Penicillin Acylase Activity Research Articles

Bile Salt Hydrolase Degrades β-Lactam Antibiotics and Confers Antibiotic Resistance on Lactobacillus paragasseri.

Bile salt hydrolase (BSH) is a well-characterized probiotic enzyme associated with bile detoxification and colonization of lactic acid bacteria in the human gastrointestinal tract. Here, we isolated a putative BSH (LpBSH) from the probiotic bacterium Lactobacillus paragasseri JCM 5343T and demonstrated its bifunctional activity that allows it to degrade not only bile salts but also the antibiotic (penicillin). Although antibiotic resistance and bile detoxification have been separately recognized as different microbial functions, our findings suggest that bifunctional BSHs simultaneously confer ecological advantages to host gut bacteria to improve their survival in the mammalian intestine by attaining a high resistance to bile salts and β-lactams. Strain JCM 5343T showed resistance to both bile salts and β-lactam antibiotics, suggesting that LpBSH may be involved in this multi-resistance of the strain. We further verified that such bifunctional enzymes were broadly distributed among the phylogeny, suggesting that the bifunctionality may be conserved in other BSHs of gut bacteria. This study revealed the physiological role and phylogenetic diversity of bifunctional enzymes degrading bile salts and β-lactams in gut bacteria. Furthermore, our findings suggest that the hitherto-overlooked penicillin-degrading activity of penicillin acylase could be a potential new target for the probiotic function of gut bacteria.

Studying the Possibilities of Using 2-Halogen-Substituted Acetamides As Acyl Donors in Penicillin Acylase-Catalyzed Reactions.

The possibility of using amides of halogen-substituted acetic acids as acyl donors in penicillin acylase-catalyzed reactions has been investigated, and the ability of this group of compounds to inactivate enzymes in the course of the catalytic conversion has been established. The strongest inactivating effect was demonstrated by iodoacetamide and bromoacetamide. However, the negative contribution of this side activity can be minimized by decreasing the temperature, when the rate of acyl donor conversion by penicillin acylases is still high enough, but the impact of enzyme inactivation becomes less significant. The catalytic activity of penicillin acylase from Alcaligenes faecalis in the conversion of 2-haloacetamides was significantly (5–8 times) higher than that of penicillin acylase from Escherichia coli.

Medium Optimization for Penicillin Acylase (PAc) Production by Recombinant B. megaterium MS941 Containing pac Gene from B. thuringiensis BGSC BD1 Using Response Surface Methodology

Penicillin G acylase (PAc) hydrolyses of the amide bond of benzylpenicillin (Pen-G) releasing PAA and 6-APA, key intermediate in the production of various semisynthetic penicillins. In this study, we optimised the production medium of PAc by RSM using two variables (xylose as inducer and CaCl 2 as divalent cations) to obtain the optimum PAc specific activity from Bacillus megaterium bt pac BD1. For this purpose, combinations of five different xylose concentrations (0.13 – 0.87 %) and five different CaCl 2 concentrations (0.64 – 4.36 mM) were analysed, in a total of 22 experiments. CCD used for the analysis showed that in shake flask cultivations, xylose and CaCl 2 showed significant effects on PAc volumetric activity and the quadratic model was in good agreement with the experimental results (R 2 = 0.86 (p-value < 0.0001)). The maximum specific activity (130.669 ± 50.241 units mg protein -1 ) was reached when xylose and CaCl 2 concentrations were 0.49% and 2.4 mM, respectively, and medium pH was around 7. Under such conditions, the activity of PAc and protein concentration achieved were 1.318 ± 0.406 units mL -1 and 0.0101 ± 0.01 mg mL -1 . The shake flask validation experiments demonstrated that with such medium composition the volumetric activity, protein concentration and specific activity achieved were 1.294 ± 0.171 units mL -1 , 0.0102 ± 0.0003 mg mL -1 and 125.91 ± 13.309 units mg -1 , respectively. When the optimum medium composition was applied in 10 L bioreactor, the optimum volumetric activity (2.0687 ± 0.0820 units mL -1 ) and protein concentration (0.0078 ± 0.0008 mg mL -1 ) were achieved 48 h after the start of the cultivation. However, the optimum PAc specific acivity (1260.52 ± 27.5711 units mg protein -1 ) was achieved 18 h after the start of the cultivation.

Purification and characterization of YxeI, a penicillin acylase from Bacillus subtilis

The paper reports the purification and characterization of the first penicillin acylase from Bacillus subtilis. YxeI, the protein annotated as hypothetical, coded by the gene yxeI in the open reading frame between iol and hut operons in B. subtilis was cloned and expressed in Eshcherichia coli, purified and characterized. The purified protein showed measurable penicillin acylase activity with penicillin V. The enzyme was a homotetramer of 148kDa. The apparent Km of the enzyme for penicillin V and the synthetic substrate 2-nitro-5-(phenoxyacetamido)-benzoic acid was 40mM and 0.63mM, respectively, and the association constants were 8.93×102M−1 and 2.51×105M−1, respectively. It was inhibited by cephalosporins and conjugated bile salts, substrates of the closely related bile acid hydrolases. It had good sequence homology with other penicillin V acylases and conjugated bile acid hydrolases, members of the Ntn hydrolase family. The N-terminal nucleophile was a cysteine which is revealed by a simple removal of N-formyl-methionine. The activity of the protein was affected by high temperature, acidic pH and the presence of the denaturant guanidine hydrochloride.

Post-immobilization of modified macromolecular reagents using assembled penicillin acylase for microenvironmental regulation of nanopores and enhancement of enzyme stability

Penicillin acylase (PA) is known to regulate the microenvironment of nanospores. In this study, nanopores containing chemically-modified macromolecules co-assembled with immobilized PA were constructed. We also investigated the various types of functionalized mesocellular siliceous foams (MCFs) commonly used for the immobilization of PA by measuring the catalytic performance and stability of each PA preparation. Amino-MCF activated by p-benzoquinone was chosen as the optimum support for PA immobilization. Successful modification of macromolecules was verified by FT-IR and ultraviolet (UV) spectroscopy. The specific activity of PA co-assembled with dextran 10 k was 99.1 U/mg, which was 1.5-fold that of pristine immobilized PA, while the optimum pH was shifted to neutral. Compared to pristine immobilized and free PA, the optimum temperatures for the modified PA were 5 and 10°C higher, respectively. The residual activity of the ficoll derivative of PA after treatment at 50°C for 6 h was 70%, and this was later increased to 214.5% compared to that of pristine immobilized PA. The dextran 10 k derivative of PA exhibited 90.2% residual activity after 25 times of continuous use. The results show that chemically-modified macromolecules co-assembled with PA in amino-MCF provided a suitable microenvironment for enzyme stability.

Fluorimetric determination of ampicillin by use of non-toxic catalysts. Estimation of beta-lactamase activity and parameters.

A fluorescence assay based on the use of biological reducing agents as catalysts rather than heavy metal ions has been developed for estimation of ampicillin concentrations. The assay is based on the conversion of ampicillin to its penicilloate, by treatment with sodium hydroxide, then neutralization, dilution with 0.5 M acetate buffer at pH4 and heating for 30 min at 100 degrees C in the presence of ascorbic acid (0.5 mg) and EDTA (50 microM). Reduced glutathione, cysteine and N-acetylcysteine also catalysed the development of fluorescence. A practical sensitivity range of 0.5-50 microM ampicillin was used. The assay was used to estimate ampicillin in some biological solutions to which the antibiotic has been added. Milk, blood serum, trypticase soy broth and nutrient broth containing 25 microM antibiotic assayed at 18.5, 21.7, 16.5 and 14.7 microM, respectively, with standard deviations between 1.2 and 0.7%. The low results were attributed to binding of some ampicillin by proteins or peptides which were removed by pretreatment. Urine containing 5 mM ampicillin assayed at 4.97 mM with a standard deviation of 3%. A modification of the procedure was used to measure beta-lactamase activity against ampicillin in several organisms in a fixed time assay. Kinetic parameters of a commercial beta-lactamase preparation from Bacillus cereus could also be determined by an additional modification. In both instances a correction was required for the intrinsic fluorescence of ampicillin remaining in the solution. The preparation examined had a Michaelis constant (Km) of 0.32 mM, a maximum velocity (Vmax) of 5398 micromol ampicillin hydrolysed mg(-1) min(-1), an apparent catalytic constant (Kcat, turnover number) of 20,220 s(-1) and a Kcat/Km ratio of 0.63 x 10(7) M(-1) s(-1). The major advantage of using this assay technique is that toxic metals are not used in the development of fluorescence so it is more environmentally acceptable. The technique is useful for examining beta-lactamase activity against ampicillin and might be useful for pharmaceutical products-for determining available therapeutic levels and for monitoring the activity of penicillin acylase against the penicilloate of ampicillin.

Improvement of the catalytic performance of immobilized penicillin acylase through assembly of macromolecular reagents in nanopore to create a crowding environment

Macromolecular reagents were co-assembled with penicillin acylase (PA) and immobilized in mesocellular siliceous foams (MCFs) to resemble living cells. Types and concentrations of macromolecules were studied. The catalytic characteristic and stability of PA preparations were also investigated. PA assembled with dextran 10 k in MCFs showed maximum specific activity, 1.32-fold of that of the solely immobilized PA. The optimum pH of dextran and BSA derivatives shifted to neutrality, and the optimum temperature increased by 10 °C. Also, Km of BSA derivative of PA declined 56.7% compared to solely immobilized PA, while the Kcat/Km of PA assembled with BSA was enhanced to 147%. After incubation at 50 °C for 6 h, residual activity of PA assembled with BSA exhibited 53.0%. The ficoll derivative showed 82.8% of its initial activity at 4 °C after 8-week storage. The results indicated that macromolecular reagents assembled with PA in MCFs could dramatically improve the catalytic performance and stability of immobilized enzyme.

Penicillin acylase immobilization depending on macromolecular crowding and catalysis in aqueous–organic medium

To enhance penicillin acylase (PA) performance, it was immobilized in mesocellular silica foams (MCFs) depended on macromolecular crowding and applied to catalysis in non-aqueous medium. Ficoll 70, dextran 10,000, dextran 40,000 and bovine serum albumin were co-assembled with PA. It was observed that specific activity of PA assembled in MCFs with dextran 10,000 in 80% cyclohexane (v/v) was 233.2 U/mg, 200% as that of PA assembled in MCFs in 80% cyclohexane and 323.5% as that of free PA in full aqueous medium. As content of alkane increased, activity of PA in MCFs with macromolecules varied slightly. In addition, PA co-immobilized with dextran 10 in MCFs retained 58.2% of its initial activity after heating at 50 degrees C for 4 h, 1.2 times higher than that of PA immobilized alone in MCFs. The results showed that macromolecular crowding was favorable for immobilization of PA and its catalysis in suitable aqueous-organic medium.

Conjugation of penicillin acylase with the reactive copolymer of N-isopropylacrylamide: a step toward a thermosensitive industrial biocatalyst.

Conjugation of penicillin acylase (PA) to poly-N-isopropylacrylamide (polyNIPAM) was studied as a way to prepare a thermosensitive biocatalyst for industrial applications to antibiotic synthesis. Condensation of PA with the copolymer of NIPAM containing active ester groups resulted in higher coupling yields of the enzyme (37%) compared to its chemical modification and copolymerization with the monomer (9% coupling yield) at the same NIPAM:enzyme weight ratio of ca. 35. A 10-fold increase of the enzyme loading on the copolymer resulted in 24% coupling yield and increased by 4-fold the specific PA activity of the conjugate. Two molecular forms of the conjugate were found by gel filtration on Sepharose CL 4B: the lower molecular weight fraction of ca. 10(6) and, presumably, cross-linked protein-polymer aggregates of MW > 10(7). Michaelis constant for 5-nitro-3-phenylacetamidobenzoic acid hydrolysis by the PA conjugate (20 microM) was found to be slightly higher than that of the free enzyme (12 microM), and evaluation of V(max) testifies to the high catalytic efficiency of the conjugated enzyme. PolyNIPAM-cross-linked PA retained its capacity to synthesize cephalexin from d-phenylglycin amide and 7-aminodeacetoxycephalosporanic acid. The synthesis-hydrolysis ratios of free and polyNIPAM-cross-linked enzyme in cephalexin synthesis were 7.46 and 7.49, respectively. Thus, diffusional limitation, which is a problem in the industrial production of beta-lactam antibiotics, can be successfully eliminated by cross-linking penicillin acylase to a smart polymer (i.e., polyNIPAM).

Newly Discovered Penicillin Acylase Activity of Aculeacin A Acylase from Actinoplanes utahensis

Aculeacin A acylase from Actinoplanes utahensis produced by Streptomyces lividans revealed acylase activities that are able to hydrolyze penicillin V and several natural aliphatic penicillins. Penicillin K was the best substrate, showing a catalytic efficiency of 34.79 mM(-1) s(-1). Furthermore, aculeacin A acylase was highly thermostable, with a midpoint transition temperature of 81.5 degrees C.

Production of penicillin acylase by a recombinant Escherichia coli using cheese whey as substrate and inducer

Cheese whey (CW) is the major subproduct from cheese manufacturing and it is considered as a waste pollutant since its high content of lactose. In this work a fermentation process for the production of penicillin acylase (PA) by a recombinant Escherichia coli and using CW as unique carbon source and inducer was developed. A design factorial 3 2 was used to evaluate the influence of independent variables (dissolved oxygen and CW concentration) on the ability of E. coli W3110/pPA102 to produce PA. Maximum specific PA activity of 781 U g −1 was attained at 5 g L −1 of CW and 3% dissolved oxygen. The results showed that CW can be used successfully as unique carbon source and inducer for the production of recombinant proteins using constructions driven by the lac promoter and this way reducing the discharges of that pollutant to the environment.

Crosslinked Penicillin Acylase Aggregates for Synthesis of β-Lactam Antibiotics in Organic Medium

Crosslinked enzyme aggregates (CLEAs) of a partially purified penicillin acylase from a recombinant Escherichia coli strain have been produced as a novel type of biocatalysts well endowed to perform in organic media. Different protein precipitants were studied and glutaraldehyde was used as the crosslinking agent. Precipitation curves were obtained for all precipitants to determine the concentrations at which all the protein precipitated out of the solution. The effect of the glutaraldehyde-to-protein ratio was studied with respect to process recovery and the specific activity and stability of the biocatalyst. Recovery of penicillin acylase activity was moderately high, about 50%; major losses of enzyme activity were produced at the precipitation step. Specific activities of all CLEAs were very high, which is one of the advantages of using nonsupported biocatalysts. Ammonium sulfate and tert-butyl alcohol were the best precipitants at a glutaraldehyde-protein mass ratio of 2 and were selected to perform the kinetically controlled synthesis of ampicillin in 60% (v/v) ethylene glycol medium. At comparable conversion yields, volumetric and specific antibiotic productivity were much higher for CLEAs than for carrier-bound penicillin acylases.

Novel reactive perstraction system applied to the hydrolysis of penicillin G

The activity of penicillin acylase has been studied in aqueous and organic solvents, as free enzyme as well as immobilized within the membrane of liquid-core capsules. The activity of the enzyme is inhibited by the accumulation of the products of the hydrolysis reaction, namely phenyl acetic acid (PAA). In order to overcome this inhibition a range of organic solvents were tested for use in in situ product recovery. Of these solvents dibutyl sebacate (DBS) was chosen due to the rapid extraction rate, the high logP and to facilitate capsule production. The extraction efficiency at pH 3.5 for PAA was >80% for phase ratios of >50% free solvent with partition coefficients of 8 and 0.7 for PAA and penicillin G (PenG), respectively, thereby showing that PAA could be selectively extracted at pH 3.5 and 25 degrees C. Liquid-core capsules containing DBS were shown to efficiently remove PAA selectively and the PAA could be effectively back-extracted and the capsules re-used in a three-stage process resulting in high product separation. Immobilization of penicillin acylase onto the capsule membranes resulted in increased operational stability of the enzyme and a very high enzyme activity. Over 53.3% of the PAA formed could be recovered in the capsule core with a concentration over sevenfold higher than in the aqueous phase. Higher extraction efficiencies could be obtained by varying the substrate concentration and number of capsules. The enzyme immobilized on capsules could be stored for over 4 months at pH 8 and 4 degrees C with no loss of activity. Over 80% of the initial activity could be recovered over five repeated batch cycles of the bioconversion process. The importance of capsular perstraction and reactive capsular perstraction has been clearly demonstrated.

Effects of dissolved oxygen tension on the production of recombinant penicillin acylase in Escherichia coli

The effect of dissolved oxygen tension (DOT) on over-expression of penicillin acylase (PA) by a recombinant Escherichia coli was studied. Relevant kinetic and stoichiometric parameters were evaluated in batch cultures performed in 1-l bioreactors maintained at constant DOT values in the range of 0–40%. Specific growth rate, biomass concentration, and biomass yield from glucose increased in a Monod-type fashion, whereas acetate and formate concentration decreased as DOT increased. As DOT decreased, specific PA activity at glucose depletion increased from 0.09 to 0.16 U mg −1 dry cell weight (DCW), whereas volumetric PA activity decreased in a Monod-type behavior from 0.25 to 0 U ml −1. The PA activity increased 1.5–3.3-fold when prolonging the culture for 10–25 h after growth cessation. Maximum PA activity was obtained at 1% DOT. Kinetics of PA activity followed a combined growth-associated and non-growth-associated behavior. It is proposed that the Luedeking–Piret coefficients can be expressed as a function of DOT. The growth-associated production coefficient increased with DOT following a Monod-type behavior, whereas the non-growth-associated production coefficient showed a maximum between 1 and 5% of DOT. Moreover, the non-growth-associated coefficient was importantly affected by DOT. Novel control and optimization strategies for DOT can be proposed based in the determined Luedeking–Piret coefficients.

Penicillin Acylase Catalysed Synthesis of Ampicillin in Hydrophilic Organic Solvents

AbstractPenicillin acylase (EC 3.5.1.11) from Alcaligenes faecalis, immobilised as a cross‐linked enzyme aggregate (CLEA), catalysed the synthesis of ampicillin in water‐miscible organic solvents at low water concentrations. Below 4% water (v/v) no reaction was observed, showing the crucial role of water in maintaining the activity of penicillin acylase. The initial value of S/H was strongly affected by the nature of the solvent, but the effect of the water content was slight in the studied range of 4 to 15%. A reaction in acetonitrile containing 8% water afforded ampicillin in up to 86% yield.

Roles of DegP in prevention of protein misfolding in the periplasm upon overexpression of penicillin acylase in Escherichia coli.

Enhancement of the production of soluble recombinant penicillin acylase in Escherichia coli via coexpression of a periplasmic protease/chaperone, DegP, was demonstrated. Coexpression of DegP resulted in a shift of in vivo penicillin acylase (PAC) synthesis flux from the nonproductive pathway to the productive one when pac was overexpressed. The number of inclusion bodies, which consist primarily of protein aggregates of PAC precursors in the periplasm, was highly reduced, and the specific PAC activity was highly increased. DegP was a heat shock protein induced in response to pac overexpression, suggesting that the protein could possibly suppress the physiological toxicity caused by pac overexpression. Coexpression of DegP(S210A), a DegP mutant without protease activity but retaining chaperone activity, could not suppress the physiological toxicity, suggesting that DegP protease activity was primarily responsible for the suppression, possibly by degradation of abnormal proteins when pac was overexpressed. However, a shortage of periplasmic protease activity was not the only reason for the deterioration in culture performance upon pac overexpression because coexpression of a DegP-homologous periplasmic protease, DegQ or DegS, could not suppress the physiological toxicity. The chaperone activity of DegP is proposed to be another possible factor contributing to the suppression.

Unexpected enantioselectivity and activity of penicillin acylase in the resolution of methyl 2,2-dimethyl-1,3-dioxane-4-carboxylate

The penicillin acylase (PA) from E. coli catalyzes the hydrolytic kinetic resolution of methyl 2,2-dimethyl-1,3-dioxane-4-carboxylate with remarkably high enantioselectivity and catalytic efficiency. This result is highly unusual as this ester does not contain the phenylacetic acid residue, normally considered to be a prerequisite for high activity and enantioselectivity in PA catalyzed resolutions. The apparent enantioselectivity ( E app) was found to be high (>50) at neutral or slightly acidic pH and to decrease at more alkaline pH (>7.5) due to significant non-specific chemical hydrolysis. Similarly, enantioselectivity increased with decreasing temperature. The substrate concentration had only a slight effect on enantioselectivity and activity. The rate of hydrolysis of ester 1 is comparable to that for PA’s “natural” substrate, penicillin G.

The role of hydrophobic active‐site residues in substrate specificity and acyl transfer activity of penicillin acylase

Penicillin acylase of Escherichia coli catalyses the hydrolysis and synthesis of beta-lactam antibiotics. To study the role of hydrophobic residues in these reactions, we have mutated three active-site phenylalanines. Mutation of alphaF146, betaF24 and betaF57 to Tyr, Trp, Ala or Leu yielded mutants that were still capable of hydrolysing the chromogenic substrate 2-nitro-5-[(phenylacetyl)amino]-benzoic acid. Mutations on positions alphaF146 and betaF24 influenced both the hydrolytic and acyl transfer activity. This caused changes in the transferase/hydrolase ratios, ranging from a 40-fold decrease for alphaF146Y and alphaF146W to a threefold increase for alphaF146L and betaF24A, using 6-aminopenicillanic acid as the nucleophile. Further analysis of the betaF24A mutant showed that it had specificity constants (kcat/Km) for p-hydroxyphenylglycine methyl ester and phenylglycine methyl ester that were similar to the wild-type values, whereas the specificity constants for p-hydroxyphenylglycine amide and phenylglycine amide had decreased 10-fold, due to a decreased kcat value. A low amidase activity was also observed for the semisynthetic penicillins amoxicillin and ampicillin and the cephalosporins cefadroxil and cephalexin, for which the kcat values were fivefold to 10-fold lower than the wild-type values. The reduced specificity for the product and the high initial transferase/hydrolase ratio of betaF24A resulted in high yields in acyl transfer reactions.

Penicillin acylase release from Escherichia coli cells by mechanical cell disruption and permeabilization

AbstractThe release of Penicillin acylase from Escherichia coli cells through mechanical cell disruption using high‐pressure homogenization and sonication was studied. From these cell disruption processes, the enzyme activity was totally released although with low specific activities, 0.1–0.3 IU(mg prot)−1. Intracellular total soluble protein release was quantified and modelled by a first order kinetic model. The effect of the driving force for each mechanical method, namely acoustic power input and homogenization pressure, on the respective kinetic disruption constants was also analysed. The release of Penicillin acylase by cell permeabilization using osmotic shock was also evaluated. The effects of cell concentration, penicillin acylase activity in E coli cells, type of buffer, pH, hypertonic solution composition, temperature and time used for osmotic shock were evaluated. Using cold osmotic shock, highly selective penicillin acylase release was attained with specific enzyme activities of about 4 IU(mg prot)−1 and enzyme activity release yields higher than 90%. The high purity of the penicillin acylase was a consequence of the optimized differential enzyme release method which was validated by SDS gel electrophoresis.© 2002 Society of Chemical Industry

An integrated downstream processing strategy for the recovery and partial purification of penicillin acylase from crude media

An integrated process strategy for the recovery of penicillin acylase was developed, based on precipitation of non-enzymatic proteins directly from Escherichia coli homogenates or crude extracts using Rolquat (quaternary ammonium salt) and adsorption of the enzyme on Amp-Seph (3.8 µmole ampicillin cm−3) under pseudo-affinity conditions. The effect of pH, concentrations of ammonium sulfate and Rolquat, and also concentrations of protein and cell debris on the precipitation of non-enzymatic proteins from homogenates and crude extracts of penicillin acylase were analysed. The method of addition of Rolquat to homogenates and crude extracts significantly influenced the size of the precipitated particles. Improved results on the specific activity of penicillin acylase were obtained for 22% and 1% (w/v) of ammonium sulfate and Rolquat, respectively, added sequentially to enzyme solutions and at room temperature. Under these experimental conditions, the specific activity of penicillin acylase in homogenates and crude extracts was enhanced 2.5–3.0-fold. Finally, the integrated process strategy was implemented first by precipitation of non-enzymatic proteins and recovery of penicillin acylase directly from the enzyme solution treated with Rolquat using an adsorption/filtration system with an overall yield of 86%. This system allows simultaneously the filtration of cell debris and fine precipitated particles, in situ recovery of penicillin acylase by its adsorption on Amp-Seph, and selective desorption of the enzyme with a specific activity of 11 IU (mg prot)−1 and a desorption yield of 95%. © 2002 Society of Chemical Industry