Expression, purification and crystallization of penicillin G acylase from Escherichia coli ATCC 11105

The penicillin acylase gene (pac) from Escherichia coli ATCC 11105 was cloned into pUC 9 and the resulting vector (pUPA-9), when transformed into E. coli strain 5K, allowed the constitutive overproduction of mature penicillin acylase when grown at 28 degrees C. The enzyme was purified from the periplasmic fraction of E. coli pUPA-9 by hydrophobic interaction chromatography and anion exchange. Crystals of penicillin acylase were grown in batch using polyethylene glycol 8000 as a precipitant. The crystals (space group P1) diffracted to beyond 2.3 A.     

Effects of site-directed mutations on processing and activities of penicillin G acylase from Escherichia coli ATCC 11105.

Penicillin G acylase from Escherichia coli ATCC 11105 is synthesized from its precursor polypeptide into a catalytically active heterodimer via a complex posttranslational processing pathway. Substitutions in the pair of aminoacyl residues at the cleavage site for processing the small and large subunits were made. Their processing phenotypes and penicillin G acylase activities were analyzed. By the introduction of a prolyl residue at either position, the processing of the small subunit was blocked without a change in enzymatic activity. Four other substitutions had no effect. At the site for processing the large subunit, four substitutions out of the seven examined blocked processing. In general, penicillin G acylase activity seemed to be proportional to the efficiency of the large-subunit-processing step. Ser-290 is an amino acid critical for processing and also for the enzymatic activity of penicillin G acylase. In the mutant pAATC, in which Ser-290 is mutated to Cys, the precursor is processed, but there is no detectable enzymatic activity. This suggests that there is a difference in the structural requirements for the processing pathway and for enzymatic activity. Recombination analysis of several mutants demonstrated that the small subunit can be processed only when the large subunit is processed first. Some site-directed mutants from which signal peptides were removed showed partial processing phenotypes and reduced enzymatic activities. Their expression showed that the prerequisite for penicillin G acylase activity is the efficient processing of the large subunit and that the maturation of the small subunit does not affect the enzymatic activity.     

A common precursor for the two subunits of the penicillin acylase from Escherichia coli ATCC11105

Penicillin acylase (PA) is an industrial enzyme that is used to convert penicillin G into a precursor for semisynthetic penicillins. We have cloned a segment of DNA that codes for the two subunits required for PA activity. We also report the nucleotide sequence of a DNA fragment that codes for (i) the small subunit, (ii) the N-terminal region of the large subunit and (iii) a putative connecting peptide. These results confirm the existence of a common precursor for both peptides.     

Primary structure requirements for the maturation in vivo of penicillin acylase from Escherichia coli ATCC 11105

The two constituent subunits of the enzyme penicillin acylase from Escherichia coli strain ATCC 11105 are derived from a single precursor polypeptide by post-translational processing. Mutant penicillin acylase precursors were constructed carrying insertions and deletions in various domains and they were analysed for their processing behaviour. It was found that an endopeptide region of appropriate size and an intact C-terminus were absolutely necessary for the maturation process. Internal deletions within the beta-subunit domain also prevented post-translational cleavage. Processing competence, therefore, was not merely determined by the amino acid sequence in the vicinity of the processing sites but relied on a correct overall conformation of the protein. The processing pathway in vivo proceeds via an intermediate comprising the alpha subunits plus endopeptide and is thus identical to the pathway which has been determined previously by in vitro analysis. The post-translational modification of the precursor is probably not carried out by a specific processing enzyme(s) as the heterologous expression of the penicillin acylase (pac) structural gene yielded processed and active enzyme in different enterobacteria and in a Pseudomonas species.     

Identification of a new active site for autocatalytic processing of penicillin acylase precursor in Escherichia coli ATCC11105

Penicillin acylase (PA) from Escherichia coli ATCC11105 is a periplasmic heterodimer consisting of a 24 kDa small subunit and a 65 kDa large subunit. It is synthesized as a single 96 kDa precursor and then matures to functional PA via a posttranslational processing pathway. The GST-PA fusion protein expression system was established for monitoring the precursor PA processing in vitro. The purified PA precursor was processed into mature PA the same way as in vivo, but pH dependently. From the primary sequence analysis, we identified a putative conserved lysine residue (K299) responsible for the pH dependent processing. The substitution of K299 residue by site-directed mutagenesis affected both the enzyme activity and the precursor PA processing in vivo. Furthermore, it was shown that the processing rates of wild-type and mutant precursor PAs depended on the pKa values of their side chain R group. These results demonstrated that the lysine residue (K299) was involved in the precursor processing of PA together with N-terminal serine residue (S290) of the large subunit.     

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.     

Improvement of catalytic properties of Escherichia coli penicillin G acylase immobilized on glyoxyl agarose by addition of a six-amino-acid tag

A tag of three lysines alternating with three glycines was added to the C-terminal end of the beta chain of penicillin G acylase (PGA). This modification improved the immobilization efficiency of PGA on glyoxyl agarose and the catalytic properties of the PGA derivative, although it impaired the posttranslational steps of overexpressed protein maturation.     

Site-directed chemical conversion of serine to cysteine in penicillin acylase from Escherichia coli ATCC 11105. Effect on conformation and catalytic activity.

Penicillin acylase (EC 3.5.1.11) was completely inactivated with equimolar phenylmethane [35S]sulphonyl fluoride (PhMe35SO2F); the stability of the sulphonyl group in the modified protein was determined by measurement of the radioactivity in ultrafiltrates. In 8 M urea, the rate of loss of the sulphonyl group was similar to that observed in PhMeSO2F-inactivated chymotrypsin [Gold, A.M. & Fahrney, D. (1964) Biochemistry 3, 783-791]. Incubation of the PhMeSO2F-inactivated acylase with 0.7 M potassium thioacetate yielded an acetylthiol enzyme which was subsequently converted to a thiol-enzyme during incubation with 10 mM 6-aminopenicillanic acid. 4-Pyridyl-ethylcysteine was released by acid hydrolysis after reaction of the thiol-protein with 4-vinylpyridine. The rates of reaction of thiol-penicillin acylase with iodoacetic acid and 2,2'-dipyridyl disulphide were consistent with the presence of an incompletely accessible cysteinyl sidechain. After carboxymethylating the thiol-enzyme with iodo[2-3H]acetic acid, the label was shown by SDS/PAGE and sequencing analysis to be associated exclusively with the beta-chain NH2-terminal residue, indicating conversion of Ser290 to S-carboxymethyl-cysteine. Near-ultraviolet CD spectra showed the conformation of thiol-penicillin acylase to be indistinguishable from that of the native protein but the catalytic activity was less than 0.02% of that of the normal enzyme. The possibility that Ser290 acts as a nucleophile in catalysis is discussed.     

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.     

Enhanced production of penicillin G acylase from a recombinant Escherichia coli

Penicillin G acylase (pac) gene was cloned into a stable asd ⁺ vector (pYA292) and expressed in Escherichia coli. This recombinant strain produced 1000 units penicillin G acylase g^–1 cell dry wt, which is 23-fold more than that produced by parental Escherichia coli ATCC11105. This enzyme was purified to 16 units mg^–1 protein by a novel two-step process.     

Modifying the substrate specificity of penicillin G acylase to cephalosporin acylase by mutating active-site residues

The penicillin G acylase (PGA) and cephalosporin acylase (CA) families, which are members of the N-terminal (Ntn) hydrolases, are valuable for the production of backbone chemicals like 6-aminopenicillanic acid and 7-aminocephalosporanic acid (7-ACA), which can be used to synthesize semi-synthetic penicillins and cephalosporins, respectively. Regardless of the low sequence similarity between PGA and CA, the structural homologies at their active-sites are very high. However, despite this structural conservation, they catalyze very different substrates. PGA reacts with the hydrophobic aromatic side-chain (the phenylacetyl moiety) of penicillin G (PG), whereas CA targets the hydrophilic linear side-chain (the glutaryl moiety) of glutaryl-7-ACA (GL-7-ACA). These different substrate specificities are likely to be due to differences in the side-chains of the active-site residues. In this study, mutagenesis of active-site residues binding the side-chain moiety of PG changed the substrate specificity of PGA to that of CA. This mutant PGA may constitute an alternative source of engineered enzymes for the industrial production of 7-ACA.     

Improved penicillin amidase production using a genetically engineered mutant of Escherichia coli ATCC 11105

Penicillin G amidase (PGA) is a key enzyme for the industrial production of penicillin G derivatives used in therapeutics. Escherichia coli ATCC 11105 is the more commonly used strain for PGA production. To improve enzyme yield, we constructed various recombinant E. coli HB101 and ATCC 11105 strains. For each strain, PGA production was determined for various concentrations of glucose and phenylacetic and (PAA) in the medium. The E. coli strain, G271, was identified as the best performer (800 U NIPAB/L). This strain was obtained as follows: an E. coli ATCC 11105 mutant (E. coli G133) was first selected based on a low negative effect of glucose on PGA production. This mutant was then transformed with a pBR322 derivative containing the PGA gene. Various experiments were made to try to understand the reason for the high productivity of E. coli G271. The host strain, E. coli G133, was found to be mutated in one (or more) gene(s) whose product(s) act(s) in trans on the PGA gene expression. Its growth is not inhibited by high glucose concentration in the medium. Interestingly, whereas glucose still exerts some negative effect on the PGA production by E. coli G133, PGA production by its transformant (E. coli G271) is stimulated by glucose. The reason for this stimulation is discussed. Transformation of E. coli G133 with a pBR322 derivative containing the Hindlll fragment of the PGA gene, showed that the performance of E. coli G271 depends both upon the host strain properties and the plasmid structure. Study of the production by the less efficient E. coli HB101 derivatives brought some light on the mechanism of regulation of the PGA gene. (c) 1993 John Wiley & Sons, Inc.     

Expression of penicillin G acylase gene from Bacillus megaterium ATCC 14945 in Escherichia coli and Bacillus subtilis.

Penicillin G acylase gene from Bacillus megaterium ATCC 14945 has been isolated. Recombinant Escherichia coli clones were screened for clear halo forming activity on the lawn of Staphylococcus aureus ATCC 6538P using the enzymatic acylating reaction of 7-aminodeacetoxycephalosporanic acid (7-ADCA) and D-(alpha)-phenylglycine methylester. The gene was contained within a 2.8 kb DNA fragment and expressed efficiently when transferred from E. coli to Bacillus subtilis. A twenty times greater amount of enzyme was produced in B. subtilis transformant than that in B. megaterium. The purified enzyme from subcloned B. subtilis showed that the native enzyme consisted of two identical subunits, each with a molecular weight of 57,000. The enzyme was able to react on various cephalosporins, i.e., cephalothin, cefamandole, cephaloridine, cephaloglycin, cephalexin and cephradine.     

粪产碱杆菌青霉素G酰化酶的分离提取

比较研究了几种破碎大肠杆菌细胞的方法,如渗透压法、超声波法、玻珠震碎法、玻珠研磨法、有机溶剂法、冻融法以及盐酸胍/EDTA法等,以确定出一种简单、快速、高效的破碎重组大肠杆菌细胞的方法获得粪产碱杆菌青霉素G酰化酶(AfPGA)用于后续试验。结果表明玻珠震碎法、超声波法和渗透压法是较优的细胞破碎方法,活力回收率分别为99.7%、78.4%、60.7%,其他方法均低于22%。而比活力以渗透压法为最高,达到4.40 U/mg。     

Leakage of recombinant penicillin G acylase from Escherichia coli by adding chloroform under fermentation conditions

A simple method was developed to release periplasmic penicillin G acylase from Escherichia coli BL21(DE3) during the fermentation process. More than 80% of the total penicillin G acylase was released into the broth when 3% (v/v) chloroform was added at 3 h after induction. The activity of extracellular penicillin G acylase reached 20699 U/l. This method was efficient and would facilitate further investigation of penicillin G acylase for industrial applications.     

Improvement of the extraction of penicillin acylase from Escherichia coli cells by a combined use of chemical methods

Several techniques for protein extraction were tested for recovering penicillin acylase from a recombinant strain of Escherichia coli. These techniques include chemical [guanidine hydrochloride, Triton X-100, ethylenediaminetetraacetic acid (EDTA), ethanol/toluene], physical (sonication, freeze-and-thawing), and enzymatic (lysozyme) treatments. Best results were obtained with the combined use of guanidine and EDTA. This extraction procedure was optimized, and it was found that 95% of the enzyme was extracted after a 10 m/M EDTA plus 10 mM guanidine treatment at room temperature for 10 h. The purification factor was 25 when compared to disruption by sonication. This extraction method could avoid purification steps for particular applications. (c) 1994 John Wiley & Sons, Inc.