Recent progress in the development of immobilized penicillin G acylase for chemical and industrial applications: A mini‐review

Immobilized penicillin G acylase (PGA) as an important industrial catalyst can catalyze penicillin G potassium (PG) to 6‐aminopenicillanic acid (6‐APA). 6‐APA is an important intermediate for semisynthetic penicillin drugs, which occupies a huge market space in the anti‐inflammatory field; as a result, immobilized PGA occupies a huge market space in the pharmaceutical field. However, at present, there are different degrees of defects in the preparation and production process of immobilized PGAs on the market because of the huge demand; therefore, the performance of immobilized PGA and its productivity will bring huge economic benefits to enterprises. Therefore, research on immobilized PGA has always been a focus. This review first introduces the source, classification, structure, and catalytic mechanism of PGA and then studies the development of immobilization methods, immobilized carriers, reaction media, enzyme activity regeneration, and reactors of immobilized PGA in recent years.     

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.     

Immobilization of whole-cell penicillin g acylase by entrapping within polymethacrylamide beads

Escherichia coli ATCC 11105 containing the periplasmic penicillin G acylase was entrapped within a copolymer of methacrylamide andN,N’- methylenebisacrylamide. A solution of monomer that was made up from methacrylamide andN,N’-methylenebisacrylamide dissolved in buffer was mixed with lyophilized cells and ammonium persulfate. This suspension was then pumped drop by drop into in soybean oil supplemented with 0.06% (v/v) 3-(dimethylamino)-propionitril. During submerging in the oil phase, the droplets were hardened and induced to polymerize within the droplets. Particles with a volume ranging from 0.013–0.017 mL per bead containing a biomass concentration up to 38.0 g/L were prepared. The optimal condition for the deacylation of penicillin G to 6-aminopencillanic acid (6-APA) catalyzed by the immobilized whole-cell penicillin G acylase was found to be 45‡C and pH 8.0. Product inhibition of this enzyme by 6-APA could be eliminated by controlling pH value at 8 during the course of penicillin G hydrolysis using a pH-stat. Conversion determined by the pH-stat method were 0.3% higher than that by p-dimethylaminobenzaldehyde method. Cell concentration in the matrix was found to be an important factor influencing the maximum velocity and the specific activity retained in the matrix. A kinetic model, in which the mass transfer resistances as a result of external film mass transfer and pore diffusion were assumed to be negligible, could properly describe the hydrolysis of penicillin G by the cells entrapped within the polymethacylamide beads.     

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 β-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 × 10⁷ spores/mL of germination medium, germination during 24 h, and 72 h for the production phase.     

TREATMENT AND RESOURSE REUSE OF INDUSTRIAL WASTEWATER FROM PRODUCTION PROCESS OF PHENYL ACETIC ACID

1INTRODUCTIONPhenylaceticacidisakeyintermediateofmedicine,pesticideandperfume.ItismainlyproducedbyhydrolysisofbenZylcyanidedomestically,whichwilldogreatharmtoenvironmentandhealthduetothetoxicityoftherawmaterialll].JininginstitUteofChemicalEngineeringDesignhassuccessfullydevelopedanewcarbonyltechnologyforproductionofPhCH,COOH.InthistechnologyNaOH,COandbenZylchlorideareusedasrawmaterialsandmethanolassolvent.ThePhCH,COOHwillbeachievedafteracidificationofphCH,COONaAtthesametime7t…     

Synthesis and screening of a molecularly imprinted polymer library targeted for penicillin G

A library of molecularly imprinted polymers (MIPs) was synthesized by radical bulk polymerization using the beta-lactam antibiotic penicillin G as the template. Diversity of the library was obtained by combining various functionalized monomers and cross-linkers and by varying the stoichiometry and the concentration of the components in the prepolymerization mixtures. The library was screened for selectivity to penicillin G by a radioligand binding assay and was compared to a corresponding control library. The best MIP candidate, showing the highest selectivity for penicillin G, was prepared from methacrylic acid and trimethylolpropane trimethacrylate as the functionalized monomer and cross-linker, respectively. Cross-reactivity studies with other beta-lactam antibiotics showed a low cross-reactivity of penicillin V (15%), ampicillin (16%), and amoxicillin (19%). Nafcillin and oxacillin showed less cross-reactivity (<1%). Cross-reaction with a cephalosporin antibiotic (cephapirin) and structurally nonrelated antibiotics (chloramphenicol, tetracycline, dapsone, and erythromycin) was less than 0.01%.     

Effect of thickening agents on the penicillin fermentation

The effects of thickening agents (polyethylene glycol [PEG] 20000 and carboxymethylcellulose [CMC]) on the penicillin fermentation byPenicillium chrysogenum were investigated. By adding the thickening agents to the fermentation medium, the growth form of the mold can be manipulated. Depending on the amount of thicking agent added, the change in morphology is from compact smooth pellets to various intermediate forms, and finally to filamentous mycelia. It was found that better penicillin production was obtained when the mold was in small, fluffy, loose pellets. The penicillin fermentation is not only affected by the thickening agents, but also the status of inoculum and agitation. Under the condition that the mold will otherwise grow in large pellets (e.g., under a low level of spore inoculum), the enhancement in the penicillin production through addition of the thickening agents may be more significant. In tank fermentation, the thickening agent was introduced in the stage of preculture, rather than main culture. The increase in the broth viscosity caused by addition of the thickening agent resulted in a decrease in dissolved oxygen level, which could be compensated in the case of PEG 20000.     

TREATMENT AND RESOURSE REUSE OF INDUSTRIAL WASTEWATER FROM PRODUCTION PROCESS OF PHENYL ACETIC ACID

The effluent from phenyl acetic acid (Ph CH2COOH) production process can be treated with NDA-999 macroporous polymeric adsorbent with about 100% remopval efficiency of Ph CH2COOH,benzyl alcohol(PhCH2OH) and benzaldehyde(PhCOHO)as well as the decrease in Total Organic Carbon(TOC)from 4691mg/l to &lt;300mg/L.3.7kg phCH2COOH and 120kg NaCl will be recovered from per m^3 wastewater and the adsorbent can be reused after being regenerated by NaOH aqueous solution and methanol.Good economic,social and environmental results can be achieved with this method.     

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 9.5 U/ml in shake flask. The experiments conducted in bioreactor with different oxygen flow rates revealed that 0.66 vvm oxygen flow rate could be sufficient for the maximum PGA activity of 12.7 U/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 10 ml of 0.1 % (w/v) PAA once in every 2 h from 6th hour of growth showed the maximum PGA activity of 32 U/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.     

Substitution of 6-aminopenicillanic acid at the 6 carbon atom

The hydroxyalkylation of the penicillin nucleus at C-6 with benzaldehyde and formaldehyde is reported. 6-aminopenicillanic acid (6-APA) ( 2 ) was used as starting material. Protection of the functional groups and activation of the C-6 position were effected by converting 6-APA successively into the Schiff base 13 , the methyl ester 14 and the copper complex 15 . The latter gave with benzaldehyde the C-6 monosubstituted complex 17 , and with formaldehyde the disubstituted complex 18 . The direct α-hydroxybenzylation of 6-APA at C-6 was also carried out with an excess of benzaldehyde at pH 7,5 leading to the SCHIFF base 19 . Mild hydrolysis of 19 gave 6-amino-6-(α-hydroxybenzyl)-penicillanic acid ( 21 ). Phenylacetylation of the latter yielded the penicillin G analogue 22 . Direct reaction of 6-APA with formaldehyde took place only in the presence of salicylaldehyde, giving the oxazolidine 24 , from which the amino acid 25 could not be obtained. The new compounds showed only weak antibacterial activity as compared with penicillin G.     

A kinetic study of synthesis of amoxicillin using penicillin G acylase immobilized on agarose

We presenta kinetic model for the synthesis of amoxicillin from p-hydroxyphenylglycine methyl ester and 6-aminopenicillanic acid, catalyzed by penicillin G acylase immobilized on agarose, at 25°C. Michaelis-Menten kinetic parameters (with and without inhibition) were obtained from initial velocity data (pH 7.5 and 6.5). Amoxicillin synthesis reactions were used to validate the kinetic model after checking mass transport effects. A reasonable representation of this system was achieved under some operational conditions, but the model failed under others. Nevertheless, it will be useful whenever a simplified model is required, e.g., in model-based control algorithms for the enzymatic reactor.     

Dissipation and residue determination of penicillin G and its two metabolites in citrus under field conditions by DSPE/UPLC–MS/MS

A rapid determination method of residual penicillin G and its two metabolites in citrus was developed and validated by dispersive solid-phase extraction and ultra-high performance liquid chromatography–tandem mass spectrometry (DSPE/UPLC–MS/MS). The samples were extracted with 80% acetonitrile and purified with octadecylsilane. High linearity was obtained with correlation coefficients (r²) >0.9981. The limits of quantification were 0.005–0.01 mg/kg. The recoveries of penicillin G and its metabolites spiked in blank citrus were within 76.7–107%, with relative standard deviations of 1.3–9.6%. The dissipation dynamics and distribution of penicillin G in citrus followed first-order kinetics, with half-life of 1.7–2.7 days. Penicillin G degraded easily in citrus and the metabolite was mainly penilloic acid, which can exist stably for long time. The terminal residues of penicillin G in pulp, whole citrus and peels were 0.015–0.701, 0.047–7.653 and 0.162–13.376 mg/kg, respectively. The hazard indexes for risk assessment of citrus were significantly <1, suggesting that the health risks to humans after consumption of citrus were insignificant and negligible. These results could provide necessary data for evaluating the safe and proper use of penicillin G in citrus.     

超高效液相色谱-串联质谱法同时测定全血中青霉素G及其主要代谢产物

建立了同时检测全血中青霉素G及其2种主要代谢产物青霉噻唑酸和脱羧青霉噻唑酸的超高效液相色谱-电喷雾串联质谱分析方法。血样经简单的蛋白沉淀提取后,目标化合物经BEH C18色谱柱(50 mm×2.1 mm, 1.7 μm),以含0.1%甲酸溶液-乙腈为流动相,梯度洗脱分离后,选用正离子电喷雾多反应监测(MRM)模式检测。方法检出限(信噪比(S/N)为3计)为0.1～2.0 ng/mL,定量限(S/N=10)为0.5～5.0 ng/mL。各被测物的线性相关系数均大于0.9974,准确度为92.3%～105.5%,日内精密度小于10%。考察了不同储存温度(18、4、-18、-80 ℃)下全血中青霉素G及其代谢物的稳定性,结果表明随着储存温度升高和时间的延长,青霉素G的质量浓度下降明显。应用建立的方法检测了给予20 mg/kg青霉素G后的大鼠血液样本,血液中青霉素G原形药物在给药后0.5 h即消除完全(低于检出限),而其代谢物的体内消除时限可延长至36 h。所建方法对司法鉴定的适用性得到了进一步扩大,同时对食品残留检测也具有一定的参考价值。     

Application of spontaneous suction phase-dispersing (SSPD) extractors in the extraction of penicillin G

The extraction of penicillin G from an aqueous solution with butylacetate (BA) and tributyl phosphate (TBP) as extractants was carried out at pH 4 with spontaneous suction phase-dispersing (SSPD) extractors under various operating conditions. Four kinds of SSPD extractors were tested with results compared to those obtained by using an extractor with mechanical stirrers. Rotation speed and different extraction systems were found to influence the penicillin recovery and the stability of emulsion formed during extraction. The percentage of extraction under optimum conditions was 91% without formation of emulsion. The laser particle size measurement instrument combined with SSPD can be used to measure the emulsion droplet size in situ.     

Immobilized penicillin G acylase as reactor and chiral selector in liquid chromatography

In this paper, the use of penicillin G acylase (PGA) as a biocatalyst and as a chiral selector is described. Penicillin G-acylase is an interesting enzyme used in the manufacture of semisynthetic antibiotics and, in particular, in the production of 6-APA by hydrolysis of penicillin G. Five PGA-based HPLC columns have been prepared by using two different silica supports by employing two immobilization methods, namely "in situ" and "in batch". The effects of the immobilization techniques and of different silica pore size on the catalytic properties of the enzyme as well as the applicability of the PGA-bonded stationary phases as chiral selectors for a number of chiral drugs have been investigated. The HPLC columns based on immobilized PGA combine the hydrolytic activity and the chiral recognition properties of PGA, therefore they have been used for the development of a combined reaction-separation system for chiral and achiral substrates.     

Determination of penicillin G in bovine plasma by high-performance liquid chromatography after pre-column derivatization.

A simple, selective, and sensitive liquid chromatographic method with ultraviolet detection was developed for the analysis of penicillin G in bovine plasma. The assay utilizes a simple extraction of penicillin G from plasma (with a known amount of penicillin V added as internal standard) with water, dilute sulphuric acid and sodium tungstate solutions, followed by concentration on a conditioned C18 solid-phase extraction column. After elution with 500 microliters of elution solution, the penicillins are derivatized with 500 microliters of 1,2,4-triazole-mercuric chloride solution at 65 degrees C for 30 min. The penicillin-mercury mercaptide complexes are separated by reversed-phase liquid chromatography on a C18 column. The method, which has a detection limit of 5 ng/ml (ppb) in bovine plasma, was used to quantitatively measure the concentrations of penicillin G in plasma of steers at a series of intervals after the intramuscular administration of a commercial formulation of procaine penicillin G.     

Integration of anodic and cathodic processes for the synergistic electrochemical production of peracetic acid

Efficient in-situ production of peracetic acid is an unreached milestone of electrochemical engineering. Previous attempts in the production of peracetic acid were focused either on the cathodic production of hydrogen peroxide and its further addition to acetic acid solutions or on the oxidation of a suitable raw material (v. g. acetic acid, acetaldehyde, ethanol). In the present work, the oxidation of acetic acid by a boron doped diamond (BDD) anode was integrated with the cathodic production of hydrogen peroxide using a carbon felt gas diffusion electrode. A marked synergistic effect (synergy coefficient of 192.0 ± 13.1%) is observed when the oxidation of acetic acid by hydroxyl radicals is performed together with the cathodic production of hydrogen peroxide. A maximum PAA production efficiency of 19.87% was obtained, a value much higher than previous works based on the oxidation of acetic acid by BDD anodes and approximately double the optimal value reported in studies based on the production of hydrogen peroxide.     

A Process to Produce Penicillin G Acylase by Surface-Adhesion Fermentation Using <Emphasis Type="Italic">Mucor griseocyanus</Emphasis> to Obtain 6-Aminopenicillanic Acid by Penicillin G Hydrolysis

The production of extracellular and mycelia-associated penicillin G acylase (maPGA) with Mucor griseocyanus H/55.1.1 by surface-adhesion fermentation using Opuntia imbricata, a cactus, as a natural immobilization support was studied. Enzyme activity to form 6-aminopencillanic acid (6-APA) from penicillin G was assayed spectrophotometrically. The penicillin G hydrolysis to 6-APA was evaluated at six different times using PGA samples recovered from the skim milk medium at five different incubation times. Additionally, the effect of varying the penicillin G substrate concentration level on the PGA enzyme activity was also studied. The maximum reaction rate, V _max, and the Michaelis constant, K _M, were determined using the Michaelis–Menten model. The maximum levels for maPGA and extracellular activity were found to be 2,126.50 international unit per liter (IU/l; equal to 997.83 IU/g of support) at 48 h and 755.33 IU/l at 60 h, respectively. Kinetics of biomass production for total biomass showed a maximum growth at 60 h of 3.36 and 2.55 g/l (equal to 0.012 g of biomass per gram of support) for the immobilized M. griseocyanus biomass. The maPGA was employed for the hydrolysis of penicillin G to obtain 6-APA in a batch reactor. The highest quantity of 6-APA obtained was 226.16 mg/l after 40-min reaction. The effect of substrate concentration on maPGA activity was evaluated at different concentrations of penicillin G (0–10 mM). K _M and V _max were determined to be 3.0 × 10⁻³ M and 4.4 × 10⁻³ mM/min, respectively.     

Penicillinase-based amperometric biosensor for penicillin G

A biosensor for penicillin G was created by immobilizing penicillinase to a gold electrode by means of a cysteine self-assembled monolayer. The biosensor amperometrically monitored the catalytic hydrolysis of penicillin in a very sensible manner. Furthermore, it was successfully used to measure the Michaelis–Menten enzymatic constant and a low limit of detection of 4.5 nM was obtained.     

Analysis of wine for penicillin

This study addresses the question of whether the antibiotic Penicillin, which is produced by the common mold Penicillium notatum, could possibly become a contaminate of wine during the fermentation process. The significance of this study is related to the potential health effects this agent might produce in those consumers who have an allergic response to Penicillin. It has been estimated that between 6% and 8% of the American population is subject to this type of allergic response. A method is developed for the detection of penicillin in wines using high-pressure liquid chromatography. We demonstrate that penicillin G hydrolyzes rapidly in wine with first-order kinetics, and the half-life of this antibiotic is 147 min in a typical commercial wine. An analysis of a number of commercial wines shows no evidence of the presence of penicillin, which should negate the question of any allergic response associated with this potential contaminate.