Organic Solvent Content Research Articles

Solvent induced conformational changes for the altered activity of laccase: A molecular dynamics study

The growing demands of solvent-based industries like paint, pharmaceutical, petrochemical, paper and pulp, etc., have directly increased the release of effluents that are rich in hazardous aromatic compounds in the environment. A sustainable biotechnological approach utilizing laccases as biocatalyst enable in biodegradation of these aromatic toxin-rich effluents. However, this enzymatic process is ineffective as laccases lose their stability and catalytic activity at high organic solvent concentrations. In this study, molecular dynamic simulations of a novel solvent tolerant laccase, DLac from Cerrena sp. RSD1 was performed to explore the molecular-level understanding of DLac in 30%(v/v) acetone and acetonitrile. Solvent-induced conformational changes were analyzed via protein structure network, which was illustrated with respect to cliques and communities. In the presence of acetonitrile, the cliques around the active site and substrate-binding site were disjoined, thus the communities lost their network integrity. Whereas with acetone, the community near the substrate-binding site gained new residues and formed a rigidified network that corresponded to enhanced DLac’s activity. Moreover, prominent solvent binding sites were speculated, which can be probable mutation targets to further improve solvent tolerance and catalytic activity. The molecular basis behind solvent induced catalytic activity will further aid in engineering laccase for its industrial application.

Biochemical properties and repression studies of an alkaline serine protease from a haloalkaliphilic actinomycete, Nocardiopsis dassonvillei subsp. albirubida OK-14

A marine haloalkaliphilic actinomycete identified as Nocar diopsis dassonvillei subsp. albirubida (strain OK-14) was screened for its ability to produce extracellular alkaline protease under various physical conditions pertinent to different applications. The strain utilized amino acids as the sole source of nitrogen, displaying repression and de-repression phenomenon above the threshold levels. The activity of the partially purified alkaline protease was 1421.7 U/ml/min. The enzyme was optimally active at pH 10 and 60 °C. The activity was assessed under various concentrations of NaCl, organic solvents and metal ions. The partially purified alkaline protease was then examined for its suitability in blood de-staining, gelatine degradation and keratinase activity, implicating its relevance in detergent industries, gelatine extraction from X-ray films and degradation of bio-wastes. • Repression and de-repression of protease by different amino acids. • Protease stability in solvents, metal ions and various other agents. • Highly efficient in gelatin deproteinization and silver recovery. • Compatible as detergent supplement.

Eco-Friendly and Scalable Synthesis of Fullerenols with High Free Radical Scavenging Ability for Skin Radioprotection.

Radiation dermatitis is a common but torturous side effect during radiotherapy, which greatly decreases the life quality of patients and potentially results in detrimental cessation of tumor treatment. Fullerenol, known as "free radical sponge," is a great choice for skin radioprotection because of its broad-spectrum free radical scavenging performance, good chemical stability, and biosafety. In this work, a facile scalable and eco-friendly synthetic method of fullerenols by catalyst assistant mechanical chemistry strategy is provided. As no organic solvent or high concentration of acid and alkali is introduced to this synthetic system, large-scale (>20g) production of fullerenols with high yield (>95%) is obtained and no complicated purification is required. Then, the skin radioprotective performance of fullerenols is systematically explored for the first time. In vitro results indicate that fullerenols significantly block the reactive oxygen species-induced damage and enhance the viability of irradiated human keratinocyte cells. In vivo experiments suggest that medical sodium hyaluronate hydrogels loaded with fullerenols are suitable for skin administration and powerfully mitigate radiodermatitis via effectively protecting epidermal stem cells. The work not only provides an efficient gram-scale and eco-friendly synthetic method of fullerenols, but also promotes the development of fullerenols as potential skin radioprotectors.

Effect of Crystallinity on the Properties of Polycaprolactone Nanoparticles Containing the Dual FLAP/mPEGS-1 Inhibitor BRP-187.

Seven polycaprolactones (PCL) with constant hydrophobicity but a varying degree of crystallinity prepared from the constitutional isomers ε-caprolactone (εCL) and δ-caprolactone (δCL) were utilized to formulate nanoparticles (NPs). The aim was to investigate the effect of the crystallinity of the bulk polymers on the enzymatic degradation of the particles. Furthermore, their efficiency to encapsulate the hydrophobic anti-inflammatory drug BRP-187 and the final in vitro performance of the resulting NPs were evaluated. Initially, high-throughput nanoprecipitation was employed for the εCL and δCL homopolymers to screen and establish important formulation parameters (organic solvent, polymer and surfactant concentration). Next, BRP-187-loaded PCL nanoparticles were prepared by batch nanoprecipitation and characterized using dynamic light scattering, scanning electron microscopy and UV-Vis spectroscopy to determine and to compare particle size, polydispersity, zeta potential, drug loading as well as the apparent enzymatic degradation as a function of the copolymer composition. Ultimately, NPs were examined for their potency in vitro in human polymorphonuclear leukocytes to inhibit the BRP-187 target 5-lipoxygenase-activating protein (FLAP). It was evident by Tukey’s multi-comparison test that the degree of crystallinity of copolymers directly influenced their apparent enzymatic degradation and consequently their efficiency to inhibit the drug target.

Thermophilic iron containing type superoxide dismutase from Cohnella sp. A01

Superoxide dismutases (SODs) (EC 1.15.1.1) are well known antioxidant enzymes that play critical roles in cellular defenses of living organisms against harmful superoxide radicals during oxidative stress. This study details on cloning, biochemical and functional characterization of an iron containing type superoxide dismutase (SOD) from a novel thermophilic bacteria Cohnella sp. A01 (CaSOD). The secondary and three dimensional structure of the protein were predicted. CaSOD gene was subsequently cloned into pET-26b(+) expression vector and expression of the recombinant protein (rCaSOD) was optimized in E. coli BL21 (DE3) and the purified recombinant SOD showed a single band with an apparent molecular weight of 26 kDa by SDS-PAGE. The half-life and thermodynamic parameters including ΔH⁎, ΔS⁎, and ΔG⁎ were 187 min at 60 °C, 7.3 kJ.mol−1, −76.8 kJ.mol−1.°K−1, and 84.1 kJ.mol−1, respectively. The rCaSOD exhibited catalytic activity in a very broad range of pH (6.0–10.0) and temperatures (35–75 °C), as well as stability in a broad pH range, from 3.0 to 11.0, and wide range of temperature, different concentrations of detergent agents, metal ions, organic solvents and other chemicals. The results suggest that this novel enzyme could be used for various industrial applications in cosmetic, food, and pharmaceutical industries.

Recovery of Valuable Solutes from Organic Solvent/Water Mixtures via Direct Contact Membrane Distillation (DCMD) as a Non-Heated Process.

Recently, the demand for the recovery of valuable solutes from organic solvents/water mixtures has increased in various fields. Furthermore, due to the abundance of heat-sensitive valuable solutes, the demand for non-heated concentration technologies has increased. In this study, the direct contact membrane distillation (DCMD) using hydrophobic polyvinylidene difluoride (PVDF) hollow fiber membranes was investigated to confirm the possibility of recovering valuable solutes from organic solvents/water mixtures as a non-heated process. The DCMD with 1000 ppm NaCl aqueous solution achieved 0.8 kg/m2·h of vapor flux and >99.9% of NaCl retention, even at feed and coolant temperatures of 25 and 10 °C, respectively. Furthermore, when DCMD was conducted under various conditions, including feed temperatures of 25, 35 and 45 °C, and organic solvent concentration of 15, 30 and 50 wt%, using ethanol/water and acetonitrile/water mixtures containing 1000 ppm NaCl. A surfactant was also used as a valuable solute, in addition to NaCl. As a result, it was found that the total vapor flux increased with increasing temperature and concentration of organic solvents, as the partial vapor pressure of the organic solvents increased. Additionally, no solute leaked under any condition, even when the surfactant was used as a valuable solute.

Analysis of adsorption energy distribution in selected hydrophilic-interaction chromatography systems with amide, amine, and zwitterionic stationary phases.

Adsorption mechanisms of caffeine, quercetin, and phenol as test substances in various chromatographic systems have been analyzed. The investigations were conducted using three different chromatographic columns packed with polar bonded stationary phases, that is, amide, amine, and zwitterionic. Methanol-water and acetonitrile-water systems with different organic solvent contents have been used as mobile phases. On the basis of adsorption isotherms obtained for the tested systems, Scatchard plots and adsorption energy distributions have been determined. The most likely retention mechanisms have been discussed. The results of investigations indicate that (i) the surfaces of tested adsorbents are energetically heterogeneous, and (ii) the main role in sorption mechanism is played by low-energy sites.

Directed evolution of unspecific peroxygenase in organic solvents.

Fungal unspecific peroxygenases (UPOs) are efficient biocatalysts that insert oxygen atoms into nonactivated C-H bonds with high selectivity. Many oxyfunctionalization reactions catalyzed by UPOs are favored in organic solvents, a milieu in which their enzymatic activity is drastically reduced. Using as departure point the UPO secretion mutant from Agrocybe aegerita (PaDa-I variant), in the current study we have improved its activity in organic solvents by directed evolution. Mutant libraries constructed by random mutagenesis and in vivo DNAshuffling were screened in the presence of increasing concentrations of organic solvents that differed both in regard to their chemical nature and polarity. In addition, a palette of neutral mutations generated by genetic drift that improved activity in organic solvents was evaluated by site directed recombination in vivo. The final UPO variant of this evolutionary campaign carried nine mutations that enhanced its activity in the presence of 30% acetonitrile (vol/vol) up to 23-fold over PaDa-I parental type, and it was also active and stable in aqueous acetone, methanol and dimethyl sulfoxide mixtures. These mutations, which are located at the surface of the protein and in the heme channel, seemingly helped to protect UPO from harmful effects of cosolvents by modifying interactions with surrounding residues and influencing critical loops.

Characteristics of Ultrasonically Enhanced Low-Temperature Thermal Regeneration of Powdered Activated Carbon: A Case Study of Acetone and Aniline

Effective regeneration of powdered activated carbon (PAC) is the key to reduce the operating cost of the PAC in wastewater treatment processes. In this study, volatile acetone and semi-volatile aniline were selected to investigate the regeneration characteristics of ultrasonically enhanced low-temperature thermal process. The results showed that the regeneration efficiency of the PAC that had adsorbed aniline or acetone increased with the increase in ultrasonic power, and optimal value of frequency and regeneration times were determined. The concentration and properties of organic solvents had a significant influence on the ultrasonic regeneration process. With the increase in heating temperature and regeneration time, the regeneration efficiency increased, but the loss of mass of the saturated PAC increased noticeably. With the combination of ultrasonic treatment in a solvent with low temperature heating, the PAC regeneration efficiency was successfully improved, and the PAC mass loss rate was noticeably reduced. The microjet, shock wave, and cavitation effects produced by ultrasonic treatment restored the specific surface area of the PAC, expanded its mesopore volume, and increased the pore diameter. A reasonable selection of the regeneration solution and optimization of the ultrasonic treatment conditions could create favorable conditions for subsequent low temperature thermal regeneration.

Hydrogen from Dark Fermentation of the Organic Fraction of Waste Diapers: Optimization Based on Response Surface Experiments

Waste diapers (WD) handling and disposal in Mexico are typically based on their burial in dumping sites and landfills. Practically reclaiming and recycling of WD are non-existent. The clean diapers are composed of cellulose fibres (37–43% db), hemicellulose (5–9%), lignin (4–7%), protein (<1), plastics (polypropylene and polyethylene) (12–16%), absorbent sodium polyacrylate (14–18%), and elastic and adhesives tapes (9–12%). The latter can be valuable resources. WD composition is similar to clean diaper, although humidity is very high, and the ranges of faeces and urine are 1.5–2.5 and 6–9% dry weight, respectively. International literature searches indicate that there is some research on composting, fungal biodegradation, and methanogenic co-digestion of waste activated sludge with the organic fraction of waste diapers (OFWD.) However, research on dark fermentation of OFWD is limited. In this work, the generation of biohydrogen from dark fermentation of OFWD was optimised. We used the response surface methodology (RSM). Independent variables were the temperature of operation (37–55°C), ratio C/N of the feed (30, 40 gC/gN), and initial total solids of the feed (TSi) (15, 25%). The dependent (response) variables examined were Y’H2 (H2 produced per initial g of dry matter), contents of low molecular weight organic solvents and acids, lactic acid, the ratio A/B (acetic-to-butyric acid), and the quotient organic acids C2 to C4-to-solvents. The predicted maximum Y’H2 occurred at the combination of factors of 43 gC/gN, 12% and 31°C; its value was 2.79 mmolH2/gTS; its experimental validation gave 2.48 mmolH2/gTS, which shows a good agreement between values (11% lower than the predicted value). The maximum of Y’H2 with OFWD compared very favourably with bioH2 values obtained from a wide variety of wastes (organic municipal residues, agricultural wastes, etc.) using the same batch type fermentation with intermittent venting. Interestingly, the predicted temperature optimum fell in the lower side of the mesophilic range. Process heating savings would be in the order of 60.0 and 27.2% for thermophilic and mesophilic operation, respectively. In this way, it would be a contribution to the sustainability of the dark fermentation of OFWD. This result was somewhat counterintuitive and strongly indicates the usefulness of the response surface methodolog for analyzing the experimental results and uncovering favourable, although unexpected conditions.

Food Properties of Lebui Bean Powder Extract Fermented by Rhizopus Sp.

The fermentation that involves the performance of microbes is very effective for breaking the glycoside bonds that bind bioactive compounds in cells without using high temperatures and chemicals, as well as to improve the quality of nutritional components. Bioactive compounds in free form, obtained from the fermentation of black bean powder (Cajanus sp.) need to be extracted and characterized to obtain the types of bioactive compounds that have the potential to be applied in functional foods. Fermented black bean powder used in this experiment is the fermented lebui bean powder that has been fermented using the fungus Rhizopus sp. for two days. These lebui beans are local beans from Lombok Island, West Nusa Tenggara, Indonesia. This experiment aimed to determine the type and concentration of organic solvents, which can maintain the quality of nutritional contents and bioactive compounds of fermented lebui bean powder. The nested design was used. The main factor was the type of solvent (n-hexane or ethanol), while the solvent concentration (70% or 90%) was the second factor and nested in the main factor. The result showed that ethanol 90% was the best organic solvent to extract the bioactive compound in fermented lebui bean powder, and this extract contained protein, lipid, moisture, ash, fiber, and carbohydrates respectively 26.33%; 13.60%; 14.24%; 3.28%; 16.03%; and 26.54%. The bioactive compounds identified were phenolic, flavonoid, and anthocyanin, respectively 78.544 mgGAE/g; 217.358 mgQE/g; and 147.665 ppm.

Citrus Pomace Biomass as a Source of Pectin and Lignocellulose Fibers: From Waste to Upgraded Biocomposites for Mulching Applications.

Citrus pomace derived from the industrial processing of juice and essential oils mostly consists of pectin, cellulose, hemicellulose, and simple sugars. In this work, citrus pomace waste from an agricultural company in South Italy was used as source of pectin. The extraction conditions of the polysaccharide were optimized using a suitable combination of time and a concentration of a mild organic solvent, such as acetic acid; thus recovering high Mw pectin and bioactive molecules (flavonoids and polyphenols). The pectin was structurally (GPC, FTIR), morphologically (SEM), thermally (TGA/DTG), and mechanically characterized, while bioactive molecules were separated and the total phenolic content (TPC) and total flavonoids content (TFC) were evaluated. With the aim to develop novel biocomposite-based materials, the pectin extracted from citrus waste was reinforced with different amounts of lignocellulose fractions also recovered from citrus waste after polysaccharide extraction, according to a “zero waste” circular economy approach. The prepared biocomposites were morphologically and mechanically characterized to be used as biodegradable mulching systems for crop protection. Thus, the citrus waste biomass was recovered, fractionated into its main raw materials, and these were recombined to develop novel upgraded biocomposites for mulching applications, by means of a cost-effective and eco-sustainable approach.

Effective separation of water-DMSO through solvent resistant membrane distillation (SR-MD)

The treatment of organic waste or wastewater with high organic solvent content has been challenging in industries as it cannot be done effectively using conventional wastewater treatment technologies such as biodegradation and advanced oxidation process. Solvent resistant membrane distillation (SR-MD) was proposed as an energy-efficient alternative to treat these waste streams but its application is hampered by the lack of solvent-resistant membranes, and there is a research gap in studying the feeds with water-solvent mixtures. In this work, ceramic tubular membranes with different pore sizes and structures were molecularly grafted with 1H,1H,2H,2H-perfluorodecyltriethoxysilane to obtain hydrophobic ceramic membranes for SR-MD. The modified membranes exhibited excellent hydrophobicity and solvent resistant properties, and they were tested for SR-MD performance with a wide range of dimethyl sulfoxide (DMSO) feed concentrations, from 3.5 to 85 wt%. The membranes exhibited a high DMSO rejection of >98% and the separation factor of >170, with permeation flux >4.4 kg m−2 h−1 when the DMSO concentration in feed was below 65 wt%. The separation performance was found strongly dependent on the evaporation step and the vapour-liquid equilibrium near the interface. The DMSO rejection was also comparable to pervaporation while the permeation flux was much higher at the feed concentration of 50 wt%. This study establishes the strategy of using SR-MD as a promising membrane process in treating complex industrial wastes with high organic solvent content.

Erratum: Dependence of the extraction efficiency of activated carbon adsorbents for work envi-ronment measurement on the concentration of organic solvents [SANGYO EISEIGAKU ZASSHI 2020; 62(5): 192-197]

Erratum: Dependence of the extraction efficiency of activated carbon adsorbents for work envi-ronment measurement on the concentration of organic solvents [SANGYO EISEIGAKU ZASSHI 2020; 62(5): 192-197]

Optimization of Two Eco-Friendly Extractions of Black Medick (Medicago lupulina L.) Phenols and Their Antioxidant, Cosmeceutical, α-Glucosidase and α-Amylase Inhibitory Properties.

Medicago lupulina is an ancient edible plant from the Fabaceae family. In this work, two eco-friendly methods for extraction of bioactive phenolics from M. lupulina were developed using mixtures of water with two non-toxic, skin- and environmentally-friendly polyol solvents: glycerol and polypropylene glycol. Ultrasound-assisted extractions were optimized using a Box–Behnken design. The independent variables were the concentration of organic solvent in water (X1), extraction temperature (X2) and time (X3), while the response was phenolic content. The optimum conditions for extraction of polyphenols were (X1, X2, X3): (45%, 70 °C, 60 min) and (10%, 80 °C, 60 min) for glycerol and polypropylene glycol extraction, respectively. The extracts prepared at optimum conditions were rich in phenolic compounds, mainly derivatives of apigenin, kaempferol, luteolin, quercetin, caffeic and ferulic acid, as well as coumestrol. Their cosmeceutical and antidiabetic activity was tested. Both extracts demonstrated notable antioxidant, anti-lipoxygenase and anti-α-amylase activity. In addition to those activities, the glycerol extract efficiently inhibited protein coagulation, elastase and α-glucosidase activity. Glycerol present in the extract displayed enzyme-inhibiting activity in several assays and supported the action of the bioactive constituents. Thus, the optimized glycerol extract is a desirable candidate for direct incorporation in antidiabetic food supplements and cosmeceutical products.

Polyaniline/titanium dioxide nanorods functionalized carbon fibers for in-tube solid-phase microextraction of phthalate esters prior to high performance liquid chromatography-diode array detection

To improve extraction performance of carbon fibers (CFs) towards phthalate esters (PAEs), titanium dioxide (TiO2) nanorods array was in-situ grown on the surface of CFs, then polyaniline (PANI) was used to modify it. PANI/TiO2 nanorods-CFs were placed into a polyetheretherketone tube for solid-phase microextraction (SPME). Combining the tube to high performance liquid chromatography (HPLC), it was evaluated and displayed good extraction performance for several PAEs. Compared with bare CFs, TiO2 nanorods and PANI, PANI/TiO2 nanorods presented best performance, attributed to the unique advantages between high surface area of TiO2 nanorods and multiple adsorption interactions (like π-π stacking, hydrogen bond) of PANI. After the optimization of the important factors (sampling volume, sampling rate, sample pH, concentrations of organic solvent and salt in sample, and desorption time), the online in-tube SPME-HPLC method was established. It provided low limits of detection (0.01-0.05 μg L−1) and wide linear ranges (0.03-30, 0.10-30, 0.17-30 μg L−1) with correlation coefficients larger than 0.9991. The relative standard deviations (n=6) between intra-day and inter-day tests were in the ranges of 3.5-10.3% and 4.7-13.9%, respectively. The method was successfully used to determine seven PAEs in real water samples. Besides of satisfactory durability, the material also exhibited superior extraction performance than some materials.

Using the ash of common water reeds as a silica source for producing high purity ZSM-5 zeolite microspheres

Abstract Utilizing of ash extracted from Iraqi common water reeds as an inexpensive effective silica source in the preparation of ZSM-5 zeolite was investigated for the first time in the present work. This study was conducted using a hydrothermal treatment at autogenous pressure. The molar content of silica (17.84–26.76), sodium oxide (1.71–2.4), water (76.92–304.6), organic template (0.55–2.73), organic solvent (23.46–140.76), and synthesis conditions (crystallization time (24–72 h) and temperature (130–180 °C)) were investigated to obtain high purity ZSM-5 zeolite microspheres. The zeolite samples were characterized using X-Ray Diffraction (XRD), Scanning Electron Microscopy (SEM), Energy Dispersive Analysis by X-Ray (EDAX), N2 adsorption/desorption isotherms, Thermogravimetric Analyzer (TGA), and Fourier transform infrared spectroscopy (FTIR). XRD pattern of all synthesized samples was compared to XRD pattern of the commercial ZSM-5 zeolite. This work allows using locally available solid waste resulted from a dry natural plant as an inexpensive silica source without using an external source of silica. Also, this is the first report of the direct synthesis of a high purity ZSM-5 zeolite microspheres from common water reeds ash containing highly reactive silica. A gel composition of 2Na2O: 26.76SiO2: Al2O3: 2.73TPAOH: 23.46EtOH: 304.6H2O gave an outstanding final product of ZSM-5 zeolite microspheres at 180 °C and 27 h. This high crystallinity ZSM-5 zeolite porous microspheres possess Si/Al ratio of 17, a crystal size of 61.419 nm, BET surface area of 277.482 m2/g, a total pore volume of 0.1659 cm3/g, micropore volume of 0.1131 cm3/g, pore width of 5.28 nm, and hierarchical factor of 0.14.

Molecular diagnostic of toxigenic Corynebacterium diphtheriae strain by DNA sensor potentially suitable for electrochemical point-of-care diagnostic

The presented study is focused on the development of electrochemical genosensor for detection of tox gene fragment of toxigenic Corynebacterium diphtheriae strain. Together with our previous studies it fulfils the whole procedure for fast and accurate diagnostic of diphtheria at its early stage of infection with the use of electrochemical methods. The developed DNA sensor potentially can be used in more sophisticated portable device. After the electrochemical stem-loop probe structure optimization the conditions for real asymmetric PCR (aPCR) product detection were selected. As was shown it was crucial to optimize the magnesium and organic solvent concentrations in detection buffer. Under optimal conditions it was possible to selectively detect as low as 20.8 nM of complementary stand in 5 min or 0.5 nM in 30 min with sensitivity of 12.81 and 0.24 1⋅μM−1 respectively. The unspecific biosensor response was elucidated with the use of new electrode blocking agent, diethyldithiocarbamate. Its application in electrochemical genosensors lead to significant higher current values and the biosensor response even in conditions with magnesium ion depletion. The developed biosensor selectivity was examined using samples containing genetic material originated from a number of non-target bacterial species which potentially can be present in the human upper respiratory tract.

Polymer Modification of Lipases, Substrate Interactions, and Potential Inhibition.

An industrially important enzyme, Candida antarctica lipase B (CalB), was modified with a range of functional polymers including hydrophilic, hydrophobic, anionic, and cationic character using a "grafting to" approach. We determined the impact of polymer chain length on CalB activity by synthesizing biohybrids of CalB with each polymer at three different chain lengths, using reversible addition-fragmentation chain transfer (RAFT) polymerization. The activity of CalB in both aqueous and aqueous-organic media mixtures was significantly enhanced for acrylamide (Am) and N,N-dimethyl acrylamide (DMAm) conjugates, with activity remaining approximately constant in 25 and 50% ethanol solvent systems. Interestingly, the activity of N,N-dimethylaminopropyl-acrylamide (DMAPA) conjugates increased gradually with increasing organic solvent content in the system. Contrary to other literature reports, our study showed significantly diminished activity for hydrophobic polymer-protein conjugates. Functional thermal stability assays also displayed a considerable enhancement of retained activity of Am, DMAm, and DMAPA conjugates compared to the native CalB enzyme. Thus, this study provides an insight into possible advances in lipase production, which can lead to new improved lipase bioconjugates with increased activity and stability.

Case Study 1: Practical Considerations with Experimental Design and Interpretation.

This chapter deals with practical considerations on key issues such as choosing an enzyme source, determining linear conditions, and choosing appropriate substrate and organic solvent concentrations. Practical solutions for working with limited resources and carrying out inhibition experiments are also addressed. Thus, after reading this chapter, the novice reader should have a better idea of how to design, develop, and interpret basic experiments using drug metabolism enzymes.