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.     

Laccase-catalyzed oxidation of phenolic compounds in organic media

Rhus vernificera laccase-catalyzed oxidation of phenolic compounds, i.e., (+)-catechin, (−)-epicatechin and catechol, was carried out in selected organic solvents to search for the favorable reaction medium. The investigation on reaction parameters showed that optimal laccase activity was obtained in hexane at 30 °C, pH 7.75 for the oxidation of (+)-catechin as well as for (−)-epicatechin, and in toluene at 35 °C, pH 7.25 for the oxidation of catechol. E_a and Q₁₀ values of the biocatalysis in the reaction media of the larger log p solvents like isooctane and hexane were relatively higher than those in the reaction media of lower log p solvents like toluene and dichloromethane. Maximum laccase activity in the organic media was found with 6.5% of buffer as co-solvent. A wider range of 0–28 μg protein/ml in hexane than that of 0–16.7 μg protein/ml in aqueous medium was observed for the linear increasing conversion of (+)-catechin. The kinetic studies revealed that in the presence of isooctane, hexane, toluene and dichloromethane, the K_m values were 0.77, 0.97, 0.53 and 2.9 mmol/L for the substrate of (+)-catechin; 0.43, 0.34, 0.14 and 3.4 mmol/L for (−)-epicatechin; 2.9, 1.8, 0.61 and 1.1 mmol/L for catechol, respectively, while the corresponding V_max values were 2.1 × 10⁻², 2.3 × 10⁻², 0.65 × 10⁻² and 0.71 × 10⁻² δA/μg protein min); 1.8 × 10⁻², 0.88 × 10⁻², 0.19 × 10⁻² and 1.0 × 10⁻² δA/μg protein min); 0.48 × 10⁻², 0.59 × 10⁻², 0.67 × 10⁻² and 0.54 × 10⁻² δA/μg protein min), respectively. FT-IR indicated the formation of probable dimer from (+)-catechin in organic solvent. These results suggest that this laccase has higher catalytic oxidation capacity of phenolic compounds in suitable organic media and favorite oligomers could be obtained.     

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.     

Synthesis of alkyl esters by cutinase in miniemulsion and organic solvent media

The main objective of this work was studying and testing the nature and influence of reaction media (organic solvent vs. miniemulsion system) on the synthesis of alkyl esters catalyzed by Fusarium solani pisi cutinase. Ester synthesis and cutinase selectivity for different chain length of acids and alcohols (ethyl and hexyl) were evaluated. In iso-octane, after 1 h of reaction, cutinase exhibits rates of esterification between 0.24 μmol x mg^–1 x min^–1 for ethyl oleate and 1.15 μmol x mg^–1 x min^–1 for ethyl butyrate, while in a miniemulsion system the rates were from 0.05 for ethyl heptanoate to 0.76 μmol x mg^–1 x min^–1 for ethyl decanoate. The reaction rate for the synthesis of hexyl esters in a miniemulsion system was from 0.19 for hexyl heptanoate to 1.07 μmol x mg^–1 x min^–1 for hexyl decanoate. High conversion yields of 95% at equilibrium after 8 h of reaction in iso-octane for pentanoic acid (C₅) with ethanol at equimolar concentration (0.1 M) was achieved. Additionally, this work showed that a significant and unexpected shift in cutinase selectivity occurred towards longer chain length carboxylic acids (C₈–C₁₀) in miniemulsion system as compared to organic solvent (iso-octane) and previous studies in reverse micellar systems. The possibility of working with higher concentration of substrates, without inhibitory effect on the enzyme, was another advantage of the miniemulsion system.     

In situ laccase-assisted overdyeing of denim using flavonoids

A laccase-mediated system for denim overdyeing using phenolic compounds was developed. Laccase from ascomycete Myceliophthora thermophila was able to oxidize phenolic compounds such as catechol and catechin and mediate their attachment to denim surfaces. Laccase-generated polymers gave rise to new coloration states from dark brown to green-yellow and replaced dyes in the overdyeing process. Process parameters, such as enzyme dosage, incubation time and presence of mediator, were studied by considering a compromise between the highest overdyeing level and lower energy/products consumption (2 U/mL laccase; 4 h incubation in the absence of mediator). Enzyme-generated polymers were followed by UV/Vis spectrophotometry and their level of attachment to denim surfaces was evaluated by means of spectral values quantification [k/s, Kubelka-Munk relationship (k=absorption coefficient, s=scattering coefficient)]. Overdyeing of denim with phenolics, such as catechol or catechin, was successfully achieved with acceptable levels in terms of durability.     

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.     

New class of alkynyl glycoside analogues as tyrosinase inhibitors

A new series of alkynyl glycoside analogues were designed and synthesized from cheap and a commercially available sugar by introduction of various alkynyl and alkyl groups at C-1 and C-6 positions of the sugar ring. The inhibitory abilities of alkynyl glycosides were investigated in vitro on mushroom tyrosinase for the catalysis of l-Tyrosine and l-DOPA as substrates and comparing with arbutin and kojic acid. Non-terminal alkyne compound 2d showed excellent tyrosinase inhibitory activity (IC₅₀ 54.0 μM) against l-Tyrosine comparable to arbutin (IC₅₀ 1.46 mM) while 2b exhibited potent activities (IC₅₀ 34.3 μM) against L-DOPA higher than kojic acid (IC₅₀ 0.11 mM) and arbutin (IC₅₀ 13.3 mM). Kinetic studies revealed that compound 2d was a non-competitive inhibitor with the best Ki value of 21 μM and formed an irreversible receptor complex with mushroom tyrosinase. The SARs results showed that the type of alkyne and alkyl groups at position C-6 on sugar and the stereoisomer played an important role in determining their inhibitory activities. The potent activity of alkynyl glycosides identified in this study highlight the importance of this scaffold and these compounds are very modestly potent to the development of new class for tyrosinase inhibitor.     

On protein solubility in organic solvent

Solubility of a model protein, hen egg-white lysozyme, was investigated in a wide range of neat nonaqueous solvents and binary mixtures thereof. All solvents that are protic, very hydrophilic, and polar readily dissolve more than 10 mg/mL of lysozyme (lyophilized from aqueous solution of pH 6.0). Only a marginal correlation was found between the lysozyme solubility in a non-aqueous solvent and the letter's dielectric constant or Hildebrand solubility parameter, and no correlation was observed with the dipole moment. Lysozyme dissolved in dimethyl sulfoxide (DMSO) could be precipitated by adding protein nondissolving co-solvents, although the enzyme had a tendency to form supersaturated solutions in such mixtures. The solubility of lysozyme, both in an individual solvent (1,5-pentanediol) and in binary solvent mixtures (DMSO/acetonitrile), markedly increased when the pH of the enzyme aqueous solution prior to lyophilization was moved away from the proteins's isoelectric point. (c) 1994 John Wiley & Sons, Inc.     

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.     

Biocatalysis in Spain: A field of success and innovation

Abstract

This is the presentation of the Special Issue “Biocatalysis in Spain”, covering the work of several Spanish groups in the field of Biocatalysis and Biotransformations. Thus, both research articles and reviews allow one to draw an accurate view of the state-of-the-art development in Spain.     

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.     

Unfolding of cardosin A in organic solvents and detection of intermediaries

In the present study the relationship between conformational stability and enzymatic activity in the presence of organic solvents (OS) was investigated. We have found that cardosin A, the model protease investigated in this work, inactivates through a biphasic mechanism, which is incipient in aqueous buffer and becomes visible in the presence of hydrophilic OS. In fact, in OS this inactivation originates stable intermediaries that were detected in acetonitrile. This biphasic mechanism can be described in two phases: an initial one, where OS induce alterations that affect the active site cleft and global mobility, but with little interference on the global protein conformation, and, a second phase, where there is a global change in protein conformation with concomitant activity loss. It is shown that in the presence of hydrophilic OS there is a larger mobility of the enzyme, revealed by limited proteolysis, probably due to a weakening of hydrophobic interactions within the protein core.