Enhanced Dissolution of 7-ADCA in the Presence of PGME for Enzymatic Synthesis of Cephalexin.

Enzymatic catalysis has been recognized as a green alternative to classical chemical route for synthesis of cephalexin (CEX). However, its industrial practice has been severely limited by the low productivity due to the low solubility of 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) and high hydrolysis of D-phenylglycine methyl ester (PGME). In this work, the enhanced dissolution of 7-ADCA in the presence of PGME for efficient enzymatic synthesis of CEX was investigated. Results showed that the solubility of 7-ADCA in water could be improved by PGME. Moreover, supersaturated solution of 7-ADCA could be created in the presence of PGME by a pH shift strategy. The supersaturated solution of 7-ADCA possess good stability, which could be explained in terms of the inhibition of 7-ADCA precipitation due to the presence of PGME. The interaction between 7-ADCA and PGME is explored by spectroscopic determination and DFT analysis and the mechanism of enhanced dissolution of 7-ADCA in the presence of PGME is discussed and proposed. The feasibility of supersaturated solution of 7-ADCA for the enzymatic synthesis of CEX is evaluated. It was demonstrated that high conversion ratio (> 95.0%) and productivity (> 240.0 mmol/L/h) was obtained under a wide range of reaction conditions, indicating that the supersaturated solution system was highly superior to conventional homogeneous solution system. The information obtained in this work will be helpful to industrial production of CEX via enzymatic route.

Transfer of certain beta-lactam antibiotics from cow's milk to fresh cheese and whey.

The aim of this study was to investigate the transfer of cephalexin, penicillin-G, and ampicillin & cloxacillin from cow's milk to cheese and whey. For this purpose, raw milk was artificially contaminated to different antibiotic levels and then heat-treated to prepare fresh cheese from it. Antibiotic levels of the milk, whey and cheese were measured with LC-MS/MS. The extent of heat degradation was not sufficient to remove the antibiotic residues from milk. Antibiotic concentrations in whey and fresh cheese were in good accordance with the concentration of the same compound in milk suggesting that contamination of the milk will result in contamination of the product. The investigated antibiotics were transferred less into the cheese curd (1.6-12.5% of the original amount), than into the whey (33.2-74.1%). For penicillin-G even 100% (complete removal) was experienced.

Efficient enzymatic synthesis of cephalexin in suspension aqueous solution system.

An efficient method for the enzymatic synthesis of cephalexin (CEX) from 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) and d-phenylglycine methyl ester (PGME) using immobilized penicillin G acylase (IPGA) as catalyst in a suspension aqueous solution system was developed, where the reactant 7-ADCA and product CEX are mainly present as solid particles. The effects of key factors on the enzymatic synthesis were investigated. Results showed that continuous feeding of PGME was more efficient for the synthesis of CEX than the batch mode. Under the optimized conditions, the maximum 7-ADCA conversion ratio of 99.3% and productivity of 200 mmol/L/H were achieved, both of which are much superior to the homogeneous aqueous solution system. Besides, IPGA still retained 95.4% of its initial activity after 10 cycles of enzymatic synthesis, indicating the excellent stability of this approach. The developed approach shows great potential for the industrial production of CEX via an enzyme-based route.

Adsorption of cephalexin in aqueous media by graphene oxide: kinetics, isotherm, and thermodynamics.

The present study proposes the synthesis and characterization of graphene oxide (GO) and its application in the adsorption of the antibiotic cephalexin (CFX) in aqueous solution. The characterization of graphene oxide was obtained by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and zeta potential. The influence of pH on the batch adsorption process was investigated by analysing adsorption equilibrium isotherms and adsorption kinetics. The images obtained by SEM and TEM presented the typical morphology attributed to GO sheets. The kinetic adsorption tests showed that equilibrium was reached in 420 min, and an adsorption capacity of 164 mg g-1 was obtained. The models that best fit the experimental data were pseudo-second as well as the Langmuir isotherm. Therefore, GO was effective for removing the CFX antibiotic from aqueous solution by using a batch adsorption process.

Photocatalytic degradation of cephalexin by ZnO nanowires under simulated sunlight: Kinetics, influencing factors, and mechanisms.

Increasing concentrations of anthropogenic antibiotics and their metabolites in aqueous environments has caused growing concerns over the proliferation of antibiotic resistance and potential adverse impacts to agro-environmental quality and human health. Photocatalysis using novel engineered nanomaterials such as ZnO nanowires may be promising for removing antibiotics from waters. However, much remains to be learned about efficiency and mechanism for photocatalytic degradation of antibiotics by ZnO nanowires. This study systematically investigated photodegradation of cephalexin using ZnO nanowires under simulated sunlight. The degradation efficiency of cephalexin was substantially increased in the presence of ZnO nanowires especially at circumneutral and alkaline condition (solution pH of 7.2-9.2). The photodegradation followed the first-order kinetics with degradation rate constants (k) ranging between 1.19 × 10-1 and 2.52 × 10-1 min-1 at 20-80 mg L-1 ZnO nanowires. Radical trapping experiments demonstrated that hydroxyl radicals (OH) and superoxide radicals (O2-) predominantly contributed to the removal of cephalexin. With the addition of HCO3- (1-5 mM) or Suwannee River natural organic matter (SRNOM, 2-10 mg L-1), the k values were substantially decreased by a factor of 1.8-70 to 1.69 × 10-3-6.67 × 10-2 min-1, probably due to screening effect of HCO3- or SRNOM sorbed on ZnO nanowires and scavenging of free radicals by free HCO3- or SRNOM in solution. Combining product identification by mass spectrometry and molecular computation, cephalexin photodegradation pathways were identified, including hydroxylation, demethylation, decarboxylation, and dealkylation. Overall, the novel ZnO nanowires have the potential to be used for removing antibiotics from contaminated waters.

Preparation, physicochemical properties, in vitro evaluation and release behavior of cephalexin-loaded niosomes.

In this study, optimized cephalexin-loaded niosomal formulations based on span 60 and tween 60 were prepared as a promising drug carrier system. The niosomal formulations were characterized using a series of techniques such as scanning electron microscopy, Fourier transformed infrared spectroscopy, dynamic light scattering, and zeta potential measurement. The size and drug encapsulation efficiency are determined by the type and composition of surfactant. The developed niosomal formulations showed great storage stability up to 30 days with low change in size and drug entrapment during the storage, making them potential candidates for real applications. Moreover, the prepared niosomes showed negligible cytotoxicity for HepG2 cells, measured by MTT assay. The antibacterial properties of cephalexin-loaded niosome were investigated using S. aureus and E. coli as gram-positive and gram-negative bacteria, respectively. The results showed that the encapsulation of antibiotic drug in niosomal formulation could enhance the antibacterial efficiency of the drug, where the minimum inhibitory concentration was droped from 8 µg/mL (cephalexin) to 4 µg/mL (cephalexin-loaded niosome) and from 4 µg/mL (cephalexin) to 1 µg/mL (cephalexin-loaded niosome) against E. coli and S. aureus, respectively. The findings of our study show that the improvement of cephalexin bioavailability and prolonged drug release profile could be obtained by niosomal formulation as a favorable antibiotic drug delivery system.

DNA binding studies of antibiotic drug cephalexin using spectroscopic and molecular docking techniques.

The purpose of this study was to explore an accurate characterization of the binding interaction of antibiotic drug cephalexin with calf thymus DNA (CT-DNA) as a relevant biological target by using UV absorption, fluorescence spectroscopy and circular dichroism (CD) in vitro under simulated physiological conditions (pH = 7.4) and also through a molecular modeling study. The results showed that the drug interacts with the DNA helix via a minor groove binding mode. The thermodynamic parameters were calculated and showed that the reaction between the drug and CT-DNA was exothermic. In addition, the drug enforced traceable changes in the viscosity of DNA. The molecular modeling results indicated that cephalexin forcefully binds to the minor groove of DNA with a relative binding energy of -21.02 kJ mol-1. The obtained theoretical results were in good agreement with those obtained from experimental studies.

Electrochemical Sensor Based on Molecularly Imprinted Polymer for the Detection of Cefalexin.

In this study, a new electrochemical sensor was developed for the detection of cefalexin (CFX), based on the use of a molecularly imprinted polymer (MIP) obtained by electro‒polymerization in an aqueous medium of indole-3-acetic acid (I3AA) on a glassy carbon electrode (GCE) and on boron-doped diamond electrode (BDDE). The two different electrodes were used in order to assess how their structural differences and the difference in the potential applied during electrogeneration of the MIP translate to the performances of the MIP sensor. The quantification of CFX was performed by using the electrochemical signal of a redox probe before and after the rebinding of the template. The modified electrode was characterized using atomic force microscopy (AFM), scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The influence of different parameters on the fabrication of the sensor was tested, and the optimized method presented high selectivity and sensitivity. The MIP-based electrode presented a linear response for CFX concentration range of 10 to 1000 nM, and a limit of detection of 3.2 nM and 4.9 nM was obtained for the BDDE and the GCE, respectively. The activity of the sensor was successfully tested in the presence of some other cephalosporins and of other pharmaceutical compounds. The developed method was successfully applied to the detection of cefalexin from real environmental and pharmaceutical samples.

Single-step pyrolysis of phosphoric acid-activated chitin for efficient adsorption of cephalexin antibiotic.

Cephalexin (CFX) antibiotic, a potent pharmaceutical water pollutant, was efficiently removed by activated carbon (AC) derived from a single-step pyrolysis of phosphoric acid-activated chitin. Experimental conditions such as temperature, CFX initial concentration, and solution pH were screened in batch adsorption. Phosphoric acid activation of chitin and subsequent pyrolysis tailored the Brunauer-Emmett-Teller surface area, total pore volume, and average pore diameter to 1199.02 m2/g, 0.641 cm3/g, and 21.37 Å, respectively. The Langmuir isotherm adequately described the equilibrium data for CFX adsorption on chitin-AC, with an R2 of 0.99 and a monolayer capacity of 245.19 mg/g at 50 °C. Chitin-AC showed higher adsorption capacity compared with other ACs derived from industrial and agricultural precursors. When activated by phosphoric acid, chitin-AC featured functional multi-sites for vast antibiotic adsorption treatment. Overall, chitin-AC could be a promising adsorbent for removal of CFX.

Effect of surface properties of activated carbon fiber cathode on mineralization of antibiotic cefalexin by electro-Fenton and photoelectro-Fenton treatments: Mineralization, kinetics and oxidation products.

Solutions of 200 mg L-1 cefalexin (CLX), an antibiotic with high usage frequency and biodegradation resistance, have been comparatively degraded by electro-Fenton (EF) and photoelectro-Fenton (PEF) processes using two kinds of activated carbon fiber (ACF) cathodes with different physical properties. These two ACFs shared similar pore volumes and pore diameters but varied BET surface areas, which were confirmed to be 0.5210 cm3 g-1, 2.26 nm and 921 m2 g-1 for ACF1, while 0.6508 cm3 g-1, 2.16 nm and 1206 m2 g-1 for ACF2, respectively. Their oxidation abilities were comparatively assessed in terms of degradation kinetics and mineralization rates, which increased in the order: ACF1-EF < ACF2-EF < ACF1-PEF < ACF2-PEF. These results confirmed the superiority of ACF with higher surface area, which was correlated to faster H2O2 and OH accumulation in more reaction sites provided. After 120 min electrolysis, ACF1 exhibited 1510 µM H2O2 and 37 µM OH accumulation, while ACF2 generated 1934 µM H2O2 and 85 µM OH. Moreover, ACF cathode with more developed pore structure also revealed faster formation of degradation by-products like inorganic ions (NH4+ and NO3- ions) and short-chain carboxylic acids (acetic, formic, oxamic and oxalic acids), as well as enhanced removal for partial acids. In order to gain a deeper understanding of degradation mechanisms for ACF2-PEF system, evolutions of six aromatic by-products generated from sulfoxidation, hydroxylation and decarboxylation were confirmed by UPLC-QTOF-MS/MS determination. Based on the above identifications of the degradation intermediates, a plausible reaction pathway for CLX removal was proposed.

Drug-Loaded Halloysite Nanotube-Reinforced Electrospun Alginate-Based Nanofibrous Scaffolds with Sustained Antimicrobial Protection.

Halloysite nanotube (HNT)-reinforced alginate-based nanofibrous scaffolds were successfully fabricated by electrospinning to mimic the natural extracellular matrix (ECM) structure which is beneficial for tissue regeneration. An antiseptic drug, cephalexin (CEF)-loaded HNT, was incorporated into the alginate-based matrix to obtain sustained antimicrobial protection and robust mechanical properties, the key criteria for tissue engineering applications. Electron microscopic imaging and drug release studies revealed that CEF had penetrated into the lumen space of the HNT and also deposited on the outer walls, with a total loading capacity of 30 wt %. Moreover, the diameter of alginate-based nanofibers of the scaffolds ranged from 40 to 522 nm with well-aligned HNTs, resulting in superior mechanical properties. For instance, the addition of 5% (w/w) HNT improved the tensile strength (σ) and elastic modulus by 3-fold and 2-fold, respectively, compared to those of the alginate-based scaffolds without HNT. The fabricated scaffolds exhibited remarkable antimicrobial properties against both Gram-negative and Gram-positive bacteria, and the cytotoxicity studies confirmed the nontoxicity of the fabricated scaffolds. Drug release kinetics showed that CEF inside HNTs diffuses within 24 h and that the diffusion of the drug is delayed by 7 days once the CEF-loaded HNTs are incorporated into the alginate-based nanofibers. These fabricated alginate-based electrospun scaffolds with enhanced mechanical properties and sustained antimicrobial protection hold great potential to be used as artificial ECM scaffolds for tissue engineering applications.

Coordination and redox interactions of ß-lactam antibiotics with Cu2+ in physiological settings and the impact on antibacterial activity.

An increase in the copper pool in body fluids has been related to a number of pathological conditions, including infections. Copper ions may affect antibiotics via the formation of coordination bonds and/or redox reactions. Herein, we analyzed the interactions of Cu2+ with eight ß-lactam antibiotics using UV-Vis spectrophotometry, EPR spectroscopy, and electrochemical methods. Penicillin G did not show any detectable interactions with Cu2+. Ampicillin, amoxicillin and cephalexin formed stable colored complexes with octahedral coordination environment of Cu2+ with tetragonal distortion, and primary amine group as the site of coordinate bond formation. These ß-lactams increased the solubility of Cu2+ in the phosphate buffer. Ceftazidime and Cu2+ formed a complex with a similar geometry and gave rise to an organic radical. Ceftriaxone-Cu2+ complex appears to exhibit different geometry. All complexes showed 1:1 stoichiometry. Cefaclor reduced Cu2+ to Cu1+ that further reacted with molecular oxygen to produce hydrogen peroxide. Finally, meropenem underwent degradation in the presence of copper. The analysis of activity against Escherichia coli and Staphylococcus aureus showed that the effects of meropenem, amoxicillin, ampicillin, and ceftriaxone were significantly hindered in the presence of copper ions. The interactions with copper ions should be taken into account regarding the problem of antibiotic resistance and in the selection of the most efficient antimicrobial therapy for patients with altered copper homeostasis.

Oxidation of Cefalexin by Permanganate: Reaction Kinetics, Mechanism, and Residual Antibacterial Activity.

The oxidation of cefalexin (CFX), a commonly used cephalosporin antibiotic, was investigated by permanganate (PM) in water. Apparent second-order rate constant of the reaction between CFX and PM was determined to be 12.71 ± (1.62) M-1·s-1 at neutral pH. Lower pH was favorable for the oxidation of CFX by PM. The presence of Cl- and HCO3- could enhance PM-induced oxidation of CFX, whereas HA had negligible effect on CFX oxidation by PM. PM-induced oxidation of CFX was also significant in the real wastewater matrix. After addition of bisulfite (BS), PM-induced oxidation was significantly accelerated owing to the generation of Mn(III) reactive species. Product analysis indicated oxidation of CFX to three products, with two stereoisomeric sulfoxide products and one di-ketone product. The thioether sulfur and double bond on the six-membered ring were the reactive sites towards PM oxidation. Antibacterial activity assessment indicated that the activity of CFX solution was significantly reduced after PM oxidation.

Oxidation of cefalexin by thermally activated persulfate: Kinetics, products, and antibacterial activity change.

While the widely used ß-lactam antibiotics, such as cephalosporins, are known to be susceptible to oxidation by sulfate radical (SO4-), comprehensive study about SO4--induced oxidation of cephalosporins is still limited, such as the impact of water matrices, and the structure and antibacterial activity of transformation products. Herein, the oxidation of cefalexin (CFX), a most frequently detected cephalosporin, was systematically investigated by thermally activated persulfate (PS). CFX oxidation followed pseudo-first-order kinetics, and SO4- dominantly contributed to the overall oxidation of CFX. The impact of water matrices, such as Cl-, HCO3- and natural organic matter, on CFX degradation was predicted using a pseudo-steady-state kinetic model. The secondary reactive species, such as chlorine and carbonate radicals, were found to contribute to CFX degradation. Product analysis indicated oxidation of CFX to six products (molecular weight of 363), with two stereoisomeric sulfoxides as the primary oxidation products. It was thus suggested that the primary amine on the side chain, and the thioether sulfur and double bond on the six-membered ring were the reactive sites of CFX towards SO4- oxidation. Antibacterial activity assessment showed that the biological activity of CFX solution was significantly diminished after treatment by the thermally activated PS.

Synthesis and characterization of basil seed mucilage coated Fe3O4 magnetic nanoparticles as a drug carrier for the controlled delivery of cephalexin.

A novel drug delivery system, loaded the drug cephalexin on the basil seed mucilage coated magnetic nanoparticles (Fe3O4@BSM-CPX) was prepared and characterized by means of X-ray diffraction (XRD), Furier Transform Infrared (FTIR), Field Emission Scanning Electron Microscope (FESEM), Vibrating Sample Magnetometer (VSM), Transmission Electron Microscopy (TEM), and Anti-bacterial, and Specific Surface (BET). By comparing the size of the uncoated nanoparticles (12nm) and the size of the coated magnetite nanoparticles (6nm), it was found that with the mucilage coating being put on the magnetite nanoparticles, the size of the nanoparticle cores has also decreased. The optimum pH results showed that the higher adsorption capacity occurs when cephalexin is cationic at pH2.5 because the NH3+ group of cephalexin interacts better with negative functional groups of the basil seed mucilage. Disk Diffusion Anti-Bacterial test showed that the loading of CPX on the Fe3O4@BSM nanocarrier, not only does not have any negative effects on the structure and performance of the drug, but also increases the antibacterial properties of CPX. Furthermore, the in vitro release of Fe3O4@BSM-CPX nanocomposites showed an initial burst release in the first 18h, followed by a more gradual and sustained release for 120h.

Detection of the iron complexes with hydrolysis products of cephalexin and cefradine upon high-performance liquid chromatography/electrospray ionization mass spectrometry analysis.

RATIONALE: Cephalosporins (e.g. cephalexin, cefradine) are a major group of widely used ß-lactam antibiotics. Hydrolysis of the ß-lactam ring is an important reaction (often undesired) which leads to deactivation of ß-lactams. To the best of our knowledge there is no electrospray ionization mass spectrometry (ESI-MS) data reported concerning the products of hydrolysis of cephalosporins. METHODS: The hydrolysis of cephalexin and cefradine was performed in aqueous NaOH solutions. After the process the solutions were analyzed by high-performance liquid chromatography (HPLC)/ESI-MS. The elemental compositions of the ions discussed were confirmed by the accurate mass measurements on a quadrupole time-of-flight (QTOF) mass spectrometer. RESULTS: Unexpectedly, complexes between the hydrolysis products of cephalexin and cefradine (CFLh and CFRh ) and iron cation were detected upon HPLC/ESI-MS analysis, namely the ions [(CFLh -H)2 +Fe]+ and [(CFRh -H)2 +Fe]+ , although iron was not added to the analyzed solutions or to the mobile phase. These ions were found to be very stable in the gas phase. CONCLUSIONS: The detection of the complexes between the hydrolysis products of cephalosporins and iron may have a positive impact on the sensitivity and specificity of HPLC/ESI-MS analyses of the hydrolysis products of some cephalosporins.

Green synthesis of nano-zero-valent iron from Nettle and Thyme leaf extracts and their application for the removal of cephalexin antibiotic from aqueous solutions.

In this study, the removal of cephalexin (CEX) antibiotic from aqueous solution was examined using a novel green adsorbent without employing any toxic chemicals or capping agents. Nettle and Thyme extracts were used to synthesize novel nano-zero-valent iron (NNZVI and TNZVI) for the adsorption of CEX. The nature and morphology of synthesized adsorbent were characterized by Transmission electron microscopy, scanning electron microscope, X-ray diffraction and Fourier transform infrared spectroscopy spectroscopy. Batch experiments were performed to study the influence of various experimental parameters such as contact time, initial concentration of the CEX, solution pH and adsorbent dosage. The adsorption isotherms of CEX by NNZVI and TNZVI were found to fit well with Freundlich and Langmuir models, respectively. The maximum adsorption capacity of CEX onto NNZVI and TNZVI were observed as 1667 and 1428 mg/g, respectively, based on the Langmuir model. The adsorption trend followed the pseudo-first-order kinetics model and equilibrium could be established in about two hours for both adsorbents. The developed nanoparticles in this study have considerable potential for the removal of CEX and could be considered as a promising adsorbent for the removal of other antibiotics also from aqueous solutions.

Surface restructuring of red mud to produce FeO x (OH) y sites and mesopores for the efficient complexation/adsorption of ß-lactam antibiotics.

In this work, iron oxide in the red mud (RM) waste was restructured to produce mesopores with surface [FeO x (OH) y ] sites for the efficient complexation/adsorption of ß-lactam antibiotics. Red mud composed mainly by hematite was restructured by an acid/base process followed by a thermal treatment at 150-450 °C (MRM150, MRM200, MRM300, and MRM450) and fully characterized by Mössbauer, XRD, FTIR, BET, SEM, CHN, and thermogravimetric analyses. The characterization data showed a highly dispersed Fe3+ oxyhydroxy phase, which was thermally dehydrated to a mesoporous α-Fe2O3 with surface areas in the range of 141-206 m2 g-1. These materials showed high efficiencies (21-29 mg g-1) for the adsorption of ß-lactam antibiotics, amoxicillin, cephalexin, and ceftriaxone, and the data was better fitted by the Langmuir model isotherm (R 2 = 0.9993) with monolayer adsorption capacity of ca. 39 mg g-1 for amoxicillin. Experiments such as competitive adsorption in the presence of phosphate and H2O2 decomposition suggested that the ß-lactamic antibiotics might be interacting with surface [FeO x (OH) y ] species by a complexation process. Moreover, the OH/Fe ratio, BET surface area and porosity indicated that this complexation is occurring especially on [FeO x (OH) y ]surf sites contained in the mesopore space.

Probing the interaction of cephalosporin with bovine serum albumin: A structural and comparative perspective.

Cephalosporins belong the largest class of antibiotics used in the treatment of a wide range of infectious diseases caused by susceptible organisms. In the present study, we chose two typical antibiotics cefalexin/cefixime based on their structure, and investigated the interaction of cephalexin/cefixime with bovine serum albumin (BSA) using UV-vis absorption spectra, fluorescence spectroscopy, circular dichroism (CD) spectroscopy and molecular modeling approaches. Spectroscopic experiments revealed the formation of a BSA - cefalexin/cefixime complex. The binding parameters calculated using a modified Stern - Volmer method and the Scatchard method reached 103 -104  L·mol-1 . Thermodynamic parameter studies revealed that binding characteristics by negative enthalpy and positive entropy changes, and electrostatic interactions play a major role. Site marker competitive displacement experiments and molecular modeling approaches demonstrated that cefalexin and cefixime bind with appropriate affinity to site I (subdomain IIA) of BSA. Furthermore, synchronous fluorescence spectra, CD spectra and molecular modeling results indicated that the secondary structure of BSA was changed in the presence of cefalexin and cefixime. Additionally, the effects of metal ions on the BSA - cefalexin/cefixime system were also assessed.

Hydrolysis of cephalexin and meropenem by New Delhi metallo-ß-lactamase: the substrate protonation mechanism is drug dependent.

Emergence of antibiotic resistance due to New Delhi metallo-ß-lactamase (NDM-1) bacterial enzymes is of great concern due to their ability to hydrolyze a wide range of antibiotics. There are ongoing efforts to obtain the atomistic details of the hydrolysis mechanism in order to develop inhibitors for NDM-1. In particular, it remains elusive how drug molecules of different families of antibiotics are hydrolyzed by NDM-1 in an efficient manner. Here we report the detailed molecular mechanism of NDM-1 catalyzed hydrolysis of cephalexin, a cephalosporin family drug, and meropenem, a carbapenem family drug. This study employs molecular dynamics (MD) simulations using hybrid quantum mechanical/molecular mechanical (QM/MM) methods at the density functional theory (DFT) level, based on which reaction pathways and the associated free energies are obtained. We find that the mechanism and the free energy barrier for the ring-opening step are the same for both the drug molecules, while the subsequent protonation step differs. In particular, we observe that the mechanism of the protonation step depends on the R2 group of the drug molecule. Our simulations show that allylic carbon protonation occurs in the case of the cephalexin drug molecule where Lys211 is the proton donor, and the proton transfer occurs via a water chain formed (only) at the ring-opened intermediate structure. Based on the free energy profiles, the overall kinetics of drug hydrolysis is discussed. Finally, we show that the proposed mechanisms and free energy profiles could explain various experimental observations.