Separation of penicillin G from phenylacetic acid in a supported liquid membrane system

The separation of penicillin G (Pen G) from phenylacetic acid (PAA) by use of a supported liquid membrane (SLM) system with Amberlite LA-2 dissolved in 1-decanol, supported on a microporous polypropylene membrane, was studied. The results show that the individual permeability of each component in mixture was lower than that in a single compartment system and, it suggests a strong transport competition between Pen G and PAA. The SLM system in this study proved to be a promising process for the selective separation of Pen G from PAA. The maximum separation factor was found to be 1.8 under a liquid membrane resistance controlled mechanism. (c) 1994 John Wiley & Sons, Inc.     

Enhancement of penicillin G hydrolysis using penicillin acylase to 6-aminopenicillanic acid by simultaneous chromatographic separation

Penicillin G (Pen-G) was hydrolyzed to 6-aminopenicillanic acid (6-APA) and phenylacetic acid (PAA) in a chromatographic reactor-separator using the mixture of immobilized Escherichia coli cells and a macroporous adsorbent as stationary phase and a phosphate buffer of pH 7.8 as eluant. Pen-G conversion of 98% was observed without adjustment of the eluant pH due to the effective separation of 6-APA from Pen-G and PAA. At a sample load of 600 mg Pen-G, the volume overload gave higher Pen-G conversion (86%) than the mass overload (68%), while their difference in product resolution (0.9 and 1.0, respectively) was insignificant.     

Simultaneous extraction and concentration of penicillin G by hollow fiber renewal liquid membrane

In this article, hollow fiber renewal liquid membrane (HFRLM) technique was used for recovery of penicillin G from aqueous solution. The organic solution of 7 vol % di‐n‐octylamine (DOA) + 30 vol % iso‐octanol + kerosene was used as liquid membrane phase, and Na₂CO₃ aqueous solution was used as stripping phase. Experiments were performed as a function of carrier concentration in the organic phase, organic/aqueous volume ratio, pH, and initial penicillin G concentration in the feed phase, pH in the stripping phase, flow rates, etc. The results showed that the HFRLM process was stable and could carry out simultaneous extraction and concentration of penicillin G from aqueous solutions. As a carrier facilitated transport process, the addition of DOA in organic phase could greatly enhance the mass transfer rate; and there was a favorable organic/aqueous volume ratio of 1:20 to 1:30 for this system. The mass transfer flux and overall mass transfer coefficient increased with decreasing pH in the feed phase and increasing pH in the stripping phase, because of variation of the mass transfer driving force caused by pH gradient and distribution equilibrium. The flow rate of the shell side had significant influence on the mass transfer performance, whereas the effect of flow rate of lumen side on the mass transfer performance was slight because of the mass transfer intensification of renewal effect in the lumen side. The results indicated that the HFRLM process was a promising method for the recovery of penicillin G from aqueous solutions. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Hydrolysis of penicillin G by combination of immobilized penicillin acylase and electrodialysis

Phenylacetic acid, as inhibitory product, was formed from a hydrolysis of penicillin G by immobilized penicillin acylase. In this article, electrodialysis was applied to remove phenylacetic acid continuously from the reaction mixture and to enhance an efficiency of the reaction. When 268 and 537 mM of penicillin G solution were used as the substrate, the concentration of phenylacetic acid in the reaction mixture could be maintained at less than 81 and 126 mM, respectively, and eventually, 86% and 88% of phenylacetic acid produced were removed from the reaction mixture at the end of the hydrolysis, respectively. Times required to reach 96% and 94.8% conversion from 268 and 537 mM of initial penicillin G could be reduced to 65% and 64% respectively, by means of electrodialysis; while 3.0% and 4.3% of initial penicillin G of 268 and 537 mM were permeated out of the reaction chamber during the hydrolysis, respectively. However, a loss of penicillin G by permeation could be reduced from 4.3% to 3.4% by a repeated addition of penicillin G.     

Separation and concentration of L-phenylalanine using a supported liquid membrane

The separation and concentration of L-phenylalanine (L-Phe) using a supported liquid membrane (SLM) is investigated. A cation complex agent, di-2-ethylhexyl phosphoric acid (D₂EHPA), is used as a carrier in the SLM with n-Heptane as a solvent. The reaction order and equilibrium constant in the formation reaction of L-Phe-carrier complex are obtained from the extraction experment. A mathematical model for a carrier mediated counter transport process is proposed to estimate the diffusion coefficient of L-Phe-carrier complex in the liquid membrane. Permeation experiments of L-Phe using a SLM are performed under various operating conditions and optimum conditions for the transport of L-Phe are obtained. Concentration of L-Phe in the strip phase against its concentration is observed. Transport rate of glucose through liquid membrane is less than that of L-Phe in the competitive transport of L-Phe and glucose. And the existence of glucose reduced the transport rate of L-Phe. The performance of separation with continuous strip phase is increased due to the dilution effect in the strip phase.     

Industrial separation of carboxylic and amino acids by liquid membranes: applicability, process considerations, and potential advantage

Liquid-liquid extraction and membrane separation are well-known separation method of extensive industrial application. Their incorporation into liquid membranes has the potential of several advantages, some of which are of particular interest for the recovery of carboxylic and amino acids: selectivities higher than those attainable by current separation methods, saving on energy costs for final concentration of separated products, high fluxes, compact installation, and low capital and operation costs. Stability of the liquid advantages, can be secured by utilizing extractant blocking polymeric membranes, Applicability, process consideration, and economic implications for recovery for carboxylic and amino acids by various extractant/membrane combinations are discussed. (c) 1993 John Wiley & Sons, Inc.     

Facilitated transport of lactic acid in a stirred transfer cell

The application of liquid membrane extraction to the recovery of lactic acid from model systems and fermentation media was investigated. An experimental study of the facilitated transport of lactic acid using ALIQUAT 336 as a mobile carrier in a stirred transfer cell is reported. The effect of stirring speed, initial lactic acid concentration, carrier concentration, and NaCl as a reagent in the acceptor phase are considered. (c) 1994 John Wiley & Sons, Inc.     

Direct chemiluminescence determination of penicillin G potassium and a chemometrical optimization approach

A highly selective and simple chemiluminescence (CL) method for determination of penicillin G potassium (PGK) was developed. In the proposed method, CL was elicited from PGK upon its oxidation with H₂O₂. The light emission was enhanced in the presence of N‐cetyl‐N,N,N‐trimethylammonium bromide (CTMAB). An experimental design, central composite design (CCD), was used to realize the optimized variables, including pH, surfactant (CTMAB) and H₂O₂ concentrations. Under optimum condition, the calibration graph was linear in the range 3.3 × 10^?3–3.3 × 10^?1 mmol/L, with a detection limit of 8.8 × 10^?4 mmol/L for PGK. The precision was calculated by analysing samples containing 1.6 × 10^?1 mmol/L PGK (n = 5) and the relative standard deviation (RSD) was 1.40%. The utility of this method was demonstrated by determining PGK in pharmaceutical formulations for injection. The proposed method was validated by a reference method. Copyright © 2011 John Wiley & Sons, Ltd.     

重组青霉素G酰化酶拆分制备（S）-邻氯苯甘氨酸

对产青霉素G酰化酶的重组枯草芽胞杆菌发酵产酶条件进行优化,确定优化后的发酵条件：可溶性淀粉10g/L、蛋白胨12g/L、酵母粉3g/L、NaCl10g,／L;pH7．5、培养温度37℃、装液量80mL（500mL三角瓶）、培养28h,青霉素G酰化酶的表达水平由最初的7．34U／mL提高至18．23U／mL。以表达青霉素G酰化酶的枯草芽胞杆菌发酵液为酶源,在水相中对映选择性催化N-苯乙酰-（R,S）-邻氯苯甘氨酸制备（S）-邻氯苯甘氨酸,当底物浓度为100mol／L时转化4h,转化率达44．2％。对底物浓度为80mmoL／L反应液中的（S）-邻氯苯甘氨酸进行分离,达到理论收率的94．29％（以N-苯乙酰-（R,S）-邻氯苯甘氨酸的0．5倍摩尔量为理论产率）,e．e．值大于99．9％。170℃条件下,N-苯乙酰-（R）-邻氯苯甘氨酸与苯乙酸共熔消旋为N-苯乙酰-（R,S）-邻氯苯甘氨酸可用于循环拆分。     

The transport of chloroquine across human erythrocyte membranes is mediated by a simple symmetric carrier

The kinetic properties of the mediated transport of chloroquine in human erythrocytes are investigated. The high rates of translocation across the cell membrane and high adsorbance properties to glass surfaces have led to the development of new techniques for measuring initial rates of transport. Three different methodological procedures are used to accomplish a complete kinetic characterization of the system. All measurements were done at 25°C. Under zero-trans conditions the system displays complete symmetry, the Michaelis constants being 39.2±2.4 μM for influx and 36.6±5.6 μM for efflux. The respective maximal velocities are 206.4±36.0 μM·min^?1 and 190.0±7.8 μM·min^?1. Under equilibrium-exchange conditions the Michaelis constant is 108.6±15.6 μM and the maximal velocity is 630.3±50.4 μM·min^?1. This 3-fold increase in both K and V over the zero-trans values indicates that the rate-limiting step in the transport of chloroquine is the movement of the unloaded carrier. The kinetic data are consistent with the prediction of a simple carrier model.     

Two-stage recovery of S-adenosylmethionine using supported liquid membranes with strip dispersion

We report the selective recovery of S-adenosylmethionine (SAM) from fermentation broths using a two-stage supported liquid membrane system with strip dispersion (SLM-SD). The system utilized two MiniModule^® hollow-fiber membrane modules as microporous supports and an organic membrane solution consisting of the extractants of sodium di-2-ethylhexyl sulfosuccinate (AOT), di-(2-ethylhexyl)phosphoric acid (DEHPA), and trioctylphosphine oxide (TOPO) in the solvent n-octanol. SAM was extracted in the first membrane module. Methionine (Met) was captured by the first stripping solution and further purified in the second membrane module. pH values in the feed phase and the first and second stripping solutions, extractant concentrations, NaCl concentration, and the SAM acceptor in the first stripping solution were optimized. Strip dispersion mixing speed, pressure differences across the membranes, and flow rates of the feed and strip dispersion phases were investigated experimentally. The optimal extractant concentrations were: AOT 2.78 wt%, DEHPA 27.0 wt%, and TOPO 1.61 wt%. The optimal pH values in the feed phase and the first and second stripping solution were 3.0, 2.5, and 1.0, respectively. SAM extraction efficiency of 98.7%, SAM recovery efficiency of 91.8% and Met removal efficiency of 85.4% were achieved within 5 h. Finally, the mass transfer analysis indicated that the mass transfer resistances from the extraction reaction and the membrane phase were predominant.     

Conversion of phenylacetyl-cysteinyl-valine in vitro into penicillin G by isopenicillin N synthase of Streptomyces lactamdurans

Abstract Purified isopenicillin N synthase of Streptomyces lactamdurans JC1843 converted phenylacetyl-cysteinyl-valine, a structural analogue of the natural substrate (α-amino-adipyl-cysteinyl-valine), into a penicillinase-sensitive β-lactam antibiotic. The antibiotic was identified as penicillin G by HPLC. Cyclization of phenylacetyl cysteinylvaline into penicillin G required dithiothreitol (DTT), ascorbic acid and Fe²⁺. The affinity of the enzyme for phenylacetyl cysteinylvaline was about 20 times lower than the affinity for α-aminoadipyl-cysteinyl-valine.     

显微注射青霉素G获得单感染Wolbachia和单感染Cardinium白背飞虱品系

Wolbachia和Cardinium都是广泛存在于节肢动物体内的一类母系遗传的共生细菌, 可以通过不同方式操纵寄主的生殖行为。Wolbachia和Cardinium感染同一寄主在自然界比较常见, 但是在某些可以同时感染Wolbachia和Cardinium的寄主中其单感染品系较难发现。本研究检测了云南文山(YN)、 海南三亚(HN)这2个不同地理种群中Wolbachia和Cardinium的感染情况; 以双感染Wolbachia和Cardinium的白背飞虱Sogatella furcifera海南种群为实验材料, 运用显微注射方法对双感染Wolbachia和Cardinium的白背飞虱若虫注射不同浓度青霉素G以获得单感染品系。结果表明： 白背飞虱自然种群中单感染Wolbachia比率极低, 本实验用到的海南种群未检测到单感染Wolbachia成虫; 通过显微注射青霉素G的方法可以从白背飞虱双感染品系中筛选获得单感染品系, 当青霉素G注射浓度为0.2%(w/v), 注射龄期为5龄时得到单感染品系效率最高; F5代的检测结果显示显微注射得到的单感染品系可以稳定遗传。本研究为获得单感染品系白背飞虱提供了快捷方法, 同时为其他双感染Wolbachia和Cardinium节肢动物不同感染品系的筛选提供参考。     

Calcium transport by pea root membranes

Calcium transport has been studied using purified endomembrane vesicles from dark-grown roots of Pisum sativum L. Membranes from a mixed microsomal (non-mitochondrial) fraction showed ATP-dependent calcium uptake which was released by the ionophore A 23187, had a pH optimum of 7.2 and required Mg²⁺ for uptake. Membranes were further purified using a rapid sucrosedensity-gradient technique yielding vesicles suitable for transport studies, and were identified using marker enzymes. Uptake by plasma membrane, tonoplast, endoplasmic reticulum and Golgi apparatus was indicated. Uptake by membranes of low density (predominantly tonoplast) had a pH optimum of 7.2–7.4 and nucleotide specificity ATP> guanosine 5-triphosphate>inosine 5-triphosphate>ADP>, while that by high-density membranes had a pH optimum of 7.5–7.9 and less specificity for ATP. The importance of regulating sucrose concentrations in calcium transport studies was demonstrated.Abbreviations ER endoplasmic reticulum - GTP guanosine 5-triphosphate - IDPase inosine diphosphatase - IIP inosine 5-triphosphate     

重组毕赤酵母产青霉素G酰化酶的分批发酵动力学研究

构建了重组毕赤酵母产青霉素G酰化酶的分批发酵动力学模型。实验考察了分批发酵过程中甘油消耗、甲醇浓度、菌体浓度、溶氧、补料时间对青霉素G酰化酶活力的影响。应用Matlab软件,对菌体生长、基质消耗和产物生成方程进行最优参数估算和非线性拟合,得到相应的动力学模型。模型的计算值与实验值能较好地拟合,表明所建模型能较好反映重组毕赤酵母产青霉素G酰化酶的分批发酵过程。     

Efficient production of S-(+)-2-chlorophenylglycine by immobilized penicillin G acylase in a recirculating packed bed reactor

(S)-(+)-2-Chlorophenylglycine 1 is an important intermediate in the synthesis of Clopidogrel. A recirculating packed bed reactor (RPBR) was constructed for efficient production of (S)-1 by kinetic resolution of racemic N-phenylacetyl-2- chlorophenylglycine 2 using immobilized penicillin G acylase (PGA). The immobilized PGA exhibited maximum activity at 50 °C and pH 8.0 with (R,S)-2 as substrate. The kinetic constants (K_m and v_max) of immobilized PGA were calculated to be 20.61 mM and 83.2 mM/min/g, respectively. The substrate displayed inhibitory effect on immobilized PGA with inhibition constant of 221.23 mM. The immobilized PGA showed a strict enantiospecificity for substrate at different temperature, pH and substrate concentration examined. The performance and productivity of RPBR were evaluated by several critical parameters, including immobilized PGA load, substrate feeding rate, height to diameter ratio and so on. The kinetic resolution process shows higher initial reaction rate and conversion by recycling 100 mL of substrate solution (80 mM) through RPBRs packed with 6.0 g immobilized PGA with a feeding rate of 1.5 mL/min while the H/D ratio was 4.0. The immobilized PGA-catalyzed kinetic resolution of (R,S)-2 was successfully operated in the RPBR for 60 batches, with an average productivity of 1.2 g/L/h for (S)-1 in high optical purity (>97% enantiomeric excess) in semi-continuous operation. The residual (R)-2 can be easily racemized and then used as substrate.     

Development of a supported emulsion liquid membrane system for propionic acid separation in a microgravity environment

Perstractive fermentation is a good way to increase the productivity of bioreactors. UsingPropionibacteria as the model system, the feasibility of using supported emulsion liquid membrane (SELM) for perstractive fermentation is assessed in this study. Five industrial solvents were considered as the solvent for preparing the SELM. The more polar a solvent is, the higher the partition coefficient. However, toxicity of a solvent also increases with its polarity. CO-1055 (industrial decanol/octanol blend) has the highest partition coefficient toward propionic acid among the solvents that has no molecular toxicity towardPropionibacteria. A preliminary extraction study was conducted using tetradecane as solvent in a hydrophobic hollow fiber contactor. The result confirmed that SELM eliminates the equilibrium limitation of conventional liquid-liquid extraction, and allows the use of a non-toxic solvent with low partition coefficient.     

On the rate limiting step in downhill transport via the LacY permease of Escherichia coli

Strains of Escherichia coli K12 were constructed for the specific purpose of evaluating the inducibility of the influx mechanism controlled by the lacY gene. These strains are heteromerodiploids characterized by a high and relatively constant level of β-D-galactosidase which is not affected significantly by induction of the Lac operon. These properties were obtained by introducing episomal lacI⁺,O^c,Z⁺,Y^? genes into the cells. In these merodiploids the rate of o-nitrophenyl-β-D-galactopyranoside (ONPG) hydrolysis of extracted cells is 50-times that of intact cells. This difference indicates that the rate limiting step in the ONPG hydrolysis by intact cells is influx. Using a set of merodiploids with and without the LacY transport system, we were able to demonstrate a specific induction of ONPG influx. However, the increase in influx due to induction was only 3.5-fold as compared to the 40-fold increase observed when the LacY permease was measured by intracellular accumulation of [¹⁴C] TMG.     

Insight into molecular mechanism underlying the transesterification catalysed by penicillin G amidase (PGA) using a combination protocol of experimental assay and theoretical analysis

Penicillin G amidase (PGA) is one of the most recognised biocatalysts because of its critical application in the antibiotic industry. Herein, the additive effects involved in transesterification catalysed by PGA are explored in detail using a combination of experimental analysis and theoretical modelling. The transesterification ability of PGA is experimentally determined with 17 N-containing compounds as additives, and, on this basis, a series of quantitative structure–activity relationship (QSAR) models are developed from various physicochemical parameters characterising structural variation over the additives. The resulting models exhibit both good stability and predictive power, from which five most important properties that highlight structural basis and reaction mechanism underlying the transesterification are extracted, revealing that the topological property and electrostatic profile of additives exert a significant effect on reaction yield; the charge distribution around additive molecules is the most significant factor controlling reaction yield, and then the topological structure. Furthermore, it is inferred that the additive imidazole might constitute the catalytic triad of Ser, Glu or Asp involved in PGA active site, which appears similar to lipase, rendering PGA with the catalytic ability of transesterfication. The study highlights the potential application of QSAR methodology in the field of enzymatic regulator design.     

Stabilization and functional properties of Escherichia coli penicillin G acylase by covalent conjugation of anionic polysaccharide carboxymethylcellulose

The stabilization of Escherichia coli penicillin G acylase (PGA) conjugated with carboxymethylcellulose (CMC) against temperature and pH was studied. The 2,3-dialdehyde derivative of CMC obtained by periodate oxidation was covalently conjugated to PGA via Schiff's base formation. The inactivation mechanism of both native and CMC-conjugated PGA appeared to obey first order inactivation kinetics during prolonged incubations at 40–60 °C and in the pH range 4–9. Inactivation rate constants of conjugated enzyme were always lower, and half-life times were always higher than that of native PGA. The activation free energy of inactivation (G _i values) of CMC-conjugated enzyme were found to be always higher than that of native PGA at all temperatures and pH values studied as another indicator of enzyme stabilization. Highest stability of CMC-conjugated enzyme was observed as nearly four-fold at 40 °C and pH 8.0. No changes were observed on the temperature and pH profiles of PGA after CMC conjugation. Lower K _m and higher k _cat values of PGA obtained after CMC conjugation indicates the improved effect of conjugation on the substrate affinity and catalytic performance of the enzyme.