Expression in Pichia pastoris of Candida antarctica Lipase B and Lipase B Fused to a Cellulose-Binding Domain

Rotticci-Mulder, Johanna C.; Gustavsson, Malin; Holmquist, Mats; Hult, Karl; Martinelle, Mats

doi:10.1006/prep.2000.1387

Cited by 96 publications

(70 citation statements)

References 27 publications

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“…In these studies carbohydrates were used as an affinity support for enzyme immobilization, with high capacity, while retaining enzymatic activity; in some instances, increased enzymatic activity was reported (19,77,93,98,101,102,119,130,155,162,166). Recent studies have shown that a CBM serving as a fusion partner may have additional values.…”

Section: Bioprocessingmentioning

confidence: 99%

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Carbohydrate Binding Modules: Biochemical Properties and Novel Applications

Shoseyov

Shani

Levy

2006

Microbiol Mol Biol Rev

498

347

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SUMMARY Polysaccharide-degrading microorganisms express a repertoire of hydrolytic enzymes that act in synergy on plant cell wall and other natural polysaccharides to elicit the degradation of often-recalcitrant substrates. These enzymes, particularly those that hydrolyze cellulose and hemicellulose, have a complex molecular architecture comprising discrete modules which are normally joined by relatively unstructured linker sequences. This structure is typically comprised of a catalytic module and one or more carbohydrate binding modules (CBMs) that bind to the polysaccharide. CBMs, by bringing the biocatalyst into intimate and prolonged association with its substrate, allow and promote catalysis. Based on their properties, CBMs are grouped into 43 families that display substantial variation in substrate specificity, along with other properties that make them a gold mine for biotechnologists who seek natural molecular “Velcro” for diverse and unusual applications. In this article, we review recent progress in the field of CBMs and provide an up-to-date summary of the latest developments in CBM applications.

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Section: Bioprocessingmentioning

confidence: 99%

“…It is well established that expression of foreign proteins fused to CBMs results, for the most part, in high expression levels (24,52,101,105,147,151,160,162,166,(181)(182)(183). As a result, expression vectors (pET34 to pET38) incorporating CBMs as fusion tags were developed (143).…”

Section: Cbm Engineering For Different Applicationsmentioning

confidence: 99%

Carbohydrate Binding Modules: Biochemical Properties and Novel Applications

Shoseyov

Shani

Levy

2006

Microbiol Mol Biol Rev

498

347

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“…[9] Both the wild-type and Ser 105 Ala variant were expressed and produced in Pichia pastoris, purified by hydrophobic interaction chromatography and gel filtration, and freeze dried. [10,28] The amount of active immobilized PalB wild-type was determined by active-site titration with methyl paranitrophenyl n-hexylphosphonate on freeze-dried enzyme before immobilization. [10,29] The PalB wild-type and PalB Ser 105 Ala were immobilized on a polypropylene carrier Accurel MP1000 A C H T U N G T R E N N U N G (<1500 mm) in potassium phosphate buffer (50 mm, pH 7.6) and equilibrated to a water activity of 0.11 by saturated lithium chloride.…”

Section: Methodsmentioning

confidence: 99%

Suppressed Native Hydrolytic Activity of a Lipase to Reveal Promiscuous Michael Addition Activity in Water

et al. 2009

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Suppression of the native hydrolytic activity of Pseudozyma antarctica lipase B (PalB) (formerly Candida antarctica lipase B) in water is demonstrated. By replacing the catalytic Ser 105 residue with an alanine unit, promiscuous Michael addition activity is favored. A Michael addition reaction between methyl acrylate and acetylacetone was explored as a model system. For the PalB Ser 105 Ala mutant, the hydrolytic activity was suppressed more than 1000 times and, at the same time, the Michael addition activity was increased by a factor of 100. Docking studies and molecular dynamics simulations revealed an increased ability of the PalB Ser 105 Ala mutant to harbor the substrates close to a catalytically competent conformation.

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“…Recombinant cells with surface-expressed CBD or hybrid enzymes/proteins containing CBD are easily and reliably bound to cellulose supports, thus providing a very simple and economical way of whole-cell or protein immobilization [14,[21][22][23]. On the other hand, use of CBD families, which reversibly bind to cellulose, in protein engineering provide specific tags for bioseparation on cellulose matrix by affinity chromatography [24].…”

Section: Introductionmentioning

confidence: 99%

Atomic force microscopy study of cellulose surface interaction controlled by cellulose binding domains

Nigmatullin

Lovitt

Wright

et al. 2004

Colloids and Surfaces B: Biointerfaces

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Colloidal probe microscopy has been used to study the interaction between model cellulose surfaces and the role of cellulose binding domain (CBD), peptides specifically binding to cellulose, in interfacial interaction of cellulose surfaces modified with CBDs.The interaction between pure cellulose surfaces in aqueous electrolyte solution is dominated by double layer repulsive forces with the range and magnitude of the net force dependent on electrolyte concentration. AFM imaging reveals agglomeration of CBD adsorbed on cellulose surface. Despite an increase in surface charge owing to CBD binding to cellulose surface, force profiles are less repulsive for interactions involving, at least, one modified surface. Such changes are attributed to irregularity of the topography of protein surface and non-uniform distribution of surface charges on the surface of modified cellulose.Binding double CBD hybrid protein to cellulose surfaces causes adhesive forces at retraction, whereas separation curves obtained with cellulose modified with single CBD show small adhesion only at high ionic strength. This is possibly caused by the formation of the cross-links between cellulose surfaces in the case of double CBD.

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Expression in Pichia pastoris of Candida antarctica Lipase B and Lipase B Fused to a Cellulose-Binding Domain

Cited by 96 publications

References 27 publications

Carbohydrate Binding Modules: Biochemical Properties and Novel Applications

Carbohydrate Binding Modules: Biochemical Properties and Novel Applications

Suppressed Native Hydrolytic Activity of a Lipase to Reveal Promiscuous Michael Addition Activity in Water

Atomic force microscopy study of cellulose surface interaction controlled by cellulose binding domains

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