Cleavage and synthesis function of high and low redox potential laccases towards 4-morpholinoaniline and aminated as well as chlorinated phenols

Laccases are able to mediate both cleavage and synthesis processes. The basis for this dual reaction capability lies in the property of the enzyme laccase to oxidize phenolic, and to some extent non-phenolic substances, to reactive radicals which can undergo on the one hand separations of small substitutents or large molecule parts from the parent compound and on the other hand coupling reactions with other radicals or molecules which are not themselves oxidizable by laccase. The cleavage of the non-phenolic compound 4-morpholinoaniline as well as the deamination of 4-aminophenol and the dechlorination of 4-chlorophenol resulted in the formation of 1,4-hydroquinone which is immediately oxidized by laccase to 1,4-benzoquinone. The formation of the 1,4-hydroquinone/1,4-benzoquinone is the rate limiting step for the synthesis of the heteromolecular dimers and trimers composed of 1,4-benzoquinone and one or two molecules of morpholine. In addition to the synthesis of new compounds from the cleavage products, 4-morpholinoaniline polymerized probably via azo groups and C-N bonds to a homomolecular dimer and trimer. Similarities and differences in cleavage and synthesis reactions catalyzed by the low redox potential laccase of Myceliophthora thermophila (0.46 V) and the high redox potential laccase of Pycnoporus cinnabarinus (0.79 V) were determined. In addition, the dependency of the cleavage and synthesis efficiencies on the (a) structure and redox potential of the laccase, (b) structure and redox potential of the substrate, (c) pH value of the buffer used, (d) incubation temperature, (e) solvent concentration, and (f) laccase activity is discussed in general.     

Isolation of myo-Inositol from Solutions by Enzymatic Biotransformation

The transferase reaction between phospholipids and inositol catalyzed by phospholipase D was studied at interfaces in water–organic solvent systems. Optimum conditions were determined for phosphatidylinositol synthesis in heterogeneous water–organic solvent systems. Hydrophobic components (phospholipids) were readily separated from water-soluble products (alcohols) in systems with organic solvents. In the hexane–water system, addition of methanol (an alcohol substrate) to the reaction medium displaced myo-inositol from the molecule of phosphatidylinositol. myo-Inositol was isolated from the mixture of its isomers using a two-step transferase reaction catalyzed by phospholipase D.     

Oxidative enzymes possess catalytic activity in systems with ionic liquids

Oxidative enzymes, laccase C from Trametes sp. and horseradish and soybean peroxidases, catalyzed oxidation reactions in systems with ionic liquids whose content varied from several volume percent to almost total non-aqueous ionic liquids. Similar to the effects produced by standard organic solvents used in non-aqueous enzymology, catalytic activity of the enzymes was decreased by adding a water-miscible ionic liquid, 4-methyl-N-butylpyridinium tetrafluoroborate, or by suspending the enzyme in a water-immiscible ionic liquid, 1-butyl-3-methylimdizaolium hexafluorophosphate. For the oxidation of anthracene, catalyzed by laccase C and assisted by a number of mediators, addition of 4-methyl-N-butylpyridinium tetrafluoroborate, instead of tert-butanol, increased the yield of the oxidation product several-fold.     

Oxidation of aromatic alcohols by laccase from Trametes versicolor mediated by the 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) cation radical and dication

Oxidation of aromatic alcohols, such as non-phenolic lignin model compounds, by oxidised species of 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) has been investigated. The cation radical and dication formed from ABTS were both capable of oxidising aromatic alcohols to aldehydes. The reactions terminated at the level of the aldehyde and no acids were formed. The cation radical and dication worked in a cycle as an electron-transfer compound between an oxidant and alcohol. In addition to the oxidation of the primary benzyl-hydroxyl group, an oxidation of the secondary α-hydroxyl group to the ketone by the dication was possible. All distinguishing features of these reactions corresponded to the results of the oxidation performed by the laccase of Trametes versicolor in the presence of ABTS. The decomposition products from the dication alone and ABTS with laccase confirmed the supposition that the dication was involved in the laccase mediator system. A reaction mechanism based on deprotonation of the alcohol cation radical was predicted to play a key role in the irreversible followup reaction and to be the driving force of the process. Received: 8 June 1998 / Received revision: 23 September 1998 / Accepted: 2 October 1998     

Inhibition of cellulose enzymatic hydrolysis by laccase‐derived compounds from phenols

The presence of inhibitors compounds after pretreatment of lignocellulosic materials affects the saccharification and fermentation steps in bioethanol production processes. Even though, external addition of laccases selectively removes the phenolic compounds from lignocellulosic prehydrolysates, when it is coupled to saccharification step, lower hydrolysis yields are attained. Vanillin, syringaldehyde and ferulic acid are phenolic compounds commonly found in wheat‐straw prehydrolysate after steam‐explosion pretreatment. These three phenolic compounds were used in this study to elucidate the inhibitory mechanisms of laccase‐derived compounds after laccase treatment. Reaction products derived from laccase oxidation of vanillin and syringaldehyde showed to be the strongest inhibitors. The presence of these products causes a decrement on enzymatic hydrolysis yield of a model cellulosic substrate (Sigmacell) of 46.6 and 32.6%, respectively at 24 h. Moreover, a decrease in more than 50% of cellulase and β‐glucosidase activities was observed in presence of laccase and vanillin. This effect was attributed to coupling reactions between phenoxyl radicals and enzymes. On the other hand, when the hydrolysis of Sigmacell was performed in presence of prehydrolysate from steam‐exploded wheat straw a significant inhibition on enzymatic hydrolysis was observed independently of laccase treatment. This result pointed out that the other components of wheat‐straw prehydrolysate are affecting the enzymatic hydrolysis to a higher extent than the possible laccase‐derived products. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:700–706, 2015     

Diverse reactions catalyzed by naphthalene dioxygenase fromPseudomonas sp strain NCIB 9816

Naphthalene dioxygenase (NDO) fromPseudomonas sp strain NCIB 9816 is a multicomponent enzyme system which initiates naphthalene catabolism by catalyzing the addition of both atoms of molecular oxygen and two hydrogen atoms to the substrate to yield enantiomerically pure (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. NDO has a relaxed substrate specificity and catalyzes the dioxygenation of many related 2- and 3-ring aromatic and hydroaromatic (benzocyclic) compounds to their respectivecis-diols. Biotransformations with a diol-accumulating mutant, recombinant strains and purified enzyme components have established that in addition tocis-dihydroxylation, NDO also catalyzes a variety of other oxidations which include monohydroxylation, desaturation (dehydrogenation),O-andN-dealkylation and sulfoxidation reactions. In several cases, the absolute stereochemistry of the oxidation products formed by NDO are opposite to those formed by toluene dioxygenase (TDO). The reactions catalyzed by NDO and other microbial dioxygenases can yield specific hydroxylated compounds which can serve as chiral synthons in the preparation of a variety of compounds of interest to pharmaceutical and specialty chemical industries. We present here recent work documenting the diverse array of oxidation reactions catalyzed by NDO. The trends observed in the oxidation of a series of benzocyclic aromatic compounds are compared to those observed with TDO and provide the basis for prediction of regio- and stereospecificity in the oxidation of related substrates. Based on the types of reactions catalyzed and the biochemical characteristics of NDO, a mechanism for oxygen activation by NDO is proposed.     

Room temperature and highly enantioselective additions of alkyltitanium reagents to aldehydes catalyzed by a titanium catalyst of (R)‐h8‐binol

Three alkyltitanium reagents of RTi(O‐i‐Pr)₃ (R = Cy ( 1a ), i‐Bu ( 1b ), and n‐Bu ( 1c )) were prepared in good yields. The high‐resolution mass spectroscopy showed that 1b and 1 c in the gas phase are monomeric species. However, the solid state of 1a revealed a dimeric structure. Asymmetric additions of 1a , 1b , 1c to aldehydes catalyzed by a titanium catalyst of (R)‐H₈‐BINOL were studied at room temperature. The reactions produced desired secondary alcohols in good yields with good to excellent enantioselectivities of up to 94% ee. Reactivity and enantioselectivity differences, in terms of steric bulkiness of the R nucleophiles, are herein described. The addition reactions of secondary c‐hexyl to aldehydes were slower than the reactions of primary i‐butyl or n‐butyl nucleophiles. For the primary alkyls, lower enantioselectivities were obtained for products from addition reactions of the linear n‐butyl as compared with the enantioselectivities of products from the addition reactions of the branched i‐butyl group. The same stereochemistry of RTi(O‐i‐Pr)₃ addition reactions as the addition reactions of organozinc, organoaluminum, Grignard, or organolithium reagents directly supports the argument of that titanium‐catalyzed addition reactions of aldehydes involve an addition of an organotitanium nucleophile. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

Adenosine diphosphoribose transfer reactions catalyzed by Bungarus fasciatus venom NAD glycohydrolase

Reactions catalyzed by purified Bungarus fasciatus venom NAD glycohydrolase were demonstrated to include ADP-ribose transfer from NAD to alcohols and to imidazole derivatives to produce a variety of ADP-ribosides. The formation of products was monitored by high performance liquid chromatography. In the enzyme-catalyzed alcoholysis of NAD, the ratio of n-alkyl-ADP-riboside formed to the hydrolytic product, ADP-ribose, increased linearly with alcohol concentration. The effectiveness of alcohols as acceptors of the ADP-ribose moiety in these reactions increased with increasing chainlength of the alcohol used. Linear positive chainlength effects extended from methanol to pentanol suggesting facilitation of these reactions by nonpolar interactions. In the methanolysis reaction, NADP, thionicotinamide adenine dinucleotide, nicotinamide-1, N6-ethenoadenine dinucleotide, and 3-acetylpyridine adenine dinucleotide were shown to be as effective as NAD as donor substrates. The NAD glycohydrolase-catalyzed ADP-ribose transfer to pyridine bases to form NAD analogs was studied at pyridine base concentrations above those determined to be saturating for the base exchange reaction. Under these conditions, the ratio of base exchange to hydrolysis of NAD was directly related to the pKa of the ring nitrogen of the pyridine base employed. In addition to alcoholysis and pyridine-base exchange reactions, the snake venom enzyme was demonstrated to catalyze an ADP-ribose transfer reaction to imidazole derivatives. Arginine methyl ester was ineffective as an ADP-ribose acceptor molecule in these reactions.     

Stereoselectivity in acid-catalyzed heteronucleophilic substitution of enantiomeric 3-O-methyloxazepam and 3-O-ethyloxazepam

Kinetics of acid-catalyzed heteronucleophilic substitution and racemization of enantiomeric MeOX in ethanol and enantiomeric EtOX in methanol were studied by quenching reaction products at various times by neutralization. Enantiomeric contents of remaining substrate and reaction product were determined by chiral stationary phase high-performance liquid chromatography. The experimental procedure allowed the determination of the stereoselectivity (i.e., the enantiomeric ratio of a substitution product formed from an enantiomerically pure substrate) involved in the heteronucleophilic substitution reactions. The stereoselectivity was found to vary between 58:42 and 87:13, depending on the acid concentration, substrate, solvent, and temperature. The enantiomeric purity of remaining substrates was identical to that of the starting substrate, indicating that the enantiomeric substrates did not undergo a ring-opening reaction. The results provided additional evidence supporting the mechanism proposed earlier in acid-catalyzed stereoselective heteronucleophilic and homonucleophilic substitutions and the resulting racemization of enantiomeric 3-alkoxy-1,4-benzodiazepines in alcoholic solvents. © 1995 Wiley-Liss, Inc.     

[1-14C] Acetate assimilation by obligate methylotrophs,Pseudomonas methanica and Methylosinus trichosporium

The oxidation of one carbon compounds (methane, methanol, formaldehyde, formate) and primary alcohols (ethanol, propanol, butanol) supported the assimilation of [1-¹⁴C]acetate by cell suspensions of type I obligate methylotroph; Pseudomonas methanica, Texas strain, and type II obligate methylotroph, Methylosinus trichosporium, strain PG. The amount of oxygen consumed and substrate oxidized correlated with the amount of [1-¹⁴C]acetate assimilated during oxidation of C-1 compounds and primary alcohols.Oxidation of methanol, formaldehyde, and primary alcohols in extracts of Pseudomonas methanica, Texas strain, and Methylosinus trichosporium, strain PG, was catalyzed by a phenazine methosulfate linked, ammonium ion dependent methanol dehydrogenase. The oxidation of aldehydes was catalyzed by a phenazine methosulfate linked, ammonium ion independent aldehyde dehydrogenase. Formate was oxidized by a NAD⁺ linked formate dehydrogenase.Deceased.This work was supported by Grant GB 8173 from the National Science Foundation and by a grant from the Robert A. Welch Foundation.     

α-Amylase-catalysed synthesis of alkyl glycosides

Alkyl glycosides were synthesised from starch and alcohols using Aspergillus oryzae α-amylase as catalyst. In the degradation of starch by α-amylase, the alcohols competed with water as glycosyl acceptors. In the reaction with methanol, methyl maltoside and methyl maltotrioside were the main alcoholysis products. Conversion of 45 g/l starch in 30% methanol resulted in a product mixture containing 26 mM maltooligosaccharides and 3.6 mM methyl glycosides. With ethanol, propanol and butanol, alkyl maltosides and alkyl maltotetraosides were detected, and with benzyl alcohol, benzyl glycosides having two, three or five glucose units were formed. No alcoholysis reaction occurred with hexanol or octanol. In conclusion, α-amylase is promising for the one-step synthesis of alkyl glycosides having more than one monosaccharide unit, which are difficult to synthesise in other ways.     

Pseudomonas cepacia lipase: Esterification reactions in AOT microemulsion systems

Summary The activity of purifiedPseudomonas cepacia lipase has been investigated in esterification reactions of various aliphatic alcohols with natural fatty acids. The reactions were carried out in microemulsions formed in isooctane by bis(2ethylhexyl)sulfosuccinate sodium salt (AOT). The optima pH, T and water content (w_o) for the enzyme activity in this type of microemulsions have been determined. Studies on the effect of various fatty acids and alcohols on the enzyme specificity have shown a preference of this lipase for palmitic and caprylic acid as well as for propanol, while reactions involving cyclic alcohols can not be catalyzed at all. The differences on the behavior of this lipase as compared to other lipases studied in microemulsion systems as well as in other systems are discussed.     

The reaction of tertiary amino alcohols with active esters: I. Acylation and deacylation steps

The reactions of triethanolamine and four other tertiary amino alcohols with six active ester substrates were studied in the pH range 6–10 at 30°C. The reaction products were in all cases the respective O-acyl-amino alcohols. Analysis of the effects of substituents in the leaving group as well as in the acyl moiety of the substrates showed that the ester product was formed by direct attack of the nucleophilic hydroxyl group. Comparison with reactions of tertiary amines with the same substrates supports this conclusion. The reactions of tertiary amino alcohols were also compared with those of zwitterionic quaternary amino alcohols and 3-quinuclidinol, a “rigid” tertiary amino alcohol. On the basis of these comparisons, it is proposed that one of the pathways for the predominant effect of the neutral species of tertiary amino alcohols involves intramolecular general base assistance by the tertiary amino group to the nucleophilic attack of the hydroxylic oxygen on the substrate. The contribution of this pathway to the rate of reaction is evaluated.In several systems the first product of the reaction, an O-acyl-amino alcohol, undergoes relatively rapid deacylation, the overall reaction being thus hydrolysis of active esters, catalyzed by the amino alcohol via an acylation-deacylation mechanism.     

Investigation of a green process for the polymerization of catechin

Flavonoids are polyphenolic secondary plant metabolites which possess antioxidant and anti-inflammatory properties. Besides, they have been shown to exhibit increased antioxidant properties in their polymerized form. Catechins are one of the attractive class of flavonoids which belong to the group of flavan-3-ols. Polymerization of catechins have been investigated in numerous studies indicating the requirement of certain amount of organic solvent to provide the solubility of the monomer. However, many research projects have been conducted recently to replace toxic organic contaminants of the processes with environmentally friendly solvents. In this aspect, deep eutectic solvents (DESs) that are regarded as “green solvents” have been studied extensively in various enzyme catalyzed reactions. In the present study, we focused on establishing a green pathway for laccase catalyzed polycatechin synthesis by replacing organic solvent content with DESs as green solvents. For this aim, various parameters were investigated, such as DES types and concentrations laccase amount and reaction time. Consequently, the highest molecular weight polycatechin was obtained using 5% (v/v) B–M, 125?U laccase in 1?hr of reaction time, at 30°C, as 4,354?±?678?g?mol^?1. Corresponding X/XO inhibitory activity and superoxide radical scavenging activities were achieved as, 59 and 50%, respectively.     

Structure-Function Studies of a Melanocarpus albomyces Laccase Suggest a Pathway for Oxidation of Phenolic Compounds

Melanocarpus albomyces laccase crystals were soaked with 2,6-dimethoxyphenol, a common laccase substrate. Three complex structures from different soaking times were solved. Crystal structures revealed the binding of the original substrate and adducts formed by enzymatic oxidation of the substrate. The dimeric oxidation products were identified by mass spectrometry. In the crystals, a 2,6-dimethoxy-p-benzoquinone and a C-O dimer were observed, whereas a C-C dimer was the main product identified by mass spectrometry. Crystal structures demonstrated that the substrate and/or its oxidation products were bound in the pocket formed by residues Ala191, Pro192, Glu235, Leu363, Phe371, Trp373, Phe427, Leu429, Trp507 and His508. Substrate and adducts were hydrogen-bonded to His508, one of the ligands of type 1 copper. Therefore, this surface-exposed histidine most likely has a role in electron transfer by laccases. Based on our mutagenesis studies, the carboxylic acid residue Glu235 at the bottom of the binding site pocket is also crucial in the oxidation of phenolics. Glu235 may be responsible for the abstraction of a proton from the OH group of the substrate and His508 may extract an electron. In addition, crystal structures revealed a secondary binding site formed through weak dimerization in M. albomyces laccase molecules. This binding site most likely exists only in crystals, when the Phe427 residues are packed against each other.     

Peroxidase-catalyzed asymmetric sulfoxidation in organic solvents versus in water

Peroxidase-catalyzed asymmetric sulfoxidations, while synthetically attractive, suffer from relatively low reaction rates due to poor substrate solubilities in water and from appreciable spontaneous oxidation of substrates (especially aryl alkyl sulfides) with H(2)O(2). In this work, we found that both of these shortcomings could be alleviated by switching from aqueous solutions to certain nearly anhydrous (99.7%) organic solvents as sulfoxidation reaction media. The rates of spontaneous oxidation of the model prochiral substrate thioanisole in several organic solvents were observed to be some 100- to 1000-fold slower than in water. In addition, the rates of asymmetric sulfoxidation of thioanisole in isopropyl alcohol and in methanol catalyzed by horseradish peroxidase (HRP) were determined to be tens to hundreds of times faster than in water under otherwise identical conditions. This dramatic activation is due to a much higher substrate solubility in organic solvents than in water and occurs even though the intrinsic reactivity of HRP in isopropyl alcohol and in methanol is hundreds of times lower than in water. Sulfoxidation of thioanisole catalyzed by four other hemoproteins (soybean peroxidase, myoglobin, hemoglobin, and cytochrome c) is also much faster in isopropyl alcohol than in water.     

Desaturation reactions catalyzed by soluble methane monooxygenase

Soluble methane monooxygenase (MMO) is shown to be capable of catalyzing desaturation reactions in addition to the usual hydroxylation and epoxidation reactions. Dehydrogenated products are generated from MMO-catalyzed oxidation of certain substrates including ethylbenzene and cyclohexadienes. In the reaction of ethylbenzene, desaturation of ethyl C-H occurred along with the conventional hydroxvlations of ethyl and phenyl C-Hs. As a result, styrene is formed together with ethylphenols and phenylethanols. Similarly, when 1,3- and 1,4-cyclohexadienes were used as substrates, benzene was detected as a product in addition to the corresponding alcohols and epoxides. In all cases, reaction conditions were found to significantly affect the distribution among the different products. This new activity of MMO is postulated to be associated with the chemical properties of the substrates rather than fundamental changes in the nature of the oxygen and C-H activation chemistries. The formation of the desaturated products is rationalized by formation of a substrate cationic intermediate, possibly via a radical precursor. The cationic species is then proposed to partition between recombination (alcohol formation) and elimination (alkene production) pathways. This novel function of MMO indicates close mechanistic kinship between the hydroxylation and desaturation reactions catalyzed by the nonheme diiron clusters.     

Microbial oxidation of methanol: Properties of crystallized alcohol oxidase from a yeast, Pichia sp

Alcohol oxidase (alcohol:oxygen oxidoreductase) was crystallized from a methanolgrown yeast, Pichia sp. The crystalline enzyme is homogenous as judged from polyacrylamide gel electrophoresis. Alcohol oxidase catalyzed the oxidation of short-chain primary alcohols (C₁ to C₆), substituted primary alcohols (2-chloroethanol, 3-chloro-1-propanol, 4-chlorobutanol, isobutanol), and formaldehyde. The general reaction with an oxidizable substrate is as follows: Primary alcohol + O₂ → aldehyde + H₂O₂ Formaldehyde + O₂ → formate + H₂O₂. Secondary alcohols, tertiary alcohols, cyclic alcohols, aromatic alcohols, and aldehydes (except formaldehyde) were not oxidized. The K_m values for methanol and formaldehyde are 0.5 and 3.5 mm, respectively. The stoichiometry of substrate oxidized (alcohol or formaldehyde), oxygen consumed, and product formed (aldehyde or formate) is 1:1:1. The purified enzyme has a molecular weight of 300,000 as determined by gel filtration and a subunit size of 76,000 as determined by sodium dodecyl sulfate-gel electrophoresis, indicating that alcohol oxidase consists of four identical subunits. The purified alcohol oxidase has absorption maxima at 460 and 380 nm which were bleached by the addition of methanol. The prosthetic group of the enzyme was identified as a flavin adenine dinucleotide. Alcohol oxidase activity was inhibited by sulfhydryl reagents (p-chloromercuribenzoate, mercuric chloride, 5,5′-dithiobis-2-nitrobenzoate, iodoacetate) indicating the involvement of sulfhydryl groups(s) in the oxidation of alcohols by alcohol oxidase. Hydrogen peroxide (product of the reaction), 2-aminoethanol (substrate analogue), and cupric sulfate also inhibited alcohol oxidase activity.     

Oxidation of Methanol,Formaldehyde and Formate by a Candida Species

1. The oxidation of methanol to carbon dioxide by Candida N–16 grown on methanol was investigated. The presence of enzymes which catalyze the following reaction was found in the cell-free extract of the yeast employed; CH₃OH→HCHO→HCOOH→CO₂. 2. Methanol was oxidized to formaldehyde by an alcohol oxidase. The reaction was as follows; CH₃OH+O₂→HCHO+H₂O₂. The alcohol oxidase was crystallized after purification by ammonium sulfate-precipitation and column chromatography using DEAE-Sephadex A-50. A prosthetic group of the enzyme was proved to be FAD. The enzyme possessed a broad specificity for alcohols such as methanol, ethanol, n-propanol, n-butanol and n-amylalcohol. The enzyme was inducibly formed only by the addition of methanol. 3. The oxidation of formaldehyde to formate was catalyzed by a NAD-linked dehydrogenase dependent on GSH. 4. Formate was oxidized by a NAD-linked dehydrogenase. 5. Catalase was also found in the extract, and methanol was chemically oxidized by the reaction of catalase and hydrogen peroxide which was generated by the alcohol oxidase system. 6. The oxidation pathway from methanol to carbon dioxide was also found in other methanol-utilizing yeasts such as Candida N-17, Saccharomyces H-1 and Torulopsis M-1.