Francisco J. Plou

Engineering and Applications of fungal laccases for organic synthesis

Laccases are multi-copper containing oxidases (EC 1.10.3.2), widely distributed in fungi, higher plants and bacteria. Laccase catalyses the oxidation of phenols, polyphenols and anilines by one-electron abstraction, with the concomitant reduction of oxygen to water in a four-electron transfer process. In the presence of small redox mediators, laccase offers a broader repertory of oxidations including non-phenolic substrates. Hence, fungal laccases are considered as ideal green catalysts of great biotechnological impact due to their few requirements (they only require air, and they produce water as the only by-product) and their broad substrate specificity, including direct bioelectrocatalysis.Thus, laccases and/or laccase-mediator systems find potential applications in bioremediation, paper pulp bleaching, finishing of textiles, bio-fuel cells and more. Significantly, laccases can be used in organic synthesis, as they can perform exquisite transformations ranging from the oxidation of functional groups to the heteromolecular coupling for production of new antibiotics derivatives, or the catalysis of key steps in the synthesis of complex natural products. In this review, the application of fungal laccases and their engineering by rational design and directed evolution for organic synthesis purposes are discussed.

Laccases and their applications: a patent review

Laccases are an interesting group of multi copper enzymes, which have received much attention of researchers in last decades due to their ability to oxidize both phenolic and non-phenolic lignin related compounds as well as highly recalcitrant environmental pollutants. This makes these biocatalysts very useful for their application in several biotechnological processes. Such applications include the detoxification of industrial effluents, mostly from the paper and pulp, textile and petrochemical industries, polymer synthesis, bioremediation of contaminated soils, wine and beverage stabilization. Laccases are also used as catalysts for the manufacture of anti-cancer drugs and even as ingredients in cosmetics. Recently, the utility of laccases has also been applied to nanobiotechnology. This paper reviews recent and important patents related to the properties, heterologous production, molecular cloning, and applications of laccases within different industrial fields as well as their potential extension to the nanobiotechnology area.

Enzymatic acylation of di- and trisaccharides with fatty acids: choosing the appropriate enzyme, support and solvent

Enzymatic synthesis of fatty acid esters of di- and trisaccharides is limited by the fact that most biological catalysts are inactivated by the polar solvents (e.g. dimethylsulfoxide, dimethylformamide) where these carbohydrates are soluble. This article reviews the methodologies developed to overcome this limitation, namely those involving control over the reaction medium, the enzyme and the support. We have proposed the use of mixtures of miscible solvents (e.g. dimethylsulfoxide and 2-methyl-2-butanol) as a general strategy to acylate enzymatically hydrophilic substrates. We observed that decreasing the hydrophobicity of the medium (i.e. lowering the percentage of DMSO) the molar ratio sucrose diesters versus sucrose monoesters can be substantially enhanced. The different regioselectivity exhibited by several lipases and proteases makes feasible to synthesise different positional isomers, whose properties may vary considerably. In particular, the lipase from Thermomyces lanuginosus displays a notable selectivity for only one hydroxyl group in the acylation of sucrose, maltose, leucrose and maltotriose, compared with lipase from Candida antarctica. We have examined three immobilisation methods (adsorption on polypropylene, covalent coupling to Eupergit C, and silica-granulation) for sucrose acylation catalysed by T. lanuginosus lipase. The morphology of the support affected significantly the reaction rate and/or the selectivity of the process.

Novel Polyphenol Oxidase Mined from a Metagenome Expression Library of Bovine Rumen BIOCHEMICAL PROPERTIES, STRUCTURAL ANALYSIS, AND PHYLOGENETIC RELATIONSHIPS

RL5, a gene coding for a novel polyphenol oxidase, was identified through activity screening of a metagenome expression library from bovine rumen microflora. Characterization of the recombinant protein produced in Escherichia coli revealed a multipotent capacity to oxidize a wide range of substrates (syringaldazine > 2,6-dimethoxyphenol > veratryl alcohol > guaiacol > tetramethylbenzidine > 4-methoxybenzyl alcohol > 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) >> phenol red) over an unusually broad range of pH from 3.5 to 9.0. Apparent Km and kcat values for ABTS, syringaldazine, and 2,6-dimetoxyphenol obtained from steady-state kinetic measurements performed at 40 °C, pH 4.5, yielded values of 26, 0.43, and 0.45 μm and 18, 660, and 1175 s-1, respectively. The Km values for syringaldazine and 2,6-dimetoxyphenol are up to 5 times lower, and the kcat values up to 40 times higher, than values previously reported for this class of enzyme. RL5 is a 4-copper oxidase with oxidation potential values of 745, 400, and 500 mV versus normal hydrogen electrode for the T1, T2, and T3 copper sites. A three-dimensional model of RL5 and site-directed mutants were generated to identify the copper ligands. Bioinformatic analysis of the gene sequence and the sequences and contexts of neighboring genes suggested a tentative phylogenetic assignment to the genus Bacteroides. Kinetic, electrochemical, and EPR analyses provide unequivocal evidence that the hypothetical proteins from Bacteroides thetaiotaomicron and from E. coli, which are closely related to the deduced protein encoded by the RL5 gene, are also multicopper proteins with polyphenol oxidase activity. The present study shows that these three newly characterized enzymes form a new family of functional multicopper oxidases with laccase activity related to conserved hypothetical proteins harboring the domain of unknown function DUF152 and suggests that some other of these proteins may also be laccases.

High-yield production of mono- and di-oleylglycerol by lipase-catalyzed hydrolysis of triolein

Abstract Lipases from several origins—in soluble form or immobilized on solid supports of different aquaphilicity—have been used for the production of mono- and di-oleylglycerol by hydrolysis of triolein. The porcine pancreatic lipase adsorbed on Celite was found to be the most effective biocatalyst tested. High activity and excellent selectivity toward the formation of intermediate acylglycerols were achieved (the conversion to diolein plus monoolein was 79% after 5 h of reaction). The time course of triolein hydrolysis with free and immobilized lipases was analyzed using a sequential kinetic model, and the apparent first-order rate constants were calculated. The differences found in the rate constants were related to differences in the regioselectivity of the enzymes and/or the aquaphilicity of the supports.

Molecular and Biochemical Characterization of a β-Fructofuranosidase from Xanthophyllomyces dendrorhous

ABSTRACT An extracellular β-fructofuranosidase from the yeast Xanthophyllomyces dendrorhous was characterized biochemically, molecularly, and phylogenetically. This enzyme is a glycoprotein with an estimated molecular mass of 160 kDa, of which the N-linked carbohydrate accounts for 60% of the total mass. It displays optimum activity at pH 5.0 to 6.5, and its thermophilicity (with maximum activity at 65 to 70°C) and thermostability (with a T50 in the range 66 to 71°C) is higher than that exhibited by most yeast invertases. The enzyme was able to hydrolyze fructosyl-β-(2→1)-linked carbohydrates such as sucrose, 1-kestose, or nystose, although its catalytic efficiency, defined by the kcat/Km ratio, indicates that it hydrolyzes sucrose approximately 4.2 times more efficiently than 1-kestose. Unlike other microbial β-fructofuranosidases, the enzyme from X. dendrorhous produces neokestose as the main transglycosylation product, a potentially novel bifidogenic trisaccharide. Using a 41% (wt/vol) sucrose solution, the maximum fructooligosaccharide concentration reached was 65.9 g liter−1. In addition, we isolated and sequenced the X. dendrorhous β-fructofuranosidase gene (Xd-INV), showing that it encodes a putative mature polypeptide of 595 amino acids and that it shares significant identity with other fungal, yeast, and plant β-fructofuranosidases, all members of family 32 of the glycosyl-hydrolases. We demonstrate that the Xd-INV could functionally complement the suc2 mutation of Saccharomyces cerevisiae and, finally, a structural model of the new enzyme based on the homologous invertase from Arabidopsis thaliana has also been obtained.

Galacto-oligosaccharide Synthesis from Lactose Solution or Skim Milk Using the β-Galactosidase from Bacillus circulans

The synthesis of galacto-oligosaccharides (GOS) catalyzed by a novel commercial preparation of β-galactosidase from Bacillus circulans (Biolactase) was studied, and the products were characterized by MS and NMR. Using 400 g/L lactose and 1.5 enzyme units per milliliter, the maximum GOS yield, measured by HPAEC-PAD analysis, was 165 g/L (41% w/w of total carbohydrates in the mixture). The major transgalactosylation products were the trisaccharide Gal-β(1→4)-Gal-β(1→4)-Glc and the tetrasaccharide Gal-β(1→4)-Gal-β(1→4)-Gal-β(1→4)-Glc. The GOS yield increased to 198 g/L (49.4% w/w of total carbohydrates) using a higher enzyme concentration (15 U/mL), which minimized the enzyme inactivation under reaction conditions. Using skim milk (with a lactose concentration of 46 g/L), the enzyme also displayed transgalactosylation activity: maximum GOS yield accounted for 15.4% (7.1 g/L), which was obtained at 50% lactose conversion.

Altering the laccase functionality by in vivo assembly of mutant libraries with different mutational spectra

The generation of diversity for directed protein evolution experiments shows an important bottleneck in the in vitro random mutagenesis protocols. Most of them are biased towards specific changes that eventually confer a predicted and conservative mutational spectrum, limiting the exploration of the vast protein space. The current work describes a simple methodology to in vivo recombine mutant libraries with different nucleotide bias created by in vitro methods. This in vivo assembly was based on the accurate physiology of Saccharomyces cerevisiae, which as host, provided its high homologous recombination frequency to shuffle the libraries in a nonmutagenic way. The fungal thermophilic laccase from Myceliophthora thermophila expressed in S. cerevisiae was submitted to this protocol under the selective pressure of high concentrations of organic solvents. Mutant 2E9 with ∼3‐fold better kinetics than parent type showed two consecutive amino acid changes (G614D ‐GGC/GAC‐ and E615K ‐GAG/AAG‐) because of the in vivo shuffling of the mutant libraries. Both mutations are located in the C‐terminal tail that is specifically processed at the Golgi during the maturation of the protein by the Kex2 protease. Notoriously, the oxygen consumption at the T2/T3 trinuclear copper cluster was altered and the catalytic copper at the T1 site was perturbed showing differences in its redox potential and geometry. The change in the isoelectric point of C‐terminal extension upon mutations seems to affect the folding of the protein at the posttranslational processing steps providing new insights in the significance of the C‐terminal tail for the functionality of the ascomycete laccases. Proteins 2008.

A Simple Procedure for the Regioselective Synthesis of Fatty Acid Esters of Maltose, Leucrose, Maltotriose and n-Dodecyl Maltosides

Abstract The enzymatic acylation of several di- and trisaccharides with acyl donors ranging from 8 to 18 carbon atoms was carried out by transesterification with the corresponding vinyl esters. The reaction was performed in 2-methyl-2-butanol/dimethylsulfoxide mixtures using the lipase from Humicola lanuginosa (immobilized on Celite). Maltose, maltotriose and n-dodecyl maltosides were specifically acylated in the primary hydroxyl of the non-reducing-end glucose moiety; the acylation of leucrose occurred preferentially in the primary hydroxyl of the glucose ring.

Immobilization of dextransucrase from Leuconostoc mesenteroides NRRL B-512F on Eupergit C supports.

Dextransucrase from Leuconostoc mesenteroides B‐512F was immobilized on epoxy‐activated acrylic polymers with different textural properties (Eupergit C and Eupergit C 250L). Prior to immobilization, dextransucrase was treated with dextranase to remove the dextran layer covering the enzyme surface, thus increasing the accessibility of its reactive groups to the epoxide centers of the support. Elimination of 99% of the initial carbohydrate content was determined by the anthrone method. To prevent enzyme inactivation, the immobilization was carried out at pH 5.4, at which the coupling to the support took place through the carboxylic groups of the enzyme. The effects of the amount (mg) of dextransucrase added per gram of support (from 0.2:1 to 30:1), temperature and contact time were studied. Maximum activity recovery of 22% was achieved using Eupergit C 250L. Using this macroporous support, the maximum specific activity (710 U/g biocatalyst) was significantly higher than that obtained with the less porous Eupergit C (226 U/g biocatalyst). The dextransucrase immobilized on Eupergit C 250L showed similar optimal temperature (30 °C) and pH (5–6) compared with the native enzyme. In contrast, a notable stabilization effect at 30 °C was observed as a consequence of immobilization. After a fast partial inactivation, the dextransucrase immobilized on Eupergit C 250L maintained more than 40% of the initial activity over the following 2 days. The features of this immobilized system are very attractive for its application in batch and fixed‐bed bioreactors.