Prevention of interfacial inactivation of enzymes by coating the enzyme surface with dextran-aldehyde

Interactions between soluble enzymes and interfaces of organic solvent drops or gas bubbles have a very negative effect on the operational stability of the soluble enzymes. In this study, the formation of a hydrophilic shell around the enzyme has been attempted using dextran-aldehyde which would prevent the interaction between enzyme and hydrophobic interfaces with minimal modification of the enzyme surface. After optimizing the size of the dextran (that was found to play a critical role), three different enzymes (glucose oxidase, d-amino acid oxidase, and trypsin) have been conjugated with dextran-aldehyde and their stability towards organic-aqueous and air-liquid interfaces has been evaluated. The treatment itself proved to be very low-cost in terms of activity and was highly stabilizing for the three enzymes assayed. The conjugated preparation of the three assayed enzymes remained fully active in the presence of air-liquid interfaces for at least 10h. However, the unmodified enzymes lost more than 50% of activity within the first hour of the experiments except for trypsin which kept 38% activity after 12h while the trypsin dextran-aldehyde conjugate maintained 100% enzyme activity. Similar results were achieved in the presence of stirred organic solvent-aqueous buffer biphasic system, although in this case some activity was lost by the action of the soluble portion of the organic solvent. In fact, this treatment seems to be also effective to improve the resistance to the action of organic solvent.     

Does biocatalysis involve inhomogeneous kinetics?

Biocatalytic reactions can occur according to two very different mechanisms: homogeneous, which is described by standard transition state theory (TST) and its modifications, and inhomogeneous (polychromatic), which is characteristic for some of the charge-transfer reactions in liquids and amorphous solids. While most data published on enzyme reactions are interpreted on the basis of homogeneous kinetics, the important recent findings suggest the involvement of inhomogeneous kinetics mechanism.     

Mechanisms of enzyme action and inhibition: Transition state analogues for acid-base catalysis

The theory of absolute reaction rates implies that the grip of a catalyst on a substrate tightens with substrate activation, relaxing later as products are formed and released. Analogs that mimic different kinds of substrate activation can, through the structural details of their complexes with enzymes, indicate how active site residues are involved in the enhancement of reactions rates. In several cases, bonds involved in general acid-base catalysis have been identified tentatively; and recent evidence points to a hydrogen bond of remarkable stability in the transition state in enzymatic deamination of adenosine. Similar approaches have been used to enzymes that act primarily by substrate distortion, nucleophilic catalysis, solvent removal and catalysis by approximation. Two recurring observations, that were not expected in theory, have been the binding of inhibitors in ionized forms that are rare in solution, and changes in enzyme configuration that accompany binding of transition state analogs. Origins and implications of these findings will be discussed with specific reference to the role of solvent water in catalytic phenomena.Supported by Grant GM-18325 from the National Institutes of Health.     

Transition state theory can be used in studies of enzyme catalysis: lessons from simulations of tunnelling and dynamical effects in lipoxygenase and other systems

The idea that enzyme catalysis involves special factors such as coherent fluctuations, quantum mechanical tunnelling and non-equilibrium solvation (NES) effects has gained popularity in recent years. It has also been suggested that transition state theory (TST) cannot be used in studies of enzyme catalysis. The present work uses reliable state of the art simulation approaches to examine the above ideas. We start by demonstrating that we are able to simulate any of the present catalytic proposals using the empirical valence bond (EVB) potential energy surfaces, the dispersed polaron model and the quantized classical path (QCP) approach, as well as the approximate vibronic method. These approaches do not treat the catalytic effects by phenomenological treatments and thus can be considered as first principles approaches (at least their ability to compare enzymatic reaction to the corresponding solution reactions). This work will consider the lipoxygenase reaction, and to lesser extent other enzymes, for specific demonstration. It will be pointed out that our study of the lipoxygenase reaction reproduces the very large observed isotope effect and the observed rate constant while obtaining no catalytic contribution from nuclear quantum mechanical (NQM) effects. Furthermore, it will be clarified that our studies established that the NQM effect decreases rather than increases when the donor-acceptor distance is compressed. The consequences of these findings in terms of the temperature dependence of the kinetic isotope effect and in terms of different catalytic proposals will be discussed. This paper will also consider briefly the dynamical effects and conclude that such effects do not contribute in a significant way to enzyme catalysis. Furthermore, it will be pointed out that, in contrast to recent suggestions, NES effects are not dynamical effects and should therefore be part of the activation free energy rather than the transmission factor. In view of findings of the present work and our earlier works, it seems that TST provides a quantitative tool for studies of enzyme catalysis and that the key open questions are related to the nature of the factors that lead to transition state stabilization.     

Hydrogen tunnelling in enzyme-catalysed H-transfer reactions: flavoprotein and quinoprotein systems

It is now widely accepted that enzyme-catalysed C-H bond breakage occurs by quantum mechanical tunnelling. This paradigm shift in the conceptual framework for these reactions away from semi-classical transition state theory (TST, i.e. including zero-point energy, but with no tunnelling correction) has been driven over the recent years by experimental studies of the temperature dependence of kinetic isotope effects (KIEs) for these reactions in a range of enzymes, including the tryptophan tryptophylquinone-dependent enzymes such as methylamine dehydrogenase and aromatic amine dehydrogenase, and the flavoenzymes such as morphinone reductase and pentaerythritol tetranitrate reductase, which produced observations that are also inconsistent with the simple Bell-correction model of tunnelling. However, these data-especially, the strong temperature dependence of reaction rates and the variable temperature dependence of KIEs-are consistent with other tunnelling models (termed full tunnelling models), in which protein and/or substrate fluctuations generate a configuration compatible with tunnelling. These models accommodate substrate/protein (environment) fluctuations required to attain a configuration with degenerate nuclear quantum states and, when necessary, motion required to increase the probability of tunnelling in these states. Furthermore, tunnelling mechanisms in enzymes are supported by atomistic computational studies performed within the framework of modern TST, which incorporates quantum nuclear effects.     

Recent advances of enzymatic reactions in ionic liquids

The tremendous potential of room temperature ionic liquids as an alternative to environmentally harmful ordinary organic solvents is well recognized. Ionic liquids, having no measurable vapor pressure, are an interesting class of tunable and designer solvents, and they have been used extensively in a wide range of applications including enzymatic biotransformation. In fact, ionic liquids can be designed with different cation and anion combinations, which allow the possibility of tailoring reaction solvents with specific desired properties, and these unconventional solvent properties of ionic liquids provide the opportunity to carry out many important biocatalytic reactions that are impossible in traditional solvents. As compared to those observed in conventional organic solvents, the use of enzymes in ionic liquids has presented many advantages such as high conversion rates, high enantioselectivity, better enzyme stability, as well as better recoverability and recyclability. To date, a wide range of pronounced approaches have been taken to further improve the performance of enzymes in ionic liquids. This review presents the recent technological developments in which the advantages of ionic liquids as a medium for enzymes have been gradually realized.     

The fluorinase, the chlorinase and the duf-62 enzymes

The fluorinase from Streptomyces cattleya and chlorinase from Salinispora tropica have a commonality in that they mediate nucleophilic reactions of their respective halide ions to the C-5' carbon of S-adenosyl-L-methionine (SAM). These enzyme reactions fall into the relatively small group of S(N)2 substitution reactions found in enzymology. These enzymes have some homology to a larger class of proteins expressed by the duf-62 gene, of which around 200 representatives have been sequenced and deposited in databases. The duf-62 genes express a protein which mediates a hydrolytic cleavage of SAM to generate adenosine and L-methionine. Superficially this enzyme operates very similarly to the halogenases in that water/hydroxide replaces the halide ion. However structural examination of the duf-62 gene product reveals a very different organisation of the active site suggesting a novel mechanism for water activation.     

Enzyme catalysis in ionic liquids

Ionic liquids offer new possibilities for the application of solvent engineering to biocatalytic reactions. Although in many cases ionic liquids have simply been used to replace organic solvents, they have often led to improved process performance. Unlike conventional organic solvents, ionic liquids possess no vapor pressure, are able to dissolve many compounds, and can be used to form two-phase systems with many solvents. To date, reactions involving lipases have benefited most from the use of ionic liquids, but the use of ionic liquids with other enzymes and in whole-cell processes has also been described. In some cases, remarkable results with respect to yield, (enantio)selectivity or enzyme stability were observed.     

Oxidative enzymes in the urinary apparatus of several freshwater fishes

Synopsis The distribution and activities of several oxidative enzymes in the urinary apparatus of five freshwater fish species (river lamprey, lobe finned eel, Prussian carp, rainbow trout and three-spined stickleback) have been studied. Species were selected from three main taxonomic groups: Cyclostomata, Polypterini, Teleostei. Distinctly positive enzyme reactions were only found in the tubular elements of the kidney and the collecting duct-archinephric duct system, with the exception of the generally weak staining intensities of lactate dehydrogenase. The distal tubule normally showed strong to very strong reactions for most of the enzymes investigated. In the epithelial cells of the collecting tubule-collecting duct system, stronger reactions were observed for most of the mitochondrial-bound enzymes, especially succinate dehydrogenase and NADH-diaphorase. For these enzymes, the cells of the archinephric duct reacted strongly positive in Lampetra, Carassius and Gasterosteus.The enzyme patterns of various types of urinary tubules and ducts are compared with results of several morphological studies. In addition, the histochemical findings are discussed in relation to kidney function in different vertebrate groups.     

The water effect on the kinetic of the bovine liver catalase

Catalase is an enzyme that occurs in almost all aerobic organisms. Its main metabolic function is to prevent oxidative damage to tissues induced by hydrogen peroxide which is a strong oxidizing agent. Catalase is very effective in performing this task, since it has the highest turnover rate among all the enzymes. The properties of catalase have been investigated extensively for many years; however, the role of the solvent molecules in the catalytic reaction of this enzyme has not yet been investigated. Therefore, the objective of this work was to investigate the contribution of the solvent molecules on the catalytic reaction of bovine liver catalase with its substrate H2O2 by the osmotic stress method. As a probe for protein structural changes in solution, the differential number of water molecules released during the transition from free to bound form of the enzyme was measured. These assays were correlated with protein structural data provided by the SAXS technique and crystallographic structures of free and CN(-) bonded enzymes. The results showed that the difference in surface accessible area of the crystal structures does not reflect the variation that is observed in solution. Moreover, catalase is not influenced by the solvent during the catalytic reaction, which represents a lower energy barrier to be crossed in the overall energetics of the reaction, a fact that contributes to the high turnover rate of catalase.     

Expanding the Halohydrin Dehalogenase Enzyme Family: Identification of Novel Enzymes by Database Mining

Halohydrin dehalogenases are very rare enzymes that are naturally involved in the mineralization of halogenated xenobiotics. Due to their catalytic potential and promiscuity, many biocatalytic reactions have been described that have led to several interesting and industrially important applications. Nevertheless, only a few of these enzymes have been made available through recombinant techniques; hence, it is of general interest to expand the repertoire of these enzymes so as to enable novel biocatalytic applications. After the identification of specific sequence motifs, 37 novel enzyme sequences were readily identified in public sequence databases. All enzymes that could be heterologously expressed also catalyzed typical halohydrin dehalogenase reactions. Phylogenetic inference for enzymes of the halohydrin dehalogenase enzyme family confirmed that all enzymes form a distinct monophyletic clade within the short-chain dehydrogenase/reductase superfamily. In addition, the majority of novel enzymes are substantially different from previously known phylogenetic subtypes. Consequently, four additional phylogenetic subtypes were defined, greatly expanding the halohydrin dehalogenase enzyme family. We show that the enormous wealth of environmental and genome sequences present in public databases can be tapped for in silico identification of very rare but biotechnologically important biocatalysts. Our findings help to readily identify halohydrin dehalogenases in ever-growing sequence databases and, as a consequence, make even more members of this interesting enzyme family available to the scientific and industrial community.     

A comparison of the catalytic properties of cellobiose:quinone oxidoreductase and cellobiose oxidase from Phanerochaete chrysosporium.

Several catalytic properties of the FAD enzyme cellobiose:quinone oxidoreductase (CBQ) and the heme/FAD enzyme, cellobiose oxidase (CBO) have been investigated and compared. Dichlorophenol-indophenol was found to be a very good electron acceptor for cellobiose oxidation by both enzymes. The optimal pH value for this oxidation with dichlorophenol-indophenol as a co-substrate was observed around pH 4 for both enzymes. The turnover numbers of this reaction were also very similar. The Km values for cellobiose oxidation were identical, whereas the Km for CBO with dichlorophenol-indophenol is lower than that of CBQ. Atmospheric oxygen is a very poor electron acceptor for both CBO and CBQ, however, CBO can utilize cytochrome c as an effective electron acceptor, while CBQ cannot. The specific activity of CBO for cytochrome c is thus about 200-times higher than for oxygen. Thus, one way to distinguish the two enzymes is by the cytochrome-c-reducing ability of CBO. Therefore, we propose that the nomenclature for CBO is tentatively changed to cellobiose:cytochrome c oxidoreductase until a rational name can be installed. Both enzymes have radical-reducing activities. The cation radical, derived from 1,2,4,5-tetramethoxybenzene, was reduced by both enzymes at almost the same reaction rate. The phenoxyradical produced by lignin peroxidase, catalyzing the oxidation of acetosyringon, was also reduced by both enzymes. The reduction of phenoxyradicals formed by phenoloxidases (lignin peroxidases, as well as laccases) may be important in preventing repolymerization reactions which we suggest would significantly facilitate lignin degradation.     

Enzyme-catalysed reactions in non-aqueous media

The discovery that enzymes can function in apolar solvents has dramatically expanded the range of reactions which can be approached through biocatalysis. Key factors which influence both the activity and stability of enzymes in organic solvents include the ionic state of the enzyme, support characteristics, and the extent of hydration of biocatalyst and solvent. Industrial applications have begun to emerge in the areas of fat and oil processing.     

Phosphatases and oxidative enzymes in the kidney of the Prussian carp (Carassius auratus gibelio Bloch) adapted to salt water

The distribution and activities of phosphatases and oxidative enzymes have been determined with the help of histochemical methods in the kidney of the Prussian Carp, a stenohaline freshwater-fish. In addition to fish maintained in freshwater aquaria, a group of the animals used has been adapted to seawater of moderate salinity. The following pattern of enzyme reaction intensities has been observed in the various kidney structures: Strong reactions of alkaline phosphatase in the nephron are confined to the glomerular capillary convolute and the brush border of proximal segments. Equally enzyme activities are observed in the connective tissue sheath of the collecting duct -- archinephric duct system. Acid phosphatase can be detected in all segments of the nephronic tubule, strong activities are found in the proximal segment (P I), in the epithelium of the archinephric duct, and, especially, in the interstitial tissue. ATPase reacts strongly positive in epithelial cells of the distal tubule and the collecting duct -- archinephric duct system. ATPase reactions are inhibited by Ouabain, and therefore can be regarded as reactions of Na--K-ATPase. Mitochondrially bound oxidative enzymes, connected with the citric acid cycle and the respiratory chain, show very strong reaction intensities in the distal tubule and the collecting duct- archinephric duct system, while the glomeruli generally exhibit negative reactions. Lactate -- and malate dehydrogenases are found to react weakly to negatively throughout the whole kidney. Maintenance in seawater does not deeply affect the enzyme pattern of the kidney of the Prussian carp, with exception of some oxidative enzymes, reacting weaker in the distal tubule and the collecting duct-archinephric duct system. In addition, the epithelial cells of the archinephric duct of seawater adapted fish show a marked apical localization of reaction products for these enzymes. Possible relations between enzyme histochemistry and fish kidney physiology are discussed, in connection with comparative aspects of the enzyme histochemistry of the vertebrate kidney. A short review of normal histology and function of the kidney of the Prussian carp is added.     

Stabilization of Candida antarctica lipase B in hydrophilic organic solvent by rational design of hydrogen bond

Enzymatic reactions conducted in organic solvents have many advantages. However, organic solvent molecules may replace water molecules at the protein surface and penetrate into the enzyme, which could lead to the denaturation of the enzyme or changes in its reaction kinetics and substrate specificity. Thus, it is important to enhance the stability of enzymes in organic solvents. To date, there has been no efficient rational approach developed to enhance enzyme stability in hydrophilic solvents. We developed a rational approach to enzyme design. The design rules were established by investigating stable mutants from previous studies of directed evolution. Candida antarctica lipase B (CalB) was used as a target enzyme due to its versatile applications in organic solvents. The N97Q, N264Q, and D265E mutants of CalB showed higher organic solvent stability than the wild type.     

Examining the promiscuous phosphatase activity of Pseudomonas aeruginosa arylsulfatase: a comparison to analogous phosphatases

Pseudomonas aeruginosa arylsulfatase (PAS) is a bacterial sulfatase capable of hydrolyzing a range of sulfate esters. Recently, it has been demonstrated to also show very high proficiency for phosphate ester hydrolysis. Such proficient catalytic promiscuity is significant, as promiscuity has been suggested to play an important role in enzyme evolution. Additionally, a comparative study of the hydrolyses of the p-nitrophenyl phosphate and sulfate monoesters in aqueous solution has demonstrated that despite superficial similarities, the two reactions proceed through markedly different transition states with very different solvation effects, indicating that the requirements for the efficient catalysis of the two reactions by an enzyme will also be very different (and yet they are both catalyzed by the same active site). This work explores the promiscuous phosphomonoesterase activity of PAS. Specifically, we have investigated the identity of the most likely base for the initial activation of the unusual formylglycine hydrate nucleophile (which is common to many sulfatases), and demonstrate that a concerted substrate-as-base mechanism is fully consistent with the experimentally observed data. This is very similar to other related systems, and suggests that, as far as the phosphomonoesterase activity of PAS is concerned, the sulfatase behaves like a "classical" phosphatase, despite the fact that such a mechanism is unlikely to be available to the native substrate (based on pK(a) considerations and studies of model systems). Understanding such catalytic versatility can be used to design novel artificial enzymes that are far more proficient than the current generation of designer enzymes.     

Biotechnological production of prostaglandin

Prostaglandins (PGs) are the oxidation products of PG endoperoxide (PGH) synthase and other tissue enzymes. They occur in a tissue-specific manner and act as local hormones. Biotechnological production of PGs has been of interest, but not yet fully established. Biological tissues have been used as PG sources, but this disturbs ecological balance, and the cost of production is very high for commercial purposes. On the other hand, various microorganisms have been shown to synthesize them de novo, or biotransform precursors to active molecules, but these processes have not been further evaluated. Using mammalian enzymes in free or immobilized form is a promising new approach to synthesize PG from fatty acid substrates. Rapid enzyme inactivation during the catalysis is the main problem to be solved. Optimization of factors in the reactions and the design of special reactors that will allow removal of products continuously from the reaction medium without affecting enzyme activity need immediate attention from researchers and the pharmaceutical industry.     

The thermodynamic landscape of testosterone binding to cytochrome P450 3A4: ligand binding and spin state equilibria

Human cytochrome P450 (CYP) 3A4 catalyzes the oxygen-dependent metabolism of greater than 60% of known drugs. CYP3A4 binds multiple ligands simultaneously, and this contributes to complex allosteric kinetic behavior. Substrates that bind to this enzyme change the ferric spin state equilibrium of the heme, which can be observed by optical absorbance and electron paramagnetic resonance (EPR) spectroscopy. The ligand-dependent spin state equilibrium has not been quantitatively understood for any ligands that exhibit multiple binding. The CYP3A4 substrate testosterone (TST) has been shown previously by absorbance spectroscopy to induce spin state changes that are characteristic of a low spin to high spin conversion. Here, EPR was used to examine the equilibrium binding of TST to CYP3A4 at [CYP3A4] > K(D), which allows for characterization of the singly occupied state (i.e., CYP3A4.TST). We also have used absorbance spectroscopy to examine equilibrium binding, where [CYP3A4] < K(D), which allows for determination of K(D)'s. The combination of absorbance and EPR spectroscopy at different CYP3A4 concentrations relative to K(D) and curve fitting of the resultant equilibrium binding titration curves to the Adair-Pauling equations, and modifications of it, reveals that the first equivalent of TST binds with higher affinity than the second equivalent of TST and its binding is positively cooperative with respect to ligand-dependent spin state conversion. Careful analysis of the EPR and absorbance spectral results suggests that the binding of the second TST induces a shift to the high spin state and thus that the second TST binding causes displacement of the bound water. A model involving six thermodynamic states is presented and this model is related to the turnover of the enzyme.     

High pressure enhancement of enzymes: A review

While most current applications of high pressure (HP) are for inactivating deleterious enzymes, there is evidence that high pressure can induce stabilization and activation of some enzymes. Various other strategies have been employed to enhance enzyme stability, including; genetic engineering, immobilization, and operating in non-aqueous media. While each of these strategies has provided varying degrees of stability or activity enhancement, the application of high pressure may be a complementary, synergistic, or an additive enzyme enhancement technique. Over 25 enzymes that have exhibited high pressure stabilization and/or activation were compiled. Each enzyme discussed responds differently to high pressure depending on the pressure range, temperature, source, solvent or media, and substrate. Possible mechanisms for pressure-induced stabilization and activation are discussed and compared with current enzyme enhancement techniques. The compiled evidence of high pressure enzyme enhancement in this review indicates that pressure is an effective reaction parameter with potential for greater utilization in enzyme catalysis.     

Kinetics of carbonic anhydrase catalysis in solvents of increased viscosity: a partially diffusion-controlled reaction

Steady-state kinetic studies of the bovine carbonic anhydrase B-catalyzed hydration of CO2, dehydration of HCO3-, and hydrolysis of p-nitrophenylacetate were made in glycerol/water solvents of increased viscosity in order that the effect of diffusion-control on the substrate association reactions could be determined. The minimum association rate constants (kmin = V/(Km[E0])) were obtained at low substrate concentrations. The esterase activity did not depend upon the solvent viscosity. However, both the CO2 hydration and HCO3- dehydration reactions depended upon the solvent viscosity consistent with partial diffusion control. Thus both chemical activation and diffusion control processes contribute to the observed kmin. In low-viscosity aqueous solutions both hydration and dehydration are largely controlled by chemical activation. However, at higher viscosities, equal to that found in the interior of the erythrocyte, both reactions are largely diffusion controlled. This result can be interpreted to mean that carbonic anhydrase is a highly evolved enzyme that has approached its maximum efficiency. The extent of diffusion control observed rules out H2CO3 as a significant reactant with the enzyme. Several models that yield minimum steric requirements for access of substrate to the active site are examined. Minimum steric constraints are less for the smaller CO2. The slower esterase reaction is not influenced by diffusion.