Guadalajara, Mexico
Guadalajara, Mexico

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Casas-Godoy L.,National Polytechnic Institute of Toulouse | Casas-Godoy L.,French National Center for Scientific Research | Casas-Godoy L.,French National Institute for Agricultural Research | Duquesne S.,National Polytechnic Institute of Toulouse | And 7 more authors.
Methods in Molecular Biology | Year: 2012

Lipases are ubiquitous enzymes, widespread in nature. They were first isolated from bacteria in the early nineteenth century and the associated research continuously increased due to the particular characteristics of these enzymes. This chapter reviews the main sources, structural properties, and industrial applications of these highly studied enzymes. © 2012 Springer Science+Business Media New York.


Castillo E.,National Autonomous University of Mexico | Torres-Gavilan A.,National Autonomous University of Mexico | Sandoval G.,Industrial Biotechnology Unit | Marty A.,National Polytechnic Institute of Toulouse | And 2 more authors.
Methods in Molecular Biology | Year: 2012

A basic insight on different thermodynamical strategies reported for the optimization of lipase-catalyzed reactions is presented. The significance of selecting the appropriate reaction media in order to enhance selectivity and operational stability of enzymes is discussed. From this analysis, the importance of developing thermodynamic strategies for controlling both the reaction kinetics and equilibrium is emphasized. A theoretical model (Conductor-like Screening Model for Realistic Solvation) for calculating thermodynamic properties in fluid phases is proposed as a powerful tool for predicting equilibrium and kinetic behavior in biocatalytic processes. © 2012 Springer Science+Business Media New York.


Mateos-Diaz E.,Industrial Biotechnology Unit | Rodriguez J.A.,Industrial Biotechnology Unit | De Los Angeles Camacho-Ruiz M.,Industrial Biotechnology Unit | Mateos-Diaz J.C.,Industrial Biotechnology Unit
Methods in Molecular Biology | Year: 2012

High-throughput screening (HTS) methods for lipases and esterases are generally performed by using synthetic chromogenic substrates (e.g., p-nitrophenyl, resorufin, and umbelliferyl esters) which may be misleading since they are not their natural substrates (e.g., partially or insoluble triglycerides). In previous works, we have shown that soluble nonchromogenic substrates and p-nitrophenol (as a pH indicator) can be used to quantify the hydrolysis and estimate the substrate selectivity of lipases and esterases from several sources. However, in order to implement a spectrophotometric HTS method using partially or insoluble triglycerides, it is necessary to find particular conditions which allow a quantitative detection of the enzymatic activity. In this work, we used Triton X-100, CHAPS, and N-lauroyl sarcosine as emulsifiers, β-cyclodextrin as a fatty acid captor, and two substrate concentrations, 1 mM of tributyrin (TC4) and 5 mM of trioctanoin (TC8), to improve the test conditions. To demonstrate the utility of this method, we screened 12 enzymes (commercial preparations and culture broth extracts) for the hydrolysis of TC4 and TC8, which are both classical substrates for lipases and esterases (for esterases, only TC4 may be hydrolyzed). Subsequent pH-stat experiments were performed to confirm the preference of substrate hydrolysis with the hydrolases tested. We have shown that this method is very useful for screening a high number of lipases (hydrolysis of TC4 and TC8) or esterases (only hydrolysis of TC4) from wild isolates or variants generated by directed evolution using nonchromogenic triglycerides directly in the test. © 2012 Springer Science+Business Media New York.


Sanchez-Gonzalez M.,Autonomous University of Nuevo León | Blanco-Gamez A.,Autonomous University of Nuevo León | Parra-Saldivar R.,Monterrey Institute of Technology | Mateos-Diaz J.C.,Industrial Biotechnology Unit | Estrada-Alvarado M.I.,Sonora Institute of Technology
Methods in Molecular Biology | Year: 2012

Recently, the crystal structure of the feruloyl esterase A from Aspergillus niger (AnFaeA) was elucidated. This enzyme displays an α/β hydrolase fold and a catalytic triad similar to that found in fungal lipases (30-37% identity). Surprisingly, AnFaeA showed an overall fold similarity with the Rhizomucor miehei and other related fungal lipases. All these data strongly suggest that the ancestral function (lipase) had shifted, with molecular adaptation leading to a novel enzyme (type-A feruloyl esterase). The discovery of new feruloyl esterases could lead to get insight into the evolutionary pathways of these enzymes and into new possibilities of directed evolution of lipases. In this chapter, the production of Bacillus flexus NJY2 feruloyl esterases is described. Unlike the previously described feruloyl esterases, which mostly belong to eukaryotes (mainly fungus), this unique feruloyl esterases from a prokaryotic alkaliphile microorganism could be the starting point for new discoveries on lipase and feruloyl esterase evolutionary relationships. © 2012 Springer Science+Business Media New York.


Ramirez-Cordova J.,Medical and Pharmaceutical Biotechnology Unit | Drnevich J.,Urbana University | Madrigal-Pulido J.A.,Medical and Pharmaceutical Biotechnology Unit | Arrizon J.,Industrial Biotechnology Unit | And 3 more authors.
Antonie van Leeuwenhoek, International Journal of General and Molecular Microbiology | Year: 2012

During ethanol fermentation, yeast cells are exposed to stress due to the accumulation of ethanol, cell growth is altered and the output of the target product is reduced. For Agave beverages, like tequila, no reports have been published on the global gene expression under ethanol stress. In this work, we used microarray analysis to identify Saccharomyces cerevisiae genes involved in the ethanol response. Gene expression of a tequila yeast strain of S. cerevisiae (AR5) was explored by comparing global gene expression with that of laboratory strain S288C, both after ethanol exposure. Additionally, we used two different culture conditions, cells grown in Agave tequilana juice as a natural fermentation media or grown in yeast-extract peptone dextrose as artificial media. Of the 6368 S. cerevisiae genes in the microarray, 657 genes were identified that had different expression responses to ethanol stress due to strain and/or media. A cluster of 28 genes was found overexpressed specifically in the AR5 tequila strain that could be involved in the adaptation to tequila yeast fermentation, 14 of which are unknown such as yor343 c, ylr162 w, ygr182 c, ymr265 c, yer053 c-A or ydr415c. These could be the most suitable genes for transforming tequila yeast to increase ethanol tolerance in the tequila fermentation process. Other genes involved in response to stress (RFC4, TSA1, MLH1, PAU3, RAD53) or transport (CYB2, TIP20, QCR9) were expressed in the same cluster. Unknown genes could be good candidates for the development of recombinant yeasts with ethanol tolerance for use in industrial tequila fermentation. © Springer Science+Business Media B.V. 2012.


Rivera I.,Industrial Biotechnology Unit | Mateos-Diaz J.C.,Industrial Biotechnology Unit | Sandoval G.,Industrial Biotechnology Unit
Methods in Molecular Biology | Year: 2012

Lipases from plants have very interesting features for application in different fields. This chapter provides an overview on some of the most important aspects of plant lipases, such as sources, applications, physiological functions, and specificities. Lipases from laticifers and particularly Carica papaya lipase (CPL) have emerged as a versatile autoimmobilized biocatalyst. However, to get a better understanding of CPL biocatalytic properties, the isolation and purification of individual C. papaya lipolytic enzymes become necessary. In this chapter, a practical protocol for partial purification of the latex-associated lipolytic activity from C. papaya is given. © 2012 Springer Science+Business Media New York.


Herrera-Lopez E.J.,Industrial Biotechnology Unit
Methods in Molecular Biology | Year: 2012

Recent advances in the field of biology, electronics, and nanotechnology have improved the development of biosensors. A biosensor is a device composed of a biological recognition element and a sensor element. Biosensor applications are becoming increasingly important in areas such as biotechnology, pharmaceutics, food, and environment. Lipases and phospholipases are enzymes which have been used widely in food industry, oleochemical industry, biodegradable polymers, detergents, and other applications. In the medical industry, lipases and phospholipases are used as diagnostic tools to detect triglycerides, cholesterol, and phospholipids levels in blood samples. Therefore, the development of lipase and phospholipase biosensors is of paramount importance in the clinical area. This chapter introduces the reader into the preliminaries of biosensor and reviews recent developments of lipase and phospholipase biosensors. © 2012 Springer Science+Business Media New York.


Fan X.,Piedmont Biofuels Industrial Llc | Niehus X.,Industrial Biotechnology Unit | Sandoval G.,Industrial Biotechnology Unit
Methods in Molecular Biology | Year: 2012

The global shortages of fossil fuels, significant increase in the price of crude oil, and increased environmental concerns have stimulated the rapid growth in biodiesel production. Biodiesel is generally produced through transesterification reaction catalyzed either chemically or enzymatically. Enzymatic transesterification draws high attention because that process shows certain advantages over the chemical catalysis of transesterification and it is "greener." This paper reviews the current status of biodiesel production with lipase-biocatalysis approach, including sources of lipases, kinetics, and reaction mechanism of biodiesel production using lipases, and lipase immobilization techniques. Factors affecting biodiesel production and economic feasibility of biodiesel production using lipases are also covered. © 2012 Springer Science+Business Media New York.


PubMed | Industrial Biotechnology Unit
Type: | Journal: Methods in molecular biology (Clifton, N.J.) | Year: 2012

High-throughput screening (HTS) methods for lipases and esterases are generally performed by using synthetic chromogenic substrates (e.g., p-nitrophenyl, resorufin, and umbelliferyl esters) which may be misleading since they are not their natural substrates (e.g., partially or insoluble triglycerides). In previous works, we have shown that soluble nonchromogenic substrates and p-nitrophenol (as a pH indicator) can be used to quantify the hydrolysis and estimate the substrate selectivity of lipases and esterases from several sources. However, in order to implement a spectrophotometric HTS method using partially or insoluble triglycerides, it is necessary to find particular conditions which allow a quantitative detection of the enzymatic activity. In this work, we used Triton X-100, CHAPS, and N-lauroyl sarcosine as emulsifiers, -cyclodextrin as a fatty acid captor, and two substrate concentrations, 1 mM of tributyrin (TC4) and 5 mM of trioctanoin (TC8), to improve the test conditions. To demonstrate the utility of this method, we screened 12 enzymes (commercial preparations and culture broth extracts) for the hydrolysis of TC4 and TC8, which are both classical substrates for lipases and esterases (for esterases, only TC4 may be hydrolyzed). Subsequent pH-stat experiments were performed to confirm the preference of substrate hydrolysis with the hydrolases tested. We have shown that this method is very useful for screening a high number of lipases (hydrolysis of TC4 and TC8) or esterases (only hydrolysis of TC4) from wild isolates or variants generated by directed evolution using nonchromogenic triglycerides directly in the test.


PubMed | Industrial Biotechnology Unit
Type: | Journal: Methods in molecular biology (Clifton, N.J.) | Year: 2012

Lipases from plants have very interesting features for application in different fields. This chapter provides an overview on some of the most important aspects of plant lipases, such as sources, applications, physiological functions, and specificities. Lipases from laticifers and particularly Carica papaya lipase (CPL) have emerged as a versatile autoimmobilized biocatalyst. However, to get a better understanding of CPL biocatalytic properties, the isolation and purification of individual C. papaya lipolytic enzymes become necessary. In this chapter, a practical protocol for partial purification of the latex-associated lipolytic activity from C. papaya is given.

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