Biocatalysis of lipoxygenase in selected organic solvent media

The biocatalysis of purified soybean lipoxygenase (LOX) (EC 1.13.11.12), using linoleic acid as a substrate model, was investigated in selected organic solvent media, including chloroform, dichloromethane, hexane, iso-octane, octane and toluene. The results indicated that there was a 2.6-fold increase in LOX activity in the monophasic iso-octane medium compared to that obtained in the aqueous medium. The results also showed that there was an increase of 2.2- and 1.8-fold in LOX activity in the monophasic reaction media of octane and hexane, respectively. However, an inhibitory effect on enzyme activity was observed when the monophasic reaction media of toluene, chloroform and dichloromethane were used. In addition, the results showed that the optimum concentration of octane and iso-octane in the biphasic medium containing the organic solvent and Tris–HCl buffer solution, was determined to be 3.5% and 4%, respectively, for LOX activity. Moreover, the biocatalysis of LOX in a ternary micellar system, containing either 3.5% octane or 4% iso-octane, Tris–HCl buffer solution and an emulsifier, resulted in an overall increase in enzyme activity. The K_m and V_max values, substrate specificity, optimum protein concentration, optimum reaction temperature as well as the enzymatically catalyzed end-products were investigated for LOX biocatalysis in both ternary micellar systems.     

Biocatalysis in organic media

The potentials of using organic reaction media in biotechnological conversions have already been demonstrated in several experimental studies. Examples of possible advantages are: possibility of higher substrate and/or product concentrations, favorable shift of reaction equilibria, reduced substrate and/or product inhibition, and facilitated product recovery. Especially water/organic solvent two-phase systems seem to possess several of these advantages. The solvent type will highly affect kinetics and stability of the (immobilized) biocatalyst, solubility and partitioning of reactants/products, and product recovery. Therefore the solvent choice can have a large influence on the economics of the two-liquid-phase biocatalytic process. Immobilization of the biocatalyst may be useful to provide protection against denaturating solvent effects. The polarity of the employed support material will also be decisive for the partitioning of substrates and products among the various phases.

A classification of biphasic systems, which is based on the possible types of theoretical concentration profiles and aqueous phase configurations, is discussed. Reversed micelles and aqueous two-liquid-phase systems can be considered as special cases. The design of two-liquid-phase bioreactors is dependent on the state of the biocatalyst, free or immobilized, and on the necessity for emulsification of one of the two liquid phases in the other. Many mass-transfer resistances, e.g. across the liquid/liquid interface, in the aqueous phase, across the liquid/solid interface, and in the biocatalyst phase, can limit the overall reaction rate. The epoxidation of alkenes in water/solvent two-phase systems is discussed to give an example of the scope of biotechnological processes that is obtained by using organic media. Finally, a design calculation of a packed-bed organic-liquid-phasel immobilized-biocatalyst reactor for the epoxidation of propene is given to illustrate some of the above aspects.     

3-hydroxykynurenine as a substrate/activator for mushroom tyrosinase

3-Hydroxykynurenine is a tryptophan metabolite with an o-aminophenol structure. It is both a tyrosinase activator and a substrate, reducing the lag phase, stimulating the monophenolase activity, and being oxidized to xanthommatin. In the early stage of monophenol hydroxylation, catechol accumulation takes place, whereas 3-hydroxykynurenine is substantially unchanged and no significant amounts of the o-quinone are produced. These results suggest an activating action of 3-hydroxykynurenine toward o-hydroxylation of monophenols. 3-Hydroxykynurenine could therefore well act as a physiological device to control phenolics metabolism to catechols and quinonoids.     

Biocatalysis with hydroperoxide lyase in extracts from Penicillium camemberti in neat organic solvent media

Abstract

Biocatalysis with hydroperoxide lyase (HPL) in extracts from Penicillium camemberti, in neat organic solvent media has been investigated. The effects of reaction conditions including organic solvent mixtures, initial water activity (a_w) and reaction temperature as well as the effect of the lyoprotectants, KCl and dextran 1 kDa, on HPL activity were studied. The addition of KCl to the enzymatic extract (70:1 protein, w/w) prior to lyophilization, enhanced HPL activity 6.53-fold. In contrast, the presence of dextran at a ratio of 8:1 decreased the enzymatic activity. Using hexane as the reaction medium, with an initial a_w of 0.1 and 0.5, the HPL specific activity was determined to be as 6.3 and 65.9 nmol converted 10-HPOD/mg protein/min, for the enzymatic extract without and with KCl present, respectively. Although HPL enzymatic extract with KCl showed a relatively low optimum reaction temperature (45°C) compared to 55°C without KCl, it exhibited a 2.51- and 2.78-fold higher thermal stability at 60 and 80°C, respectively. The kinetic results indicated that the highest HPL catalytic efficiency, V_max/K_m, of 6.58 × 10^?2 mL/mg protein/min, was obtained in the presence of KCl.     

Lipase-catalyzed transamidation of non-activated amides in organic solvent

A novel biocatalytic reaction of transamidation of non-activated amides with amines is reported. Among 45 different lipolytic and proteolytic enzymes tested, only the lipase from Candida antarcticawas able to catalyze this reaction. The reaction proceeded with up to ca. 80% conversion in anhydrous methyl tert-butyl ether and worked with both N-substituted and unsubstituted amides. The biocatalytic transamidation is an equilibrium process and, therefore, higher conversions to the desired amide were achieved by using increased concentrations of the amine nucleophile.     

Reaction of manganese-dependent peroxidase from Bjerkandera adusta in aqueous organic media

The oxidation of various phenolics and aromatic amines by manganese-dependent peroxidase (MnP) of Bjerkandera adusta was examined in aqueous organic media. MnP retained its activities in several 70% (v/v) aqueous solutions of water-miscible organic solvents including ethylene glycol, diethylene glycol, acetone and acetonitrile. The absorption spectra of MnP in these aqueous organic media were similar to that observed in the reaction without solvent addition, indicating that the heme of MnP was little affected by the addition of these water-miscible organic solvents. MnP was also found to oxidize Mn(II) to Mn(III) in these 70% (v/v) aqueous organic media. The oxidation of Mn(II) by MnP was correlated with the Dimroth–Reichardt parameter, E_T(30), of the solvents. Furthermore, MnP catalyzed the oxidation of anisidines, aminophenols, phenylenediamines and phenolics in aqueous 70% (v/v) acetone, acetonitrile and diethylene glycol media. Aromatic amines that have high hydrophobicity were shown to be suitable for the reaction of MnP in aqueous water-miscible organic media.     

Study of fumarase activity in non-conventional media. Part I

Fumarase catalysed hydration of fumarate was investigated in water/organic solvent one-phase systems. The organic solvents used were ethylene glycol, glycerol and dimethylformamide. The effects of the amount of organic solvent on the maximum velocity (V_max), the Michaelis-Menten constant (K_M) and the equilibrium constant (K_eq) were studied in all the reaction media. Together with a denaturing power of the solvent evidenced by a systematic decrease of V_max also a surprising decrease of the K_M was registered as the percentage of organic solvent in the reaction media was increased. While the equilibrium constant of the reaction (K_eq = [l-malate]/[fumarate]) decreased when the percentage of organic solvent was raised. An interpretation of these facts was given. Time-dependent denaturation was also investigated and glycerol resulted the less denaturing of the solvents used, while the aprotic DMF exhibited the highest deactivation.     

Study of fumarase activity in non-conventional media. Part II

The hydration of fumarase and the dehydration ofl-malate catalysed by fumarase were investigated in water/methanol and water/formamide one phase systems. The effects of the amount of organic solvent on the maximum velocity (V_max), the Michael is-Menten constant (K_M) and the equilibrium constant (K_eq) were studied for both the reaction media. The denaturing power of both methanol and formamide was observed together with the familiar decrease of the K_M. Fumarase catalysis in water/methanol systems was further investigated by evaporating the organic solvent and evaluating the degree of reversibility of the inactivation. Reversibility of formamide denaturation was also investigated. The effects of phosphate concentration in the reaction medium with different amounts of methanol was investigated following the variation of the kinetic parameters of the hydration reaction. At high concentrations of phosphate an inhibiting effect appeared. Time-dependent denaturation was also investigated and a remarkable instability of fumarase in systems with percentages (v/v) of formamide higher than 10% was observed. 10% formamide proved to be less deactivating than the other non-conventional reaction media so far employed.     

Studies on the mechanism of crown-ether-induced activation of enzymes in non-aqueous media.

Studies on the mechanism of crown-ether-induced activation are described in this paper. Michaelis Menten kinetics of -chymotrypsin in toluene in the presence and absence of 18-crown-6 showed that only V_max is increased upon crown ether treatment. Parallel Lineweaver–Burk plots indicate that crown ethers do not activate the enzyme by specific interactions in the active site, such as transition state stabilization or facilitated transport of water molecules. Increased V_max values of crown-ether-treated enzyme most probably originate from conformational changes, which alter k_cat as well as the amount of catalytically active enzyme.     

Thermodynamic analysis of solvent effect on substrate specificity of lyophilized enzymes suspended in organic media

A simple methodology has been successfully employed to explain the solvent dependence of the substrate specificity of enzymes in organic media. This methodology, which does not require the knowledge of the enzyme structure and is thus applicable to lyophilized and other noncrystalline enzyme preparations, predicts that the k(cat)/K(M) ratio for two substrates should be proportional to their Raoult's law activity coefficients. This approach has been validated for two enzymes, subtilisin Carlsberg and alpha-chymotrypsin, catalyzing the propanolysis of unnatural (in addition to natural) ester substrates in a variety of anhydrous solvents. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 340-344, 1997.     

Optimization of calcium pectinate gel beads for sustained-release of catechin using response surface methodology

Response surface methodology was used to optimize bead preparation conditions, including CaCl₂ concentration (X₁), hydroxypropylmethylcellulose concentration (X₂), and bead-hardening time (X₃), for the sustained-release of catechin from the calcium pectinate gel beads reinforced with liposomes and hydroxypropylmethylcellulose into simulated gastric fluid (SGF) and intestinal fluid (SIF). The optimized values of X₁, X₂, and X₃ were found to be 5.82%, 0.08%, and 10.29 min, respectively. The beads prepared according to the optimized conditions released only about half of the entrapped catechin into SGF while most of the entrapped catechin was released into SIF after 24 h incubation.     

Screening of organic solvents for bioprocesses using aqueous-organic two-phase systems

The application of conventional organic solvents has been essential in several steps of bioprocesses in order to achieve sufficient economic efficiency. The use of organic solvents is frequently used either to partly or fully replace water in the reaction medium or as a process aid for downstream separation.Nowadays, manufacturers are increasingly requested to avoid and substitute solvents with hazardous potential. Therefore, the solvent selection must account for potential environmental hazards, health and safety problems, in addition to fulfilling the ideal characteristics for application in a process.For the first time, criteria including Environment, Health and Safety (EHS), as well as the technical requirements for reaction and separation have been reviewed, collected and integrated in a single organic solvent screening strategy to be used as a guideline for narrowing down the list of solvents to test experimentally. Additionally, we have also included a solvent selection guide based on the methodology developed in the Innovative Medicines Initiative CHEM21 (IMI CHEM21) project and applied specifically to water-immiscible solvents commonly used in bioprocesses.     

Organic solvent and pH induced alteration of product specificity of CGTase

Cyclodextrin glucanotransferase [CGTase, E.C.2.4.1.19] is an extracellular enzyme, which catalyzes the formation of α−, β−, γ− CDs from starch. Their proportions of formations depend on enzyme sources and reaction conditions. To understand what determines the product specificity of CGTases, we examined the alteration of product specificity of CGTase fromBacillus macerans by organic solvents and pH. At acidic pH range less than pH 6 where the enzyme was unstable, the ratio of α−/β-CD production was increased 4 times more than that at neutral pH range. As we increased the concentration of 2-butanol, α−/β-CD ratio was proportionally increased but/ratio remained constant. The α−/β-CD ratio of products was increased in the reaction media which yielded low products.     

Pressure affects enzyme function in organic media

Pressure affects enzyme function in nonaqueous media. Activation volumes have been determined and provide evidence that the primary effect of pressure is to enhance the stripping of water off an enzyme in polar organic solvents and leads to decreased enzymatic activity. Activation volumes of subtilisin Carlsberg in organic solvents, particularly with the enzyme hydrated, have a larger magnitude than activation volumes determined in aqueous solutions. This study provides further evidence that enzymatic activity in polar organic solvents is dominated by the interaction of enzyme-bound water with the solvent. From a practical standpoint, however, the results of this study suggest that enzymatic catalysis in organic solvents may be controlled by the combined effects of pressure and enzyme hydration. (c) 1993 John Wiley & Sons, Inc.     

Transition state stabilization of subtilisins in organic media

Electrostatic forces are among the stabilizing interactions that contribute to the high degree of enzyme-transition state complementarity. The active-site polarity, which can differ substaintially from that of water, is thus an important determinant of transition state stabilization. Here we pose the question of whether the rate of an enzymatic reaction proceeding through a charged transition state can be increased by increasing the active-site polarity in an organic solvent. The active-site polarity of subtilisin has been reduced by dehydration and suspension in a nonpolar solvent (tetrahydrofuran), and then increased by adding water to the solvent. Enhancing the local polarity substantially increasing the rate of catalysis, implicating polarity as an important factor in stabilizing the charged tetrahedral transition state. Studies with subtilisins whose active sites have been modified by site-directed mutagenesis support the role of polarity in transition state stabilization. (c) 1994 John Wiley & Sons, Inc.     

Enzymatic synthesis of l-ascorbyl linoleate in organic media

A novel l-ascorbyl fatty acid ester, l-ascorbyl linoleate was successfully prepared by enzymatic esterification and transesterification in a non-aqueous medium using immobilized lipase as biocatalyst. Changes in enzymatic activity and product yield were studied for the following variable: the nature of the fatty acid, the fatty acid concentration and water content. The yield of synthesis for the C18 unsaturated fatty acids were higher than for the C18 saturated fatty acid. Initial enzyme concentration does not affect the equilibrium of the reaction. And the product yield (33.5%) in the transesterification was higher than that of the esterification (21.8%) at a high-substrate concentration 0.3 M. The medium water content was found to have a distinct influence on the l-ascorbyl linoleate synthesis.These authors contributed equally to the article.     

Native subtilisin Karlsberg and modified subtilisin 72 as effective catalysts of peptide bond formation in organic media

The activity and stability of native subtilisin Karlsberg and subtilisin 72 and their complexes with sodium dodecyl sulfate (SDS) in organic solvents were studied. The kinetic constants of the hydrolysis of specific chromogenic peptide substrates Z-Ala-Ala-Leu-pNA and Glp-Ala-Ala-Leu-pNA by the subtilisins were determined. It was found that the subtilisin Karlsberg complex with SDS in anhydrous organic solvents is an effective catalyst of peptide synthesis with multifunctional amino acids in positions P ₁ and P ₁ ^′ (Glu, Arg, and Asp) containing unprotected side ionogenic groups.     

Chalcones as potent tyrosinase inhibitors: the effect of hydroxyl positions and numbers

The inhibition of tyrosinase is one of the major strategies to treat hyperpigmentation. Various limitations are associated with many of these inhibitors, such as high cytotoxicity, poor skin penetration and low stability in formulations. In continuation of our previous study [J. Agric. Food Chem. 51 (2003) 1201], showing that isoliquiritigenin chalcone (ILC) is a potent tyrosinase inhibitor, the present study aims to characterize the chalcone family as new tyrosinase inhibitors, and demonstrate their potential whitening potency. Nine mono-, di-, tri- and tetrahydroxychalcones were tested as inhibitors of tyrosinase mono- and diphenolase activities, showing that the most important factor in their efficacy is the location of the hydroxyl groups on both aromatic rings, with a significant preference to a 4-substituted B ring, rather than a substituted A ring. Neither the number of hydroxyls nor the presence of a catechol moiety on ring B correlated with increasing tyrosinase inhibition potency. 4-Hydroxychalcone (4-HC), ILC and Butein inhibited tyrosinase and shortened the lag period of enzyme monophenolase activity from about 490 min (control) to 30 min (ILC). As pigmentation also results from auto-oxidation, the antioxidant activity of 4-HC, ILC and Butein, were tested. Results showed that chalcones are also potent antioxidants, with Butein the most potent. We may conclude that chalcones are potentially potent new depigmentation agents, with their double effect of reduction and antioxidant activity. A deeper understanding of the relation between their structures to their potency will contribute to designing the optimal agents.     

Rapid and highly sensitive electrochemical determination of alkaline phosphatase using a composite tyrosinase biosensor

The use of an amperometric graphite-Teflon composite tyrosinase biosensor for the rapid monitoring of alkaline phosphatase (ALP), with no need of an incubation step and using phenyl phosphate as the substrate, is reported. Phenol generated by the action of ALP is monitored at the tyrosinase composite electrode through the electrochemical reduction of the o-quinone produced to catechol, which produces a cycle between the tyrosinase substrate and the electroactive product, giving rise to the amplification of the biosensor response and to the sensitive detection of ALP. The current was measured at -0.10 V 5 min after the addition of ALP. As a compromise between high ALP activity and high sensitivity for the detection of phenol, a pH of 8.5 was chosen. The substrate concentration was also optimized. A linear calibration plot was obtained for ALP between 2.0 x 10(-13) and 2.5 x 10(-11), with a detection limit of 6.7 x 10(-14) M. Different types of milk were analyzed with good results, using an extremely simple and rapid procedure.     

Biocatalysis of silica gel-immobilized chlorophyllase in a ternary micellar system

A partially purified enzymic extract from Phaeodactylum tricornutum was immobilized on silica gel and the specific activity of chlorophyllase in its free and immobilized states were compared in a ternary micellar system. The storage stability of the free and immobilized chlorophyllase extracts, maintained at temperatures ranging from 4 to 35°C for a period of 0–20 h, was temperature-dependent. The results also showed that the specific activity of the free and immobilized chlorophyllase extracts was highest at 30°C for long-term incubation, using chlorophyll and pheophytin as substrates and that a three-fold increase in the specific activity of the immobilized chlorophyllase was observed in comparison to that obtained with the free counterpart. The findings indicated that when free and immobilized chlorophyllase extracts were recovered and reused with both substrates, the immobilized chlorophyllase extract could be recycled for longer periods of time, while the free enzyme extract showed no activity after the first cycle.