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Dive into the research topics where Humberto García-Arellano is active.

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Featured researches published by Humberto García-Arellano.


Journal of Biological Chemistry | 2006

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

Ana Beloqui; Marcos Pita; Julio Polaina; A. Martínez-Arias; Olga V. Golyshina; Miren Zumárraga; Michail M. Yakimov; Humberto García-Arellano; Miguel Alcalde; Victor M. Fernandez; Kieran Elborough; Antonio Ballesteros; Francisco J. Plou; Kenneth N. Timmis; Manuel Ferrer; Peter N. Golyshin

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.


Journal of Biological Chemistry | 2006

Novel Polyphenol Oxidase Mined from a Metagenome Expression Library of Bovine Rumen

Ana Beloqui; Marcos Pita; Julio Polaina Molina; Arturo Martínez Arias; Miren Zumárraga; Humberto García-Arellano; Miguel Alcalde Galeote; Antonio Ballesteros Olmo; Francisco José Plou Gasca; Manuel Ferrer; Peter N. Golyshin

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.


Journal of Biomolecular Screening | 2005

Screening Mutant Libraries of Fungal Laccases in the Presence of Organic Solvents

Miguel Alcalde; Thomas Bulter; Miren Zumárraga; Humberto García-Arellano; Mario Mencía; Francisco J. Plou; Antonio Ballesteros

Reliable screening methods are being demanded by biocatalysts’ engineers, especially when some features such as activity or stability are targets to improve under nonnatural conditions (i.e., in the presence of organic solvents). The current work describes a protocol for the design of a fungal laccase—expressed in Saccharomyces cerevisiae—highly active in organic cosolvents. A high-throughput screening assay based on ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) oxidation was validated. The stability of the ABTS radical cation was not significantly altered in the presence of acetonitrile, ethanol, or DMSO. With a coefficient of variance below 10% and a sensitivity limit of 15 pg laccase/μL, the assay was reproducible and sensitive. The expression system of Myceliophthora thermophila laccase variant T2 in S. cerevisiae was highly dependent on the presence of Cu2+. Copper concentration was limited up to 10 μM CuSO4 where expression levels (~14-18 mg/L) were acceptable without compromising the reliability of the assay. A mutant library was created by error-prone PCR with 1.1 to 3.5 mutations per kb. After only 1 generation of directed evolution, mutant 6C9 displayed about 3.5-fold higher activities than parent type in the presence of 20% acetonitrile or 30% ethanol. The method provided here should be generally useful to improve the activity of other redox enzymes in mixtures of water/cosolvents.


Biocatalysis and Biotransformation | 2007

Bioremediation of polycyclic aromatic hydrocarbons by fungal laccases engineered by directed evolution

Miren Zumárraga; Francisco J. Plou; Humberto García-Arellano; Antonio Ballesteros; Miguel Alcalde

Fungal laccases are useful for several remarkable transformations, such as bioremediation of polycyclic aromatic hydrocarbons (PAHs), synthesis of phenolic-based resins, oxidation of lignin derivatives and others. Most of these substrates are barely water-soluble, and although polar organic co-solvents may be added to enhance their solubility, transformation rates dramatically decrease due to the negative effect of organic solvents on the protein structure. Laccase from Myceliophthora thermophila variant T2 (MtLT2) has been submitted to laboratory evolution in Saccharomyces cerevisiae with the aim of improving activity and stability in organic co-solvents. Some 4500 clones created by random mutagenesis were screened in two rounds of directed evolution. Libraries were explored under increasing concentrations of acetonitrile and ethanol, and several mutants with improved features were purified and further characterised. Turnover rates of MtLT2 in 30% (v/v) acetonitrile and 50% (v/v) ethanol were increased up to 6.5- and 7.5-fold, respectively. The best variants showed similar rates in 20% (v/v) acetonitrile or 30% (v/v) ethanol as the parent type in aqueous media. Mutant laccases were also tested for the oxidation of anthracene in the presence of 20% (v/v) acetonitrile.


Microbial Cell Factories | 2004

Use and improvement of microbial redox enzymes for environmental purposes

Humberto García-Arellano; Miguel Alcalde; Antonio Ballesteros

Industrial development may result in the increase of environmental risks. The enzymatic transformation of polluting compounds to less toxic or even innocuous products is an alternative to their complete removal. In this regard, a number of different redox enzymes are able to transform a wide variety of toxic pollutants, such as polynuclear aromatic hydrocarbons, phenols, azo dyes, heavy metals, etc. Here, novel information on chromate reductases, enzymes that carry out the reduction of highly toxic Cr(VI) to the less toxic insoluble Cr(III), is discussed. In addition, the properties and application of bacterial and eukaryotic proteins (lignin-modifying enzymes, peroxidases and cytochromes) useful in environmental enzymology is also discussed.


Journal of Molecular Catalysis B-enzymatic | 2003

Chemical modification of carboxylic residues in a cyclodextrin glucanotransferase and its implication in the hydrolysis/transglycosylation ratio of the α-amylase family

Miguel Alcalde; Francisco J. Plou; Marta Pérez-Boada; Humberto García-Arellano; Israel Valdés; Enrique Méndez; Antonio Ballesteros

Abstract The product selectivity varies notably in the enzymes of family 13 of the glycosyl hydrolases, α-amylase family, despite their similar catalytic site (three COOH groups involved) and overall architecture. For example, α-amylases are strongly hydrolytic enzymes, whereas cyclodextrin glycosyltransferases (CGTases) are essentially transglycosylases. Chemical modification of the carboxylic groups (using a water soluble carbodiimide and three different amino nucleophiles) of CGTase from Thermoanaerobacter in absence or presence of a reversible inhibitor has been carried out. In contrast with most hydrolytic enzymes of the α-amylase family, in which carbodiimide modification produces an inactivation effect, the resulting CGTases kept residual activities in the range 22–50% for cyclization and coupling, and 39–80% for disproportionation and hydrolysis. In addition, the selectivity to cyclodextrins and the production of oligosaccharides were not significantly altered when tested under industrial conditions. By amino acid analysis and MALDI-TOF mass spectrometry, it was determined that 4–5 COOH residues were modified. The three carboxylic residues implicated in the active-site (Asp230, Glu258 and Asp329) have a very low water accessibility ( 2 ). This may help to explain the high transglycosylation/hydrolysis ratio of CGTases in comparison with other α-amylase enzymes.


Journal of Biotechnology | 2007

Purification and kinetic characterization of a fructosyltransferase from Aspergillus aculeatus

Iraj Ghazi; Lucia Fernandez-Arrojo; Humberto García-Arellano; Manuel Ferrer; Antonio Ballesteros; Francisco J. Plou


Methods of Molecular Biology | 2012

Functional-Based Screening Methods for Lipases, Esterases, and Phospholipases in Metagenomic Libraries

Dolores Reyes-Duarte; Manuel Ferrer; Humberto García-Arellano


ChemInform | 2008

Enzymatic Synthesis of Sugar Esters and Oligosaccharides from Renewable Resources

Antonio Ballesteros; Francisco J. Plou; Miguel Alcalde; Manuel Ferrer; Humberto García-Arellano; Dolores Reyes-Duarte; Iraj Ghazi


Archive | 2018

Functional-Based Screening Methods for Detecting Esterase and Lipase Activity Against Multiple Substrates

Dolores Reyes-Duarte; Cristina Coscolín; Mónica Martínez-Martínez; Manuel Ferrer; Humberto García-Arellano

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Antonio Ballesteros

Spanish National Research Council

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Francisco J. Plou

Spanish National Research Council

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Manuel Ferrer

Spanish National Research Council

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Miguel Alcalde

Spanish National Research Council

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Miren Zumárraga

Spanish National Research Council

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Dolores Reyes-Duarte

Spanish National Research Council

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Iraj Ghazi

Spanish National Research Council

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Ana Beloqui

Spanish National Research Council

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Lucia Fernandez-Arrojo

Spanish National Research Council

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Marcos Pita

Spanish National Research Council

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