Rational Strategies for Directed Evolution of Biocatalysts –Application to <i>Candida antarctica</i> lipase B (CALB)

Chodorge, Matthieu; Fourage, Laurent; Ullmann, Christophe; Duvivier, Vincent; Masson, Jean-Michel; Lefèvre, Fabrice

doi:10.1002/adsc.200505055

Cited by 42 publications

(20 citation statements)

References 15 publications

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“…Chodorge et al used error-prone PCR to generate CALB variants, which were screened for enhanced thermostability. [18] The variant N292Y showed 7.5-fold increased activity after incubation at 90 8C for 15 min. In a series of publications, Hult and co-workers report the effect of rational single point mutations in the CALB active site.…”

Section: Introductionmentioning

confidence: 96%

Understanding the Plasticity of the α/β Hydrolase Fold: Lid Swapping on the Candida antarctica Lipase B Results in Chimeras with Interesting Biocatalytic Properties

et al. 2009

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The best of both worlds. Long molecular dynamics (MD) simulations of Candida antarctica lipase B (CALB) confirmed the function of helix alpha5 as a lid structure. Replacement of the helix with corresponding lid regions from CALB homologues from Neurospora crassa and Gibberella zeae resulted in new CALB chimeras with novel biocatalytic properties. The figure shows a snapshot from the MD simulation. The Candida antarctica lipase B (CALB) has found very extensive use in biocatalysis reactions. Long molecular dynamics simulations of CALB in explicit aqueous solvent confirmed the high mobility of the regions lining the channel that leads into the active site, in particular, of helices alpha5 and alpha10. The simulation also confirmed the function of helix alpha5 as a lid of the lipase. Replacing it with corresponding lid regions from the CALB homologues from Neurospora crassa and Gibberella zeae resulted in two new CALB mutants. Characterization of these revealed several interesting properties, including increased hydrolytic activity on simple esters, specifically substrates with C(alpha) branching on the carboxylic side, and much increased enantioselectivity in hydrolysis of racemic ethyl 2-phenylpropanoate (E>50), which is a common structure of the profen drug family.

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Section: Introductionmentioning

confidence: 96%

Understanding the Plasticity of the α/β Hydrolase Fold: Lid Swapping on the Candida antarctica Lipase B Results in Chimeras with Interesting Biocatalytic Properties

et al. 2009

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“…One of the most frequently used lipases is Candida antarctica lipase B, which has three disulphide bonds. Recently it has been cloned and expressed in lower eukaryotes as well as in E. coli (Hoegh et al 1995;Rotticci-Mulder et al 2001;Zhang et al 2003;Chodorge et al 2005;Blank et al 2006;Liu et al 2006). Recently it has been cloned and expressed in lower eukaryotes as well as in E. coli (Hoegh et al 1995;Rotticci-Mulder et al 2001;Zhang et al 2003;Chodorge et al 2005;Blank et al 2006;Liu et al 2006).…”

Section: Introductionmentioning

confidence: 99%

Improving the expression yield of Candida antarctica lipase B in Escherichia coli by mutagenesis

Jung

Park

2007

Biotechnol Lett

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Increasing the expression yield of active Candida antarctica lipase B (CAL-B) in Escherichia coli was achieved by using a codon-optimized synthetic gene and by mutagenesis to introduce hydrophilic residues on the surface of CAL-B. Five residues (four leucines and one isoleucine) on the surface of CAL-B were selected and changed with aspartate after codon optimization. While the codon-optimized synthetic gene of CAL-B did not increase the expression yield, the mutation increased the activity of the enzyme three-fold (3.3 mg/l of culture) compared to the wild type. The mutant enzyme had similar hydrolytic activity toward hydrolysis of p-nitrophenyl acetate or p-nitrophenyl butyrate and enantioselectivity toward hydrolysis of (R, S)-1-phenylethyl acetate compared to the wild-type enzyme.

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“…The random mutagenesis EvoSight TM method was designed in order to rationalize the directed evolution strategies and make it possible to run enzyme optimization programs within timelines that are compatible with industrial requirements (Fourage et al, 2006;Chodorge et al, 2005). EvoSight TM is a three-step method.…”

Section: Random Mutagenesismentioning

confidence: 99%

How Molecular Evolution Technologies can Provide Bespoke Industrial Enzymes: Application to Biofuels

Fourage

Sonet²,

Monot

et al. 2013

Oil Gas Sci. Technol. – Rev. IFP Energies nouvelles

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Re´sume´-Comment les technologies d'e´volution mole´culaire peuvent fournir des enzymes industrielles sur mesure : application aux biocarburants -L'hydrolyse enzymatique de la lignocellulose est l'un des principaux goulets d'e´tranglement dans le de´veloppement de la conversion biologique de la biomasse lignocellulosique en biocarburants. L'un des organismes les plus efficaces pour la production d'enzymes cellulolytiques est le champignon Trichoderma reesei, principalement graˆce a`sa capacite´importante de se´cre´tion. La conversion de la cellulose en glucose implique trois types de cellulases travaillant en synergie : les endoglucanases (EC 3.2.1.4) clivant de fac¸on ale´atoire les liaisons glycosidiques en b-1,4, les cellobiohydrolases (EC 3.2.1.91) attaquant la chaıˆne de cellulose aux deux extre´mite´s afin de produire le cellobiose, dime`re qui sera converti en glucose par l'action des b-glucosidases (EC 3.2.1.21). De fac¸on inattendue, la quantite´de b-glucosidase (BGL1) se´cre´te´e par les souches de T. reesei repre´sente un tre`s faible pourcentage de la quantite´totale des prote´ines se´cre´te´es qui en fait donc une activite´limitante du cocktail. Cette faible activite´limite d'autant plus les performances du cocktail que le cellobiose repre´sente le principal inhibiteur de la re´action cellulolyse par les cellobiohydrolases. Ce goulot d'e´tranglement peut eˆtre atte´nue´soit par une surexpression de la b-glucosidase chez T. reesei, soit par une ame´lioration de son activiteś pe´cifique. Apre`s un bref aperc¸u des principales technologies existantes, cet exemple sera utilise´dans cette revue pour illustrer le potentiel des technologies d'e´volution dirige´e pour de´velopper des enzymes re´pondant aux besoins de l'industrie des biotechnologies. Nous de´crivons comment la mise en oeuvre d'une strate´gie d'e´volution dirige´e par le L-Shuffling TM avec trois ge`nes parentaux provenant de la biodiversite´microbienne permet d'obtenir des activite´s b-glucosidases tre`s ame´liore´es par rapport a`la Cel3a b-glucosidase de T. reesei (activite´spe´cifique 242 fois plus e´leve´e pour le substrat pNPGlc). Cette ame´lioration de l'activite´glucosidasique, apre`s expression du ge`ne codant pour la b-glucosidase ame´liore´e chez T. reesei et se´cre´tion du nouveau cocktail, permet une diminution d'un facteur 4 de la quantite´de cocktail ne´cessaire a`la saccharification d'une biomasse industrielle pre´traite´e (paille de ble´).Abstract -How Molecular Evolution Technologies can Provide Bespoke Industrial Enzymes: Application to Biofuels -Enzymatic hydrolysis of lignocellulose is one of the major bottlenecks in the development of biological conversion of lignocellulosic biomass to biofuels. One of the most efficient organisms for the production of cellulolytic enzymes is the fungus Trichoderma reesei, Oil & Gas Science and Technology -Rev. IFP Energies nouvelles, Vol. 68 (2013), No. 4, pp. 681-691 Copyright Ó 2013, IFP Energies nouvelles DOI: 10.2516 mainly thanks to its high secretion capacity. The conversion of ...

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Rational Strategies for Directed Evolution of Biocatalysts –Application to Candida antarctica lipase B (CALB)

Cited by 42 publications

References 15 publications

Understanding the Plasticity of the α/β Hydrolase Fold: Lid Swapping on the Candida antarctica Lipase B Results in Chimeras with Interesting Biocatalytic Properties

Understanding the Plasticity of the α/β Hydrolase Fold: Lid Swapping on the Candida antarctica Lipase B Results in Chimeras with Interesting Biocatalytic Properties

Improving the expression yield of Candida antarctica lipase B in Escherichia coli by mutagenesis

How Molecular Evolution Technologies can Provide Bespoke Industrial Enzymes: Application to Biofuels

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