Isolation and mapping of a mutant penicillin G acylase with altered substrate specificity from Escherichia coli

Summary Penicillin G acylase of Escherichia coli ATCC 11105 catalyzes hydrolysis as wellas synthesis of penicillin G. In this work a recombinant penicillin G acylase genewas mutagenized in vivo. A mutant with altered penicillin G acylase was selectedby its ability to grow with phthalyl-L-leucine as sole source of leucine. Themutant enzyme obtained was deficient in hydrolyzing penicillin G. A mutation ofGly359 to aspartic acid was mapped first by construction of chimeric pac genescomposed of wild type and mutant DNA, followed by nucleotide sequencing.     

An improved method to clone penicillin acylase genes: Cloning and expression in Escherichia coli of penicillin G acylase from Kluyvera citrophila

A simple and versatile procedure to clone penicillin acylase genes has been developed. It involves the construction of a plasmid library in a host presenting an amino acid auxotrophy. Recombinant clones carrying the acylase gene were selected on a minimal medium containing instead of the required amino acid its phenylacetyl derivative. Penicillin acylase genes from Escherichia coli ATCC 11105 and Kluyvera citrophila ATCC 21285 have been cloned in E. coli using this technique. The restriction map of the region containing the E. coli penicillin acylase gene was found to be similar to that described by H. Mayer et al. (in: Plasmids of Medical, Environmental and Commercial Importance (Timmis, K.M. and Paler, A., eds.), pp. 459–470, Elsevier, Amsterdam 1979). K. citrophila acylase gene was located within a 3.0 kb Hind III-PvuI fragment. Some differences were observed between the partial restriction maps of both genes. In addition, the production of those clones carrying the E. coli acylase was more sensitive to the growth temperature than that of the clones containing the K. citrophila gene. Bacteria harbouring plasmids containing the K. citrophila acylase sequence were able to produce about 30 fold more enzyme than the parental strain. A 60 000 dalton polypeptide corresponding to the K. citrophila acylase has been detected in a maxicell system. The industrial applications of the procedure are discussed.     

The PaaX repressor, a link between penicillin G acylase and the phenylacetyl-coenzyme A catabolon of Escherichia coli W

The pac gene, encoding the penicillin G acylase from Escherichia coli W, is regulated by the PaaX repressor of the phenylacetate catabolic pathway. pac expression depends on the synthesis of phenylacetyl-coenzyme A. PaaX and the cyclic AMP receptor protein (CRP) bind in vitro to the Ppac promoter region. A palindromic sequence proposed as the PaaX operator is located upstream of the -35 box overlapping a CRP binding site, an unusual position that suggests a novel regulatory mechanism.     

Host dependenet inactivation by IS2 of induced E.coli penicillin amidase gene cloned on multicopy plasmids

Structural instability of the cloned penicillin acylase gene (pac) from E.coli ATCC11105 was studied under various physiological conditions. Structural changes which adversely affect the expression of penicillin acylase gene were selected for only under conditions in which the gene was active and fully induced. In E.coli strain YMC the predominant mutations were the insertions of the IS2 element at various sites within the 700 bp proximal portion of the pac gene. The results indicated that the induction of the plasmid cloned gene was the main factor which rendered it a target for inactivation by insertions of the host encoded IS2 elements. The mutational events were host specific and they were not influenced by mutual positions and orientations of key marker genes on the plasmid.     

Cloning of E. coli penicillin G acylase gene with mini-Mu containing a plasmid replicon

Summary An in vivo cloning system based on mini-Mu derivatives was used for cloning of E. coli penicillin G acylase gene (pac). We have constructed several recombinant clones producing penicillin G acylase and some of them exhibit approximately two times higher activity than original strains.     

Improved activity and pH stability of E. coli ATCC 11105 penicillin acylase by error-prone PCR

Penicillin G acylase is the key enzyme used in the industrial production of β-lactam antibiotics. This enzyme hydrolyzes penicillin G and related β-lactam antibiotics releasing 6-aminopenicillanic acid, which is an intermediate in the production of semisynthetic penicillins. To improve the enzymatic activity of Escherichia coli penicillin acylase, sequential rounds of error-prone polymerase chain reaction were applied to the E. coli pac gene. After the second round of evolution, the best mutant M2234 with enhanced activity was selected and analyzed. DNA sequence analyses of M2234 revealed that one amino acid residue (K297I), located far from the center of the catalytic pocket, was changed. This mutant (M2234) has a specific activity 4.0 times higher than the parent enzyme and also displayed higher stability at pH 10.     

Selection of Pseudomonas melanvgenum KY 3987 as a New Ampicillin-producing Bacteria

Further selection for a better strain capable of producing D(?)-α-aminobenzylpenicillin (APc) from 6-aminopenicillanic acid (6–APA) was carried out. Pseudomonas melanogenum KY 3987 was consequently selected as a new strain possessing an APc-specific penicillin acylase.

The acylase could synthesize APc in good yields from 6–APA and phenylglycine ester and form 6–APA only from APc, not from other common penicillins. Since the Pseudomonas acylase was found incapable of forming penicillin G (Pc–G) from 6–APA and phenylacetic acid, in contrast with E. coli and Kluyvera citrophila enzymes, the enzymatic hydrolysate of Pc–G, for example by K. citrophila cells, which contained 6–APA and phenylacetate, became employed as a source of 6–APA instead of purified 6–APA to synthesize APc by the cells of P. melanogenum.     

Genetic construction of catalytically active cross-species heterodimer penicillin G amidase

Summary A cross-species penicillin G amidase (PGA) gene (pac) coding for an -peptide and a linker peptide fromK. citrophila ATCC 21285 and a -peptide fromE. coli ATCC 11105 has been constructed and cloned inE. coli. The naturally occurring PGA specific processing pathway led to the formation of a hybrid enzyme which was catalytically active. In comparison with the two wild-type enzymes the hybrid PGA was found to have higherk _cat values for the three tested substrates benzylpenicillin, ampicillin and 6-nitro-3-phenylacetamido-benzoic acid (NIPAB).K _m was between the values of the wild-type enzymes or close to that ofK. citrophila. The presented method for protein design of processed enzymes, like PGA, can be applied to combine enzyme properties from different species for special applications.     

Penicillin Acylase of Pseudomonas melanogenum KY 3987

Partially purified penicillin acylases (EC 3.5.1.11) were prepared from Pseudomonas melanogenum KY 3987 and Kluyvera citrophila KY 3641 capable of synthesizing d(–)-α-amino-benzylpenicillin (APc) from 6-aminopenicillanic acid (6-APA) and phenylglycine methyl ester. As the cell-free extract of P. melanogenum contained high levels of penicillinase (EC 3.5.2.6), the acylase was separated completely from the penicillinase by use of Sephadex column chromatography or electrofocusing. The most salient property of the P. melanogenum penicillin acylase was its substrate specificity to penicillin substrates: it could form 6-APA only from APc but not from penicillin G, penicillin V and p-aminobenzylpenicillin, whereas the K. citrophila acylase acted on all of these penicillins. The P. melanogenum enzyme is hence considered a novel type of penicillin acylase.     

Engineering the Substrate Specificity of a Thermophilic Penicillin Acylase from Thermus thermophilus

A homologue of the Escherichia coli penicillin acylase is encoded in the genomes of several thermophiles, including in different Thermus thermophilus strains. Although the natural substrate of this enzyme is not known, this acylase shows a marked preference for penicillin K over penicillin G. Three-dimensional models were created in which the catalytic residues and the substrate binding pocket were identified. Through rational redesign, residues were replaced to mimic the aromatic binding site of the E. coli penicillin G acylase. A set of enzyme variants containing between one and four amino acid replacements was generated, with altered catalytic properties in the hydrolyses of penicillins K and G. The introduction of a single phenylalanine residue in position α188, α189, or β24 improved the K_m for penicillin G between 9- and 12-fold, and the catalytic efficiency of these variants for penicillin G was improved up to 6.6-fold. Structural models, as well as docking analyses, can predict the positioning of penicillins G and K for catalysis and can demonstrate how binding in a productive pose is compromised when more than one bulky phenylalanine residue is introduced into the active site.     

Penicillin G acylase fromKluyvera citrophila new choice as industrial enzyme

Summary We propose a new and integrated method for the evaluation of industrial enzymes. The application of this method to the enzyme penicillin G acylase fromKlyvera citrophila shows very interesting industrial propects. This acylase presents a much better stability agains heat, pH or organic cosovents as compared with the more popular enzyme fromEscherichia coli. In addition, this enzyme is very easy to immobilize through its amine groups and to stabilize through multipoint covalent attachment on activated pre-existing supports.     

Modified penicillin acylase signal peptide allows the periplasmic production of soluble human interferon-γ but not of soluble human interleukin-2 by the Tat pathway in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Production of periplasmic human interferon-γ (hINF-γ) and human interleukin-2 (hIL-2) by the Tat translocation pathway in Escherichia coli BL21-SI was evaluated. The expression was obtained using the pEMR vector which contains the Tat-dependent modified penicillin acylase signal peptide (mSPpac) driven by the T7 promoter. The mSPpac-hINF-γ was processed and the protein was transported to periplasm. Up to 30.1% of hINF-γ was found in the periplasmic soluble fraction, whereas only 15% of the mSPpac-hIL-2 was processed, but hIL-2 was not found in the periplasmic soluble fraction.     

Changing glycine 21 for glutamic acid in the β-subunit of penicillin G acylase from Kluyvera citrophila prevents protein maturation

Summary Active penicillin acylase from Kluyvera citrophila strain ATCC 21 285 consists of two different -and -subunits derived from a single precursor by post-translational processing. Using the chemical mutagen hydroxylamine we have treated plasmid pYKD59 containing the active penicillin acylase gene (pga) from K. citrophila and have generated different point mutant penicillin acylase genes, one producing a muturation deficient precursor. This point mutation has changed the gylcine 310 residue of the precursor for a glutamic acid (residue number 21 of the mature -subunit). The introduction of a charged residue in this position did not prevent translocation of the precursor to the periplasm but the resultant molecule was not able to undergo subsequent post-translational modification to yield the active protein.     

Penicillin acylase mutants with altered site-directed activity from Kluyvera citrophila

Summary Oligonucleotide-directed mutagenesis has been used to obtain specific changes in the penicillin acylase gene from Kluyvera citrophila. Wild-type and mutant proteins were purified and the kinetic constants for different substrates were determined. Mutations in Met168 highly decreased the specificity constant of the enzyme for penicillin G, penicillin V and phenylacetyl-4-aminobenzoic acid and the catalytic constant k _cat for phenylacetyl-4-aminobenzoic acid. Likewise, the phenylmethylsulphonyl-fluoride sensitivity was significantly decreased. It is concluded that the 168 residue is involved in binding by interaction with the acid moiety of the substrate. A putative penicillin-binding domain was located in penicillin acylase by sequence homology with other penicillin-recognizing enzymes. Lys374 and His481, the conserved amino acid residues that are essential for catalysis in these enzymes, can be changed in penicillin acylase with no changes to the k _cat and phenylmethylsulphonyl fluoride reactivity, but change the K _m.The likelihood of the existence of this proposed penicillin binding site is discussed. The reported results might be used to alter the substrate specificity of penicillin acylase in order to hydrolyse substrates of industrial significance other than penicillins. Offprint requests to: I. Prieto