Enzyme stability in the presence of organic solvents

Summary The thermal stability of vacuum-dried acid-phosphatase has been investigated, both in the absence and in the presence of pure hexadecane. Preliminary experimental results indicate that: i) in both solid-phase runs, acid-phosphatase is much more stable than the free enzyme in aqueous solution, ii) the presence of the organic solvent slightly reduces thermal stability of the solid-phase enzyme. As regards the deactivation mechanism, when acid-phosphatase operates in free aqueous solution it follows a two-step in series deactivation. Initially the native configuration decays towards an intermediate, still active form. This, in turn, irreversibily yields a totally inactive structure. In the thermal deactivation of solid-phase enzyme it has been observed that: i) the first step is substantially retarded, ii) the final transition is completely hindered, iii) the intermediate configuration is more active than that produced in aqueous solution, by more than one order of magnitude.     

Aldolase stability in the presence of organic solvents

Rabbit muscle aldolase (RAMA) and trout muscle aldolase (TRMA) retained 100% activity in the presence of hexane, cyclohexane and toluene. Both enzymes retained greater than 80% activity in the presence of 20% (v/v) methanol. In the presence of 20% (v/v) N,N-dimethylformamide RAMA and TRMA were inactive, but at least 50% activity could be restored by returning the enzymes to an aqueous environment.     

On the stability of peptide nucleic acid duplexes in the presence of organic solvents

Nucleic acid double helices are stabilized by hydrogen bonding and stacking forces (a combination of hydrophobic, dispersive and electrostatic forces) of the base pairs in the helix. One would predict the hydrogen bonding contributions to increase and the stacking contributions to decrease as the water activity in the medium decreases. Study of nucleobase paired duplexes in the absence of water and ultimately in pure aprotic, non-polar organic solvents is not possible with natural phosphodiester nucleic acids due to the ionic phosphate groups and the associated cations, but could be possible with non-ionic nucleic acid analogues or mimics such as peptide nucleic acids. We now report that peptide nucleic acid (PNA) (in contrast to DNA) duplexes show almost unaffected stability in up to 70% dimethylformamide (DMF) or dioxane, and extrapolation of the data to conditions of 100% organic solvents indicates only minor (or no) destabilization of the PNA duplexes. Our data indicate that stacking forces contribute little if at all to the duplex stability under these conditions. The differences in behaviour between the PNA and the DNA duplexes are attributed to the differences in hydration and counter ion release rather than to the differences in nucleobase interaction. These results support the possibility of having stable nucleobase paired double helices in organic solvents.     

Electric and hydrodynamic properties of polypeptides in solution. II. Conformation of poly(L-glutamic acid) in various organic solvents

The electric birefringence of poly(L -glutamic acid) (PLGA) in methanol, dimethyl sulfoxide, dimethylformamide, N-methylacetamide, trifluoroacetic acid, dioxane–water mixtures (3:1 and 4:1 by volume), and dioxane–formamide mixture (1:1 by volume) has been measured by the use of the rectangular pulse technique at 30 °C. The intrinsic viscosity has also been measured at the same temperature. The magnitude of the specific Kerr constant and the intrinsic viscosity suggests that PLGA is helical and has a large dipole moment in methanol, dimethyl sulfoxide, dimelhylformamide, N-methylacetamide, and dioxane–water mixtures. In this case we have obtained the length distribution curve and the mean length of PLGA molecules from the decay of the electric birefringence, by applying the method recently developed for helical polypeptides. Furthermore, we have proposed and applied a method of obtaining the mean dipole moment and the optical anisotropy factor from the field strength dependence of the electric birefringence for polydisperse systems on the basis of the knowledge on the length distribution. The results show that PLGA may have a different helical conformation in dimethyl sulfoxide. The specific Kerr constant of PLGA in trifluoroacetic acid is very small, which suggests that PLGA is a random coil in this solvent.     

两液相培养中有机溶剂对细胞毒性的研究进展

在生物化学工程中 ,细胞培养 -分离耦合过程的研究越来越引起人们的重视[1] ,其主要原因一是为了减少代谢产物的反馈作用 ,提高代谢产物产率 ,促进更多的细胞培养工业化进程 ;二是为了协调上游和下游加工过程 ,使整个过程的研究得到优化 ,以提高生产效率 ,降低生成成本。因此近十年细胞培养 -分离耦合过程的研究有了较大的发展。可以说几乎所有主要的生化分离技术都有用在细胞培养 -分离耦合过程的研究上。但研究较多的是细胞培养与萃取分离耦合 ,即两液相培养 ,并证明这是一项很有用的技术。两液相培养研究所涉及的内容很多 ,但最重要的是…     

Enhancement of stability and activity of phospholipase A(1) in organic solvents by directed evolution.

We attempted to apply the directed evolution approach to enhancing enzyme properties in the presence of organic solvents, in which enzyme stability and activity were often drastically reduced. Stability and catalytic activity of phospholipase A(1) in the presence of an organic solvent were enhanced by error-prone polymerase chain reaction (PCR) and DNA shuffling followed by a filter-based visual screening. Three mutants (SA8, SA17 and SA20) were isolated on indicator plates (i.e., 1% phosphatidylcholine gels containing 30% dimethyl sulfoxide (DMSO)) after a second mutant library was treated in 50% DMSO for 36 h. The half-life values of the three mutants exhibited an approximately 4-fold increase. The three mutants also exhibited increased stability in all organic solvents tested compared with the wild-type enzyme. Thus, an enzyme variant having superior catalytic efficiency in most of the organic solvents could be obtained by using any solvent suitable for designing the efficient screening system, regardless of the properties of the particular solvent.     

Role of organic solvents on Pa-hydroxynitrile lyase interfacial activity and stability

Catalytic activity and adsorption of Pa-hydroxynitrile lyase (Pa-Hnl) was investigated at various organic solvent/water interfaces. We focused on the role of solvent polarity in promoting activity and stability in two-phase systems, specifically for the solvents heptane, dibutyl ether (DBE), diisopropyl ether (DIPE), butylmethyl ether (BME), and methyl tert-butyl ether (MTBE). Enzyme activity towards mandelonitrile cleavage was determined in a recycle reactor with a well-defined interfacial area as described by Hickel, et al. 1999. Here the recycle reactor was modified to permit exchange of the aqueous phase. With this modification, irreversibility of enzyme adsorption was determined as a function of the adsorption time at the interface. Irreversibility of enzyme adsorption was also investigated by measuring the surface pressure of a sessile-drop upon washout. We find that Pa-Hnl exhibits the highest stability but the lowest initial catalytic activity at the aqueous/organic solvent interface with the most polar organic solvents. Thus, DIPE and MTBE display no loss in enzyme activity over a period of several hours. However, the more apolar the solvent is the higher the initial Pa-Hnl activity, but the faster the loss of activity. Dynamic tensiometry reveals that Pa-Hnl adsorbs more strongly at the interface of the more apolar solvents. Surprisingly, Pa-Hnl develops some irreversible adsorption after 30 min at the DIPE/water interface, but does not lose catalytic activity.     

Biocatalysis using an organic-soluble enzyme for the preparation of poly(lactic acid) in organic solvents

Proleather from Bacillus sp. was chemically modified with decanoyl chloride for enhanced activity for the preparation of poly(lactic acid) in organic solvents. The modified enzyme was highly soluble (up to 44 mg-protein/ml) and active in various organic solvents including chloroform, tetrahydrofuran (THF), pyridine and acetone. The organic-soluble proleather efficiently catalyzed the polymerization of ethyl lactate. The reaction rate was 4-22 times that of native proleather, depending upon the solvent applied. The solubilized enzyme showed a highest activity at 50 degrees C, the same optimum temperature for both the native proleather and an immobilized enzyme, Novozyme-435. Denaturation of the enzymes' protein structures appeared to be the critical factor regulating the optimum activity temperature. Differential scanning calorimetry (DSC) analyses of the enzymes showed endothermic peaks around 55 degrees C, indicating the proteins' structures altered in that temperature range. Interestingly, the activity of the solubilized enzyme showed a more complicated water dependence as compared to native proleather.     

On the thermal stability of DNA in solution of mixed solvents

The study of the changes in UV absorbance of DNA solutions in water/dioxane and water/ethylene glycol mixture at different concentrations shows that the thermal denaturation of DNA is sensitive to the electrical permittivity of the media and the water content. At relative low concentrations of co-solvent the dominant feature is the electrical permittivity. When water content is lower than a critical value, the electrical permittivity is no longer the determinant of the denaturation temperature but the partial volume fraction of water. The critical water content is about 0.69 partial volume fraction of water.     

Operational stability of high initial activity protease catalysts in organic solvents

The first studies on the operational stability of cross-linked enzyme crystals (CLECs) in organic media are described. Although these catalysts display high initial specific activity, they inactivate rapidly, losing more than 50% of the initial activity within the first 4 h under continuous flow. Furthermore, the inactivation is not reversible when returned to an aqueous medium. The same rapid inactivation occurs with adsorbed protease preparations that show similar high initial specific activity (propanol-rinsed enzyme preparations (PREPs) of subtilisin and alpha-chymotrypsin).     

Effect of organic solvents on the activity and stability of halophilic alcohol dehydrogenase (ADH2) from Haloferax volcanii

The effect of various organic solvents on the catalytic activity, stability and substrate specificity of alchohol dehydrogenase from Haloferax volcanii (HvADH2) was evaluated. The HvADH2 showed remarkable stability and catalysed the reaction in aqueous?Corganic medium containing dimethyl sulfoxide (DMSO) and methanol (MeOH). Tetrahydrofuran and acetonitrile were also investigated and adversely affected the stability of the enzyme. High concentration of salt, essential to maintain the enzymatic activity and structural integrity of the halophilic enzyme under standard conditions may be partially replaced by DMSO and MeOH. The presence of organic solvents did not induce gross changes in substrate specificity. DMSO offered a protective effect for the stability of the enzyme at nonoptimal pHs such as 6 and 10. Salt and solvent effects on the HvADH2 conformation and folding were examined through fluorescence spectroscopy. The fluorescence findings were consistent with the activity and stability results and corroborated the denaturing properties of some solvents. The intrinsic tolerance of this enzyme to organic solvent makes it highly attractive to industry.     

Effect of heat treatments on stability of altemariol, alternariol monomethyl ether and tenuazonic acid in sunflower flour

A study was carried out to evaluate the effect of heat treatment on the stability of alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TeA) in sunflower flour and the effectiveness of this treatment by a biological assay in rats. The concentrations of AOH and AME remained constant during heating at 100°C for up to 90 minutes while TeA concentration decreased with time to 50% after 90 minutes. The most effective treatment in reducing AOH and AME levels was heating at 121°C for 60 minutes. Histopathological evaluation in the biological assay in rats fed withAlternaria toxins showed marked atrophy and fusion of villi in the intestines and liver cell damage; these lesions were less severe in rats fed heat-treated sunflower flour in line with the reduced toxin content. However, a lower weight gain and a noticeable renal damage in rats were produced when they fed decontaminated flour.     

Improved activity and stability of an immobilized recombinant laccase in organic solvents

Laccase (E.C. 1.10.3.2) from Trametes versicolor was immobilized (adsorbed) by drying on various supports (glass, glass powder, silica gel, and Nylon 66 membrane). The enzyme activity and stability were determined in diethyl ether, ethyl acetate, and methylene chloride. The initial rate for the oxidation of syringaldazine varied up to 245-fold depending on the solvent and support, the best results being obtained with Nylon 66 membrane. No inactivation of immobilized laccase over 72 h was observed in diethyl ether and ethyl acetate, while exposure to methylene chloride resulted in significant activity decreases regardless of the support material.     

Demineralization in organic solvents by alkylammonium salts of ethylenediaminetetra-acetic acid.

1. Insoluble salts of alklaine earth metals (e. g. calcium phosphate) dissolve in non-aqueous solvents containing EDTA salts of organic cations (e.g. tetraethylammonium). 2. Bone is efficiently demineralized in ethanolic trimethylammonium EDTA. Retention in the matrix of water-soluble materials (e.g. proteoglycan) is much better than in standard aqueous EDTA demineralization procedures.     

Engineering subtilisin E for enhanced stability and activity in polar organic solvents

We examined the effect of a novel disulfide bond engineered in subtilisin E from Bacillus subtilis based on the structure of a thermophilic subtilisin-type serine protease aqualysin I. Four sites (Ser163/Ser194, Lys170/Ser194, Lys170/Glu195, and Pro172/Glu195) in subtilisin E were chosen as candidates for Cys substitutions by site-directed mutagenesis. The Cys170/Cys195 mutant subtilisin formed a disulfide bond in B. subtilis, and showed a 5-10-fold increase in specific activity for an authentic peptide substrate for subtilisin, N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide, compared with the single-Cys mutants. However, the disulfide mutant had a 50% decrease in catalytic efficiency due to a smaller k(cat) and was thermolabile relative to the wild-type enzyme, whereas it was greatly stabilized relative to its reduced form. These results suggest that an electrostatic interaction between Lys170 and Glu195 is important for catalysis and stability in subtilisin E. Interestingly, the disulfide mutant was found to be more stable in polar organic solvents, such as dimethylformamide and ethanol, than the wild-type enzyme, even under reducing conditions; this is probably due to the substitution of uncharged Cys by charged surface residues (Lys170 and Glu195). Further, the amino-terminal engineered disulfide bond (Gly61Cys/Ser98Cys) and the mutation Ile31Leu were introduced to enhance the stability and catalytic activity. A prominent 3-4-fold increase in the catalytic efficiency occurred in the quintet mutant enzyme over the range of dimethylformamide concentration (up to 40%).     

Enzymatic transesterification of monosaccharides and amino acid esters in organic solvents

Summary Lipases and proteases from different sources were screened for their ability to catalyze the transesterification of glucose and activated N-blocked phenylalanine. A commercial protease from Bacillus licheniformis was found to be most effective for this purpose. On a basis of ¹³C-NMR analysis, glucose was acylated at the C-6 position. The enzyme showed a broad substrate specificity toward various monosaccharides.     

Assessment of ochratoxin A and tenuazonic acid in Canadian ice-wines

Twenty-six samples of commercial ice-wine made from late-harvested grapes were assayed for the mycotoxins ochratoxin A and tenuazonic acid. Canadian wines originated in British Columbia (18) and Ontario (8). For comparison two German wines from Hesse were also studied. Four additional samples of research ice-wine originating in were also studied. In all wine samples, assays using immuno-affinity chromatography and fluorescence liquid chromatography indicated ochratoxin A below 0.15 μg/L, the limit of determination of the method. Tenuazonic acid was determined by solidphase micro-extraction and liquid chromatography and was below the limit of determination (70 μg/L) in all samples. The European Union food tolerance limit for ochratoxin A in wine is 2 μg/L. A tolerance for tenuazonic acid has not yet been established.     

Improved stability and catalytic activity of chemically modified papain in aqueous organic solvents

Papain was modified with the anhydrides of various monocarboxylic (acetic or propionic) and dicarboxylic (citraconic, maleic or succinic) acids. 7–10 of the 11 primary amino groups of the enzyme were modified. The organic solvent tolerances of the modified enzyme forms were increased (especially in the concentration range of 10–60%) in comparison with the unmodified enzyme. Acylation enhanced the catalytic activity and stability of papain both in buffer and in aqueous organic solvents (ethanol and acetonitrile). Decrease of the positive charges on the surface of papain resulted in a higher enzyme stability than when they were replaced by negative charges. The kinetic parameters revealed that in aqueous ethanol the maximum rates (V_max) and Michaelis constants (K_M) of the modified papain forms were increased, and higher catalytic efficiencies (k_cat/K_M) were detected as compared with the native enzyme. The results of near-UV circular dichroism and tryptophan fluorescence spectroscopic studies suggested that the modifications caused only local changes around the aromatic residues. The modified enzyme forms led to higher N-acetyl-l-tyrosine ethyl ester synthesis conversions in aqueous ethanol; acetyl and propionyl papain furnishing the highest productivity.