The resistance of cellulases and xylanases to proteolytic inactivation

Fontes, C.M.G.A.; Hall, Judith G.; Hirst, Barry H.; Hazlewood, Geoffrey P.; Gilbert, Harry J.

doi:10.1007/bf00170622

Cited by 75 publications

(34 citation statements)

References 17 publications

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“…Because the extracellular environment of microbial ecosystems displays high proteinase activity (3), resistance to proteolytic degradation exerts a strong selection pressure on the evolution of extracellular enzymes such as xylanases. This view is consistent with the observation that extracellular xylanases from both mesophilic and thermophilic hosts are resistant to proteinases (3,5), whereas intracellular forms of these enzymes are susceptible to proteolytic attack (1). It is interesting to note that only three amino acid substitutions generate an enzyme that is more thermostable than the wild type xylanase, and thus, the dependence on calcium for this phenotype can easily be selected out.…”

Section: Discussionsupporting

confidence: 84%

The Use of Forced Protein Evolution to Investigate and Improve Stability of Family 10 Xylanases

Andrews

Taylor

Pell

et al. 2004

Journal of Biological Chemistry

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Metal ions such as calcium often play a key role in protein thermostability. The inclusion of metal ions in industrial processes is, however, problematic. Thus, the evolution of enzymes that display enhanced stability, which is not reliant on divalent metals, is an important biotechnological goal. Here we have used forced protein evolution to interrogate whether the stabilizing effect of calcium in an industrially relevant enzyme can be replaced with amino acid substitutions. Our study has focused on the GH10 xylanase CjXyn10A from Cellvibrio japonicus, which contains an extended calcium binding loop that confers proteinase resistance and thermostability. Three rounds of error-prone PCR and selection identified a treble mutant, D262N/A80T/R347C, which in the absence of calcium is more thermostable than wild type CjXyn10A bound to the divalent metal. D262N influences the properties of the calcium binding site, A80T fills a cavity in the enzyme, increasing the number of hydrogen bonds and van der Waals interactions, and the R347C mutation introduces a disulfide bond that decreases the free energy of the unfolded enzyme. A derivative of CjXyn10A (CfCjXyn10A) in which the calcium binding loop has been replaced with a much shorter loop from Cellulomonas fimi CfXyn10A was also subjected to forced protein evolution to select for thermostablizing mutations. Two amino acid substitutions within the introduced loop and the A80T mutation increased the thermostability of the enzyme. This study demonstrates how forced protein evolution can be used to introduce enhanced stability into industrially relevant enzymes while removing calcium as a major stability determinant.

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Section: Discussionsupporting

confidence: 84%

The Use of Forced Protein Evolution to Investigate and Improve Stability of Family 10 Xylanases

Andrews

Taylor

Pell

et al. 2004

Journal of Biological Chemistry

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“…In this respect, two possible explanations are worth mentioning. On one hand, the concept of stabilizing domains has emerged from studies of proteins such as xylanases (44), in which the increased stability of one domain promotes the stability of the whole molecule. It is therefore tempting to consider the heatlabile unit of PGK as a destabilizing domain, providing the required flexibility around the active site in order to increase the catalytic rate at low temperature.…”

Section: Resultsmentioning

confidence: 99%

Structural, Kinetic, and Calorimetric Characterization of the Cold-active Phosphoglycerate Kinase from the AntarcticPseudomonas sp. TACII18

Bentahir

Feller

Aittaleb

et al. 2000

Journal of Biological Chemistry

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The gene encoding the phosphoglycerate kinase (PGK) from the Antarctic Pseudomonas sp. TACII18 has been cloned and found to be inserted between the genes encoding for glyceraldhyde-3-phosphate dehydrogenase and fructose aldolase. The His-tagged and the native recombinant PGK from the psychrophilic Pseudomonas were expressed in Escherichia coli. The wild-type and the native recombinant enzymes displayed identical properties, such as a decreased thermostability and a 2-fold higher catalytic efficiency at 25°C when compared with the mesophilic PGK from yeast. These properties, which reflect typical features of cold-adapted enzymes, were strongly altered in the His-tagged recombinant PGK. The structural model of the psychrophilic PGK indicated that a key determinant of its low stability is the reduced number of salt bridges, surface charges, and aromatic interactions when compared with mesophilic and thermophilic PGK. Differential scanning calorimetry of the psychrophilic PGK revealed unusual variations in its conformational stability for the free and substrate-bound forms. In the free form, a heatlabile and a thermostable domain unfold independently. It is proposed that the heat-labile domain acts as a destabilizing domain, providing the required flexibility around the active site for catalysis at low temperatures.

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“…The domains were initially called cellulose-binding domains, but subsequently were changed slightly to stand for carbohydrate-binding modules (CBMs), as several have now been shown to bind polysaccharides other than cellulose (Boraston et al 2004;Gilbert et al 2013). The CBMs were separated from the catalytic domain by the serine rich linker region identify by Wolff et al Crystallographic data later indicated these serine linker regions were glycosylated, which conferred protection from proteolysis (Fontes et al 1995). These CBMs were essential for effective hydrolysis of crystalline cellulose, as shown by biochemical experiments with the CelE (Cel5B, locus ID CJA_2983) enzyme.…”

Section: Cellulose Degradationmentioning

confidence: 94%

Polysaccharide degradation systems of the saprophytic bacterium Cellvibrio japonicus

Gardner

2016

World J Microbiol Biotechnol

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Study of recalcitrant polysaccharide degradation by bacterial systems is critical for understanding biological processes such as global carbon cycling, nutritional contributions of the human gut microbiome, and the production of renewable fuels and chemicals. One bacterium that has a robust ability to degrade polysaccharides is the Gram-negative saprophyte Cellvibrio japonicus. A bacterium with a circuitous history, C. japonicus underwent several taxonomy changes from an initially described Pseudomonas sp. Most of the enzymes described in the pre-genomics era have also been renamed. This review aims to consolidate the biochemical, structural, and genetic data published on C. japonicus and its remarkable ability to degrade cellulose, xylan, and pectin substrates. Initially, C. japonicus carbohydrate-active enzymes were studied biochemically and structurally for their novel polysaccharide binding and degradation characteristics, while more recent systems biology approaches have begun to unravel the complex regulation required for lignocellulose degradation in an environmental context. Also included is a discussion for the future of C. japonicus as a model system, with emphasis on current areas unexplored in terms of polysaccharide degradation and emerging directions for C. japonicus in both environmental and biotechnological applications.

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The resistance of cellulases and xylanases to proteolytic inactivation

Cited by 75 publications

References 17 publications

The Use of Forced Protein Evolution to Investigate and Improve Stability of Family 10 Xylanases

The Use of Forced Protein Evolution to Investigate and Improve Stability of Family 10 Xylanases

Structural, Kinetic, and Calorimetric Characterization of the Cold-active Phosphoglycerate Kinase from the AntarcticPseudomonas sp. TACII18

Polysaccharide degradation systems of the saprophytic bacterium Cellvibrio japonicus

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