Marty A

References (27)

Title : Exploring the pH dependence of an improved PETase - Charlier_2024_Biophys.J__
Author(s) : Charlier C , Gavalda S , Grga J , Perrot L , Gabrielli V , Lohr F , Schorghuber J , Lichtenecker R , Arnal G , Marty A , Tournier V , Guy L
Ref : Biophysical Journal , : , 2024
Abstract : Enzymatic recycling of plastic and especially of polyethylene terephthalate (PET) has shown great potential to reduce its negative impact on our society. PET hydrolases (PETases) have been optimized using rational design and machine learning, but the mechanistic details of the PET depolymerization process remain unclear. Belonging to the carboxylic-ester hydrolase family with a canonical Ser-His-Asp catalytic triad, their observed alkaline pH optimum is generally thought to be related to the protonation state of the catalytic His. Here, we explore this aspect in the context of LCC(ICCG), an optimized PETase, derived from the Leaf-branch Compost Cutinase (LCC) enzyme. We use NMR to identify the dominant tautomeric structure of the six histidines. Five show surprisingly low pKa values below 4.0 while the catalytic H242 in the active enzyme displays a pKa value that varies from 4.9 to 4.7 when temperatures increase from 30 degreesC to 50 degreesC. Whereas the hydrolytic activity of the enzyme towards a soluble substrate can be modeled by the corresponding protonation/deprotonation curve, an important discrepancy is found when the substrate is the solid plastic. This opens the way to further mechanistic understanding of the PETase activity, and underscores the importance of studying the enzyme at the liquid/solid interface.
ESTHER : Charlier_2024_Biophys.J__
PubMedSearch : Charlier_2024_Biophys.J__
PubMedID: 38664965
Gene_locus related to this paper: 9bact-g9by57

Title : An NMR look at an engineered PET depolymerase - Charlier_2022_Biophys.J__
Author(s) : Charlier C , Gavalda S , Borsenberger V , Duquesne S , Marty A , Tournier V , Lippens G
Ref : Biophysical Journal , : , 2022
Abstract : Plastic environmental pollution is a major issue that our generation must face to protect our planet. Plastic recycling not only has the potential to reduce the pollution but also to limit the need for fossil fuel-based production of new plastics. Enzymes capable of breaking down plastic could thereby support such a circular economy. Poly-ethylene terephthalate (PET) degrading enzymes have recently attracted considerable interest and were subjected to intensive enzyme engineering to improve their characteristics. A quadruple mutant of Leaf-branch Compost Cutinase (LCC) was identified as a most efficient and promising enzyme (Tournier et al., Nature 2020). Here, we use Nuclear Magnetic Resonance (NMR) to follow the initial LCC enzyme through its different mutations that lead to its improved performance. We experimentally define the two calcium binding sites and show their importance on the all-or-nothing thermal unfolding process, which occurs at a temperature of 72 degreesC close to the PET glass transition temperature. Using various NMR probes such as backbone amide, methyl group and histidine side chain resonances, we probe the interaction of the enzymes with mono-(2-hydroxyethyl)terephthalic acid (MHET). The latter experiments are interpreted in terms of accessibility of the active site to the polymer chain. STATEMENT OF SIGNIFICANCE: Plastic pollution is a persistent challenge worldwide. The PET polymer, used for plastic bottles, bags and textiles, is not easily degraded. Novel processes aimed at not only destroying the polymer but truly recycling it in a form that gives access again to the same high-quality plastics are needed. Biobased methods relying on enzymes that can depolymerize the PET might constitute an alternative to chemical catalysts to fully recover the monomers needed for renewed production of high quality PET. Here, we follow by solution NMR spectroscopy the LCC enzyme through its four mutations that turn it into a PETase that outperforms all other enzymes so far in a close-to-industrial process, and provide an experimental basis for understanding its improved characteristics.
ESTHER : Charlier_2022_Biophys.J__
PubMedSearch : Charlier_2022_Biophys.J__
PubMedID: 35794828
Gene_locus related to this paper: 9bact-g9by57

Title : An engineered PET depolymerase to break down and recycle plastic bottles - Tournier_2020_Nature_580_216
Author(s) : Tournier V , Topham CM , Gilles A , David B , Folgoas C , Moya-Leclair E , Kamionka E , Desrousseaux ML , Texier H , Gavalda S , Cot M , Guemard E , Dalibey M , Nomme J , Cioci G , Barbe S , Chateau M , Andre I , Duquesne S , Marty A
Ref : Nature , 580 :216 , 2020
Abstract : Present estimates suggest that of the 359 million tons of plastics produced annually worldwide(1), 150-200 million tons accumulate in landfill or in the natural environment(2). Poly(ethylene terephthalate) (PET) is the most abundant polyester plastic, with almost 70 million tons manufactured annually worldwide for use in textiles and packaging(3). The main recycling process for PET, via thermomechanical means, results in a loss of mechanical properties(4). Consequently, de novo synthesis is preferred and PET waste continues to accumulate. With a high ratio of aromatic terephthalate units-which reduce chain mobility-PET is a polyester that is extremely difficult to hydrolyse(5). Several PET hydrolase enzymes have been reported, but show limited productivity(6,7). Here we describe an improved PET hydrolase that ultimately achieves, over 10 hours, a minimum of 90 per cent PET depolymerization into monomers, with a productivity of 16.7 grams of terephthalate per litre per hour (200 grams per kilogram of PET suspension, with an enzyme concentration of 3 milligrams per gram of PET). This highly efficient, optimized enzyme outperforms all PET hydrolases reported so far, including an enzyme(8,9) from the bacterium Ideonella sakaiensis strain 201-F6 (even assisted by a secondary enzyme(10)) and related improved variants(11-14) that have attracted recent interest. We also show that biologically recycled PET exhibiting the same properties as petrochemical PET can be produced from enzymatically depolymerized PET waste, before being processed into bottles, thereby contributing towards the concept of a circular PET economy.
ESTHER : Tournier_2020_Nature_580_216
PubMedSearch : Tournier_2020_Nature_580_216
PubMedID: 32269349
Gene_locus related to this paper: 9bact-g9by57

Title : Lipases: An Overview - Casas-Godoy_2018_Methods.Mol.Biol_1835_3
Author(s) : Casas-Godoy L , Gasteazoro F , Duquesne S , Bordes F , Marty A , Sandoval G
Ref : Methods Mol Biol , 1835 :3 , 2018
Abstract : 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 characteristics of these enzymes. This chapter reviews the main sources, structural properties, and industrial applications of these highly studied enzymes.
ESTHER : Casas-Godoy_2018_Methods.Mol.Biol_1835_3
PubMedSearch : Casas-Godoy_2018_Methods.Mol.Biol_1835_3
PubMedID: 30109644

Title : Cloning, expression, and characterization of Aureobasidium melanogenum lipase in Pichia pastoris - Wongwatanapaiboon_2016_Biosci.Biotechnol.Biochem_80_2231
Author(s) : Wongwatanapaiboon J , Klinbunga S , Ruangchainikom C , Thummadetsak G , Chulalaksananukul S , Marty A , Chulalaksananukul W
Ref : Biosci Biotechnol Biochem , 80 :2231 , 2016
Abstract : cDNA of Aureobasidium melanogenum lipase comprises 1254 bp encoding 417 amino acids, whereas genomic DNA of lipase comprises 1311 bp with one intron (57 bp). The lipase gene contains a putative signal peptide encoding 26 amino acids. The A. melanogenum lipase gene was successfully expressed in Pichia pastoris. Recombinant lipase in an inducible expression system showed the highest lipase activity of 3.8 U/mL after six days of 2% v/v methanol induction. The molecular mass of purified recombinant lipase was estimated as 39 kDa using SDS-PAGE. Optimal lipase activity was observed at 35-37 degreesC and pH 7.0 using p-nitrophenyl laurate as the substrate. Lipase activity was enhanced by Mg(2+), Mn(2+), Li(+), Ca(2+), Ni(2+), CHAPS, DTT, and EDTA and inhibited by Hg(2+), Ag(+), SDS, Tween 20, and Triton X-100. The addition of 10% v/v acetone, DMSO, p-xylene, and octanol increased lipase activity, whereas that of propanol and butanol strongly inhibited it.
ESTHER : Wongwatanapaiboon_2016_Biosci.Biotechnol.Biochem_80_2231
PubMedSearch : Wongwatanapaiboon_2016_Biosci.Biotechnol.Biochem_80_2231
PubMedID: 27427953
Gene_locus related to this paper: 9pezi-a0a0h4af14

Title : Comprehensive Analysis of a Yeast Lipase Family in the Yarrowia Clade - Meunchan_2015_PLoS.One_10_e0143096
Author(s) : Meunchan M , Michely S , Devillers H , Nicaud JM , Marty A , Neuveglise C
Ref : PLoS ONE , 10 :e0143096 , 2015
Abstract : Lipases are currently the subject of intensive studies due to their large range of industrial applications. The Lip2p lipase from the yeast Yarrowia lipolytica (YlLIP2) was recently shown to be a good candidate for different biotechnological applications. Using a combination of comparative genomics approaches based on sequence similarity, synteny conservation, and phylogeny, we constructed the evolutionary scenario of the lipase family for six species of the Yarrowia clade. RNA-seq based transcriptome analysis revealed the primary role of LIP2 homologues in the assimilation of different substrates. Once identified, these YlLIP2 homologues were expressed in Y. lipolytica. The lipase Lip2a from Candida phangngensis was shown to naturally present better activity and enantioselectivity than YlLip2. Enantioselectivity was further improved by site-directed mutagenesis targeted to the substrate binding site. The mono-substituted variant V232S displayed enantioselectivity greater than 200 and a 2.5 fold increase in velocity. A double-substituted variant 97A-V232F presented reversed enantioselectivity, with a total preference for the R-enantiomer.
ESTHER : Meunchan_2015_PLoS.One_10_e0143096
PubMedSearch : Meunchan_2015_PLoS.One_10_e0143096
PubMedID: 26580812

Title : Production of stable isotope labelled lipase Lip2 from Yarrowia lipolytica for NMR: investigation of several expression systems - Nars_2014_Protein.Expr.Purif_101_14
Author(s) : Nars G , Saurel O , Bordes F , Saves I , Remaud-Simeon M , Andre I , Milon A , Marty A
Ref : Protein Expr Purif , 101 :14 , 2014
Abstract : Extracellular lipase Lip2 from Yarrowia lipolytica is a promising biocatalyst with unusual structural features, as indicated by X-ray crystallography. These features comprise a mobile domain called the lid that controls access to the catalytic site. Conformational rearrangements of the lid have been suggested to regulate lipase enzymatic activities. We used nuclear magnetic resonance to investigate the dynamics of Lip2 by exploring four expression systems, Escherichia coli, cell-free, Pichia pastoris and Y. lipolytica to produce uniformly labelled enzyme. The expression of Lip2 was assessed by determining its specific activity and measuring (15)N-(1)H HSQC spectra. Y. lipolytica turned out to be the most efficient expression system. Here, we report the first use of Y. lipolytica as an expression host for the production of uniform stable isotopic labelled protein for further structural and dynamics studies using NMR.
ESTHER : Nars_2014_Protein.Expr.Purif_101_14
PubMedSearch : Nars_2014_Protein.Expr.Purif_101_14
PubMedID: 24859677

Title : Lipases: an overview - Casas-Godoy_2012_Methods.Mol.Biol_861_3
Author(s) : Casas-Godoy L , Duquesne S , Bordes F , Sandoval G , Marty A
Ref : Methods Mol Biol , 861 :3 , 2012
Abstract : 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.
ESTHER : Casas-Godoy_2012_Methods.Mol.Biol_861_3
PubMedSearch : Casas-Godoy_2012_Methods.Mol.Biol_861_3
PubMedID: 22426709

Title : Thermodynamical methods for the optimization of lipase-catalyzed reactions - Castillo_2012_Methods.Mol.Biol_861_383
Author(s) : Castillo E , Torres-Gavilan A , Sandoval G , Marty A
Ref : Methods Mol Biol , 861 :383 , 2012
Abstract : 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.
ESTHER : Castillo_2012_Methods.Mol.Biol_861_383
PubMedSearch : Castillo_2012_Methods.Mol.Biol_861_383
PubMedID: 22426730

Title : The yeast Yarrowia lipolytica as a generic tool for molecular evolution of enzymes - Duquesne_2012_Methods.Mol.Biol_861_301
Author(s) : Duquesne S , Bordes F , Fudalej F , Nicaud JM , Marty A
Ref : Methods Mol Biol , 861 :301 , 2012
Abstract : It has been 20 years since strains of the yeast Yarrowia lipolytica were developed for the expression of recombinant proteins as alternative host to the commonly used yeasts, Pichia pastoris and Saccharomyces cerevisiae. Recently, a new strain, JMY1212, was engineered for protein evolution. With this new strain, a very high reproducibility in protein expression level was demonstrated, thus enabling its use for both rational and directed evolution strategies. Indeed, the coefficient of variation was shown to be of 10.7% for the whole process when all the steps are optimized, i.e. transformation of this strain with the gene of interest, cell growth, and protein production under oleic acid induction, and until activity screening assay. The object of this article is to summarize the fruit of these works and show the interest of Y. lipolytica strain JMY1212 for molecular evolution of enzymes, for both rational and directed evolution strategy. Lipase Lip2 from Y. lipolytica is taken here as an example to describe both strategies of molecular evolution. In these two methods, a first step of PCR creates either one targeted (rational design) or various random mutations (directed evolution), and is followed by the incorporation of the expression cassette in the genome of Y. lipolytica. An easy and direct comparison of variant properties is then allowed thanks to the extracellular and reproducible production of variants.
ESTHER : Duquesne_2012_Methods.Mol.Biol_861_301
PubMedSearch : Duquesne_2012_Methods.Mol.Biol_861_301
PubMedID: 22426726

Title : Isolation of a thermostable variant of Lip2 lipase from Yarrowia lipolytica by directed evolution and deeper insight into the denaturation mechanisms involved - Bordes_2011_J.Biotechnol_156_117
Author(s) : Bordes F , Tarquis L , Nicaud JM , Marty A
Ref : J Biotechnol , 156 :117 , 2011
Abstract : Lip2 lipase from Yarrowia lipolytica is a very promising lipase with many potential applications (e.g. resolution of racemic mixtures, production of fine chemicals). Unfortunately this potential is impeded by a very low thermostability for temperatures higher than 40 degrees C. Error-prone PCR and screening of the library in a high-performance yeast expression system (Y. lipolytica) enabled a thermostable variant to be identified. This variant presents only one mutation, the free cysteine 244 is changed into an alanine. At 60 degrees C, the half-life time of the purified variant was 127-fold increased compared to the WT enzyme (from 1.5 min to 3 h). Saturation mutagenesis experiment at position 244 demonstrated that the presence of a cysteine at this position was responsible for the thermal denaturation. It was demonstrated that WT Lip2 and the thermostable variant are both inactivated through aggregation mechanisms, but that the kinetics and the nature of the aggregation were different. For the WT enzyme, rapid intermolecular disulphide bridge interchanges triggered by the free cysteine 244 mediates aggregation. For the variant C244A, aggregation still occurred but much slower than for the WT lipase and was mainly driven by hydrophobic forces.
ESTHER : Bordes_2011_J.Biotechnol_156_117
PubMedSearch : Bordes_2011_J.Biotechnol_156_117
PubMedID: 21763359

Title : Enantioselectivity of Candida rugosa lipases (Lip1, Lip3, and Lip4) towards 2-bromo phenylacetic acid octyl esters controlled by a single amino acid - Piamtongkam_2011_Biotechnol.Bioeng_108_1749
Author(s) : Piamtongkam R , Duquesne S , Bordes F , Barbe S , Andre I , Marty A , Chulalaksananukul W
Ref : Biotechnol Bioeng , 108 :1749 , 2011
Abstract : Enantiomer discrimination by enzymes is a very accurate mechanism, which often involves few amino acids located at the active site. Lipase isoforms from Candida rugosa are very good enzymatic models to study this phenomenon as they display high sequence homology (>80%) and their enantioselectivity is often pointed out. In the present work, we investigated three lipases from C. rugosa (Lip1, Lip3, and Lip4, respectively) towards the resolution of 2-bromo-arylacetic acid esters, an important class of chemical intermediates in the pharmaceutical industry. All exhibited a high enantioselectivity, with Lip4 preferring the R-enantiomer (E-value = 15), while Lip1 and Lip3 showed an S-enantioselectivity >200. A combination of sequence and structure analysis of the three C. rugosa lipases suggested that position 296 could play a role in S- or R-enantiomer preference of C. rugosa lipases. This led to the construction by site-directed mutagenesis of Lip1 and Lip4 variants in which position 296 was, respectively, exchanged by a Gly, Ala, Leu, or Phe amino acid. Screening of these variants for their enantioselectivity toward 2-bromo phenyl acetic acid octyl esters revealed that steric hindrance of the amino acid residue introduced at position 296 controls both the enantiopreference and the enantioselectivity value of the lipase: bulkier is the amino acid at position 296, larger is the selectivity towards the S-enantiomer. To investigate further these observations at an atomic level, we carried out a preliminary modeling study of the tetrahedral intermediates formed by Lip1 and Lip4 with the (R, S)-2-bromo-phenylacetic acid octyl ester enantiomers that provides some insight regarding the determinants responsible for lipase enantiodiscrimination.
ESTHER : Piamtongkam_2011_Biotechnol.Bioeng_108_1749
PubMedSearch : Piamtongkam_2011_Biotechnol.Bioeng_108_1749
PubMedID: 21391204

Title : Continuous lipase-catalyzed production of esters from crude high-oleic sunflower oil - Severac_2011_Bioresour.Technol_102_4954
Author(s) : Severac E , Galy O , Turon F , Monsan P , Marty A
Ref : Bioresour Technol , 102 :4954 , 2011
Abstract : The objective of this work was to develop an economically relevant enzymatic process of butyl ester production using crude high-oleic sunflower oil. Novozym 435, a non-regiospecific biocatalyst, provided the best compromise between activity and butyl-ester yield. The inhibition caused by the presence of phopholipids in crude oil was eliminated by using tert-butanol. It demonstrates the key role of the medium polarity in order to insure the stability of a process. Initial substrate concentrations and their molar ratio were optimized in a continuous packed-bed reactor to maximize product yield and productivity. The best compromise was obtained for an initial oil concentration of 500 mM and a molar ratio of 5. It enabled a high productivity of 13.8 tons year(-1)kg Novozym 435(-1) with a butyl-ester purity of 96.5% to be obtained. Experiments with the continuous reactor were performed over 50 days without any loss of enzyme activity.
ESTHER : Severac_2011_Bioresour.Technol_102_4954
PubMedSearch : Severac_2011_Bioresour.Technol_102_4954
PubMedID: 21354788

Title : The lipases from Yarrowia lipolytica: genetics, production, regulation, biochemical characterization and biotechnological applications - Fickers_2011_Biotechnol.Adv_29_632
Author(s) : Fickers P , Marty A , Nicaud JM
Ref : Biotechnol Adv , 29 :632 , 2011
Abstract : Lipases are serine hydrolases that catalyze in nature the hydrolysis of ester bonds of long chain triacylglycerol into fatty acid and glycerol. However, in favorable thermodynamic conditions, they are also able to catalyze reactions of synthesis such as esterification or amidation. The non-conventional yeast Yarrowia lipolytica possesses 16 paralogs of genes coding for lipase. However, little information on all those paralogs has been yet obtained and only three isoenzymes, namely Lip2p, Lip7p and Lip8p have been partly characterized so far. Microarray data suggest that only a few of them could be expressed and that lipase synthesis seems to be dependent on the fatty acid or oil used as carbon source confirming the high adaptation of Y. lipolytica to hydrophobic substrate utilization. This review focuses on the biochemical characterization of those enzymes with special emphasis on the Lip2p lipase which is the isoenzyme mainly synthesized by Y. lipolytica. Crystallographic data highlight that this latter is a lipase sensu stricto with a lid covering the active site of the enzyme in its closed conformation. Recent findings on enzyme conditioning in dehydrated or liquid formulation, in enzyme immobilization by entrapment in natural polymers from either organic or mineral origins are also discussed together with long-term storage strategies. The development of various biotechnological applications in different fields such as cheese ripening, waste treatment, drug synthesis or human therapeutics is also presented.
ESTHER : Fickers_2011_Biotechnol.Adv_29_632
PubMedSearch : Fickers_2011_Biotechnol.Adv_29_632
PubMedID: 21550394

Title : Exploring the conformational states and rearrangements of Yarrowia lipolytica Lipase - Bordes_2010_Biophys.J_99_2225
Author(s) : Bordes F , Barbe S , Escalier P , Mourey L , Andre I , Marty A , Tranier S
Ref : Biophysical Journal , 99 :2225 , 2010
Abstract : We report the 1.7 A resolution crystal structure of the Lip2 lipase from Yarrowia lipolytica in its closed conformation. The Lip2 structure is highly homologous to known structures of the fungal lipase family (Thermomyces lanuginosa, Rhizopus niveus, and Rhizomucor miehei lipases). However, it also presents some unique features that are described and discussed here in detail. Structural differences, in particular in the conformation adopted by the so-called lid subdomain, suggest that the opening mechanism of Lip2 may differ from that of other fungal lipases. Because the catalytic activity of lipases is strongly dependent on structural rearrangement of this mobile subdomain, we focused on elucidating the molecular mechanism of lid motion. Using the x-ray structure of Lip2, we carried out extensive molecular-dynamics simulations in explicit solvent environments (water and water/octane interface) to characterize the major structural rearrangements that the lid undergoes under the influence of solvent or upon substrate binding. Overall, our results suggest a two-step opening mechanism that gives rise first to a semi-open conformation upon adsorption of the protein at the water/organic solvent interface, followed by a further opening of the lid upon substrate binding.
ESTHER : Bordes_2010_Biophys.J_99_2225
PubMedSearch : Bordes_2010_Biophys.J_99_2225
PubMedID: 20923657
Gene_locus related to this paper: yarli-lip2

Title : Rationally engineered double substituted variants of Yarrowia lipolytica lipase with enhanced activity coupled with highly inverted enantioselectivity towards 2-bromo phenyl acetic acid esters - Cambon_2010_Biotechnol.Bioeng_106_852
Author(s) : Cambon E , Piamtongkam R , Bordes F , Duquesne S , Andre I , Marty A
Ref : Biotechnol Bioeng , 106 :852 , 2010
Abstract : Inverting enzyme enantioselectivity by protein engineering is still a great challenge. Lip2p lipase from Yarrowia lipolytica, which demonstrates a low S-enantioselectivity (E-value = 5) during the hydrolytic kinetic resolution of 2-bromo-phenyl acetic acid octyl esters (an important class of chemical intermediates in the pharmaceutical industry), was converted, by a rational engineering approach, into a totally R-selective enzyme (E-value > 200). This tremendous change in selectivity is the result of only two amino acid changes. The starting point of our strategy was the prior identification of two key positions, 97 and 232, for enantiomer discrimination. Four single substitution variants were recently identified as exhibiting a low inversion of selectivity coupled to a low-hydrolytic activity. On the basis of these results, six double substituted variants, combining relevant mutations at both 97 and 232 positions, were constructed by site-directed mutagenesis. This work led to the isolation of one double substituted variant (D97A-V232F), which displays a total preference for the R-enantiomer. The highly reversed enantioselectivity of this variant is accompanied by a 4.5-fold enhancement of its activity toward the preferred enantiomer. The molecular docking of the R- and S-enantiomers in the wild-type enzyme and the D97A-V232F variant suggests that V232F mutation provides a more favorable stacking interaction for the phenyl group of the R-enantiomer, that could explain both the enhanced activity and the reversal of enantioselectivity. These results demonstrate the potential of rationally engineered mutations to further enhance enzyme activity and to modulate selectivity.
ESTHER : Cambon_2010_Biotechnol.Bioeng_106_852
PubMedSearch : Cambon_2010_Biotechnol.Bioeng_106_852
PubMedID: 20506522
Gene_locus related to this paper: yarli-lip2

Title : New efficient recombinant expression system to engineer Candida antarctica lipase B - Emond_2010_Appl.Environ.Microbiol_76_2684
Author(s) : Emond S , Montanier C , Nicaud JM , Marty A , Monsan P , Andre I , Remaud-Simeon M
Ref : Applied Environmental Microbiology , 76 :2684 , 2010
Abstract : Here, we report the use of Yarrowia lipolytica as a versatile expression host for developing protein engineering approaches to modify the properties of Candida antarctica lipase B. A reliable screening protocol was defined and validated using a saturation mutagenesis library, yielding mutants displaying higher catalytic efficiencies than the wild-type enzyme.
ESTHER : Emond_2010_Appl.Environ.Microbiol_76_2684
PubMedSearch : Emond_2010_Appl.Environ.Microbiol_76_2684
PubMedID: 20173074

Title : Improvement of Yarrowia lipolytica lipase enantioselectivity by using mutagenesis targeted to the substrate binding site - Bordes_2009_Chembiochem_10_1705
Author(s) : Bordes F , Cambon E , Dossat-Letisse V , Andre I , Croux C , Nicaud JM , Marty A
Ref : Chembiochem , 10 :1705 , 2009
Abstract : Lip2p lipase from Yarrowia lipolytica was shown to be an efficient catalyst for the resolution of 2-bromo-arylacetic acid esters, an important class of chemical intermediates in the pharmaceutical industry. Enantioselectivity of this lipase was improved by site-directed mutagenesis targeted to the substrate binding site. To guide mutagenesis experiments, the three-dimensional model of this lipase was built by homology modelling techniques by using the structures of lipases from Rhizomucor miehei and Thermomyces lanuginosa as templates. On the basis of this structural model, five amino acid residues (T88, V94, D97, V232, V285) that form the hydrophobic substrate binding site of the lipase were selected for site-directed mutagenesis. Position 232 was identified as crucial for the discrimination between enantiomers. Variant V232A displayed an enantioselectivity enhanced by one order of magnitude, whereas variant V232L exhibited a selectivity inversion. To further explore the diversity, position 232 was systematically replaced by the 19 possible amino acids. Screening of this library led to the identification of the V232S variant, which has a tremendously increased E value compared to the parental enzyme for the resolution of 2-bromo-phenylacetic acid ethyl ester (58-fold) and 2-bromo-o-tolylacetic acid ethyl ester (16-fold). In addition to the gain in enantioselectivity, a remarkable increase in velocity was observed (eightfold increase) for both substrates.
ESTHER : Bordes_2009_Chembiochem_10_1705
PubMedSearch : Bordes_2009_Chembiochem_10_1705
PubMedID: 19504508
Gene_locus related to this paper: yarli-lip2

Title : A new recombinant protein expression system for high-throughput screening in the yeast Yarrowia lipolytica - Bordes_2007_J.Microbiol.Methods_70_493
Author(s) : Bordes F , Fudalej F , Dossat V , Nicaud JM , Marty A
Ref : J Microbiol Methods , 70 :493 , 2007
Abstract : Development of a high-throughput eukaryotic screening procedure is important to increase success in obtaining improved enzymes through directed enzyme evolution. This procedure was developed for the yeast Yarrowia lipolytica which becomes the second eukaryotic host for this purpose. The extracellular lipase Lip2 was used as expressed enzyme but this system will be easily adjusted for other enzymes. We adapted and optimized the protocol for protein expression by Y. lipolytica in 96-well microplates. Yeast transformation efficiency and expression cassette insertion were increased by constructing a strain containing a zeta docking platform for targeted integration into the genome. The coefficient of variance of the full process was reduced from 36.3% to 18.9%. The main part of the variability (11.7%) arises from the specific lipase enzyme assay whereas the coefficient of variance concerning transformation, growth and expression steps represents only 7.2%. The rate of clone with no activity was reduced from 5.8% to 0.2%. Both transformation efficiency and variability are then compatible with high-throughput screening in the yeast Y. lipolytica.
ESTHER : Bordes_2007_J.Microbiol.Methods_70_493
PubMedSearch : Bordes_2007_J.Microbiol.Methods_70_493
PubMedID: 17669530

Title : Analysis of Yarrowia lipolytica extracellular lipase Lip2p glycosylation - Jolivet_2007_FEMS.Yeast.Res_7_1317
Author(s) : Jolivet P , Bordes F , Fudalej F , Cancino M , Vignaud C , Dossat V , Burghoffer C , Marty A , Chardot T , Nicaud JM
Ref : FEMS Yeast Res , 7 :1317 , 2007
Abstract : Wild-type (WT) Yarrowia lipolytica strain secretes a major extracellular lipase Lip2p which is glycosylated. In silico sequence analysis reveals the presence of two potential N-glycosylation sites (N113IS and N134NT). Strains expressing glycosylation mutant forms were constructed. Esterase activities for the different forms were measured with three substrates: p-nitrophenol butyrate (p-NPB), tributyrin and triolein. Sodium dodecyl sulfate polacrylamide gel electrophoresis analysis of supernatant indicated that the suppression of the two sites of N-glycosylation did not affect secretion. S115V or N134Q mutations led to lipase with similar specific activity compared with WT lipase while a T136V mutation reduced specific activity toward p-NPB and tributyrin. Electrospray ionization MS of the WT entire protein led to an average mass of 36 950 Da, higher than the mass deduced from the amino acid sequence (33 385 Da) and to the observation of at least two different mannose structures: Man(8)GlcNAc(2) and Man(9)GlcNAc(2). LC-tandem MS analysis of the WT Lip2p after trypsin and endoproteinase Asp-N treatments led to high coverage (87%) of protein sequence but the peptides containing N113 and N134 were not identified. We confirmed that the presence of N-glycosylation occurred at both N113 and N134 by MS of digested proteins obtained after enzymatic deglycosylation or from mutant forms.
ESTHER : Jolivet_2007_FEMS.Yeast.Res_7_1317
PubMedSearch : Jolivet_2007_FEMS.Yeast.Res_7_1317
PubMedID: 17784853

Title : Hydrophobic substrate utilisation by the yeast Yarrowia lipolytica, and its potential applications - Fickers_2005_FEMS.Yeast.Res_5_527
Author(s) : Fickers P , Benetti PH , Wache Y , Marty A , Mauersberger S , Smit MS , Nicaud JM
Ref : FEMS Yeast Res , 5 :527 , 2005
Abstract : The alkane-assimilating yeast Yarrowia lipolytica degrades very efficiently hydrophobic substrates such as n-alkanes, fatty acids, fats and oils for which it has specific metabolic pathways. An overview of the oxidative degradation pathways for alkanes and triglycerides in Y. lipolytica is given, with new insights arising from the recent genome sequencing of this yeast. This includes the interaction of hydrophobic substrates with yeast cells, their uptake and transport, the primary alkane oxidation to the corresponding fatty alcohols and then by different enzymes to fatty acids, and the subsequent degradation in peroxisomal beta-oxidation or storage into lipid bodies. Several enzymes involved in hydrophobic substrate utilisation belong to multigene families, such as lipases/esterases (LIP genes), cytochromes P450 (ALK genes) and peroxisomal acyl-CoA oxidases (POX genes). Examples are presented demonstrating that wild-type and genetically engineered strains of Y. lipolytica can be used for alkane and fatty-acid bioconversion, such as aroma production, for production of SCP and SCO, for citric acid production, in bioremediation, in fine chemistry, for steroid biotransformation, and in food industry. These examples demonstrate distinct advantages of Y. lipolytica for their use in bioconversion reactions of biotechnologically interesting hydrophobic substrates.
ESTHER : Fickers_2005_FEMS.Yeast.Res_5_527
PubMedSearch : Fickers_2005_FEMS.Yeast.Res_5_527
PubMedID: 15780653

Title : Continuous operation of lipase-catalyzed reactions in nonaqueous solvents: Influence of the production of hydrophilic compounds - Marty_1997_Biotechnol.Bioeng_56_232
Author(s) : Marty A , Dossat V , Condoret JS
Ref : Biotechnol Bioeng , 56 :232 , 1997
Abstract : In the field of biocatalysis in nonaqueous media, water has been identified as a crucial parameter which has to be carefully controlled. This article studies the continuous operation of a water-producing enzymatic reaction, here the esterification of oleic acid by ethanol in n-hexane catalyzed by Lipozyme(TM). The conversion decreased significantly over time, eventually coming to a lower steady-state level. This would be due to the accumulation of the produced water into the enzyme fixed-bed reactor, n-hexane being unable to evacuate this water out of the reaction vessel, because of the low polarity of this solvent. Therefore the conversion decreased until the produced water could be eliminated by the solvent achieving a steady state with a lower conversion. In supercritical carbon dioxide, a more hydrophilic solvent, steady state is at once obtained. This approach has been extended to reaction producing a hydrophilic compound, here glycerol during the transesterification between triolein and ethanol, and similar conclusions can be made. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 232-237, 1997.
ESTHER : Marty_1997_Biotechnol.Bioeng_56_232
PubMedSearch : Marty_1997_Biotechnol.Bioeng_56_232
PubMedID: 18636628

Title : Selective effector coupling of muscarinic acetylcholine receptor subtypes - Fukuda_1989_Trends.Pharmacol.Sci_Suppl_4
Author(s) : Fukuda K , Kubo T , Maeda A , Akiba I , Bujo H , Nakai J , Mishina M , Higashida H , Neher E , Marty A , et al.
Ref : Trends in Pharmacological Sciences , Suppl :4 , 1989
Abstract : Attempts have been made by means of recombinant DNA technology to understand the molecular basis of the functional heterogeneity of the muscarinic acetylcholine receptor (mAChR). Molecularly defined mAChR subtypes have been produced from the cloned DNAs in Xenopus oocytes and NG108-15 neuroblastoma-glioma hybrid cells as transient and stable expression systems, respectively, and agonist-induced cellular responses have been examined. The results obtained provide evidence that mAChR subtypes are selectively coupled with different effector systems, albeit not exclusively.
ESTHER : Fukuda_1989_Trends.Pharmacol.Sci_Suppl_4
PubMedSearch : Fukuda_1989_Trends.Pharmacol.Sci_Suppl_4
PubMedID: 2694521

Title : Intracellular calcium release mediated by two muscarinic receptor subtypes - Neher_1988_FEBS.Lett_240_88
Author(s) : Neher E , Marty A , Fukuda K , Kubo T , Numa S
Ref : FEBS Letters , 240 :88 , 1988
Abstract : Four subtypes of muscarinic acetylcholine receptor (mAChR) were stably expressed in neuroblastoma-glioma hybrid cells (NG108-15). By combining fluorescent indicator dye (fura-2) studies with electrophysiological measurements it is shown that stimulation of mAChR I and mAChR III readily leads to release of calcium from intracellular stores and to associated conductance changes, whereas stimulation of mAChR II and mAChR IV exerts no such effect. Dose-response curves describing the amplitude or the delay of the calcium rise induced by acetylcholine suggest that the apparent affinity of mAChR III for its agonist is higher by about one order of magnitude than that of mAChR I. Ionic substitution experiments and current fluctuation analysis indicate that calcium activates a K+-specific conductance of 'small' single-channel amplitude similar to the SK type. Furthermore, an outward current (M current) suppressed by activation of mAChR I and mAChR III has a single-channel amplitude corresponding to a conductance of approximately 3 pS.
ESTHER : Neher_1988_FEBS.Lett_240_88
PubMedSearch : Neher_1988_FEBS.Lett_240_88
PubMedID: 3192003

Title : Ionic channels for signal transmission and propagation -
Author(s) : Neher E , Marty A , Fenwick E
Ref : Prog Brain Res , 58 :39 , 1983
PubMedID: 6138813

Title : A patch-clamp study of bovine chromaffin cells and of their sensitivity to acetylcholine - Fenwick_1982_J.Physiol_331_577
Author(s) : Fenwick EM , Marty A , Neher E
Ref : The Journal of Physiology , 331 :577 , 1982
Abstract : 1. Bovine chromaffin cells were enzymatically isolated and kept in short term tissue culture. Their electrical properties were studied using recent advances of the patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). 2. When a patch pipette was sealed tightly to a chromaffin cell ('cell-attached configuration') current wave forms due to intracellular action potentials could be observed. The frequency of the wave forms was altered by changing the pipette potential. When acetylcholine was present in the pipette solution, acetylcholine-induced single channel currents were evident in the patch recording. Action potential wave forms were then often seen to follow acetycholine-induced single channel currents. 3. In the cell-attached configuration, large single channel current events did not resemble square pulses but showed exponential relaxations with time constants of the order of 50 ms. 4. After rupture of the patch of membrane, the pipette--cell seal remained stable ('whole-cell recording', Hamill et al. 1981). Chromaffin cells were found to have a resting potential of -50 to -80 mV, and an input resistance around 5 G omega. The high cell resistance accounts for the relaxing currents evident in the cell-attached configuration. 5. In the best cases, the effective time constant of the voltage clamp in the whole-cell recording mode was 15 microseconds. Exchange of small ions such as Na+ ions between pipette and cell interior solutions was then complete within 15 s. 6. Acetylcholine-induced currents were obtained at various acetylcholine concentrations. Single acetylcholine-induced channels had a slope conductance of 44 pS between -100 and -55 mV, and a mean duration of 27 ms at -80 mV (at room temperature).
ESTHER : Fenwick_1982_J.Physiol_331_577
PubMedSearch : Fenwick_1982_J.Physiol_331_577
PubMedID: 6296371

Title : Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches - Hamill_1981_Pflugers.Arch_391_85
Author(s) : Hamill OP , Marty A , Neher E , Sakmann B , Sigworth FJ
Ref : Pflugers Arch , 391 :85 , 1981
Abstract : 1. The extracellular patch clamp method, which first allowed the detection of single channel currents in biological membranes, has been further refined to enable higher current resolution, direct membrane patch potential control, and physical isolation of membrane patches. 2. A description of a convenient method for the fabrication of patch recording pipettes is given together with procedures followed to achieve giga-seals i.e. pipette-membrane seals with resistances of 10(9) - 10(11) omega. 3. The basic patch clamp recording circuit, and designs for improved frequency response are described along with the present limitations in recording the currents from single channels. 4. Procedures for preparation and recording from three representative cell types are given. Some properties of single acetylcholine-activated channels in muscle membrane are described to illustrate the improved current and time resolution achieved with giga-seals. 5. A description is given of the various ways that patches of membrane can be physically isolated from cells. This isolation enables the recording of single channel currents with well-defined solutions on both sides of the membrane. Two types of isolated cell-free patch configurations can be formed: an inside-out patch with its cytoplasmic membrane face exposed to the bath solution, and an outside-out patch with its extracellular membrane face exposed to the bath solution. 6. The application of the method for the recording of ionic currents and internal dialysis of small cells is considered. Single channel resolution can be achieved when recording from whole cells, if the cell diameter is small (less than 20 micrometer). 7. The wide range of cell types amenable to giga-seal formation is discussed.
ESTHER : Hamill_1981_Pflugers.Arch_391_85
PubMedSearch : Hamill_1981_Pflugers.Arch_391_85
PubMedID: 6270629