Evidence for the involvement of arginyl residue at the active site of penicillin G acylase from Kluyvera citrophila

Penicillin G acylase (PGA) is used for the commercial production of semi-synthetic penicillins. It hydrolyses the amide bond in penicillin producing 6-aminopenicillanic acid and phenylacetate. 6-Aminopenicillanic acid, having the beta-lactam nucleus, is the parent compound for all semi-synthetic penicillins. Penicillin G acylase from Kluyvera citrophila was purified and chemically modified to identify the role of arginine in catalysis. Modification with 20 mM phenylglyoxal and 50 mM 2,3-butanedione resulted in 82% and 78% inactivation, respectively. Inactivation was prevented by protection with benzylpenicillin or phenylacetate at 50 mM. The reaction followed psuedo-first order kinetics and the inactivation kinetics (V(max), K(m), and k(cat)) of native and modified enzyme indicates the essentiality of arginyl residue in catalysis.     

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.     

Mutant Escherichia coli penicillin acylase with enhanced stability at alkaline pH

Increased stability at alkaline pH should be a valuable attribute for the utilization of penicillin acylase in bioreactors employed to convert penicillins into 6-aminopenicillanic acid, a precursor of semisynthetic penicillins. In these systems, base is added for pH control, which results in local alkaline conditions that promote enzyme inactivation. Hydrolysis and synthesis reactions are also pH dependent. Here, we report work in which the gene coding for Escherichia coli penicillin acylase was subjected to oligonucleotide-directed random mutagenesis at regions coding for amino acids predicted to be at the surface of the enzyme. The resulting mutant library, cloned in E. coli, was screened by a filter paper assay of the colonies for the presence of penicillin acylase activity with enhanced stability at alkaline pH. Characterization of one of the selected clones revealed the presence of a mutation, Trp431-Arg, which would presumably alter the surface charge of the protein. In vitro experiments demonstrated a near twofold increase in the half-life of the mutant enzyme when stored at pH 8.5 as compared with the wild-type enzyme, with a comparable specific activity at several pH values. In general, the mutant displayed increased stability toward the basic side in the pH-stability profile. (c) 1995 John Wiley & Sons, Inc.     

Evidence for an essential arginine residue at the active site of Escherichia coli acetate kinase

Escherichia coli acetate kinase (ATP: acetate phosphotransferase, EC 2.7.2.1.) was inactivated in the presence of either 2,3-butanedione in borate buffer or phenylglyoxal in triethanolamine buffer. When incubated with 9.4 mM phenylglyoxal or 5.1 mM butanedione, the enzyme lost its activity with an apparent rate constant of inactivation of 0.079 min-1, respectively. The loss of enzymatic activity was concomitant with the loss of an arginine residue per active site. Phosphorylated substrates of acetate kinase, ATP, ADP and acetylphosphate as well as AMP markedly decreased the rate of inactivation by both phenylglyoxal and butanedione. Acetate neither provided any protection nor affected the protection rendered by the adenine nucleotides. However, it interfered with the protection afforded by acetylphosphate. These data suggest that an arginine residue is located at the active site of acetate kinase and is essential for its catalytic activity, probably as a binding site for the negatively charged phosphate group of the substrates.     

Evidence for an arginine residue at the coenzyme-binding site of Escherichia coli isocitrate dehydrogenase.

Immobilization of biological systems in solid matrices is presently of great interest, in view of the many potential advantages associated with both the higher stability of the immobilized macromolecules and the potential utilization for biotechnology. In the present paper the electrochemical behaviour of the undecapeptide from cytochrome c (called microperoxidase) tightly entrapped in cellulose triacetate membrane is reported; its utilization as 'solid-state' promoter in the electrochemistry of soluble metalloproteins is presented. The results obtained indicate that: (i) membrane-entrapped microperoxidase undergoes rapid reversible electron transfer at a glassy carbon electrode; (ii) the electrochemical process is diffusion-controlled; (iii) entrapped microperoxidase acts as 'solid-state' promoter in the electrochemistry of soluble cytochrome c and of azurin.     

Permeabilization of Escherichia coli cells for enhanced penicillin acylase activity

Summary Escherichia coli cells with penicillin acylase activity were permeabilized with aqueous solutions of the cationic detergent N-cetyl-N,N,N-trimethylammonium bromide (CTAB), at pH 8.0 and the activity was found to have almost doubled. The concentration of CTAB, the time and temperature of treatment were optimised for maximum enzyme activity and were found to be 0.2%, 20 min and 5°C respectively. Subsequently, the cell bound activity was retained for a longer period by chemical cross-linking with 0.1% glutaraldehyde.     

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。     

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.     

Factors affecting the synthesis of penicillins by the immobilized penicillin acylase from Escherichia coli

The effect of pH, temperature, reactant concentration, and reaction time has been investigated for the synthesis of N-benzhydryl-N′-acetamidopiperazyl-6-penicillanic acid and N-benzyl-N′-acetamidopiperazyl-6-penicillanic acid from 6-aminopenicillanic acid by the immobilized penicillin acylase from Escherichia coli. The synthesis of penicillins from carboxylic acids proceeds most rapidly at pH 5; with ethyl ester derivatives of carboxylic acids the pH optimum is higher (6–7). The most rapid synthesis of penicillins was obtained with ethyl ester derivatives of carboxylic acids. The optimum temperatures were 25–35°C.     

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.     

ADPglucose pyrophosphorylase: Evidence for a lysine residue at the activator site of the Escherichia coli B enzyme

Pyridoxal-P can be covalently linked to $\text{E. coli}$ B ADPglucose pyrophosphorylase by reduction with sodium borohydride. The modified enzyme is almost fully active when less than 1 mole of pyridoxal-P is incorporated per mole of enzyme subunit and is no longer dependent on the presence of allosteric activators in reaction mixtures for high activity. The allosteric activators, fructose-P₂ or hexanediol 1,6 bisphosphate, decrease the incorporation of pyridoxal-P into enzyme suggesting that the pyridoxal-P is linked at or near the allosteric activator binding site. Acid hydrolysis of the modified enzyme yields pyridoxyllysine suggesting that the epsilon amino group of lysine is functional in the binding of the allosteric activators of the enzyme.     

Studies on the isolation of penicillin acylase from Escherichia coli by aqueous two-phase partitioning

A method of enzyme release and aqueous two-phase extraction is described for the separation of penicillin acylase from Escherichia coli cells. Butyl acetate, 12% (v/v), treatment combined with freeze-thawing gives up to 70% enzyme release. For polyethylene glycol (PEG) + phosphate two-phase extraction systems the enzyme purity and yield were rather low. Modified PEG, including PEG-ampicillin, PEG-aniline, PEG-phosphate, and PEG-trimethylamine, were synthesized and used in aqueous two-phase systems; PEG-trimethylamine is the most satisfactory. A system containing 12% (w/w) PEG4000, 8% (w/w) of which is PEG-trimethylamine, with 0.7M potasium phosphate at pH 7.2, resulted in the enzyme selective partition being greatly enhanced by charge directed effects. Possible mechanisms for the separation process are discussed. (c) 1992 John Wiley & Sons, Inc.     

Arabinose-induction of lac-derived promoter systems for penicillin acylase production in Escherichia coli

Arabinose was shown to serve as an effective inducer for induction of the lac-derived promoters in Escherichia coli using penicillin acylase (PAC) as a model protein. Upon the induction with a conventional inducer, isopropyl-beta-d-thiogalactopyranoside (IPTG), for pac overexpression, which is regulated by the trc or (DE3)/T7 promoter, the production of PAC was limited by the accumulation of PAC precursors (proPAC) as inclusion bodies. Negative cellular responses, such as growth inhibition and cell lysis, were frequently observed, resulting in a low pac expression level and poor culture performance. Interestingly, these technical hurdles can be overcome simply through the use of arabinose as an inducer. The results indicate that arabinose not only induced the lac-derived promoter systems (i.e., trc and (DE3)/T7) for pac (or LL pac) overexpression but also facilitated the posttranslational processing of proPAC for maturation. However, the arabinose-inducibility appears to be host-dependent and becomes less observable in the strains with a mutation in the ara operon. The arabinose-inducibility was also investigated in the expression system with the coexistence of the trc promoter system regulating pac expression and another arabinose-inducible promoter system of araB regulating degP coexpression.     

Efficient two-step chromatographic purification of penicillin acylase from clarified Escherichia coli ultrasonic homogenate

A two-step chromatographic purification procedure from clarified Escherichia coli ultrasonic homogenate was evaluated. The capture step included immobilized metal affinity chromatography with Cu²⁺ as metal ion. Two elution methods were performed: 1 M NH₄Cl and 0.01 M imidazole. Respectively, we obtained a different purification fold (16.5 to 3.15) and a similar result for the recovery of activity (90–99%). The best elution method was chosen for the procedure. The second step, hydrophobic interaction chromatography, gave a 3.8-fold purification with 77.7% of activity. The total procedure gave a 66-fold purification in relation to the initial crude extract with 70% for the recovery of activity and was performed without any conditioning step and at the same pH value.     

Separation and purification of penicillin acylase from Escherichia coli using AOT reverse micelles

Summary The extraction of penicillin acylase by reverse micellar solutions of a surfactant was studied. A 50 mM solution of dioctyl sodium sulphosuccinate in isooctane extracted 46% of the enzyme activity in a crude periplasmic extract of induced cells of E. coli ATCC 9637. The increase in the specific activity of the final enzyme preparation, after stripping of the organic phase at pH 7.5, in the presence of 1 M KCl, was 8 - fold.Abbreviations PA penicillin acylase (penicillin amidohydrolase EC 3.5.1.11) - AOT Aerosol OT (dioctyl sodium sulphosuccinate) - NIPAB 6-nitro-3-(phenylacetamido)-benzoic acid - NABA 6-nitro-3-aminobenzoic acid - BSA bovine serum albumin - SDS sodium dodecylsulphate     

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.