pf3 are selected from phage pf3 (Luiten, supra). The DNA sequences of these promoters are depicted in Sequence listings 4 and 5, respectively, and the structures of these promoters in FIGS. 7 and 8, respectively.

Stability of A. Faecalis Penicillin Acylase

A. faecalis penacylase and E. coli were tested for thermostability using the following protocol: Enzyme solutions are incubated at various temperatures for 5 minutes in a solution of 100 mM sodium phosphate, pH 7.5. Residual activity was measured at 35.degree. C. with 50 mM penicillin G as substrate. The temperatures with 100%, 50% and 0% residual activity after 5 minutes were determined. From Table 2 it can be concluded that the A. faecalis enzyme is significantly more stable than the E. coli enzyme. 100%       50%      0% ______________________________________ A. faecalis 45.degree. C. 58.0.degree. C. 66.degree. C. E. coli 5K 40.degree. C. 54.8.degree. C. 60.degree. C. ______________________________________

The enzyme preparation E. coli 5K was obtained from Produktions Gesellschaft fur Biotechnologie Braunschweig (Mayer et al., supra). SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Quax, Wilhelmus J. (ii) TITLE OF INVENTION: Penicillin G Acylase, a Gene Encoding the Same and a Method for the Production of This Enzyme (iii) NUMBER OF SEQUENCES: 8 (iv) CORRESPONDENCE ADDRESS:  (A) ADDRESSEE: Cooley Godward Castro Huddleson & Tatum  (B) STREET: Five Palo Alto Square  (C) CITY: Palo Alto  (D) STATE: CA  (E) COUNTRY: USA  (F) ZIP: 94306 (v) COMPUTER READABLE FORM:  (A) MEDIUM TYPE: Floppy disc  (B) COMPUTER: IBM PC compatible  (C) OPERATING SYSTEM: PC-DOS/MS-DOS  (D) SOFTWARE: WordPerfec t 5.0 (vi) CURRENT APPLICATION DATA:  (A) APPLICATION NUMBER: 07/687,400 (B) FILING DATE: April 18, 1991  (C) CLASSIFICATION: (viii) ATTORNEY/AGE NT INFORMATION:  (A) NAME: Rae-Venter Ph.D., Barbara  (B) REGISTRATION NUMBER: 32750  (C) REFERENCE/DOCKET NUMBER: GBR0-028/00US (ix) TELECOMMUN ICATION INFORMATION:  (A) TELEPHONE: 415-494-7622  (B) TELEFAX: 415-857-0 663 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS:  (A) LENGTH: 2451 base pairs  (B) TYPE: nucleic acid  (C) STRANDEDNESS: single  (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:  (A) ORGANISM: Alcaligenes faecalis (ix) FEATURE:  (A) NAME/KEY: CDS  (B) LOACTION: 1..2451  (D) OTHER INFORMATION: /function= "enzyme"    /product= "penicillin acylase or penicillin amidase" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: ATG CAG AAA GGG CTT GTT CGT ACC GGG CTT GTG GCC GCT GGT TTG A TC 48 Met Gln Lys Gly Leu Val Arg Thr Gly Leu Val Ala Ala Gly Leu Ile 1 5     10     15 TTG GGT TGG GCG GGG GCA CCG ACC CAC GCG CAA GTG CAG TCG GTA GAG 96 Leu Gly Trp Ala Gly Ala Pro Thr His Ala Gln Val Gln Ser Val Glu    20     25     30 GTG ATG CGG GAC AGT TAT GGC GTG CCG CAC GTC TTT GCC GAC AGC CAC 144 Val Met Arg Asp Ser Tyr Gly Val Pro His Val Phe Ala Asp Ser His   35     40     45 TAT GGC TTG TAT TAC GGC TAT GGT TAT GCG GTC GCC CAA GAC CGT CTG 192 Tyr Gly Leu Tyr Tyr Gly Tyr Gly Tyr Ala Val Ala Gln Asp Arg Leu  50     55     60 TTC CAG ATG GAC ATG GCG CGT CGC TCC TTT GTC GGC ACA ACC GCC GCC 240 Phe Gln Met Asp Met Ala Arg Arg Ser Phe Val Gly Thr Thr Ala Ala 65     70     75     80 GTC TTA GGC CCT GGT GAG CAA GAT GCC TAC GTC AAG TAC GAC ATG CAG 288 Val Leu Gly Pro Gly Glu Gln Asp Ala Tyr Val Lys Tyr Asp Met Gln     85     90     95 GTG CGG CAG AAC TTC ACC CCG GCT TCC ATA CAG CGG CAG ATC GCG GCC 336 Val Arg Gln Asn Phe Thr Pro Ala Ser Ile Gln Arg Gln Ile Ala Ala    100     105 110 TTG TCC AAG GAT GAG CGC GAT ATT TTT CGT GGC TAT GCC GAT GGC TAT 384 Leu Ser Lys Asp Glu Arg Asp Ile Phe Arg Gly Tyr Ala Asp Gly Tyr   115 120     125 AAC GCC TAT CTG GAG CAG GTG CGG CGT CGC CCT GAG TTG CTG CCC AAA 432 Asn Ala Tyr Leu Glu Gln Val Arg Arg Arg Pro Glu Leu Leu Pro Lys 130     135     140 GAA TAT GTG GAT TTT GAT TTC CAG CCC GAG CCG CTG ACC GAC TTT GAT 480 Glu Tyr Val Asp Phe Asp Phe Gln Pro Glu Pro Leu Thr Asp Phe Asp 145     150     155     160 GTG GTC ATG ATC TGG GTG GGC TCC ATG GCC AAT CGC TTC TCC GAC ACG 528 Val Val Met Ile Trp Val Gly Ser Met Ala Asn Arg Phe Ser Asp Thr     165     170     175 AAT CTG GAA GTG ACG GCA CTG GCC ATG CGT CAG TCT CTG GAG AAA CAG 576 Asn Leu Glu Val Thr Ala Leu Ala Met Arg Gln Ser Leu Glu Lys Gln    180     185     190 CAC GGC CCG GAA CGA GGC CGT GCC TTG TTT GAT GAG CTG CTG TGG ATC 624 His Gly Pro Glu Arg Gly Arg Ala Leu Phe Asp Glu Leu Leu Trp Ile   195     200     205 AAT GAC ACA ACA GCT CCC ACT ACG GTT CCG GCC CCC GCT GCC GAG CAC 672 Asn Asp Thr Thr Ala Pro Thr Thr Val Pro Ala Pro Ala Ala Glu His  210     215 220 AAG CCG CAG GCA CAA GCA GGG ACG CAG GAT CTG GCT CAT GTT TCC TCG 720 Lys Pro Gln Ala Gln Ala GLy Thr Gln Asp Leu Ala His Val Ser Ser 225 230     235     240 CCA GTA CTG GCT ACC GAG CTA GAG CGC CAG GAC AAG CAC TGG GGC GGC 768 Pro Val Leu Ala Thr Glu Leu Glu Arg Gln Asp Lys His Trp Gly Gly     245     250     255 CGT GGC CCG GAC TTC GCG CCC AAG GCT AGC AAC CTG TGG AGC ACT CGC 816 Arg Gly Pro Asp Phe Ala Pro Lys Ala Ser Asn Leu Trp Ser Thr Arg    260     265     270 CCC GAG CGA GTG CAG GAG GGC TCG ACC GTA CTG ATC AAC GGC CCA CAG 864 Pro Glu Arg Val Gln Glu Gly Ser Thr Val Leu Ile Asn Gly Pro Gln   275     280     285 TTT GGC TGG TAC AAC CCG GCC TAC ACC TAT GGC ATT GGC TTG CAT GGC 912 Phe Gly Trp Tyr Asn Pro Ala Tyr Thr Tyr Gly Ile Gly Leu His Gly  290     295     300 GCC GGC TTC GAT GTG GTG GGT AAT ACG CCT TTT GCC TAT CCG ATC GTA 960 Ala Gly Phe Asp Val Val Gly Asn Thr Pro Phe Ala Tyr Pro Ile Val 305     310 315     320 CTG TTT GGC ACC AAT AGC GAG ATT GCC TGG GGG GCG ACT GCT GGC CCG 1008 Leu Phe Gly Thr Asn Ser Glu Ile Ala Trp Gly Ala Thr Ala Gly Pro 325     330     335 CAA GAT GTG GTG GAC ATA TAT CAG GAA AAA TTG AAC CCC TCG CGT GCC 1056 Gln Asp Val Val Asp Ile Tyr Gln Glu Lys Leu Asn Pro Ser Arg Ala    340     345     350 GAT CAG TAC TGG TTC AAC AAT GCC TGG CGC ACG ATG GAG CAG CGC AAG 1104 Asp Gln Tyr Trp Phe Asn Asn Ala Trp Arg Thr Met Glu Gln Arg Lys   355     360     365 GAA CGT ATC CAG GTA CGC GGT CAG GCT GAT CGG GAA ATG ACG ATC TGG 1152 Glu Arg Ile Gln Val Arg Gly Gln Ala Asp Arg Glu Met Thr Ile Trp  370     375     380 CGC ACC GTG CAC GGC CCT GTG ATG CAG TTT GAT TAC GAT CAG GGC GCG 1200 Arg Thr Val His Gly Pro Val Met Gln Phe Asp Tyr Asp Gln Gly Ala 385     390     395     400 GCG TAC AGC AAG AAA CGC AGC TGG GAT GGC TAT GAG GTG CAG TCC TTG 1248 Ala Tyr Ser Lys Lys Arg Ser Trp Asp Gly Tyr Glu Val Gln Ser Leu     405 410     415 GAT CAA GCC AGC AAG ATG GCG ATT TCG ATC AAC TGG TAC TAC GCC GAC 1344 Asp Gln Ala Ser Lys Met Ala Ile Ser Ile Asn Trp Tyr Tyr Ala Asp 435     440     445 AAG CAC GGC AAT ATT GGT TAT GTC TCG CCG GCC TTC CTG CCC CAG CGT 1392 Lys His Gly Asn Ile Gly Tyr Val Ser Pro Ala Phe Leu Pro Gln Arg  450     455     460 CCT GCC GAT CAG GAC ATC CGT GTC CCT GCC AAG GGG GAT GGC AGC ATG 1440 Pro Ala Asp Gln Asp Ile Arg Val Pro Ala Lys Gly Asp Gly Ser Met 465     470     475     480 GAG TGG CTG GGC ATC AAG AGT TTC GAC GCG ATT CCC AAA GCC TAC AAT 1488 Glu Trp Leu Gly Ile Lys Ser Phe Asp Ala Ile Pro Lys Ala Tyr Asn     485     490     495 CCA CCC CAG GGC TAT CTG GTC AAC TGG AAC AAC AAG CCT GCG CCG GAC 1536 Pro Pro Gln Gly Tyr Leu Val Asn Trp Asn Asn Lys Pro Ala Pro Asp    500     505     510 AAA ACC AAT ACG GAT ACT TAC TAT TGG ACC TAT GGC GAC CGC ATG AAT 1584 Lys Thr Asn Thr Asp Thr Tyr Tyr Trp Thr Tyr Gly Asp Arg Met Asn   515 520     525 GAA CTG GTC AGT CAG TAC CAG CAG AAA GAC CTC TTC AGT GTG CAG GAG 1632 Glu Leu Val Ser Gln Tyr Gln Gln Lys Asp Leu Phe Ser Val Gln Glu 530     535     540 ATC TGG GAG TTC AAT CAA AAA GCC TCC TAT AGC GAT GTG AAC TGG CGC 1680 Ile Trp Glu Phe Asn Gln Lys Ala Ser Tyr Ser Asp Val Asn Trp Arg 545     550     555     560 TAC TTC CGC CCA CAT CTG GAA AAG CTG GCG CAA CAG CTG CCG GCC GAC 1728 Tyr Phe Arg Pro His Leu Glu Lys Leu Ala Gln Gln Leu Pro Ala Asp     565     570     575 GAT AGC AGC AAG GCG GCG CTG ACG ATG TTG CTC GCC TGG GAT GGA ATG 1776 Asp Ser Ser Lys Ala Ala Leu Thr Met Leu Leu Ala Trp Asp Gly Met    580     585     590 GAA CAG GAT CAG GGA GGG CAA AAT GCC GGA CCG GCG CGG GTG CTC TTC 1824 Glu Gln Asp Gln Gly Gly Gln Asn Ala Gly Pro Ala Arg Val Leu Phe   595     600     605 AAG ACC TGG CTG GAA GAA ATG TAC AAG CAG GTC TTG ATG CCG GTG GTG 1872 Lys Thr Trp Leu Glu Glu Met Tyr Lys Gln Val Leu Met Pro Val Val  610     615 620 CCT GAA TCG CAT CGC GCC ATG TAT AGC CAG ACT GGT TTT GCC ACG CAG 1920 Pro Glu Ser His Arg Ala Met Tyr Ser Gln Thr Gly Phe Ala Thr Gln 625 630     635     640 CAA GGT CCC AAC CCC GGT TCC ATC AAC TTG AGC ATG GGC ACC AAG GTC 1968 Gln Gly Pro Asn Pro Gly Ser Ile Asn Leu Ser Met Gly Thr Lys Val     645     650     655 TTG TTG CGT GCC TTG GTG CTG GAA GCC CAT CCC GAT CCC AAG CGT GTG 2016 Leu Leu Arg Ala Leu Val Leu Glu Ala His Pro Asp Pro Lys Arg Val    660     665     670 AAT GTC TTT GGT GAG CGT TCG TCT CAG GAA ATC ATG CAC ACA GCT TTG 2064 Asn Val Phe Gly Glu Arg Ser Ser Gln Glu Ile Met His Thr Ala Leu   675     680     685 CAA AAT GCG CAG GCC CGC TTG AGC CAG GAG CAG GGC GCT CAG ATG GCG 2112 Gln Asn Ala Gln Ala Arg Leu Ser Gln Glu Gln Gly Ala Gln Met Ala  690     695     700 CGC TGG ACC ATG CCG ACC TCC GTG CAT CGT TTC AGC GAC AAG AAC TTC 2160 Arg Trp Thr Met Pro Thr Ser Val His Arg Phe Ser Asp Lys Asn Phe 705     710 715     720 ACG GGA ACC CCG CAG ACG ATG CCT GGC AAT ACC TTT GCC TTT ACC GGC 2208 Thr Gly Thr Pro Gln Thr Met Pro Gly Asn Thr Phe Ala Phe Thr Gly 725     730     735 TAT CAG AAT CGA GGC ACG GAA AAT AAC CGC GTG GTG TTT GAT GCC AAG 2256 Tyr Gln Asn Arg Gly Thr Glu Asn Asn Arg Val Val Phe Asp Ala Lys    740     745     750 GGC GTG GAG TTC TGC GAC GCC ATG CCG CCC GGC CAA AGC GGT TTC ACC 2304 Gly Val Glu Phe Cys Asp Ala Met Pro Pro Gly Gln Ser Gly Phe Thr   755     760     765 GAC CGC AAT GGA GTG CGC AGC CCG CAT TAT GAG GAT CAG CTG AAG TTG 2352 Asp Arg Asn Gly Val Arg Ser Pro His Tyr Glu Asp Gln Leu Lys Leu  770     775     780 TAC GAG AAC TTC GAG TGC AAG ACG ATG GAT GTG ACG CAT GCG GAC ATT 2400 Tyr Glu Asn Phe Glu Cys Lys Thr Met Asp Val Thr His Ala Asp Ile 785     790     795     800 CGT CGT AAT GCG CAA AGC AGC ACG ATG CTG TTG ATT CAG CCT CAG CCT 2448 Arg Arg Asn Ala Gln Ser Ser Thr Met Leu Leu Ile Gln Pro Gln Pro     805 810     815 TAA                2451 End (2) INFORMATION SEQ ID NO:2: (i) S EQUENCE CHARACTERISTICS:  (A) LENGTH: 124 base pairs  (B) TYPE: nucleic acid  (C) STRANDEDNESS: single  (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:  (A) ORGANISM: E. coli (ix) FEATURE:  (A) NAME/KEY: TAC PROMOTER (B) LOCATION: 1..124  (D) OTHER INFORMATION: bp 35-40 = "-35 " region bp 57-62 = "-10" region   bp 110-114 = Shine Dalgarno lac gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GAATTCGAGC TCGAGCTTAC TCCCCATCCC CCTGTTGACA ATTAATCATC GGCTCGTATA  60 ATGTGTGGAA TTGTGAGCGG ATAACAATTT CACACAGGAA ACAGGATCCA AGGAAAAACA 120 TATG      124 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS:  (A) LENGTH: 151 base pairs (B) TYPE: nucleic acid  (C) STRANDEDNESS: single  (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (iii) HYPOTHETICAL: NO (iv) ORIGINAL SOURCE:  (A) ORGANISM: E. coli (ix) FEATURE:  (A) NAME/KEY: TRP PROMOTER (B) LOCATION: 1..151  (D) OTHER INFORMATION: bp 84-89 = "-35"   bp 107-112 = "-10 " (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GAATTCAAGG CGCACTCCCG TTCTGGATAA TGTTTTTTGC GCCGACATCA TAACGGTTCT  60 GGCAAATATT CTGAAATGAG CTGTTGACAA TTAATCATCG AACTAGTTAA CTAGTACGCA 120 AGTTCACGTA AAAAGGAGGT ATCGACATAT G   151 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS:  (A) LENGTH: 114 base pairs  (B) TYPE: nucleic acid  (C) STRANDEDNESS: single  (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:  (A) ORGANISM: Pseudonomas aeruginosa; phage pf3 (ix) FEATURE: (A) NAME/KEY: P78 PROMOTER  (B) LOCATION: 1..114  (D) OTHER INFORMATION: b p 23-28 = "-35"       or pf3 gene     p78 promoter  bp 46-51 = "-10" bp 100-103 = Shine Dalgarno p78 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GAATTCGATC GCAAAAAAGT ACTTGCAAGT TCCCGAAACC CTGTCTAGAG TTCTAGGTGC  60 ATCTGAATGG AGCTCGGTAC CAATCTGTTT GCTTCCATTG AGGTGCATCA TATG 114 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS:  (A) LENGTH: 105 base pairs  (B) TYPE: nucleic acid  (C) STRANDEDNESS: single  (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:  (A) ORGANISM: Pseudonomas aeruginos a and E. coli (ix) FEATURE:  (A) NAME/KEY: PF3 PROMOTER  (B) LOCATION: 1..105  (D) OTHER INFORMATION: bp 23-28 = "-35"       or pf3 gene p78 promoter bp 46-51 = "-10" bp 92-95 = Shine Dalgarno lac Z gene of E. coli (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: GAATTCGATC GCAAAAAAGT ACTTGCAAGT TCCCGAAACC CTGTCTAGAG TTCTAGGTGC  60 ATCTGAATGG AGCTCGGTAC CCGGGGATCC AAGGAAAAAC ATATG  105 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS:  (A) LENGTH: 10 amino acids  (B) TYPE: amino acid  (C) STRANDEDNESS: single  (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:  (A) ORGANISM: Alcaligenes faecalis (xi) FEATURE:  (A) NAME/KEY: DOMAIN, subunit  (B) LOCATION: 1..10  (D) OTHER INFORMATION: /note= "aa2=unknown; aa4=unknown;    aa9=unknown" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Gln Xaa Gln Xaa Val Glu Val Met Xaa Thr 10 1    5     10 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS:  (A) LENGTH: 11 amino acids  (B) TYPE: amino acid  (C) STRANDEDNESS: single  (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:  (A) ORGANISM: Alcaligenes faecalis (ix) FEATURE: (A) NAME/KEY: DOMAIN, subunit  (B) LOCATION: 1..11  (D) OTHER INFORMATION : /note= "aa7=unknown; aa10=unknown" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Ser Asn Leu Trp Ser Thr Xaa Pro Glu Xaa Val 11 1    5     10 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS:  (A) LENGTH: 32 base pairs  (B) TYPE: nucleic acid  (C) STRANDEDNESS: single  (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:  (A) ORGANISM: Alcaligenes faecalis (ix) FEATURE:  (A) NAME/KEY: OLIGONUCLEOTIDE  (B) LOCATION: 1..32  (D) OTHER INFORMATION: /note= "M: A or C; S: C or G" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: AGCAACCTG T GGAGCMSCTG CCCGGAGTGC GT 32

## Claims

1. A isolated gene encoding penicillin G acylase having essentially the nucleotide sequence depicted in SEQ ID NO: 1.

2. A gene according to claim 1 which is isolated from Alcaligenes faecalis.

3. A gene according to claim 1 or 2 under the control of a regulon comprising transcription and/or translation regulating sequences, wherein one or both of said regulating sequences have been replaced by other transcription and/or translation regulating sequences, respectively, obtained from the same or another organism.

4. A cloning vector comprising one or more penicillin G acylase genes as defined in claim 1, 2 or 3.

5. A vector according to claim 4, wherein the transcription sequences of the penicillin G acylase gene are replaced by the trp promoter.

6. A prokaryotic transformed host comprising a vector as defined in claim 4 or 5.

7. A transformed host according to claim 6 which is a gram-negative microorganism.

8. A transformed host according to claim 7 wherein the microorganism is of the genus Alcaligenes or Escherichia.

9. A transformed host according to claim 8 wherein the microorganism is Alcaligenes faecalis.

10. A method of preparing penicillin G acylase which comprises culturing a transformed host as claimed in any one of the claims 6 to 9 and recovering the penicillin G acylase in isolated form.

11. A method for producing, or enhancing the production of, penicillin G acylase in a prokaryotic host, the method comprising (i) preparing a DNA vector as defined in claim 4 or 5; (ii) transforming the host with the said vector; and (iii) cloning the resulting transformants and selecting the same.

12. A method according to claim 11, characterized by transforming a gram-negative microorganism.

13. A method according to claim 12, characterized by transforming a microorganism of the genus Alcaligenes or Escherichia.

14. A method according to claim 13, characterized by transforming Alcaligenes faecalis.