Organic solvent functional group effect on enzyme inactivation by the interfacial mechanism

We have used a bubble column apparatus to study interfacial inactivation of enzymes. The amount of enzyme inactivated was proportional to the area of organic solvent exposed, as is characteristic of the interfacial mechanism. Tests were made with a series of 12 solvents of log P close to 4.0, but with different functional groups. With - and β-chymotrypsin, inactivation was much less severe with amphiphilic molecules like decyl alcohol, than with less polar compounds (heptane as the extreme case). This corresponds to a correlation with aqueous–organic interfacial tension, and presumably reflects a more polar interface as seen by the enzyme adsorbing from the aqueous phase. A 50% mixture of decyl alcohol and heptane behaved similarly to pure decyl alcohol, which would be expected to accumulate at the interface. With pig liver esterase, the correlation was rather weak, however. Accumulated data for interfacial inactivation by alkanes was examined for the above enzymes, and also papain, trypsin, urease and ribonuclease. The differing sensitivities did not show a clear correlation with any enzyme property, although there was some relationship to adiabatic compressibility, thermal denaturation temperature and mean hydrophobicity.     

Inactivation of enzymes by organic solvents: New technique with well-defined interfacial area

A liquid-liquid bubble column apparatus allows exposure of enzyme solutions to water-immiscible organic solvents with a known total interfacial area and welldefined time scales and flow. It allows clear distinction of the different classes of inactivation mechanism. With urease as a model enzyme, octan-2-one and butylbenzene act only through the effects of solvent molecules dissolved in the aqueous phase, giving first-order inactivation at 0.34 and 0.21 h(-1), respectively. Hexane and tridecane act only through exposure to the interface. The amount of urease inactivated is proportional to the total area of interface exposed, rather than to elapsed time, and may be characterized by a rate of about 0.5 mukat m(-2). This is consistent with the formation and (partial) inactivation of a complete adsorbed monolayer of protein. With butan-1-ol, both mechanisms contribute significantly to the observed inactivation. The presence of O(2) increases the rate of interfacial inactivation, but not that by dissolved solvent. (c) 1994 John Wiley & Sons, Inc.     

Lysozyme inactivation under mechanical stirring: effect of physical and molecular interfaces

This article focuses on the role of interfaces on lysozyme inactivation and aggregation process in stirred reactor. The first order inactivation constant of this process has found to be proportional not only to the power imparted by the impeller but also to the area of glass-liquid, air-liquid and PTFE-liquid interfaces in three reactors. Both area and type of interfaces act on inactivation: PTFE and air are four more efficient than glass to promote lysozyme inactivation because of their hydrophobicity. As well as physical interfaces, molecular surfaces of inactivated enzymes -more hydrophobic than native enzymes- enhance lysozyme inactivation and aggregation. This enhancement has been found to be correlated with the properties of aggregates of inactivated enzymes, especially their number. Then, under mechanical stirring, inactivation-aggregation process is induced by physical interfaces and self-catalyzed by increasing hydrophobic surfaces of inactivated enzymes.     

Resistance of vaccinia virus to inactivation by solvent/detergent treatment of blood products.

The inactivation of enveloped viruses by two different solvent/detergent combinations, i.e. tri-n-butyl phosphate (TNBP)/Triton X-100 or TNBP/Tween 80, has been investigated using a high purity factor VIII (Replenate) and factor IX (Replenine) respectively. Treatment with TNBP/Triton X-100 rapidly inactivated all the typical enveloped viruses tested, i.e. Sindbis, semliki forest virus (SFV), herpes simplex virus type-1 (HSV-1) and vesicular stomatitis virus (VSV), by 3.7-5.8 log within 15 seconds. While virus inactivation with TNBP/Tween 80 was slower, effective inactivation of Sindbis, HSV-1, VSV and human immunodeficiency virus type-1, i.e. 4.1-->6.3 log, occurred within 30 minutes. In contrast, vaccinia virus was relatively resistant to inactivation in either of these solvent/detergent combinations. Incubation times of 10 minutes for TNBP/Triton X-100 or 6-24 hours for TNBP/Tween 80, were required to reach inactivation levels of about 4 log.     

Effect of immiscible organic solvents on activity/stability of native chymotrypsin and immobilized-stabilized derivatives

We have developed different activity/stability tests to evaluate the possibilities of fully dispersed chymotrypsin derivatives as industrial catalysts in biphasic systems. We have tested different immiscible organic solvents (log P ranged from 0.65 to 2.8) and used different enzyme derivatives (soluble chymotrypsin and one-point and multipoint covalent attached derivatives). Special emphasis has been given to the role of the "exact composition of the aqueous phase."High phosphate concentrations largely protect every hymotrypsin derivative from the distorting effects of dissolved solvent molecules. The effects on the activity and stability of soluble chymotrypsin due to saturating solvent concentrations in an aqueous solution, and the much more severe effects of contact with the phase interface in a stirred biphasic system, all show the opposite trend for the influence of solvent polarity to that generally observed for biocatalysts. For example, deleterious effects decline in the order chloroform, dichloromethane, ethyl acetate. On the contrary, with or without stirring, our stabilized chymotrypsin-agarose derivatives are much more stable against these water-immiscible solvents, and their relative effects follow the normal trend. From these integrated activity and stability tests we can conclude that fully dispersed immobilized-stabilized derivatives seem to be an interesting alternative to develop industrial biphasic processes catalyzed by chymotrypsin.     

Virus inactivation by solvent/detergent treatment using Triton X-100 in a high purity factor VIII

Virus inactivation by solvent/detergent treatment using 0.3% tri-n-butyl phosphate and 1% Triton X-100 in the high purity factor VIII concentrate Replenate((R)) has been investigated. A wide range of model enveloped viruses were confirmed to be inactivated by >4 to >6log after 30min at 22 degrees C under standard conditions. Using Sindbis as a representative enveloped virus, the effect of various parameters on the inactivation process was tested. Virus inactivation was confirmed to be effective in different batches of product and was not influenced by changing the process conditions with regard to protein and salt concentration or pH. Virus inactivation was effective even at a temperature as low as 4-5 degrees C. Although solvent/detergent concentration was the most critical parameter, a concentration as low as 0.15% TnBP/0.5% Triton X-100 was still completely effective. At a lower concentration an extended incubation period was required. These studies demonstrate the robustness of this solvent/detergent procedure based on Triton X-100 and allow suitable process limits to be set for this manufacturing step.     

Stability of the lipase immobilized on DEAE-Sephadex for continuous lipid hydrolysis in organic solvent

Summary Continuous hydrolysis reaction was carried out in glass-column by using lipase fromCandida rugosa immobilized on DEAE-Sephadex A50. Substrate olive oil dissolved in hydrophobic organic solvent (isooctane) was supplied into the reactor with a concurrent stream of aqueous buffer. Stability of the immobilized enzyme was greatly increased in higher substrate concentration. Glyccrol added in the aqueous stream showed stabilizing effect against the organic solvent; half life was extended from 220 hrs to 450 hrs by 15% glycerol supplemented at 30°C.     

Inactivation of animal viruses during sewage sludge treatment

Using a previously developed filter adsorption technique, the inactivation of a human rotavirus, a coxsackievirus B5, and a bovine parvovirus was monitored during sludge treatment processes. During conventional anaerobic mesophilic digestion at 35 to 36 degrees C, only minor inactivation of all three viruses occurred. The k' values measured were 0.314 log10 unit/day for rotavirus, 0.475 log10 unit/day for coxsackievirus B5, and 0.944 log10 unit/day for parvovirus. However, anaerobic thermophilic digestion at 54 to 56 degrees C led to rapid inactivation of rotavirus (k' greater than 8.5 log10 units/h) and of coxsackievirus B5 (k' greater than 0.93 log10 unit/min). Similarly, aerobic thermophilic fermentation at 60 to 61 degrees C rapidly inactivated rotavirus (k' = 0.75 log10 unit/min) and coxsackievirus B5 (k' greater than 1.67 log10 units/min). Infectivity of parvovirus, however, was only reduced by 0.213 log10 unit/h during anaerobic thermophilic digestion and by 0.353 log10 unit/h during aerobic thermophilic fermentation. Furthermore, pasteurization at 70 degrees C for 30 min inactivated the parvovirus by 0.72 log10 unit/30 min. In all experiments the contribution of temperature to the total inactivation was determined separately and was found to be predominant at process temperatures above 54 degrees C. In conclusion, the most favorable treatment to render sludge hygienically safe from the virological point of view would be a thermal treatment (60 degrees C) to inactivate thermolabile viruses, followed by an anaerobic mesophilic digestion to eliminate thermostable viruses that are more sensitive to chemical and microbial inactivations.     

Inactivation of animal viruses during sewage sludge treatment.

Using a previously developed filter adsorption technique, the inactivation of a human rotavirus, a coxsackievirus B5, and a bovine parvovirus was monitored during sludge treatment processes. During conventional anaerobic mesophilic digestion at 35 to 36 degrees C, only minor inactivation of all three viruses occurred. The k' values measured were 0.314 log10 unit/day for rotavirus, 0.475 log10 unit/day for coxsackievirus B5, and 0.944 log10 unit/day for parvovirus. However, anaerobic thermophilic digestion at 54 to 56 degrees C led to rapid inactivation of rotavirus (k' greater than 8.5 log10 units/h) and of coxsackievirus B5 (k' greater than 0.93 log10 unit/min). Similarly, aerobic thermophilic fermentation at 60 to 61 degrees C rapidly inactivated rotavirus (k' = 0.75 log10 unit/min) and coxsackievirus B5 (k' greater than 1.67 log10 units/min). Infectivity of parvovirus, however, was only reduced by 0.213 log10 unit/h during anaerobic thermophilic digestion and by 0.353 log10 unit/h during aerobic thermophilic fermentation. Furthermore, pasteurization at 70 degrees C for 30 min inactivated the parvovirus by 0.72 log10 unit/30 min. In all experiments the contribution of temperature to the total inactivation was determined separately and was found to be predominant at process temperatures above 54 degrees C. In conclusion, the most favorable treatment to render sludge hygienically safe from the virological point of view would be a thermal treatment (60 degrees C) to inactivate thermolabile viruses, followed by an anaerobic mesophilic digestion to eliminate thermostable viruses that are more sensitive to chemical and microbial inactivations.     

Chlorination of indicator bacteria and viruses in primary sewage effluent

Wastewater disinfection is used in many countries for reducing fecal coliform levels in effluents. Disinfection is therefore frequently used to improve recreational bathing waters which do not comply with microbiological standards. It is unknown whether human enteric viruses (which are responsible for waterborne disease) are simultaneously inactivated alongside fecal coliforms. This laboratory study focused on the chlorination of primary treated effluent with three doses (8, 16, and 30 mg/liter) of free chlorine as sodium hypochlorite. Seeding experiments showed that inactivation (>5 log(10) units) of Escherichia coli and Enterococcus faecalis was rapid and complete but that there was poor inactivation (0.2 to 1.0 log(10) unit) of F(+)-specific RNA (FRNA) bacteriophage (MS2) (a potential virus indicator) at all three doses. However, seeded poliovirus was significantly more susceptible (2.8 log(10) units) to inactivation by chlorine than was the FRNA bacteriophage. To ensure that these results were not artifacts of the seeding process, comparisons were made between inactivation rates of laboratory-seeded organisms in sterilized sewage and inactivation rates of organisms occurring naturally in sewage. Multifactorial analysis of variance showed that there was no significant difference (P > 0.05) between the inactivation rates for seeded and naturally occurring FRNA bacteriophage. However, laboratory-grown poliovirus was inactivated much more rapidly than were naturally occurring, indigenous enteroviruses (P < 0.001). This may reflect differences in the way indigenous virus is presented to the disinfectant. Inactivation rates for indigenous enteroviruses were quite similar to those seen for FRNA bacteriophage at lower doses of chlorine. These results have significance for the effectiveness of chlorination as a sewage treatment process, particularly where virus contamination is of concern, and suggest that FRNA bacteriophage would be an appropriate indicator of such viral inactivation under field conditions.     

Immobilized lipase Candida sp. 99-125 catalyzed methanolysis of glycerol trioleate: solvent effect

The immobilized lipase Candida sp. 99-125 catalyzed methanolysis of glycerol trioleate was studied in twelve different solvents in order to deduce the solvent effect through an attempt to correlate the highest yield with such solvent properties as hydrophobicity (log P), dielectric constant (epsilon), and Hildebrand solubility parameter (delta). The results showed that the conversion of glycerol trioleate and yield of oleic acid methyl ester were quite dependent on the solvent. The catalyst lipase in various solvents also needed different optimum amount of water to keep its maximum activity, and generally this lipase in more hydrophobic solvents required more water. The correlation between the highest yield and log P value was found to be reasonable except deviation of data points of certain solvents, while no obvious correlation existed between the other two parameters, dielectric constant (epsilon) and Hildebrand solubility parameter (delta), and the enzyme activity. The study revealed that more hydrophobic solvents such as n-hexane or cyclohexane were more suitable solvents for Candida sp. 99-125 catalyzed transesterification of glycerol trioleate to oleic acid methyl ester.     

Interfacial inactivation of epoxide hydrolase in a two-liquid-phase system

Enantioselective epoxide hydrolases are useful biocatalysts for the preparation of enantiopure epoxides and diols. The kinetic resolution of racemic epoxides can be carried out in an organic/aqueous biphasic system to allow use of high epoxide concentrations. Enzyme inactivation in such a system, however, may occur by contact with the interface. In this study, we investigated the factors which influence the interfacial inactivation of Agrobacterium radiobacter epoxide hydrolase in an octane/water biphasic system. Rates of interfacial inactivation were measured both in a stirred-cell, which has a planar interface, and in an emulsion reactor. Interfacial inactivation rates measured in the stirred-cell at a fixed interfacial area increased with mixing intensity. Interfacial inactivation rates per unit area were lower in the emulsion reactor than in the stirred-cell and increased with bulk aqueous enzyme concentration. Circular dichroism measurements showed that during biphasic incubation all unadsorbed soluble enzyme existed in the native conformation. Activity assays showed that the dissolved enzyme was also fully active, indicating that inactivated enzyme precipitated from solution. Using an inactive epoxide hydrolase mutant structurally similar to the wild-type enzyme in order to avoid the conversion of the epoxide, it was found that high concentrations of epoxide in the organic phase increased the rate of interfacial inactivation.     

Self-assembling of polymer-enzyme conjugates at oil/water interfaces

Interface-binding enzymes are desirable for biphasic reactions in that they offer simultaneous access to substrates dissolved in both phases across the interface. It has been shown that conjugating water-soluble enzymes with hydrophobic polymers facilitated the assembling of enzymes at oil/water interfaces. In this work, the interfacial assembling of alpha-chymotrypsin conjugated with polystyrene, poly(methyl methacrylate), and poly(l-lactic acid) was examined using the pendant drop method. The interface-assembling process of the conjugates from the organic phase followed a similar pattern of that of native alpha-chymotrypsin from the aqueous buffer phase, i.e., the interfacial tension decreased gradually with time. However, when the conjugates were dispersed in the form of particulates in the aqueous phase, in which the conjugate was insoluble, the assembling occurred faster and the interfacial tension quickly approached zero. It was suspected that the assembling in this case involved two steps, i.e., the adsorption of the particulates and the subsequent rearrangement, dissociation, and redispersion of the conjugates at the interface. The effect of other factors, including the polarity of organic solvent and pH and ionic strength of the aqueous phase, was evaluated. It was found that the polar solvent slightly facilitated the assembling, whereas pH and ionic strength showed minimal effects.     

A protease stable in organic solvents from solvent tolerant strain of Pseudomonas aeruginosa

A solvent tolerant strain of Pseudomonas aeruginosa (PseA) was isolated from soil samples by cyclohexane enrichment in medium. The strain was able to sustain and grow in a wide range of organic solvents. The adaptation of P. aeruginosa cell towards solvents was seen at membrane level in transmission electron micrographs. It also secreted a novel protease, which exhibited remarkable solvent stability and retained most of the activity at least up to 10 days in the presence of hydrophobic organic solvents (log P > or = 2.0) at 25% (v/v) concentrations. The protease was able to withstand as high as 75% concentration of solvents at least up to 48 h. P. aeruginosa strain and its protease, both seem promising for solvent bioremediation, wastewater treatment and carrying out biotransformation in non-aqueous medium.     

Structural and conformational differences of acylated hyaluronan modified in protic and aprotic solvent system

Acylated hyaluronan (HA) in aqueous (DMSO/H₂O) and nonaqueous (DMSO) solutions was studied by means of nuclear magnetic resonance, differential scanning calorimetry (DSC), mass spectrometry and UV/vis spectroscopy. It has been demonstrated that structural and conformational properties of the acylated hyaluronan derivates are strongly dependent on the nature of reaction solvent. Acylation in DMSO was more selective than that carried out in DMSO/H₂O, though in both cases in average a maximum of one acyl chain was detected per HA dimer. The hydrophobic functionalization of hyaluronan induced its interaction with hydrophobic dye as a consequence of acyl chain aggregation. The higher the degree of acylation the more hydrophobic dye was interacting with HA. For concentrated samples, aggregation was more evident in case of acylated HA in aqueous solution. This phenomenon was explained by its different conformational arrangement in solution which was further supported by DSC data indicating an existence of hydrophobic cavities. The formation of self-aggregated assemblies indicates potential applications of this type of HA derivate as drug delivery system.     

Hydrolytic reactions in two-phase systems. Effect of water-immiscible organic solvents on stability and activity of acid phosphatase, beta-glucosidase, and beta-fructofuranosidase.

The stability and activity of three hydrolytic enzymes, acid phosphatase (EC 3.1.3.2), beta-fructofuranosidase (EC 3.2.1.26), and beta-glucosidase (EC 3.2.1.4), were studied at 30 degrees C in two-phase systems. They were prepared with equal quantities of buffered water and a water-immiscible organic solvent. Low-molecular-weight acetates and paraffins were tested in this investigation. The kinetic constant of storage inactivation was correlated with the logarithm of solvent polarity. Enzyme stability in the presence of organic phases, whose log P value was included in 1.2-2.2, was greater than the one measured in pure buffered aqueous media. On the other hand, a dramatic enzyme denaturation took place making use of solvents at higher log P-value. Experiments carried out during the 24-h operation clarified that the reaction yield does not depend solely on solvent polarity. Acid phosphatase and beta-glucosidase, which are less resistant than beta-fructofuranosidase to temperature and shear in buffered solutions, showed especially significant enhancement of catalytic activity when hydrolysis was performed with the addition of acetates (50% v/v).     

Aspects Of Biocatalyst Stability In Organic Solvents

The stability of biocatalysis in systems containing organic solvents is reviewed. Among the examples presented are homogeneous mixtures of water and water-miscible organic solvents, aqueous/organic two-phase systems, solid biocatalysts suspended in organic solvents, enzymes in reverse micelles and modified enzymes soluble in water immiscible solvents. The stability of biocatalysts in organic solvents depends very much on the conditions. The hydrophobicity or the polarity of the solvent is clearly of great importance. More hydrophobic solvents (higher log P values) are less harmful to enzymes than less hydrophobic solvents. The water content of the system is a very important parameter. Some water is essential for enzymatic activity; however, the stability of enzymes decreases with increasing water content. Mechanisms of enzyme inactivation are discussed.     

Solvent-tolerance of fungi located on an interface between an agar plate and an organic solvent

While 6 by 20 of type culture fungi could grow on an interface between organic solvent (log P, 4.12) and agar plate, 13 by 20 of strains could form a large colony after the removal of more toxic solvent, such as styrene (log P, 2.95) and tert-butyl acetate (log P, 1.76) because of viability of spores on the interface.     

Chemical Inactivation of Lipase in Organic Solvent: A Lipase from Pseudomonas aeruginosa TE3285 is More Like a Typical Serine Enzyme in an Organic Solvent than in Aqueous Media

A microbial lipase from Pseudomonas aeruginosa TE3285 was treated in anhydrous diisopropyl ether with three kinds of serine-reactive reagents, ethyl p-nitrophenyl methylphosphonate (ENMP), diisopropyl fluorophosphate (DFP), and phenylmethylsulfonyl fluoride (PMSF) to lose its catalytic activity for both transesterification in an organic solvent and ester hydrolysis in aqueous system. In contrast with the facile inactivation in an organic solvent, no or very slow inactivation was observed in an aqueous solution. The lipase was shown to behave more like a typical serine enzyme in an organic solvent than in aqueous solution with regard to the chemical inactivation by serine-reactive reagents. The unique behavior of the lipase in an organic solvent may be associated with inferfacial activation of the lipase, which is one of the most distinct characteristics of the lipase family, and the activiation of lipase could be induced by a hydrophobic interaction with an organic solvent.     

Evaluation of the growth inhibition strength of hydrocarbon solvents against Escherichia coli and Pseudomonas putida grown in a two-liquid phase culture system consisting of a medium and organic solvent

The growth of microorganisms is often inhibited in a two-liquid phase culture system consisting of an aqueous medium and a large volume of hydrophobic solvent. Escherichia coli and Pseudomonas putida were cultured in a two-phase system containing a solvent with a log Pow value in a range of 2.1 to 6.0. The increase in the cell mass was monitored by increase in turbidity of the medium phase. We devised a semiquantitative method to evaluate the growth inhibition strength of solvents based on the relative amount of bacterial growth occurring in the two-phase system. Analyses of growth of the bacteria by this method showed that the growth inhibition strength of a given solvent was usually but not always correlated inversely with its polarity. It is clear that growth inhibition strength is not determined simply by polarity of the solvent.