Penicillin G Acylase Production Research Articles

Penicillin G acylase-responsive near-infrared fluorescent probe: Unravelling biofilm regulation and combating bacterial infections

Antibiotic resistance poses a critical threat to human healthcare, largely driven by bacterial biofilms. These biofilms resist the immune system and antibiotics, rendering enclosed microbial cells 10–1000 times more antibiotic-resistant than planktonic cells, leading to severe infections. Therefore, there is an urgent need to develop innovative tools for investigating biofilm regulators and devising novel antibacterial strategies. In this study, we developed Cy-NEO-PA, a near-infrared (NIR) fluorescent probe responsive to penicillin G acylase (PGA), with bacteria-targeting ability. This probe was designed to visualize the influence of environmental factors on biofilm formation in Acinetobacter baumannii (A. baumannii). Our findings demonstrated that glucose suppressed PGA production, leading to enhanced biofilm formation, whereas phenylacetic acid (PAA) stimulated PGA production and inhibited biofilm formation in A. baumannii. These observations highlight the remarkable capability of Cy-NEO-PA to accurately measure PGA dynamics, shedding light on the critical role of PGA in biofilm development. Additionally, Cy-NEO-PA exhibited excellent biocompatibility, potent reactive oxygen species (ROS) generation, efficient photothermal conversion, and bacteria-targeting abilities, making it a promising agent for combating bacterial infections and promoting wound healing through photothermal (PTT)/photodynamic (PDT) therapy. These discoveries emphasize the significant role of PGA in antibacterial therapy and offer valuable insights for the design of effective strategies targeting PGA to combat biofilm-associated infections.

Expression of Synthetic pac Gene Encoding Penicillin G Acylase (PGA) Enzyme in E. coli BL21(DE3) and HB101

High bacterial infection cases in Indonesia cause a high need for antibiotic drugs. Unfortunately, most of the raw materials used for antibiotic production in Indonesia are still imported. For this reason, the government is eager to find better ways to produce penicillin and its derivatives, which are widely used in society. The production of penicillin-derivative requires penicillin G acylase (PGA) as a catalyst. In previous studies, the expression of the syn-pac gene in E. coli BL21(DE3) to produce a recombinant PGA enzyme was performed, but the enzyme activity was low (0.01754 U/mg). Thus, the expression was carried out in different hosts and inducers. The purpose of this research was to obtain the production of PGA with higher enzyme activity. The transformation was carried out in the pET22b-pacEc in E. coli BL21(DE3) and HB101. For enzyme expression, the recombinant hosts were induced by 0.05 mM IPTG, 176 mM lactose, and 1998 mM arabinose at a temperature of 20°C and 150 rpm of shaking for 17 h. Protein isolation was performed by sonication and freeze-thawing to recover biologically active PGA. Verification of PGA was performed by SDS-PAGE and the enzyme activity was tested by pDAB. E. coli HB101 produced PGA with higher activity (10.17 U/mg) than BL21(DE3) (6.67 U/mg), and arabinose was the strongest inducer for enzyme expression.

Optimization of a medium composition for the heterologous production of Alcaligenes faecalis penicillin G acylase in Bacillus megaterium

Penicillin G acylase (PGA) is a strategic enzyme in the production processes of beta-lactam antibiotics. High demand for β-lactam semisynthetic antibiotics explain the genetic and biochemical engineering strategies devoted towards novel ways for PGA production and application. This work presents a fermentation process for the heterologous production of PGA from Alcaligenes faecalis in Bacillus megaterium with optimization. The thermal stability from A. faecalis PGA is considerably higher than other described PGA and the recombinant enzyme is secreted to the culture medium by B. megaterium, which facilitates the separation and purification steps. Media optimization using fractional factorial design experiments was used to identify factors related to PGA activity detection in supernatant and cell lysates. The optimized medium resulted in almost 6-fold increased activity in the supernatant samples when compared with the basal medium. Maximum enzyme activity in optimized medium composition achieves values between 135 and 140 IU/ml. The results suggest a promising model for recombinant production of PGA in B. megaterium with possible extracellular expression of the active enzyme.

Investigations to Enhance Production of Penicillin G Acylase from Recombinant Bacillus badius pac Expressed in Escherichia coli DH5α

Optimization of the M9 medium for production of Penicillin G Acylase (PGA) from recombinant E. coli DH5α carrying a plasmid encoding pac from Bacillus badius was carried out based on response surface methodology (RSM). For the first time M9 medium was used for enhancing the production of PGA from recombinant B. badius pac expressed in E. coli DH5α. RSM is a useful method for studying the effect of several variables influencing the responses by varying them simultaneously and carrying out a limited number of experiments. The initial screening method of Plackett-Burman design gave rise to identification of fructose, yeast extract, and magnesium sulfate as significant medium components. A 23 full-factorial central composite design (CCD) was applied to further optimize concentration of each significant variable. The optimal concentration for production of PGA consisted of 10 g/l fructose, 6 g/l yeast extract, 1.85 ml/l MgSO4 · 7H2O (100 mM), 9 g/l disodium hydrogen phosphate, 4 g/l potassium dihydrogen phosphate, 0.75 g/l sodium chloride, 2 ml/l thiamine HCl (100 mM), and 2 ml/l calcium chloride (10 mM). The model prediction of PGA activity (14.11 U/ml) at optimum conditions was verified experimentally (13.94 U/ml). Through statistically designed optimization, the production of PGA was found to go up from 2.2 U/ml to an average of 13.94 ± 0.03 U/ml. Thus the modified M9 media has shown a 7.74-fold increase in PGA activity.

Biological Real-Time Reaction Calorimeter Studies for the Production of Penicillin G Acylase from Bacillus badius

Penicillin G acylase (PGA) is a commercially important enzyme that cleaves penicillin G to 6-amino penicillanic acid (6-APA) and phenyl acetic acid (PAA). The strain Bacillus badius has been identified as potential producer of PGA. A detailed calorimetric investigation on PGA production was carried out to enable generation of thermokinetic data possible for commercial application. Reaction calorimetric studies coupled with respirometric studies suggested that enzyme activity of the species B. badius was calorimetrically traceable. Three phases of growth were distinctly noticeable in the metabolic heat-time curve. Increase in enzymatic activity with restricted growth confirmed intracellular nature of the production process. The estimated heat yields due to biomass growth, 10.026 kJ/g, substrate consumption 22.761 kJ/g, and oxygen uptake 383 ± 10 kJ/mol helped to understand the energetic of the organism under study. Low oxycalorific coefficient confirmed the existence of fermentation-coupled metabolism of B. badius.

Strategies for Enhancing the Production of Penicillin G Acylase from Bacillus badius: Influence of Phenyl Acetic Acid Dosage

Bacillus badius isolated from soil has been identified as potential producer of penicillin G acylase (PGA). In the present study, batch experiments performed at optimized inoculum size, temperature, pH, and agitation yielded a maximum PGA of 9.5U/ml in shake flask. The experiments conducted in bioreactor with different oxygen flow rates revealed that 0.66vvm oxygen flow rate could be sufficient for the maximum PGA activity of 12.7U/ml. From a detailed investigation on the strategies of the addition of phenyl acetic acid (PAA) for increasing the production of PGA, it was found that the controlled addition of 10ml of 0.1% (w/v) PAA once in every 2h from 6th hour of growth showed the maximum PGA activity of 32U/ml. Thus, our studies for the first time showed that at concentration above 0.1% (w/v) PAA, the PGA production decreased. This selective condition paves the way for less costly bioprocess for the production of PGA.

A computational model for enhancing recombinant Penicillin G Acylase production from Escherichia coli DH5α

An attempt was made to develop a computational model based on artificial neural network and ant colony optimization to estimate the composition of medium components for maximizing the productivity of Penicillin G Acylase (PGA) enzyme from Escherichia coli DH5α strain harboring the plasmid pPROPAC. As a first step, an artificial neural network (ANN) model was developed to predict the PGA activity by considering the concentrations of seven important components of the medium. Design of experiments employing central composite design technique was used to obtain the training samples. In the second step, ant colony optimization technique for continuous domain was employed to maximize the PGA activity by finding the optimal inputs for the developed ANN model. Further, the effect of a combination of ant colony optimization for continuous domain with a preferential local search strategy was studied to analyze the performance. For a comparative study, the training samples were fed into the response surface methodology optimization software to maximize the PGA production. The obtained PGA activity (56.94U/mL) by the proposed approach was found to be higher than that of the obtained value (45.60U/mL) with the response surface methodology. The optimum solution obtained computationally was experimentally verified. The observed PGA activity (55.60U/mL) exhibited a close agreement with the model predictions.

Mutations in the translation initiation region of the pac gene resulting in increased levels of activity of penicillin G acylase

Penicillin G acylase (PA) is an important enzyme used in the industrial production of b-lactam antibiotics. In this study, the effects of mutations in the translation initiation region of the Escherichia coli pac gene, encoding periplasmic PA, were examined. Several mutations led to increased amounts of PA activity, including those that lengthened the spacer region between the ribosome binding site and the ATG start codon, and those with altered codons on positions +2 and +4 relative to the start codon. These results indicated that the wild-type sequence of the pac gene does not provide maximum expression levels and that the strategies applied in this study can be used to improve production of PA in E. coli. Unexpectedly, our study also suggested that translocation of PA was, in contrast to earlier reports, shown not to require the Twin-arginine translocation pathway for transport into the periplasm.

Heterologous production of Escherichia coli penicillin G acylase in Pseudomonas aeruginosa

Penicillin G acylase (PGA) is a widely studied bacterial enzyme of great industrial importance. Since its overproduction in the original organisms is mostly limited to the intracellular bacterial spaces which may lead to aggregation and cell toxicity, we have set out to explore the host organism Pseudomonas aeruginosa that possesses the Xcp machinery for secretion of folded proteins to the extracellular medium. We have made fusion proteins, consisting of Pseudomonas Sec- or Tat-specific signal peptides, the elastase propeptide and the mature penicillin G acylase. With all constructs we obtained production of PGA in P. aeruginosa, but we observed that processing of the PGA was temperature dependent and that the active enzyme could only be found after growth at 25 °C or lower temperatures. Remarkably, the mature protein, expressed from a TatProPGA hybrid, was not only found in the extracellular medium and the periplasm, but also in the cytoplasm as assessed by comparison to the reporter beta-lactamase protein. The unusual cytoplasmic localization of the mature protein strongly suggests that processing of PGA can also occur in the cytoplasm of P. aeruginosa. The extracellular localization of the TatProPGA hybrid was found not to be dependent on the tatABC-genes. The elastase signal sequence/propeptide combination appeared to be an inadequate carrier for transporting penicillin G acylase across the outer membrane of P. aeruginosa.

Development and Application of a Novel Signal Peptide Probe Vector with PGA as Reporter in Bacillus subtilis WB700: Twenty-Four Tat Pathway Signal Peptides from Bacillus subtilis were Monitored

In this study, we have developed a novel, versatile signal peptide probe vector driven by promoter P43 in Bacillus subtilis WB700, using Penicillin G Acylase (PGA) as reporter. Twenty-four signal peptides considered belonging to twin-arginine translocation (Tat) pathway were cloned into the probe vector to direct the secretion expression of PGA, respectively. Through 6-nitro-3-phenylacetamidobenzoic acid (NIPAB) filter paper assay, four signal peptides (AmyX, AlbB, LipA, and YmzC) were chosen for further investigation. The extracellular production of PGA demonstrated that these recombinants mediated efficient secretion expression in B. subtilis WB700, in which the maximum activity reached 0.11, 0.21, 0.08, and 0.26 U/mL, respectively. Thus, we provided an efficient tool for easy detection of the signal peptides in B. subtilis, and demonstrated the efficiency of Tat pathway signal peptides via PGA secretion in B. subtilis WB700.

6-Aminopenicillanic Acid Production by Intact Cells of E. coli Containing Penicillin G Acylase (PGA)

The aim of present study was to optimize conditions for conversion of penicillin G into 6-APA using intact crude cells of locally collected PGA producing bacterial strains as biocatalyst. Corn steep liquor medium supplemented with phenylacetic acid was used for PGA production. For enzymatic conversion of penicillin G into 6-APA by PGA impregnated bacterial cells, a maximum reaction time of 4 h was found adequate. The procedure for extraction and crystallization of 6-APA from the enzyme reaction mixture was standardized. Isolation process was carried out under controlled pH conditions and 6-APA crystals were recovered from the reaction mixture via filtration, concentration and drying. The maximum PGA activity was observed in Escherichia coli strain BDCS-N-FMu12 (6.4 mg 6-APA h(-1) mg(-1) wet cells) whereas Bacillus megaterium (ATCC 14945 used as check) exhibited only 2.4 mg 6-APA h(-1) mg(-1) wet cells. The overall yield of 6-APA crystals obtained after enzymatic conversion of penicillin G ranged between 37-55 and 47-68% in foreign and local strains, respectively. BDCS-N-FMu12 was identified as the best PGA producer with 68% 6-APA conversion whereas ATCC 14945 showed the lowest conversion (37%). The recovery of 6-APA (68%) obtained by using crude intact cells as cheap biocatalyst appeared promising. The process of enzyme fermentation and 6-APA crystallization optimized during this study seems cost-effective and environment-friendly. However, further studies are required to scale up the 6-APA biosynthesis reaction for achieving 80-90% conversion of penicillin G into 6-APA by PGA hyper-producing locally collected strains of E. coli.

Comparing the performance of multilayer perceptrons networks and neuro-fuzzy systems for on-line inference of Bacillus megaterium cellular concentrations

Penicillin G acylase (PGA) is one of the most important enzymes for the pharmaceutical industry. Bacillus megaterium has the advantage of producing extra-cellular PGA. This work compares two neural networks (NNs) architectures for on-line inference of B. megaterium cell mass in an aerated stirred tank bioreactor, during the production of PGA. Nowadays, intelligent computing tools such as artificial NNs and fuzzy logic are commonly applied for state inference and modeling of bioreactors. Combining these two approaches in hybrid, neuro-fuzzy systems, may be advantageous. Our results indicate that a neuro-fuzzy inference system showed a better performance to infer cell concentrations, when compared to multilayer perceptrons networks.

The penicillin G acylase production by B. megaterium is amino acid consumption dependent

Aiming at to enhance the production of penicillin G acylase (PGA) by Bacillus megaterium, we have performed flasks experiments using different medium composition. Using 51 g/L of casein hydrolyzed with Alcalase and 2.7 g/L of phenylacetic acid (PhAc), the following carbon substrates were tested, individually and combined: glucose, glycerol, and lactose (present in cheese whey). Glycerol and glucose showed to be effective nutrients for the microorganism growth but delayed the PGA production. Cheese whey always increased enzyme production and cell mass. However, lactose (present in cheese whey) was not a significant carbon source for B. megaterium. PhAc, amino acids, and small peptides present in the hydrolyzed casein were the actual carbon sources for enzyme production. Replacement of hydrolyzed casein by free amino acids, 10.0 g/L, led to a significant increase in enzyme production (app. 150%), with a preferential consumption of alanine, aspartic acid, glycine, serine, arginine, threonine, lysine, and glutamic acid. A decrease of the enzyme production was observed when 20.0 g/L of amino acids were used. Using the single omission technique, it was shown that none of the 18 tested amino acids was essential for enzyme production. The use of a medium containing eight of the preferentially consumed amino acids lead to similar enzyme production level obtained when using 18 amino acids. PhAc, up to 2.7 g/L, did not inhibit enzyme production, even if added at the beginning of the cultivation.

Production of Alcaligenes faecalis penicillin G acylase in Bacillus subtilis WB600 (pMA5) fed with partially hydrolyzed starch

In this study, a recombinant strain Bacillus subtilis WB600 (pMA5) was used to produce Alcaligenes faecalis penicillin G acylase (PGA). The PGA activity and overall PGA productivity were respectively 378 and 7.13 U/l h in a batch culture using a complex medium containing 25 g/l of soluble starch. Fed-batch cultures indicated that glucose had a negative effect on the production of PGA. Starch is a good carbon source, but it could not be directly used as a feed due to high viscosity after heat sterilization. A feed was prepared by limited hydrolysis of starch with a small amount of α-amylase, which greatly improved the fluidity with little formation of glucose. The production of PGA increased to 546 U/l by feeding of the partially hydrolyzed starch. When a mixture containing the partially hydrolyzed starch and Tryptone was added by using a pH-stat strategy, the PGA activity reached 1960 U/l, over five-fold of that obtained in the batch culture, and the overall productivity reached 19.6 U/l h, which was more than two-fold of that in the batch culture.

Mutagenic effect of acridine orange on the expression of penicillin G acylase and beta-lactamase in Escherichia coli

The present work aimed to improve the production of penicillin G acylase (PGA) and reduce the beta-lactamase activity through acridine orange (AO) induced mutation in Escherichia coli. Three wild E. coli strains BDCS-N-FMu10, BDCS-N-S21 and BDCS-N-W50, producing both the enzymes PGA and beta-lactamase were treated by AO. Minimum inhibitory concentration of AO was 10 microg ml(-1) and it was noted that bacterial growth was gradually suppressed by increasing the concentration of AO from 10 to 100 microg ml(-1). The highest concentration that gave permissible growth rate was 50 microg ml(-1). The isolated survivals were screened on the bases of PGA and beta-lactamase activities. Among the retained mutants, the occurrence of beta-lactamase deficient ones (91%) was significantly higher than penicillin acylase deficient ones (27%). In seven of the mutants, PGA activity was enhanced with considerable decrease in beta-lactamase activity. One of the mutant strains (BDCS-N-M36) exhibited very negligible expression of beta-lactamase activity and twofold increase in PGA activity [12.7 mg 6-amino-penicillanic acid (6-APA) h(-1) mg(-1) wet cells] compared with that in the wild-type strain (6.3 mg 6-APA h(-1) mg(-1) wet cells). The treatment of E. coli cells with AO resulted in mutants with enhanced production of PGA and inactivation of beta-lactamase. These mutants could be used for industrial production of PGA.

A fuzzy logic algorithm for identification of the harvesting threshold during PGA production by Bacillus megaterium

Penicillin G acylase (PGA) is an important enzyme used as biocatalyst in the production of semisynthetic beta-lactam antibiotics. Many microorganisms produce this enzyme and recombinant Escherichia coli has been preferred use for industrial applications. Bacillus megaterium is one of the microorganisms that excretes this enzyme into the medium. As a consequence, separation and purification steps are simplified. On-line measurement of enzyme activity during cultivation using in-situ sensors is a difficult task in the industrial environment due to the lack of robust and inexpensive instrumentation. This work presents the results of a fuzzy logic algorithm used to determine the moment of maximum enzyme concentration during Bacillus megaterium cultivations in an aerated and stirred, automated lab-scale bioreactor. The fuzzy algorithm was written in Fortran, compiled as a dynamic link library and implemented on a platform developed in MS-Visual Basic. Data were exchanged in real time between the platform and the supervisory system, which was coupled to the bioreactor. It was possible to determine the moment at which maximum enzyme activity was reached in several bioreactor assays. At this point, the end of the process was indicated to the operator. The results illustrate the importance of using reliable computational intelligence-based algorithms in biochemical reactions.

Optimization of the host–plasmid interaction in the recombinant Escherichia coli strains overproducing penicillin G acylase

Segregational stability of the recombinant plasmid pKA18 bearing the pga gene encoding penicillin G acylase (PGA) and the production stability for PGA was studied in Escherichia coli strain W and its descendants. The hosts differ in the number of indigenous plasmids (pRK1–3) and the specific activity of PGA. The production of PGA by the recombinant strain RE3(pKA18) is the highest among the hosts and the increase of activity is proportional to the increase of copy number (CN) of the plasmid pKA18. The plasmid is segregationally stable in spite of the presence of the indigenous plasmid pRK2 that belong to the same ColE1 plasmid family. Both plasmids (total sum of CN equals 271) are maintained without selection pressure during 102 generations. Subculture-based selection experiments taking advantage of a very high segregational and production stability of plasmid pKA18 in the host RE3 were used to study the optimization of the host–plasmid interaction. The clones with increased synthesis of the enzyme average value of 833 U/g DM were isolated: the expression of PGA per plasmid molecule increased in these strains from 3 to 4 U. Once this expression is reached, the population of segregationally stable, high-producing strains becomes heterogeneous: segregationally stable, low-producing clones and segregationally unstable, high-producing clones appear in population.

Optimization of the host?plasmid interaction in the recombinant Escherichia coli strains overproducing penicillin G acylase

Segregational stability of the recombinant plasmid pKA18 bearing the pga gene encoding penicillin G acylase (PGA) and the production stability for PGA was studied in Escherichia coli strain W and its descendants. The hosts differ in the number of indigenous plasmids (pRK1–3) and the specific activity of PGA. The production of PGA by the recombinant strain RE3(pKA18) is the highest among the hosts and the increase of activity is proportional to the increase of copy number (CN) of the plasmid pKA18. The plasmid is segregationally stable in spite of the presence of the indigenous plasmid pRK2 that belong to the same ColE1 plasmid family. Both plasmids (total sum of CN equals 271) are maintained without selection pressure during 102 generations. Subculture-based selection experiments taking advantage of a very high segregational and production stability of plasmid pKA18 in the host RE3 were used to study the optimization of the host–plasmid interaction. The clones with increased synthesis of the enzyme average value of 833 U/g DM were isolated: the expression of PGA per plasmid molecule increased in these strains from 3 to 4 U. Once this expression is reached, the population of segregationally stable, high-producing strains becomes heterogeneous: segregationally stable, low-producing clones and segregationally unstable, high-producing clones appear in population.

Production of penicillin G acylase from Bacillus sp.: Effect of medium components

A Bacillus sp. producing a high level of intracellular penicillin G acylase (PAC) was isolated. The PAC production in this strain was induced by phenylacetic acid. Various carbon and nitrogen sources were evaluated for their effect on growth and PAC production at 28 °C and pH 7.0. Cells grown in medium supplemented with sucrose as carbon source and tryptone as nitrogen source produced maximum activity of 6.45 and 8.92 U mg−1, respectively. Maximum concentration of PAC (10.1 Umg−1) was produced by the cells grown in the medium containing sucrose and tryptone, which was twofold higher than the production in basal medium.

Inoculum studies in production of penicillin G acylase by Bacillus megaterium ATCC 14945.

This article reports studies concerning the production of penicillin G acylase (PGA) by Bacillus megaterium. This enzyme has industrial use in the hydrolysis of penicillin G to obtain 6-aminopenicillanic acid, an essential intermediate for the production of semisynthetic beta-lactam antibiotics. Although most microorganisms produce the enzyme intracellularly, B. megaterium provides extracellular PGA. The enzyme production by microorganisms involves several steps, resulting in a many operational variables to be studied. The study of the inoculum is an important step to be accomplished, before addressing other issues such as culture optimization and downstream processing. In this study, using a standard inoculum as reference, several runs were performed aiming at the definition of operational conditions in the PGA production. Cell concentration and PGA activity in the production medium were measured after 24, 48, and 72 h of the beginning of the production phase. This study encompasses the duration of the inoculum germination phase and the concentration of cells used to startup the germination. Based on these results, PGA productivity during the production phase was maximized. The selected values for these variables were 1.5 x 10(7) spores/mL of germination medium, germination during 24 h, and 72 h for the production phase.