Efficient degradation of tetracycline antibiotics using a novel rGO/Ag/g-C3N4 photocatalyst for hospital wastewater treatment.

This study focuses on the development of an efficient photocatalyst for degrading hospital wastewater, specifically targeting the degradation of the antibiotic tetracycline (TC). We introduce a novel 2D/2D heterostructure photocatalyst composed of graphitic carbon nitride (g-CN), functionalized with silver nanoparticles (Ag NPs) and reduced graphene oxide (rGO). The primary aim is to enhance the photocatalytic performance of g-CN through the synergistic effects of Ag NPs and rGO. The rGO/Ag/g-CN nanocomposites demonstrated remarkable photocatalytic activity, achieving over 97% TC degradation within 60 min under commercial LED light irradiation. Additionally, these photocatalysts were used to remove other antibiotics, such as doxycycline hydrochloride and ofloxacin, and it was observed that the nanocomposite effectively removed these antibiotics as well. This enhanced performance is attributed to the surface plasmon resonance (SPR) effects of Ag NPs and the electron sink properties of rGO, which were confirmed through comprehensive physicochemical characterization. Various concentrations of Ag NPs and rGO were tested to optimize the nanocomposite synthesis, with optical and electrical characterizations, including photoluminescence (PL), electrochemical impedance spectroscopy (EIS), and Mott-Schottky (M-S) measurements, revealing higher electron-hole pair generation rates and carrier concentrations in the rGO/Ag/g-CN nanocomposites compared to pristine g-CN, Ag/g-CN, and rGO/g-CN. The results demonstrate the potential of the rGO/Ag/g-CN photocatalyst as a cost-effective and scalable solution for the treatment of medical pollutants in wastewater.

Agrobacterium tumefaciens-mediated genetic transformation of a recalcitrant grain legume, lentil (Lens culinaris Medik).

A simple and reproducible Agrobacterium-mediated transformation protocol for a recalcitrant legume plant, lentil (Lens culinaris M.) is reported. Application of wounding treatments and efficiencies of three Agrobacterium tumefaciens strains, EHA105, C58C1, and KYRT1 were compared for T-DNA delivery into lentil cotyledonary node tissues. KYRT1 was found to be on average 2.8-fold more efficient than both EHA105 and C58C1 for producing transient beta-glucuronidase (GUS) gene (gus) expression on cotyledonary petioles. Wounding of the explants, use of an optimized transformation protocol with the application of acetosyringone and vacuum infiltration treatments in addition to the application of a gradually intensifying selection regime played significant roles in enhancing transformation frequency. Lentil explants were transformed by inoculation with Agrobacterium tumefaciens strain, KYRT1 harboring a binary vector pTJK136 that carried neomycin phosphotransferase gene (npt-II) and an intron containing gusA gene on its T-DNA region. GUS-positive shoots were micrografted on lentil rootstocks. Transgenic lentil plants were produced with an overall transformation frequency of 2.3%. The presence of the transgene in the lentil genome was confirmed by GUS assay, PCR, RT-PCR and Southern hybridization. The transgenic shoots grafted on rootstocks were successfully transferred to soil and grown to maturity in the greenhouse. GUS activity was detected in vegetative and reproductive organs of T(0), T(1), T(2) and T(3) plants. PCR assays of T(1), T(2) and T(3) progenies confirmed the stable transmission of the transgene to the next generations.

Vancomycin-loaded HPMC microparticles embedded within injectable thermosensitive chitosan hydrogels.

Antibiotic use is an essential method for the treatment of bacterial infections. In certain cases, antibiotics are not effective because of the distribution problems caused by physiological barriers in the body. Such problems are thought to be minimized by development of sustained release systems which involve implantation of antibiotic loaded polymeric systems directly to the site of infection. In this work, a new composite vancomycin hydrochloride release system based on HPMC microparticles and chitosan/glycerophosphate (Ch/Gp) thermosensitive hydrogel was designed for the aim of local treatment of osteomyelitis. Vancomycin-loaded HPMC microparticles (Van-HPMCs) were prepared by spray drying method. The SEM results showed that these particles had a mean diameter of 1.5-6.4 µm with a narrow size distribution and homogeneous particle production. Their drug encapsulation efficiency was 72.6%. The Van-HPMCs were embedded in an injectable Ch/Gp solution to introduce a composite drug release platform (Van/HPMC-Ch/Gp). In vitro release studies indicated that inclusion of the Van-HPMCs into the Ch/Gp hydrogel caused a reduction in both the release rate and total amount of vancomycin release, which suggests that HPMC microparticles entrapped into the Ch/Gp hydrogels showed more suitable sustained release kinetics for local antibiotics delivery.

Synthesis and characterization of Co3O4 ultra-nanosheets and Co3O4 ultra-nanosheet-Ni(OH)2 as non-enzymatic electrochemical sensors for glucose detection.

The present study examines the synthesis of Co3O4 ultra-nanosheets (Co3O4 UNSs) and Co3O4 ultra-nanosheet-Ni(OH)2 (Co3O4 UNS-Ni(OH)2) via solvothermal process and their application as non-enzymatic electrochemical sensors for glucose detection. X-ray diffraction and transmission electron microscopy results confirmed the Co3O4 UNS deposition on Ni(OH)2 surface. The presence of Co3O4 UNSs on Ni (OH) 2 surface improved the sensitivity of glucose detection, from the increase of glucose oxidation peak current at the Co3O4 UNS-Ni(OH)2/glassy carbon electrode (current density: 2000µA·cm(-2)), compared to the Co3O4 UNSs. These results confirmed that Ni(OH)2 on glassy carbon electrode is a sensitive material for glucose detection, moreover the Co3O4 UNSs can increase the interaction and detection of glucose due to their high surface area. The estimated limit of detection (S/N=3) and limit of quantification (S/N=10) of the linear segment (5-40µM) are 1.08µM and 3.60µM respectively. The reproducibility experiments confirmed the feasibility of Co3O4 UNS-Ni(OH)2 for the quantitative detection of certain concentration ranges of glucose.

In vitro binding of dantrolene to sarcoplasmic reticulum of rabbit skeletal muscle.

Dantrolene upon binding to microsomes containing sarcoplasmic reticulum of rabbit thigh muscle exhibits a fluorescence with emission at 490 nm, which shows a blue shift of 35 nm compared with its fluorescence in ethylacetate. Using fluorescence techniques, dantrolene binding to microsomes isolated from rabbit thigh muscle was investigated. From Scatchard plots of binding studies, the association constant (Kass) and the number of binding sites of dantrolene to sarcoplasmic reticulum were calculated, which was found to be 9.6 X 10(4) M-1 and 1.71 mumole/g of membrane proteins, respectively. In the presence of verapamil (1.25 X 10(-4) M), another calcium antagonist, the binding of dantrolene to microsomes was enhanced. However, at a high concentration of verapamil (3.75 X 10(-4) M), the Scatchard plot of dantrolene binding was found to be biphasic.

The mechanism of action and mode of inhibition of dihydroorotate dehydrogenase. A quantum chemical study.

Semiemperical quantum chemical calculations have been applied to study the reaction mechanism and mode of inhibition of dihydroorotate dehydrogenase. The structure of substrate, intermediate, product and various inhibitors of dihydroorotate dehydrogenase were optimized using MNDO method and the geometry, heat of formation and the net atomic partial charges of optimized molecules, as well as the energy of the reaction path were calculated. This study shows that the carbanion intermediate of this reaction is rather stable (heat of formation = -134.5 kcal) and readily forms upon nucleophilic attack by groups such as hydroxyl ion. There is good correlation between electronic properties and the biological activities of various inhibitors of this enzyme and the geometry of the most active inhibitor resembles closely that of the intermediate of the reaction. Therefore, it is concluded that the enzymatic oxidation of dihydroorotate dehydrogenase proceeds via formation of an intermediate and the inhibitors bind to the active site of this enzyme in the place of this intermediate.

Effect of dantrolene sodium on the contractility of rabbit jejunum in vitro.

The effect of dantrolene sodium on the spontaneous contractions of rabbit jejunum was studied in vitro. Dantrolene sodium (4.5 x 10(6) to 4.5 10(4) M) reversibly decreased the amplitude of contractions in a dose-dependent manner. ED50 was found to be about 7.9 x 10(-5) M. Its effect was biphasic in that a period of potentiation preceded that of suppression of contractions. Lowering or increasing (2.5 fold in each direction) the calciumm concentration of bathing media did not affect the suppression of contraction caused by dantrolene sodium to any significant degree. Caffeine but not quinine was found to be able to restore the activity of the intestine to normal after a 50% inhibition caused by dantrolene sodium. Dantrolene sodium, verapamil and nifedipine were able to shift the dose-response curves of calcium in potassium-polarized rabbit jejunum to the right and pA2 values were found to be 4.18, 7.76 and 8.47 respectively. These data indicate that the effect of dantrolene on smooth muscle is mediated via inhibition of calciu movement across the membrane.

The cannabinoid receptor: computer-aided molecular modeling and docking of ligand.

A three-dimensional model of human cannabinoid receptor is constructed using computer-aided molecular modeling techniques. The helices of bacteriorhodopsin were used as the initial template to construct the transmembrane helices. The extracellular and intracellular loops were added using the SYBYL molecular modeling package. The extracellular N terminus was modeled on the basis of its similarity to rat oncomodulin. Similarly, the C terminus was constructed on the basis of similarity to bovine prothrombin fragment 1. The final structure was refined by several runs of minimization and dynamics calculation using the CHARMm package. delta 9-Tetra hydrocannabinol was docked into the internal cavity using the AUTODOCK program. Our study snows that there may be a calcium-binding site in the extracellular N terminus of this receptor. The ligand binds mainly to a hydrophobic site, which consists of residues Met-240, Trp-241 (TMH-4), Trp-356, Leu-359, Leu-360 (TMH-6), and Ala-283 (TMH-5). Its phenolic hydroxyl group forms a hydrogen bond with the carboxy group of Ala-198 (TMH-3). The results of modeling agree well with experimental QSAR studies.

Diabetes-induced changes in the contractility of the aorta and pA2 of nifedipine in the rat.

Diabetes-induced changes in the calcium influx and contractile responses of aortic rings to various drugs were investigated in streptozotocin-treated rats. Diabetes is associated with calcium influx into the aortic rings (1.5-and 2.5-fold, respectively, after either KCl or noradrenaline stimulation compared with normal). The maximum KCl-induced contraction of the arorta in diabetic rats was reduced by 38%, but the EC50 of KCl remained unchanged. The pA2 of nifedipine for inhibiting the contractile response of aorta to KCl decreased one order of magnitude in the diabetic rats (8.26 vs 9.03 for non-diabetic rats). It is concluded that diabetes reduces the sensitivity of aortic tissue to nifedipine and may affect the stimulation-contraction coupling of vascular smooth muscle in such a way that a higher influx of calcium results after stimulation and that this may be responsible for diabetes-induced vascular complications.

Homology-based molecular modelling of PLP-dependent histidine decarboxylase from Mmorganella morganii.

The 3-D structural information is a prerequisite for a rational ligand design. In the absence of experimental data, model building on the basis of a known 3-D structure of a homologous protein is at present the only reliable method to obtain structural information. A homology model building study of the pyridoxal 5'-phosphate (PLP)-dependent histidine decarboxylase from Morganella morganii (HDC-MM) has been carried out based on the crystal structure of the aspartate aminotransferase from Escherichia coli (AAT-EC). The primary sequences of AAT-EC and HDC-MM were aligned by automated alignment procedure. A 3-D model of HDC-MM was constructed by copying the coordinates of the residues from the crystal structure of AAT-EC into the corresponding residues in HDC-MM. After energy-minimization of the resulting 3-D model of HDC-MM, possible active site residues were identified by fitting the substrate (l-histidine) into the proposed active-site. In our model, several residues, which have an important role in the AAT-EC active-site, are located in positions spatially identical to those in AAT-EC structure. The back-bone of the modelled active site pocket is constructed by residues; Gly-92, Gly-93, Thr-93, Ser-115, Asp-200, Ala-202, Ser-229 and Lys-232 together with residues Asn-8, His-119, Thr-171, His-198, Leu-203, His-231, Ser-236 and Ile-238. In the ligand binding site, it appears that the HDC-MM model will position l-histidine (substrate) in the area consisting of the residues; Glu-29, Ser-30, Leu-38, His-231 and Lys-232. The nitrogen atom of the imidazole ring (N2) of the substrate is predicted to interact with the carboxylate group of Ser-30. The alpha-carboxylate of histidine points toward the Lys-232 to have electrostatic interaction with its side chain nitrogen atom (N(Z)). In conclusion, this combination of sequence and 3-D structural homology between AAT-EC and HDC-MM model could provide insight in assigning the probable active site residues.

High performance liquid chromatography of mebudipine: application to pharmacokinetic study.

PURPOSE: To develop a high performance liquid chromatography system for the determination of a new 1,4-dihydropyridine, mebudipine, in rabbit plasma. METHODS: To 1 ml of rabbit plasma was added internal standard (dibudipine) and 0.5 ml of 1 M NaOH. Mebudipine and internal standard were extracted to 5 ml ethyl acetate, evaporated under slow stream of nitrogen. The residue was reconstituted in 200 microl mobile phase and 20 microl of aliquots were injected into a HPLC system equipped with 4.6 x 250 mm i.d. C18 analytical column. Mobile phase consisted of methanol (70%), water (25%) and acetonitril (5%) and its flow rate was 1 ml/min. RESULTS: There were no interfering peaks from endogenous components in blank plasma chromatograms. Standard curves were linear (r(2)>0.99) over 10 to 500 ng/ml. The extraction efficiency was >90% and the minimum quantifiable concentration was 10 ng/ml (CV<10%). CONCLUSION: A suitable, convenient and simple HPLC assay for pharmacokinetic study of mebudipine in rabbits was developed.

A quantitative thin layer chromatography method for determination of theophylline in plasma.

A simple assay method for theophylline in plasma using thin layer chromatography (TLC) was developed. The method involves extraction of the drug and internal standard (acetaminophen) by chloroform-isopropanol (75:25) followed by separation on TLC silica plates using a mixture of acetic acid, isopropanol, toluene (1: 12: 6), as the eluting solvent. Both peak height ratios and peak are ratios showed high correlation coefficient (r>0.98, p<0.001). However we used peak heights for the determinations. Within-day and between-day coefficients of variation were less than 4.4% and 7.8% respectively. The assay proved inexpensive, accurate and reproducible with a limit of detection of 100 ng/ml that makes it suitable for bioavailability studies.

Determination of captopril in human serum by high performance liquid chromatography using solid-phase extraction.

A rapid, simple and sensitive assay was developed for determination of captopril in human serum. We employed silica gel cartridge for efficient extraction of captopril adduct from human serum. Captopril was trapped with p-bromophenacyl bromide (pBPB) to give captopril-pBPB adduct. A 4-ml benzene extract of 1 ml acidified serum was passed through 1 ml silica gel cartridge. Potential interfering compounds were removed with 4-ml benzene wash. The captopril-pBPB adduct was eluted with 0.5 ml acetonitrile. Of this acetonitrile solution (100 microliters) was injected on an ODS reverse phase HPLC column (chromatography conditions; mobile phase; acetonitrile-water-acetic acid (225:270:5, v/v/v), flow rate; 1 ml min-1, detection; UV at 263 nm). It is found that this method is accurate and does not require time consuming evaporation-concentration steps. Recovery exceeds 94% and analytical responses are linear over captopril concentration range from 50 up to 1000 ng ml-1. The coefficients of variation from 108 ng ml-1 to 605 ng ml-1 varied between 3.7-7.7% and the relative error did not exceed 3.7%. Therefore, this method can be used for routine clinical monitoring and in pharmacokinetic studies of captopril.

QSAR of binding of dihydropyridine-type calcium antagonists to their receptor on ileal smooth muscle preparations.

Quantitative structure-activity analysis (Hansch analysis) is applied to elucidate the structural requirement for the binding of dihydropyridine-type calcium antagonists (DHPs) to their receptor in the guinea-pig ileal muscle preparations. It is found that various steric (B1, L), electronic (sigma) and hydrophobic (pi) parameters or their combinations correlate well with the potency of various DHPs to inhibit the binding of [3H]nitrendipine to the microsomal preparations of the guinea-pig ileal muscle. The potency of DHPs increases with the minimum width (B1) of substituent at ortho- or meta-positions, but decreases with the increase in the length of substituent at the meta-position. The potency of DHPs decreases with the increase in both minimum width or length of substituent at the para-position and the optimal values were found to be those for hydrogen. The hydrophobicity (pi) of substituents at different positions in the 4-phenyl ring affects the potency differently, indicating that a different environment exists around each position at the binding site. From the slopes of the pi variable in the regression equations, it is concluded that the receptive environment of the ortho-position of the 4-phenyl ring of DHPs is lipophilic, and for that of the para-position hydrophilic. A good correlation is also observed between the Hammett electronic parameter (sigma) and biological activity of meta-substituted DHPs. It is suggested that in the binding of the substituted 4-phenyl DHPs to their receptor, both electronic and hydrophobic interactions should be considered.

Synthesis and biological activity of two new calcium-channel blockers, mebudipine and dibudipine.

Dihydropyridine derivative calcium-channel blockers are widely used in the therapy of hypertension, angina pectoris and other cardiovascular diseases. Because the prototype of dihydropyridine derivatives, nifedipine, does not have the optimum pharmacokinetic and pharmacodynamic characteristics, attempts have been made to synthesize other drugs in this class with improved properties. The synthesis and biological activity of two new calcium-channel blockers, non-symmetrical (mebudipine) and symmetrical (dibudipine) analogues of nifedipine, is described herein. The pharmacological potencies of the compounds were evaluated by studying their effects on the contractions of isolated guinea-pig ileum and rat aortic rings. Results were compared with those obtained from nifedipine. The new analogues and nifedipine inhibited the contractile response of guinea-pig ileum to electrical stimulation and the pIC50 value of the compounds did not differ significantly from each other. The compounds also antagonized the contractile responses of K+-depolarized guinea-pig ileum to cumulative concentrations of calcium. The inhibitory effect of mebudipine was significantly higher than that of nifedipine whereas the inhibitory effects of dibudipine and nifedipine were not different. All three compounds relaxed KCl (40 mM)-treated isolated aortic rings; the pIC50 values for relaxation were: mebudipine > nifedipine > dibudipine. It is concluded that these new dihydropyridine derivatives are potent relaxants of vascular and ileal smooth muscles and therefore have high potential for use as antihypertensive and anti-anginal agents.

Effects of mebudipine and dibudipine, two new calcium-channel blockers, on rat left atrium, rat blood pressure and human internal mammary artery.

Mebudipine and dibudipine are two new dihydropyridine calcium-channel blockers that have been synthesized in our laboratory. In a previous study, they showed considerable relaxant effect on vascular and ileal smooth muscle. Here, the pharmacological effects of mebudipine and dibudipine on isolated rat left atrium, rat blood pressure and isolated human internal mammary artery are described. Results are compared with those obtained for nifedipine. Mebudipine and dibudipine reduced contraction force of rat left atrium (pIC30 values: 5.37+/-0.13 and 5.49+/-0.15, respectively) but their negative inotropic effects were significantly weaker than that of nifedipine (pIC30 value: 6.63+/-0.11). Mebudipine and dibudipine lowered rat blood pressure. The hypotensive effect of mebudipine was similar to that of nifedipine while dibudipine was weaker than nifedipine. It was found that the half-life of the hypotensive action of dibudipine (41.91+/-3.77 min, 31.13+/-2.26 min and 28.20+/-4.37 min at 2, 4 and 8 mg kg(-1) orally administered doses, respectively) was longer than that of nifedipine (11.85+/-2.88 min, 16.65+/-2.42 min and 14.03+/-0.10 min at the same doses, respectively). Also, it appeared that mebudipine had a slower rate of absorption compared with nifedipine (the time to reach peak hypotensive action at 2, 4 and 8 mg kg(-1) orally administered doses were, respectively, 24.00+/-6.96 min, 23.75+/-2.39 min and 15.00+/-2.04 min for mebudipine and 7.80+/-0.86 min, 13.75+/-3.15 min and 833+/-0.88 min for nifedipine). The two new compounds, as well as nifedipine, relaxed KCl-treated isolated human internal mammary artery (pEC50 values; 7.87+/-0.12, 7.22+/-0.24 and 7.67+/-0.12 for mebudipine, dibudipine and nifedipine, respectively). The relaxant effects of mebudipine and dibudipine did not show any significant difference compared with that of nifedipine. It is concluded that these new compounds are weak cardiodepressants and, with due attention to its significant vasorelaxant action, mebudipine is a vasoselective compound. In addition, these two compounds have potent blood pressure lowering effects. Also, their vasorelaxant action can be reproduced in human vascular preparations.

Metabolism of the dihydropyridine calcium channel blockers mebudipine and dibudipine by isolated rat hepatocytes.

The prototype 1,4-dihydropyridine (1,4-DHP) nifedipine, indicated for the management of hypertension and angina pectoris, has drawbacks of rapid onset of vasodilating action and a short half-life. Several newer analogues have been designed to offset these problems and these include mebudipine and dibudipine. These analogues contain t-butyl substituents that have been selected to alter the fast metabolism without altering pharmacological activity. In this study, the metabolism of mebudipine and dibudipine by isolated rat hepatocytes has been investigated. These compounds were extensively metabolized in 2 h by oxidative pathways, analogous to those known for nifedipine, and by O-glucuronidation after hydroxylation of the t-butyl substituents. The in-vitro half-lives of mebudipine (22 +/- 7.1 min) and dibudipine (40 +/- 9.8 min) were significantly longer than that of nifedipine (5.5 +/- 1.1 min), which was investigated in parallel in this study. These newer 1,4-DHPs address the problem of the short half-life of nifedipine and have potential for further development in view of their comparable potency to nifedipine.

Resolution of 4-amino-cyclopentanecarboxylic acid methyl esters using hydrolytic enzymes.

A number of esterases (EC 3.1.1.1) and lipases (EC 3.1.1.3) of microbial and mammalian origin were screened for the ability to resolve racemic 4-amino-cyclopentanecarboxylic acid methyl ester derivatives as potential intermediates in the production of carbocyclic nucleosides. Surprisingly, functionalization of the remote amino group had a profound effect on both the rate and enantioselectivity of hydrolysis of the methyl ester. 4-(Benzoylamino)-2-cyclopentenecarboxylic acid, methyl ester (V) with pig liver esterase gave the highest enantioselectivity. The residual ester, which was of the correct absolute stereochemistry [(+) 1S, 4R] for carbocyclic nucleoside synthesis, could be obtained in high optical purity. Optimization of pH, solvent type, and concentration improved the enantioselectivity of the process by a further twofold.

An efficient process for production of N-acetylneuraminic acid using N-acetylneuraminic acid aldolase.

N-acetyl-D-neuraminic acid (Neu5Ac) aldolase (EC 4.1.3.3) has bee reported for synthesis of Neu5Ac,1-5 but there are no reports of processes which do not have significant drawbacks for large-scale operation. Here, Neu5Ac aldolase from an overexpressing recombinant strain of Escherichia coli has been used to develop an immobilized enzyme process for production of Neu5Ac. The enzyme was immobilized onto Eupergit-C and could be reused many times in the reaction. Base-catalyzed epimerization of N-acetyl-D-glucosamine (GlcNAc) yielded GlcNAc/N-acetyl-D-mannosamine (ManNAc) mixtures (c 4:1) which could be used directly in the aldolase reaction; however, inhibition of the enzyme by GlcNAc limited the concentration of ManNAc which could be used in the reaction by this approach. This necessitated the addition of a large molar excess of pyruvate (five- to seven-fold) to drive the equilibrium over to Neu5Ac; nevertheless, a method has been developed to remove the excess pyruvate effectively by complexation with bisulfite, thus allowing Neu5Ac to be recovered by absorption onto an anion-exchange resin. In a second approach, a method has been developed to enrich GlcNAc/ManNAc mixtures for ManNAc. ManNAc can be used at high concentrations in the reaction, thus obviating the need to use a large molar excess of pyruvate. Neu5Ac can be isolated from such reaction mixtures by a simple crystallization. This work shows the importance of integrated process solutions for the effective scale-up of biotransformation reactions.

Enzymatic production of optically pure (2'R-cis)-2'-deoxy-3'-thiacytidine (3TC, lamivudine): a potent anti-HIV agent.

Although equipotent in terms of antiviral activity, the two enantiomers of 2'-deoxy-3'-thiacytidine (BCH 189) differ markedly in their cytotoxicity. (2'R-cis)-2'-deoxy-3'-thiacytidine (3TC) is substantially less toxic than its optical antipode, and is undergoing development for the therapy of HIV infection. Cytidine deaminase from Escherichia coli is shown here to deaminate 2'-deoxy-3'-thiacytidine enantioselectively to leave 3TC essentially optically pure. This reaction has been used to develop a process for production of 3TC in multikilogram amounts. The production of cytidine deaminase was enhanced by strain improvement, fermentation development, and finally by cloning and overexpression of the gene. The enzyme was immobilized on Eupergit-C, which allowed it to be reused many times. The biotransformation conditions were optimized so that the best use could be made of the catalyst. A robust scaleable product isolation process was developed to yield the crystalline product. Overall, yields through the resolution process of 76% were obtained. All aspects of this process are capable of substantial further scaleup with only minor modifications.