Activity of penicillin acylase from E. coli in the reversed-micelle system AOT--H2O--octane

The behavior of a penicillin acylase from E. coli was studied in the reversed-micelle system AOT--H2O--octane. Kinetic studies of the enzymatic hydrolysis of the m-carboxy-p-nitroanilide of phenylacetic acid, titration of the penicillin acylase active site with an irreversible specific inhibitor (phenylmethylsulfonyl fluoride), sedimentation analysis at different hydration degrees, and chemical modification showed that the enzyme loses no more than 20% of its initial activity during 3-4 h in the reversed-micelle systems of different hydration degrees and retains its catalytically active structure.     

大肠杆菌青霉素G酰化酶基因及其邻近区域的核苷酸全序列

Some of microorganisms have been known to possess penicillin G acylase activity. The E. coli derived penicillin G acylase (PGA) can catalyze the conversion of penicillin G into phenylacetic acid and 6-amino-penicillanic acid, the latter is used as the starting compound for the industrial formation of semi-synthetic penicillins. Apart from its industrial importance, the enzyme PGA displays a number of interesting properties. Catalytically active enzyme is localized in the periplasmic space of E. coli cells and composed of two dissimilar subunits. The two subunits are apparently produced from a precursor protein, via a processing pathway hitherto unique in its features for a prokaryotic enzyme. The studies on processing of the precursor and on the relationship between structure and function of the mature enzyme are important theoretically. Previously we cloned a 3.5 kb DNA fragment from a strain (E. coli AS 1.76), which displays PGA activity. In this paper, we report a nucleotide sequence of the 3.5 kb DNA fragment containing PGA gene. After insertion of the DNA fragment into EcoR I and Hind III sites in pWR 13, pPGA 20 had been obtained. We subcloned the Hind III and Bg1 II treated fragment of 1.6 kb in length from pPGA 20 into Hind III and BamH I sites of pWR 13 to get a pPGA 1.6, and Bg1 II and EcoR I treated fragment of 1.9 kb in length into BamH I and EcoR I sites of pWR 13 to get a pPGA 1.9. The linearized pPGA 1.9 which were digested with appropriate restriction enzymes were progressively shortened from both ends respectively by digestion with Bal 31 nuclease, followed by cleavage of shortened target DNA off vector DNA molecules with appropriate restriction enzymes. The series of the DNA fragments shortened from EcoR I end were then cloned into plasmid pWR 13 which had previously digested with Hind III and Sma I enzymes (Fig. 1). The DNA fragment cloned in pWR 13 were directly sequenced on the resulted plasmids by using primer I and primer II. Thus we have obtained the complete nucleotide sequence of the 3.5 kb DNA fragment. The 3.5 kb fragment contains an intact PGA gene which is 2.6 kb.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reconstitution in vivo of penicillin G acylase activity from separately expressed subunits.

Penicillin G acylase from Escherichia coli ATCC11105 is synthesized as a precursor polypeptide with a signal sequence for secretion into the periplasm and an endopeptide separating two subunit domains. Proteolytic processing leads to mature, heterodimeric penicillin G acylase. We have shown that the alpha- and beta-subunits of the enzyme, which have no detectable enzymatic activity on their own, can reconstitute enzyme activity when their genes are put into an E. coli host on separate plasmids. Activity is reconstituted in the cytoplasm whereas normally processing and formation of the active heterodimer occurs in the periplasm. Enzyme activity can reach levels close to wild type in the strain used. The activity recovered from a combination of alpha-subunit linked to a 54-amino-acid endopeptide and beta-subunit was lower than with the subunits alone.     

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.     

Role of protein subunits in Proteus rettgeri penicillin G acylase.

Penicillin G acylase from Proteus rettgeri is an 80,000- to 90,000-dalton enzyme composed of two nonidentical subunits. Both subunits were required for enzymatic activity. The 65,000-dalton beta subunit contained a phenylmethylsulfonyl fluoride-sensitive residue required for enzymatic activity, and the 24,500-dalton alpha subunit contained the domain that imparts specificity for the penicillin side chain.     

A postfermentative stage improves penicillin acylase production by a recombinant E. coli

Active penicillin acylase is formed only after an in vivo post-translational processing of the polypeptide precursor. Such a maturation process is rare in procaryotes. In this work, incubation under aerated conditions, of whole recombinant E. coli cells after glucose depletion and growth cessation, i.e., during the postfermentative stage, consistently resulted in 2- to 4-fold increases in penicillin acylase activity. Such results suggest that penicillin acylase maturation occurs to a high extent even during the postfermentative stage. Accordingly, the effect of different incubation conditions, during the postfermentative stage, on penicillin acylase was determined. Incubation under anaerobic conditions resulted only in a 1.27-fold increase of enzyme activity, with respect to the end of the batch culture, whereas a 3- and 4- fold increase occurred during incubation under dissolved oxygen concentrations of 100 and 43% (with respect to air sat.), respectively. Only a small negative effect, on the maturation process, was observed during incubation with acetate concentrations above 0.6 g/L. No effect of pH, in the range of 6.0 to 8.0, was observed.     

Electron microscopic study of the localization of penicillin acylase in E. coli cells]

The paper describes the studies on definition of penicillinacylase localizations in the cells of E. coli with the help of ferritin labeled immune sera and electron microscopy. Both the intact cells and the cells treated with the substances affecting the cell wall intactness were used. The study showed relation between penicillinacylase and the surface structures of the cell, i.e. the cell wall and the periplasmic areas. It was found that penicillinacylase got into the environmental medium with the splitted cell fragments which corresponded to the general mechanism of excretion of large high molecular compounds by gramnegative organisms.     

Thermodynamic and kinetic stability of penicillin acylase from Escherichia coli

Thermal denaturation of penicillin acylase (PA) from Escherichia coli has been studied by high-sensitivity differential scanning calorimetry as a function of heating rate, pH and urea concentration. It is shown to be irreversible and kinetically controlled. Upon decrease in the heating rate from 2 to 0.1 K min(-1) the denaturation temperature of PA at pH 6.0 decreases by about 6 degrees C, while the denaturation enthalpy does not change notably giving an average value of 31.6+/-2.1 J g(-1). The denaturation temperature of PA reaches a maximum value of 64.5 degrees C at pH 6.0 and decreases by about of 15 degrees C at pH 3.0 and 9.5. The pH induced changes in the denaturation enthalpy follow changes in the denaturation temperature. Increasing the urea concentration causes a decrease in both denaturation temperature and enthalpy of PA, where denaturation temperature obeys a linear relation. The heat capacity increment of PA is not sensitive to the heating rate, nor to pH, and neither to urea. Its average value is of 0.58+/-0.02 J g(-1) K(-1). The denaturation transition of PA is approximated by the Lumry-Eyring model. The first stage of the process is assumed to be a reversible unfolding of the alpha-subunit. It activates the second stage involving dissociation of two subunits and subsequent denaturation of the beta-subunit. This stage is irreversible and kinetically controlled. Using this model the temperature, enthalpy and free energy of unfolding of the alpha-subunit, and a rate constant of the irreversible stage are determined as a function of pH and urea concentration. Structural features of the folded and unfolded conformation of the alpha-subunit as well as of the transition state of the PA denaturation in aqueous and urea solutions are discussed.     

Instability of the plasmid carrying active penicillin acylase gene from Escherichia coli: conditions inducing insertional inactivation

Abstract The marker instability of the recombinant plasmid coding penicillin acylase production was investigated in Escherichia coli . In the absence of any selective pressure and under fully induced production of penicillin acylase the number of cells lacking plasmids increased during cultivation. Cells still harboring plasmids fully maintained their ability to synthesize the enzyme. However, when a selection for Tet^r was applied, the number of selected cells containing the actively synthesizing gene decreased. Changes occurring in plasmid DNA revealed high frequency of insertions affecting expression of penicillin acylase gene.     

Promotion of multipoint covalent immobilization through different regions of genetically modified penicillin G acylase from E. coli

A novel approach is proposed to prepare a set of immobilized derivatives of a enzyme covalently rigidified through different regions of its surface. Six different variants of penicillin G acylase (PGA) from Escherichia coli (which lacks Cys) were prepared by introducing a unique Cys residue via site-directed mutagenesis in six different enzyme regions which were rich in Lys residues. All variants exhibited a similar activity and stability compared to those of the native enzyme. Each variant was immobilized on supports having a low concentration of reactive disulfide moieties and a high concentration of poorly reactive epoxy groups. After immobilization at pH 7.0 by site-directed thiol-disulfide intermolecular exchange, derivatives were further incubated at pH 10.0 for 48 h to promote an additional intramolecular reaction between Lys residues of enzyme and epoxy groups of the support. The establishment of at least three covalent attachments per PGA molecule was determined for all immobilized enzyme variants. The different derivatives exhibited diverse stability against several distorting agents and different selectivity in two interesting reactions. The derivative of the PGA variant obtained by replacement of GlnB380 by Cys was the most stable against heat and organic cosolvents: it preserved 90% of the initial activity and was 30-fold more stable than soluble PGA. This derivative also exhibited an improved enantioselectivity in the hydrolysis of chiral esters (E was improved from 8 to 16) and in kinetically controlled synthesis of amides (synthetic yields were increased from 31 to 49%).     

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.     

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.     

Comparative characteristics of the metabolism of E. coli cells with various activity of penicillin acylase

A correlation between the synthesis and secretion of penicillin acylase (PA; EC 3.5.1.11) and the membrane phospholipid composition was observed in three E. coli strains. In cells with overproduction of PA, the phospholipid/protein ratio decreases, while the cardiolipin/phosphatidylglycerol ratio increases. The differences in the functioning of the electron transport system were revealed in cells with different levels of PA synthesis and secretion. The O2 consumption rate was 3 times lower in the cells with overproduction of PA than in those of less productive strains. On the contrary, membrane particles isolated from the cells of PA producers had no significant differences in the O2-reduction rate. The sensitivity of the strains to the inhibitor of terminal oxidases, sodium cyanide, and to the uncoupler of redox phosphorylation, chlorocarbonyl-phenylhydrazone, was different. Thus the E. coli cells with PA overproduction are characterized by significant changes in energetics and constructive metabolism. The interrelations between PA overproduction, phospholipid metabolism and the respiratory chain activity are discussed.