Characterization of a thermostable and solvent-tolerant laccase produced by Streptomyces sp. LAO

Objectives

Decaying wood samples were collected, and actinomycetes were isolated and screened for laccase production. The identity of the efficient laccase-producing isolate was confirmed by using a molecular approach. Fermentation conditions for laccase production were optimized, and laccase biochemical properties were studied.

Results

Based on the 16S rRNA gene sequencing and phylogenetic analysis, the isolate coded as HWP₃ was identified as Streptomyces sp. LAO. The time-course study showed that the isolate optimally produced laccase at 84 h with 40.58?±?2.35 U/mL activity. The optimized physicochemical conditions consisted of pH 5.0, ferulic acid (0.04%; v/v), pine back (0.2 g/L), urea (1.0 g/L), and lactose (1 g/L). Streptomyces sp. LAO laccase was optimally active at pH and temperature of 8.0 and 90 °C, respectively, with remarkable pH and thermal stability. Furthermore, the enzyme had a sufficient tolerance for organic solvents after 16 h of preincubation, with laccase activity?>?70%. Additionally, the laccase maintained considerable residual activity after pretreatment with 100 mM of chemical agents, including sodium dodecyl sulphate (69.93?±?0.89%), ethylenediaminetetraacetic acid (93.1?±?7.85%), NaN₃ (96.28?±?3.34%) and urea (106.03?±?10.72%).

Conclusion

The laccase's pH and thermal stability; and robust catalytic efficiency in the presence of organic solvents suggest its industrial and biotechnological application potentials for the sustainable development of green chemistry.

     

Effect of organic solvents on penicillin acylase-catalyzed reactions: interaction of organic solvents with enzymes

The effects of various organic solvents on penicillin acylase-catalyzed synthesis of β-lactam antibiotics (pivampicillin and ampicillin) have been investigated in water-solvent mixtures. The rates of penicillin acylase-catalyzed reactions were found to be significantly reduced by the presence of a small amount of organic solvent. In particular, the rate of enzyme catalysis was extremely low in the presence of ring-structured solvents and acids while enzyme activities were fully restored after removing the solvents. This indicates that interactions between the solvents and the enzyme are specific and reversible. To correlate the inhibitory effects of organic solvents with solvent properties the influence of solvent hydrophobicities and solvent activity on the rate of pivampicillin synthesis was examined. The reaction rate was found to decrease with increasing solvent hydrophobicities, and a better correlation was observed between the reaction rate and solvent activity. The effects of ionic strength on the synthesis of pivampicillin and ampicillin were also examined. The ionic strength dependence indicates that electrostatic interactions are involved in the binding of ionic compounds to the enzyme. On the basis of the active site structure of penicillin acylase, a possible mechanism for molecular interactions between the enzyme and organic solvents is suggested.     

Characterization and catalytic property of surfactant-laccase complex in organic media

The oxidation of o-phenylenediamine catalyzed in anhydrous organic solvents by surfactant-laccase complex was investigated. The complex was prepared by utilizing a novel preparation technique in water-in-oil (W/O) emulsions. The surfactant-laccase complex effectively catalyzed the oxidation reaction in various dry organic solvents, while laccase, lyophilized from an aqueous buffer solution in which its activity was optimized, exhibited no catalytic activity in nonaqueous media. To optimize the preparation and reaction conditions for the surfactant-enzyme complexes, we examined the effects of pH in the water pool of W/O emulsions, the concentration of enzyme and surfactant at the preparation stage, and the nature of organic solvents at the reaction stage on the laccase activity in organic media. Surfactant-laccase complex showed a strong pH-dependent catalytic activity in organic media. Its optimum activity was obtained when the complex was prepared at a pH of about 3. Interestingly, native laccase in an aqueous buffer solution exhibited an optimum activity at the same pH of 3. The optimum preparation conditions of surfactant-laccase complex were [laccase] = 0.8 mg/mL and [surfactant] = 10 mM, and the complex showed the highest catalytic activity in toluene among nine anhydrous organic solvents. The effect of a cosolubilized mediator (1-hydroxybenzotriazole (HBT)) on the reaction was also investigated. The addition of HBT at the preparation stage of the enzyme complex did not accelerate the catalytic reaction because HBT was converted to an inactive benzotriazole (BT) by laccase. However, the addition of HBT at the reaction stage enhanced the catalytic performance by a factor of five compared to that without HBT.     

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.     

Antioxidant activity of brown alga Saccharina bongardiana from Kamchatka (Pacific coast of Russia). A methodological approach

The present study aims to investigate the levels of polyphenols and antioxidant activity in one of the most important commercial species of seaweeds in Kamchatka, an edible brown seaweed Saccharina bongardiana. Six extracts of S. bongardiana, acetone, methanol, ethanol, and the respective 70 % aqueous solutions, were assessed for total phenol content in order to determine the most efficient extracting solvent. The total phenol content was measured by the Folin–Ciocalteu method and expressed as phloroglucinol equivalents (PGE). The antioxidant tests used were 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, linoleic acid-β carotene oxidation inhibiting assay, and Fe²⁺ ion chelating method. Higher phenolic contents were obtained using aqueous organic solvents, as compared to the respective absolute solvents; 70 % acetone was found to be the most efficient solvent (1.039 mg PGE 100 mg^?1 dry algal powder). High significant correlations were noted between total phenol content and the tested antioxidant activities; so the aqueous organic extracts exhibited the highest antioxidant activities versus DPPH radicals (EC₅₀ values of 0.6–1.1 mg dry weight (DW) mL^?1), linoleic acid-β carotene oxidation (74–78 % at 0.8 mg DW mL^?1), as well as ferrous ions (EC₅₀ values of 5.0–7.9 mg DW mL^?1). Some methodological recommendations regarding the assays used and the expression of results are proposed.     

Evaluation of deep eutectic solvents as new media for Candida antarctica B lipase catalyzed reactions

This study aimed at analyzing the advantages and limitations of several deep eutectic solvents (DESs) as ‘green solvents’ for biotransformation using immobilized Candida antarctica lipase B as catalyst. The transesterification of vinyl laurate was chosen as model reaction and the influence of substrate polarity was assessed using alcohols of various chain lengths. Results showed that grinding of immobilized lipase was essential parameters for good lipase activity. Moreover, in our model reaction some hydrogen-bond donor component from the DES can compete with the alcoholysis reaction. Indeed, side reactions were observed with DES based on dicarboxylic acid or ethylene glycol, leading to some limitations of their use. However, the results showed that other DESs such as choline chloride:urea and choline chloride:glycerol could exhibit high activity and selectivity making them promising solvents for lipase-catalyzed reactions. Finally, the best DES's specific activity – and stability up to five days incubation time – were analyzed and compared with conventional organic solvents. Experiments revealed that iCALB is less influenced by the chain length of alcohol in DES than organic solvents and it is preserves its activity with minimally destructive to protein structure.     

Evaluation of antioxidant and anticancer activities of chemical constituents of the Saururus chinensis root extracts

Evaluation of antioxidant and anticancer activities were screened by various Saururus chinensis root extracts. Four solvents (ethyl acetate, methanol, ethanol, and water) extracts were investigated for their total flavonoids, phenol contents and their antioxidant activity of DPPH (2,2-diphenyl-1-picrylhydrazyl), NO (nitric oxide), H₂O₂ (hydrogen peroxide), ABTS 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid)diammonium assays, FRAP (ferric reducing ability of plasma) assays and anticancer activity. The total phenolic and flavonoid content of extracts were determined by using FC (Folin–Ciocalteu) and AlCl₃ colorimetric assay method. Total flavonoid content in these plants ranged from 24.7 to 72.1 mg g^?1 and amount of free phenolic compounds was between 11.2 and 67.1 mg g^?1 extract. The all extracts have significant levels of phenolics and flavonoids content. Anticancer activity was screened for MCF-7 breast cancer cell line. Ethanol extract shows significant of antioxidant activity and water extract shows significant of anticancer activity compared with standard (BHT) butylated hydroxy toluene. These ethanol and water extracts could be considered as a natural source for using antioxidant, and anticancer agents compared to commercial available synthetic drugs.     

Effects of alkyl chain lengths of gallates upon enzymatic wool functionalisation

The covalent grafting of alkyl gallates on wool through a laccase catalysed reaction in 80/20 (v/v, %) aqueous–ethanol mixture provided in a one-step process a multifunctional textile material with antioxidant, antibacterial and water repellent properties. Gallic acid and its alkyl esters ethyl, propyl, octyl and dodecyl gallate have been enzymatically grafted on wool fibres in order to study the effect of alkyl chain length on wool functional modification. The capacity of laccase to oxidise these phenolic compounds in an aqueous–organic medium has been verified by electrochemical techniques. The increase of CH₂, CH₃ groups in the FTIR spectra, together with the XPS analysis of the enzymatically modified fabrics confirmed the covalent grafting of ester gallates on wool. The result obtained in this work for antibacterial, water repellent as well as antioxidant properties show that the length of the alkyl chain of gallates molecule play an important role on wool functionalisation.     

Phytochemical analysis and antioxidant activity of Tamarix africana,Arthrocnemum macrostachyum and Suaeda fruticosa,three halophyte species from Algeria

Abstract

Extracts and fractions using six solvents of increasing polarities from Northwest Algeria (Tamarix africana, Arthrocnemum macrostachyum and Suaeda fruticosa) were studied for phytochemical analysis and in vitro antioxidant properties. Methanol and water fractions were found to be the more suitable solvents used for extraction of polyphenolic compounds. Aqueous leaf fraction of T. africana showed the highest content of phenolics (61.06?±?0.40?mg GAE/g DW) and condensed tannins (118.43?±?11.79?mg CE/g DW). Dichloromethane stem fraction of T. africana had the highest 2,2-diphenyl-1-picrylhydrazil radical scavenging ability (0.34?±?0.00?mg/ml). Methanol leaf fraction of the same plant exhibited the highest antioxidant power against the inhibition of β-carotene bleaching, while the maximum total antioxidant capacity was recorded in the leaf extract of S. fruticosa. Phenolic content was not influenced by the species but very affected by the extraction solvent, while antioxidant activities were not influenced by these two parameters. High-performance liquid chromatography with a diode-array detector analysis of methanol and aqueous leaf fractions of T. africana revealed the presence of six phenolic acids; chlorogenic and gallic acids were predominant and 10 flavonoid compounds among which rutin and quercetin-3-O-arabonoside were the major constituents. These findings suggest that these species may be considered as an interesting source of antioxidants.     

Peculiarities of direct bioelectrocatalysis by laccase in aqueous-nonaqueous mixtures

The effect of concentration of ethanol and dimethyl sulfoxide on the catalytic activity of laccase is studied for the enzymatic reaction of catechol oxidation and bioelectrocatalytic reaction of oxygen reduction under the conditions of direct electron transfer. Laccase-Nafion composite is elaborated ensuring the enzyme stability in a wide potential range and a content of organic solvents. Based on the STM measurements, the structure of composite layer is proposed. It is shown that the mechanism of oxygen reduction reaction by laccase in organo-aqueous mixtures is similar to that earlier proposed for aqueous solutions. A decrease in the electrocatalytic activity of laccase in the oxygen reduction correlates with a decrease in the laccase enzymatic activity in the substrate oxidation. However, a decrease in the laccase activity in the composite is observed at a higher content of organic solvent in the mixture. The mechanism of laccase inactivation by organic solvents is proposed.     

Influence of the extraction solvent on antioxidant activity of Althaea officinalis L. root extracts

Althaea officinalis (Malvaceae) is a well-known plant that is widely distributed throughout the world. Aqueous and hydroalcoholic extracts from A. officinalis root are used mainly because of their antitussive and expectorant activity. It is well known that these activities are based on the polysaccharide composition, but little is known about the possible antioxidant activity of root extract. The present study evaluated antioxidant activity of root extracts prepared with different extraction solvents applying ABTS^·+ (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), hypochlorous acid scavenging assay and iron-induced lipid peroxidation. The results showed that the extract prepared with water as extraction solvent did not possess antioxidant activity, whereas the extracts obtained using ethanol:water as extraction agent showed well pronounced antioxidant activity. In particular, the extracts obtained at low concentration of ethanol in the mixed solvent (50:50 and 70:30, v/v) showed higher scavenging activity for ABTS^·+ radicals and hypochlorite ions than the extract obtained with the higher ethanol concentration (90:10, v/v). These results correlated very well with phenolic and flavonoid content of the extracts. The extracts did not show cytotoxic effect on human BV-173 leukemic cells but may have immunomodulating effects due to their antioxidant properties.     

Enzyme-mediated coupling of a bi-functional phenolic compound onto wool to enhance its physical,mechanical and functional properties

Wool fibres have been modified with nordihydroguaiaretic acid (NDGA) to improve their performance at use. This water insoluble bi-functional phenolic compound has been grafted on wool through a laccase enzyme catalyzed reaction in an aqueous–ethanol mixture. The capacity of laccase to oxidise NDGA in this aqueous–organic medium has been studied electrochemically. The increase of CH₂, CH₃ and aromatic groups signal in the DRIFT spectra, together with SEM images of the enzymatically modified fabrics confirmed the covalent grafting of NDGA on wool. This one step enzymatic process for grafting of NDGA improved the physical and mechanical properties of wool fabrics such as shrink resistance, crease recovery and tensile strength. Furthermore, the NDGA imparted to the textile material strong antioxidant activity and UV protection.     

Effects of organic solvents on the enzyme activity of Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase in calorimetric assays

In drug discovery programs, dimethyl sulfoxide (DMSO) is a standard solvent widely used in biochemical assays. Despite the extensive use and study of enzymes in the presence of organic solvents, for some enzymes the effect of organic solvent is unknown. Macromolecular targets may be affected by the presence of different solvents in such a way that conformational changes perturb their active site structure accompanied by dramatic variations in activity when performing biochemical screenings. To address this issue, in this work we studied the effects of two organic solvents, DMSO and methanol (MeOH), in the isothermal titration calorimetry (ITC) kinetic assays for the catalyzed reaction of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Trypanosoma cruzi. The solvent effects on T. cruzi GAPDH had not yet been studied. This enzyme was shown here to be affected by the organic solvents content up to 5.0% for MeOH and up to 7.5% for DMSO. The results show that when GAPDH is assayed in the presence of DMSO (5%, v/v) using the ITC experiment, the enzyme exhibits approximately twofold higher activity than that of GAPDH with no cosolvent added. When MeOH (5%, v/v) is the cosolvent, the GAPDH activity is sixfold higher. The favorable effects of the organic solvents on the Michaelis-Menten enzyme-substrate complex formation ensure the consistency of the biological assays, structural integrity of the protein, and reproducibility over the measurement time. The reaction was also kinetically monitored by standard spectrophotometric assays to establish a behavioral performance of T. cruzi GAPDH when used for screening of potential inhibitors.     

Combinatorial evaluation of laccase-mediator system in the oxidation of veratryl alcohol

Laccases play an important role in the biological break down of lignin and have great potential in the deconstruction of lignocellulosic feedstocks. We examined 16 laccases, both commercially prepared and crude extracts, for their ability to oxidize veratryl alcohol in the presence of various solvents and mediators. Screening revealed complete conversion of veratryl alcohol to veratraldehyde catalyzed by a crude preparation of the laccase from Trametes versicolor ATCC 11235 and the mediator TEMPO in 20 % (v/v) tert-butanol.     

Modification of the Microenvironment of Enzymes in Organic Solvents. Substitution of Water by Polar Solvents

Enzyme catalysis in water-immiscible organic solvents is strongly influenced by the amount of water present in the reaction mixture. Effects of substitution of part of the water by other polar solvents were studied. In an alcoholysis reaction catalyzed by chymotrypsin deposited on celite, it was possible to exchange half of the water by formamide, ethylene glycol or dimethyl sulfoxide with often increased initial reaction rate. Furthermore, these substitutions caused the suppression of the competing hydrolysis reaction. However, formamide caused enzyme inactivation, and ethylene glycol participated as a reactant in the alcoholysis to some extent, hence dimethyl sulfoxide was considered the best water substitute among the solvents tested. These effects were noted for chymotrypsin catalyzed alcoholysis in several water immiscible solvents and also for interesterification reactions catalyzed by Candida cylindracea lipase on celite. In the latter case a change in the stereoselectivity was observed. At a low water content a high stereoselectivity was observed; when the amount of polar solvent was increased, either by doubling the water content or adding an equal amount of DMSO, the stereoselectivity decreased.     

Catalytic activity and stability of xanthine oxidase in aqueous-organic mixtures

In the present study, bovine milk xanthine oxidase activity in various aqueous-organic mixtures and the effects of pH, temperature, and lyophilization on the enzyme activity have been investigated. The enzyme was incubated with xan-thine as the substrate in Sorenson’s phosphate buffer (pH 7.0) containing 0.1 mM EDTA, and the activity was determined spectrophotometrically in the absence and presence of different fractions of nine water-miscible organic solvents at 27–50°C and at different pH values ranging from 6 to 9. The organic solvents reduced the enzyme activity to different extents. In spite of these inhibitory effects, the enzyme showed relatively good stability in the aqueous-organic mixtures compared with the aqueous medium. A significant increase in the activity of the lyophilized enzyme was observed in pure organic solvents. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 1, pp. 124–130.     

Application of organic mono-phase and organic–aqueous two-liquid-phase systems in microalgal conversion of androst-4-en-3,17-dione by Nostoc muscorum

Organic mono-phase and organic–aqueous two-phase systems were applied for 17-carbonyl reduction of androst-4-en-3,17-dione to testosterone by whole cells of the microalga Nostoc muscorum (Nostocaceae). To investigate the correlation between solvent hydrophobicity and biotransformation yield in mono- and biphasic systems, a range of 16 organic solvents with log P_octanol values (logarithm of the solvent partition coefficient in the n-octanol/water system) between ? 1.1 and 8.8 were examined. Organic solvents with log P_octanol values greater than 7, such as hexadecane and tetradecane, provided the best biocompatibility with the bioconversion by algal cells. The data also indicated that the highest yields were obtained using organic–aqueous (1:1, v/v) biphasic systems. The optimum volumetric phase ratio, reaction temperature and substrate concentration were 1:1, 30°C and 0.5 mg mL^?1, respectively. Under the mentioned conditions a fourfold increase in biotransformation yield (from 7.8±2.3 to 33.4±1.8%) was observed.     

Environmentally Friendly Approach to the Synthesis of 3-[Benzylideneamino]-2-methylquinazolin-4(3H)-one Derivatives and Calculation of Their Toxicity

Application of deep eutectic solvents in synthesis of different heterocyclic compounds was proven very efficient. These solvents are a new generation of green solvents showing excellent potential for different purposes, where they are used as environmentally acceptable substitute for toxic and volatile organic solvents. This research describes their application in the synthesis of series of quinazolinone Schiff bases in combination with microwave, ultrasound-assisted and mechanochemical methods. First, a model reaction was performed in 20 different deep eutectic solvents to find the best solvent and then reaction conditions (solvent, temperature and reaction time) were optimized for each method. Afterwards, 40 different quinazolinone derivatives were synthesized in choline chloride/malonic acid (1 : 1) DES by each method and compared by their yields. Here we show that deep eutectic solvents can be very efficient in the synthesis of quinazolinone derivatives as an excellent substitution for volatile organic solvents. With green chemistry approach in mind, we have also performed a calculation on compounds’ toxicity and solubility, showing that most of them possess toxic and mutagenic properties with low water solubility.     

Toward advanced ionic liquids. Polar,enzyme-friendly solvents for biocatalysis

Ionic liquids, also called molten salts, are mixtures of cations and anions that melt below 100°C. Typical ionic liquids are dialkylimidazolium cations with weakly coordinating anions such as (MeOSO₃) or (PF₆). Advanced ionic liquids such as choline citrate have biodegradable, less expensive, and less toxic anions and cations. Deep eutectic solvents are also included in the advanced ionic liquids. Deep eutectic solvents are mixtures of salts such as choline chloride and uncharged hydrogen bond donors such as urea, oxalic acid, or glycerol. For example, a mixture of choline chloride and urea in 1:2 molar ratio liquefies to form a deep eutectic solvent. Their properties are similar to those of ionic liquids. Water-miscible ionic liquids as cosolvents with water enhance the solubility of substrates or products. Although traditional water-miscible organic solvents also enhance solubility, they often inactivate enzymes, while ionic liquids do not. The enhanced solubility of substrates can increase the rate of reaction and often increases the regioor enantioselectivity. Ionic liquids can also be solvents for non-aqueous reactions. In these cases, they are especially suited to dissolve polar substrates. Polar organic solvent alternatives inactivate enzymes, but ionic liquids do not even when they have similar polarities. Besides their solubility properties, ionic liquids and deep eutectic solvents may be greener than organic solvents because ionic liquids are nonvolatile, and can be made from nontoxic components. This review covers selected examples of enzyme catalyzed reaction in ionic liquids that demonstrate their advantages and unique properties, and point out opportunities for new applications. Most examples involve hydrolases, but oxidoreductases and even whole cell reactions have been reported in ionic liquids.     

Protein-protein interaction in low water organic media

Trypsin either modified with polyethylene glycol or as a suspended powder was used to catalyze digestion of protein substrates in benzene in order to get insight into protein-protein interactions in water-immiscible organic media. Depending on whether suspended or soluble trypsin was used, catalysis was found to proceed differently. In the first case, the amount of water in the reaction mixture (up to 1% v/v) appeared to be critical, and adsorption of water from the reaction medium by the protein substrate allowed it to behave as a hydrophilic support material comparable to that involved in immobilized enzymes. In the latter case, the presence of an additional nucleophile was a prerequisite for catalysis to proceed, and thus both water and nucleophile concentrations had some influence on trypsin activity. Phe-NH(2) was the most potent nucleophile for proteolysis catalyzed by polyethylene glycol-modified trypsin in organic media containing 1-2% water (v/v). The organic solvent-soluble enzyme was found to bind reversibly to the protein substrate as a function of both extent of hydration of the reaction medium and time of incubation. The overall results strongly suggested that modified trypsin catalyzed peptide bond hydrolysis at the protein substrate-organic solvent interface. Peptide mapping of bovine insulin digest by reversed-phase high-performance liquid chromatography definitely showed that enzyme-catalyzed proteolysis did occur in organic solvents with a concomitant and significant transpeptidation reaction.