非水酶学和非水相生物催化研究进展

近年来工业生物技术飞速发展,酶学和生物催化领域也取得突破性进展,特别在酶在非水相中活性及稳定性研究,耐溶剂生物催化剂的筛选、构建、修饰和改造,生物相容性和环境相容性好的绿色介质等方面取得了较大的进展。最近的研究热点和未来几年的研究方向主要为:基于基因组信息的耐溶剂酶的虚拟筛选和构建;基于自然界筛选新酶基因的耐溶剂酶重构和改造;离子液体等环境友好的绿色介质系统等几个方面。     

A three-dimensional solubility parameter approach to nonaqueous enzymology

Widespread commercial application of enzymes as catalysts for specialty or commodity chemical synthesis will require their use in nonaqueous systems. While a number of non-aqueous enzyme applications have been demonstrated, the lack of useful rules for predicting enzyme-solvent interactions has hindered the development of this technology. Both Hildebrand and solvent hydrophobicity (octanol-water partition coefficient) parameters have been used previously to correlate and predict enzyme activity in nonaqueous systems, with some success, but any single-parameter approach is inherently limited in its ability to reflect the spectrum of possible enzyme-solvent interactions. Therefore, this study evaluates the three-dimensional solubility parameter space, as proposed by Hansen, to correlate and predict enzyme activity in microaqueous, miscible, and biphasic nonaqueous systems. Preliminary results suggest that Hansen parameters may be useful for correlating nonaqueous enzyme activity, and that the dispersive and polar parameters may be disproportionately important in single-phase microaqueous systems. The Hansen hydrogen-bonding parameter appears to be the only parameter yet evaluated capable of correlating the water requirement for enzyme activity in microaqueous systems, suggesting that water affects protein structure through enthalpic rather than entropic processes in nonaqueous systems. Insufficient data are available for miscible and biphasic systems, but it is proposed that enzyme activity may correlate with the average solubility parameters of miscible systems and of the aqueous phase in biphasic systems.     

Dry enzyme-polymer complexes: Stable organosoluble biocatalysts for nonaqueous enzymology

Summary Complexes of chymotrypsin and laccase with amphiphilic alkylated poly(ethyleneimine) (CEPEI) were obtained in a dry state by evaporation of nonaqueous solutions of the polymer containing solubilized enzymes. Enzyme-polymer complexes could be readily redissolved in organic solvent with full restoration of catalytic activity and showed remarkably high storage stability in the dry state.     

Non-aqueous enzymology.

Compelling evidence has been obtained during the past year that enzymes retain their native active-site structure in organic solvents, and yet the properties of the solvent significantly affect enzyme kinetics. Fundamental advances in enzymatic catalysis in monophasic organic media are discussed and selected applications in the areas of asymmetric, polymer and chemoenzymatic syntheses are highlighted.     

Role of collateral paths in long-range diffusion in lungs.

The long-range apparent diffusion coefficient (LRADC) of (3)He gas in lungs, measured over times of several seconds and distances of 1-3 cm, probes the connections between the airways. Previous work has shown the LRADC to be small in health and substantially elevated in emphysema, reflecting tissue destruction, which is known to create collateral pathways. To better understand what controls LRADC, we report computer simulations and measurements of (3)He gas diffusion in healthy lungs. The lung is generated with a random algorithm using well-defined rules, yielding a three-dimensional set of nodes or junctions, each connected by airways to one parent node and two daughters; airway dimensions are taken from published values. Spin magnetization in the simulated lung is modulated sinusoidally, and the diffusion equation is solved to 1,000 s. The modulated magnetization decays with a time constant corresponding to an LRADC of approximately 0.001 cm(2)/s, which is smaller by a factor of approximately 20 than the values in healthy lungs measured here and previously in vivo and in explanted lungs. It appears that collateral gas pathways, not present in the simulations, are functional in healthy lungs; they provide additional and more direct routes for long-range motion than the canonical airway tree. This is surprising, inasmuch as collateral ventilation is believed to be physiologically insignificant in healthy lungs. We discuss the effect on LRADC of small collateral connections through airway walls and rule out other possible mechanisms. The role of collateral paths is supported by measurements of smaller LRADC in pigs, where collateral ventilation is known to be smaller.     

Gamma-glutamyl-glutathione. Natural occurrence and enzymology

The natural occurrence of gamma-glutamyl-glutathione (gamma-glutamyl-gamma-glutamylcysteinylglycine) in bile was established by analytical and chromatographic studies on the isolated and chemically synthesized materials. Evidence that it is formed in kidney was obtained. The origin of gamma-glutamyl-glutathione was explored through studies on the interaction of glutathione with gamma-glutamyl transpeptidase. When purified gamma-glutamyl transpeptidase was incubated with various concentrations (4 microM-50 mM) of glutathione, the initial rates of formation of gamma-glutamyl-glutathione were substantial at all concentrations of glutathione studied and were greater than the rates of formation of glutamate at physiological levels of glutathione (1-10 mM). The findings indicate that gamma-glutamyl transpeptidase catalyzes transpeptidation in vivo. That gamma-glutamyl-glutathione is formed in vivo and that it is a significant product of the reaction between glutathione and gamma-glutamyl transpeptidase under physiological conditions suggest that this polyanionic tetrapeptide may have a physiological role. gamma-Glutamyl-glutathione is not a substrate of glutathione reductase or of glutathione S-transferase, but it is a substrate of gamma-glutamyl-cyclotransferase. That gamma-glutamyl-glutathione has an additional negative charge as compared to glutathione suggests that it may be more effective than glutathione in forming complexes with certain metal ions and other cations.     

Kinetics of gas diffusion in mammalian cell culture systems. II. Theory

Mathematical models have been constructed which relate the depth of the culture fluid overlay to the oxygen available to mammalian cells cultured under static conditions. These models suggest that the maintenance of a given rate of oxygen utilization by some culture systems may be critically depended on this fluid depth and on the solubility and rate of diffusion of oxygen in the culture fluid. The importance of these concepts as applied to the isolation and growth of differentiated cells representative of the tissue of origin are noted.     

Traceability in clinical enzymology

The primary goal of standardisation for measurements of catalytic concentrations of enzymes is to achieve comparable results in human samples, independent of the reagent kits, instruments and laboratory where the assay is carried out. In order to pursue this objective, the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) has established reference systems for the most important clinical enzymes. These systems are based on three requirements: a) reference measurement procedures that are extensively evaluated and carefully described; b) certified reference materials; and c) a network of reference laboratories operating in a highly controlled manner. Using these reference systems and the manufacturer's standing procedures, industry can assign traceable values to commercial calibrators. Clinical laboratories, which use routine procedures with validated calibrators to measure human specimens, can finally obtain values which are traceable to higher-order reference procedures. These reference systems constitute the structure of the traceability chain to which the routine methods can be linked via an appropriate calibration process, provided that they have a comparable specificity (i.e. they are measuring the same quantity).     

The enzymology of apurinic/apyrimidinic endonucleases.

Studies on the enzymology of apurinic/apyrimidinic (AP) endonucleases from procaryotic and eucaryotic organisms are reviewed. Emphasis will be placed on the enzymes from Escherichia coli from which a considerable portion of our knowledge has been derived. Recent studies on similar enzymes from eucaryotes will be discussed as well. In addition, we will discuss the chemical and physical properties of AP sites and review studies on peptides and acridine derivatives which incise DNA at AP sites.