The technological utility of enzymes can be enhanced greatly by using them in organic solvents rather than their natural aqueous reaction media. Studies over the past 15 years have revealed not only that this change in solvent is feasible, but also that in such seemingly hostile environments enzymes can catalyse reactions impossible in water, become more stable, and exhibit new behaviour such as 'molecular memory'. Of particular importance has been the discovery that enzymatic selectivity, including substrate, stereo-, regio- and chemoselectivity, can be markedly affected, and sometimes even inverted, by the solvent. Enzyme-catalysed reactions in organic solvents, and even in supercritical fluids and the gas phase, have found numerous potential applications, some of which are already commercialized.

Poly(4-vinyl-N-alkylpyridinium bromide) was covalently attached to glass slides to create a surface that kills airborne bacteria on contact. The antibacterial properties were assessed by spraying aqueous suspensions of bacterial cells on the surface, followed by air drying and counting the number of cells remaining viable (i.e., capable of growing colonies). Amino glass slides were acylated with acryloyl chloride, copolymerized with 4-vinylpyridine, and N-alkylated with different alkyl bromides (from propyl to hexadecyl). The resultant surfaces, depending on the alkyl group, were able to kill up to 94 ؎ 4% of Staphylococcus aureus cells sprayed on them. A surface alternatively created by attaching poly(4-vinylpyridine) to a glass slide and alkylating it with hexyl bromide killed 94 ؎ 3% of the deposited S. aureus cells. On surfaces modified with N-hexylated poly(4-vinylpyridine), the numbers of viable cells of another Gram-positive bacterium, Staphylococcus epidermidis, as well as of the Gram-negative bacteria Pseudomonas aeruginosa and Escherichia coli, dropped more than 100-fold compared with the original amino glass. In contrast, the number of viable bacterial cells did not decline significantly after spraying on such common materials as ceramics, plastics, metals, and wood. Because of the ever-growing demand for healthy living, there is a keen interest in materials capable of killing harmful microorganisms. Such materials could be used to coat the surfaces of common objects touched by people in everyday life (e.g., door knobs, children's toys, computer keyboards, telephones, etc.) to render them antiseptic and thus unable to transmit bacterial infections.Because ordinary materials are not antimicrobial, they require modification. For example, surfaces chemically modified with poly(ethylene glycol) and certain other synthetic polymers can repel (although not kill) microorganisms (1-6). Alternatively, materials can be impregnated with antimicrobial agents, such as antibiotics, quaternary ammonium compounds, silver ions, or iodine, that are released gradually into the surrounding solution over time and kill microorganisms therein (6-9). Although these strategies have been verified in aqueous solutions containing bacteria, they would not be expected to be effective against airborne bacteria in the absence of a liquid medium; this situation is especially true for release-based materials (6), which are also liable to become impotent when the leaching antibacterial agent is exhausted.It has been reported (10-13) that various polycations possess antibacterial properties in solution, presumably by interacting with and disrupting bacterial cell membranes. However, this antibacterial activity vanishes when these polycations are crosslinked or otherwise insolubilized (12,14,15). We have hypothesized that their antibacterial properties can be preserved, even after insolubilization, and expressed in a dry state, if the immobilized polycationic chains are sufficiently long and flexible to be able to penetrate the bacterial...

The results obtained are consistent with the proton sponge hypothesis and strongly suggest that the transfection activity of PEI vectors is due to their unique ability to avoid acidic lysosomes.

The explosive growth in our knowledge of genomes, proteomes, and metabolomes is driving ever-increasing fundamental understanding of the biochemistry of life, enabling qualitatively new studies of complex biological systems and their evolution. This knowledge also drives modern biotechnologies, such as molecular engineering and synthetic biology, which have enormous potential to address urgent problems, including developing potent new drugs and providing environmentally friendly energy. Many of these studies, however, are ultimately limited by their need for even-higher-throughput measurements of biochemical reactions. We present a general ultrahigh-throughput screening platform using drop-based microfluidics that overcomes these limitations and revolutionizes both the scale and speed of screening. We use aqueous drops dispersed in oil as picoliter-volume reaction vessels and screen them at rates of thousands per second. To demonstrate its power, we apply the system to directed evolution, identifying new mutants of the enzyme horseradish peroxidase exhibiting catalytic rates more than 10 times faster than their parent, which is already a very efficient enzyme. We exploit the ultrahigh throughput to use an initial purifying selection that removes inactive mutants; we identify ∼100 variants comparable in activity to the parent from an initial population of ∼10 7 . After a second generation of mutagenesis and high-stringency screening, we identify several significantly improved mutants, some approaching diffusion-limited efficiency. In total, we screen ∼10 8 individual enzyme reactions in only 10 h, using < 150 μL of total reagent volume; compared to state-of-the-art robotic screening systems, we perform the entire assay with a 1,000-fold increase in speed and a 1-million-fold reduction in cost.

Three different lipases (porcine pancreatic, yeast, and mold) can vigorously act as catalysts in a number of nearly anhydrous organic solvents. Various transesterification reactions catalyzed by porcine pancreatic lipase in hexane obey Michaelis-Menten kinetics. The dependence of the catalytic activity of the enzyme in organic media on the pH of the aqueous solution front which it was recovered is bell-shaped, with the maximum coinciding with the pH optimum of the enzymatic activity in water. The catalytic power exhibited by the lipases in organic solvents is comparable to that displayed in water. In addition to transesterification, lipases can catalyze several other processes in organic media including esterification, aminolysis, acyl exchange, thiotransesterification, and oximolysis; some of these reactions proceed to an appreciable extent only in nonaqueous solvents.In nature, enzymes function in aqueous solutions. Therefore, it is not surprising that virtually all studies in enzymology thus far have used water as the reaction medium. However, from the biotechnological standpoint there are numerous advantages of conducting enzymatic conversions in organic solvents as opposed to water: (i) high solubility of most organic compounds in nonaqueous media; (ii) ability to carry out new reactions impossible in water because of kinetic or thermodynamic restrictions; (iii) greater stability of enzymes; (iv) relative ease of product recovery from organic solvents as compared to water; and (v) the insolubility of enzymes in organic media, which permits their easy recovery and reuse and thus eliminates the need for immobilization.Conventional wisdom dictates that water is required for enzyme action. This conclusion stems from the fact that water participates (directly or indirectly) in all noncovalent interactions maintaining the native, catalytically active enzyme conformation (1-5); hence, removal of water should drastically distort that conformation and inactivate the enzyme. Although this reasoning is undoubtedly correct, the real question is not whether water is indeed required but how much water. It is hard to imagine that an enzyme molecule can "see" more than just a few monolayers of water around it. As long as this water is localized about enzyme molecules, the rest (i.e., the bulk) of water can probably be replaced with an organic solvent without adversely affecting the enzyme.Since the absolute amount of water contained in those few monolayers is very small, this situation is tantamount to an enzyme functioning in a nearly anhydrous organic medium.There is some experimental confirmation of the above rationale. Price and co-workers have shown that chymotrypsin (6) and xanthine oxidase (7,8) are catalytically active when suspended in organic solvents. We recently have found (9) that porcine pancreatic lipase vigorously acts as a catalyst in the 99.98% organic medium; in addition, upon dehydration the enzyme acquires some remarkable new properties-e.g., it becomes extremely thermostable and more selective...

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The stability of protein-based pharmaceuticals (e.g., insulin) is important for their production, storage, and delivery. To gain an understanding ofinsulin's aggregation mechanism in aqueous solutions, the effects of agitation rate, interfacial interactions, and insulin concentration on the overall aggregation rate were examined. Ultraviolet absorption spectroscopy, high-performance liquid chromatography, and quasielastic light scattering analyses were used to monitor the aggregation reaction and identify intermediate species. The reaction proceeded in two stages; insulin stability was enhanced at higher concentration. Mathematical modeling of proposed kinetic schemes was employed to identify possible reaction pathways and to explain greater stability at higher insulin concentration.The stability of protein-based pharmaceuticals is essential for the efficacy of conventional therapeutic preparations (1), continuous infusion pumps, and controlled release polymeric devices. Insulin aggregation, accompanied by drastic reduction of biological potency and obstruction of delivery routes, creates serious problems for drug delivery systems (2-4). Although insulin aggregation has been investigated (5-16), its molecular mechanism remains speculative.This study aims at elucidating the fundamental nature of this phenomenon using a rigorous kinetic analysis. Based on experimental observations, a reaction mechanism was formulated and possible destabilizing pathways were identified. Mathematical modeling was used to verify the predictive powers of the proposed scheme.MATERIALS AND METHODS Solution Preparation. Bovine Zn-insulin (specific activity, 24.4 international units/mg; Zn2+ content, <0.5%) was used.Phosphate-buffered saline (PBS) (0.14 M NaCI/0.1% NaN3 preservative, pH 7.4) was sterilized by filtration through a 0.45-,um Millipore HV filter and degassed by sonication. Stock solutions were prepared by adding Zn-insulin to PBS; the resulting cloudy mixture was sealed with Parafflm, placed in a shaker, and gently agitated for 3 hr at 37°C. Zn-insulin dissolved completely at concentrations up to 0.6 mg/ml. The stock solutions were filtered through sterile 0.22-,m Millex GV low-protein binding filters; lower concentrations were obtained by dilution with PBS prior to final filtration. All glassware was rinsed with 0.01 M HCl, followed by drying at 100°C. The initial concentrations of Zn-insulin solutions were determined by UV absorbance at 280 nm (e = 5.53 mM-'cm-1).Concentration-Dependence Studies. Air-water interface. Glass 1.1-ml HPLC vials were filled with 0.75 ml of insulin solution, capped, sealed with Parafilm, taped horizontally to the shaker platform, and agitated at 250 rpm and 370C. Every 20 min, one vial was removed and the extent of aggregation was determined by size-exclusion isochratic HPLC analysis [Bio-Rad's SEC-125 column; mobile phase consisting of 10% acetonitrile and 90% aqueous solution containing 0.02 M NaH2PO4 and 0.05 M Na2SO4 (pH 6.8), flow rate of 1.2 ml/min, detection at 280 and 217 nm]. Ins...

Porcine pancreatic lipase catalyzes the transesterification reaction between tributyrin and various primary and secondary alcohols in a 99 percent organic medium. Upon further dehydration, the enzyme becomes extremely thermostable. Not only can the dry lipase withstand heating at 100 degrees C for many hours, but it exhibits a high catalytic activity at that temperature. Reduction in water content also alters the substrate specificity of the lipase: in contrast to its wet counterpart, the dry enzyme does not react with bulky tertiary alcohols.