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      Expression of a Deschampsia antarctica Desv. Polypeptide with Lipase Activity in a Pichia pastoris Vector

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          Abstract

          The current study isolated and characterized the Lip3F9 polypeptide sequence of Deschampsia antarctica Desv. (GeneBank Accession Number JX846628), which was found to be comprised of 291 base pairs and was, moreover, expressed in Pichia pastoris X-33 cells. The enzyme was secreted after 24 h of P. pastoris culture incubation and through induction with methanol. The expressed protein showed maximum lipase activity (35 U/L) with an optimal temperature of 37 °C. The lipase-expressed enzyme lost 50% of its specific activity at 25 °C, a behavior characteristic of a psychrotolerant enzyme. Recombinant enzyme activity was measured in the presence of ionic and non-ionic detergents, and a decrease in enzyme activity was detected for all concentrations of ionic and non-ionic detergents assessed.

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          Molecular Cloning : A Laboratory Manual

          Joseph Sambrook, David William Russell (2001)
          <p>The first two editions of this manual have been mainstays of molecular biology for nearly twenty years, with an unrivalled reputation for reliability, accuracy, and clarity.<br>In this new edition, authors Joseph Sambrook and David Russell have completely updated the book, revising every protocol and adding a mass of new material, to broaden its scope and maintain its unbeatable value for studies in genetics, molecular cell biology, developmental biology, microbiology, neuroscience, and immunology.<br>Handsomely redesigned and presented in new bindings of proven durability, this three–volume work is essential for everyone using today’s biomolecular techniques.<br>The opening chapters describe essential techniques, some well–established, some new, that are used every day in the best laboratories for isolating, analyzing and cloning DNA molecules, both large and small.<br>These are followed by chapters on cDNA cloning and exon trapping, amplification of DNA, generation and use of nucleic acid probes, mutagenesis, and DNA sequencing.<br>The concluding chapters deal with methods to screen expression libraries, express cloned genes in both prokaryotes and eukaryotic cells, analyze transcripts and proteins, and detect protein–protein interactions.<br>The Appendix is a compendium of reagents, vectors, media, technical suppliers, kits, electronic resources and other essential information.<br>As in earlier editions, this is the only manual that explains how to achieve success in cloning and provides a wealth of information about why techniques work, how they were first developed, and how they have evolved. </p>
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            Multifunctionality and diversity of GDSL esterase/lipase gene family in rice (Oryza sativa L. japonica) genome: new insights from bioinformatics analysis

            Hanna Chepyshko, Chia-Ping Lai, LI-MING HUANG … (2012)
            Background GDSL esterases/lipases are a newly discovered subclass of lipolytic enzymes that are very important and attractive research subjects because of their multifunctional properties, such as broad substrate specificity and regiospecificity. Compared with the current knowledge regarding these enzymes in bacteria, our understanding of the plant GDSL enzymes is very limited, although the GDSL gene family in plant species include numerous members in many fully sequenced plant genomes. Only two genes from a large rice GDSL esterase/lipase gene family were previously characterised, and the majority of the members remain unknown. In the present study, we describe the rice OsGELP (Oryza sativa GDSL esterase/lipase protein) gene family at the genomic and proteomic levels, and use this knowledge to provide insights into the multifunctionality of the rice OsGELP enzymes. Results In this study, an extensive bioinformatics analysis identified 114 genes in the rice OsGELP gene family. A complete overview of this family in rice is presented, including the chromosome locations, gene structures, phylogeny, and protein motifs. Among the OsGELPs and the plant GDSL esterase/lipase proteins of known functions, 41 motifs were found that represent the core secondary structure elements or appear specifically in different phylogenetic subclades. The specification and distribution of identified putative conserved clade-common and -specific peptide motifs, and their location on the predicted protein three dimensional structure may possibly signify their functional roles. Potentially important regions for substrate specificity are highlighted, in accordance with protein three-dimensional model and location of the phylogenetic specific conserved motifs. The differential expression of some representative genes were confirmed by quantitative real-time PCR. The phylogenetic analysis, together with protein motif architectures, and the expression profiling were analysed to predict the possible biological functions of the rice OsGELP genes. Conclusions Our current genomic analysis, for the first time, presents fundamental information on the organization of the rice OsGELP gene family. With combination of the genomic, phylogenetic, microarray expression, protein motif distribution, and protein structure analyses, we were able to create supported basis for the functional prediction of many members in the rice GDSL esterase/lipase family. The present study provides a platform for the selection of candidate genes for further detailed functional study.
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              Exploring the specific features of interfacial enzymology based on lipase studies.

              Frédéric Carrière, Ahmed Aloulou, Dirk Van Gansbeke … (2006)
              Many enzymes are active at interfaces in the living world (such as in the signaling processes at the surface of cell membranes, digestion of dietary lipids, starch and cellulose degradation, etc.), but fundamental enzymology remains largely focused on the interactions between enzymes and soluble substrates. The biochemical and kinetic characterization of lipolytic enzymes has opened up new paths of research in the field of interfacial enzymology. Lipases are water-soluble enzymes hydrolyzing insoluble triglyceride substrates, and studies on these enzymes have led to the development of specific interfacial kinetic models. Structure-function studies on lipases have thrown light on the interfacial recognition sites present in the molecular structure of these enzymes, the conformational changes occurring in the presence of lipids and amphiphiles, and the stability of the enzymes present at interfaces. The pH-dependent activity, substrate specificity and inhibition of these enzymes can all result from both "classical" interactions between a substrate or inhibitor and the active site, as well as from the adsorption of the enzymes at the surface of aggregated substrate particles such as oil drops, lipid bilayers or monomolecular lipid films. The adsorption step can provide an alternative target for improving substrate specificity and developing specific enzyme inhibitors. Several data obtained with gastric lipase, classical pancreatic lipase, pancreatic lipase-related protein 2 and phosphatidylserine-specific phospholipase A1 were chosen here to illustrate these specific features of interfacial enzymology.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Int J Mol Sci
                Journal ID (iso-abbrev): Int J Mol Sci
                Journal ID (publisher-id): ijms
                Title: International Journal of Molecular Sciences
                Publisher: Molecular Diversity Preservation International (MDPI)
                ISSN (Electronic): 1422-0067
                Publication date Collection: February 2014
                Publication date (Electronic): 07 February 2014
                Volume: 15
                Issue: 2
                Pages: 2359-2367
                Affiliations
                [1 ]Laboratorio de Fisiología y Biología Molecular Vegetal, Instituto de Agroindustria, Facultad de Ciencias Agropecuarias y Forestales, Universidad de La Frontera, Casilla 54D, Temuco 4811230, Chile; E-Mails: claudia.rabert@ 123456ufrontera.cl (C.R.); hilda.gutierrez@ 123456ufrontera.cl (A.G.-M.); leon.bravo@ 123456ufrontera.cl (L.A.B.)
                [2 ]Center of Plant, Soil Interaction and Natural Resources Biotechnology, Scientific and Technological Bioresource Nucleus, Universidad de La Frontera, Casilla 54D, Temuco 4811230, Chile
                [3 ]Laboratorio de Fisiología Vegetal, Departamento de Botánica, Universidad de Concepción, Casilla 160-C, Correo 3, Concepción 3801061, Chile; E-Mails: anavarre@ 123456gmail.com (A.N.-G.); darnacos@ 123456gmail.com (D.N.-C.)
                [4 ]Vicerrectoría de Investigación y Postgrado, Universidad de La Frontera, Casilla 54D, Temuco 4811230, Chile
                Author notes
                [* ]Author to whom correspondence should be addressed; E-Mail: mgidekel@ 123456gmail.com ; Tel.: +56-2-2988149; Fax: +56-2-29492781.
                Article
                Publisher ID: ijms-15-02359
                DOI: 10.3390/ijms15022359
                PMC ID: 3958855
                PubMed ID: 24514564
                SO-VID: 985de44f-0c60-4f39-8e68-bd6f7e3f32f3
                Copyright © © 2014 by the authors; licensee MDPI, Basel, Switzerland
                License:

                This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license ( http://creativecommons.org/licenses/by/3.0/).

                History
                Date received : 13 December 2013
                Date revision received : 18 January 2014
                Date accepted : 28 January 2014
                Categories
                Subject: Short Note

                ScienceOpen disciplines: Molecular biology
                Keywords: deschampsia antarctica,lipase,pichia pastoris,detergent tolerance
                Data availability:
                ScienceOpen disciplines: Molecular biology
                Keywords: deschampsia antarctica, lipase, pichia pastoris, detergent tolerance

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