Overexpression and single-step purification of a thermostable xylanase from Bacillus stearothermophilus T-6

Lapidot, Aviva; Mechaly, Adva; Shoham, Yuval

doi:10.1016/s0168-1656(96)01604-5

Cited by 51 publications

(38 citation statements)

References 12 publications

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“…[37,38] The mutagenic primers used to produce XT6E265G were as follows (the mutated nucleotides are in bold type): 5'-GACAACCAAAT-CACTGGCCTTGATGTGAGCATG-3' and 5'-CATGCTCACATCAAGGC-CAGTGATTTGGTTGTC-3'.…”

Section: Methodsmentioning

confidence: 99%

Glycosynthase Activity of Geobacillus stearothermophilus GH52 β‐Xylosidase: Efficient Synthesis of Xylooligosaccharides from α‐D‐Xylopyranosyl Fluoride through a Conjugated Reaction

et al. 2007

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Glycosynthases are mutant glycosidases in which the acidic nucleophile is replaced by a small inert residue. In the presence of glycosyl fluorides of the opposite anomeric configuration (to that of their natural substrates), these enzymes can catalyze glycosidic bond formation with various acceptors. In this study we demonstrate that XynB2E335G, a nucleophile-deficient mutant of a glycoside hydrolase family 52 beta-xylosidase from G. stearothermophilus, can function as an efficient glycosynthase, using alpha-D-xylopyranosyl fluoride as a donor and various aryl sugars as acceptors. The mutant enzyme can also catalyze the self-condensation reaction of alpha-D-xylopyranosyl fluoride, providing mainly alpha-D-xylobiosyl fluoride. The self-condensation kinetics exhibited apparent classical Michaelis-Menten behavior, with kinetic constants of 1.3 s(-1) and 2.2 mM for k(cat) and K(M(acceptor)), respectively, and a k(cat)/K(M(acceptor)) value of 0.59 s(-1) mM(-1). When the beta-xylosidase E335G mutant was combined with a glycoside hydrolase family 10 glycosynthase, high-molecular-weight xylooligomers were readily obtained from the affordable alpha-D-xylopyranosyl fluoride as the sole substrate.

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Section: Methodsmentioning

confidence: 99%

Glycosynthase Activity of Geobacillus stearothermophilus GH52 β‐Xylosidase: Efficient Synthesis of Xylooligosaccharides from α‐D‐Xylopyranosyl Fluoride through a Conjugated Reaction

et al. 2007

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“…To overcome this barrier, a highly expressed GH10 xylanase, XynT6, from the thermophile Geobacillus stearothermophilus, was used as a solubility tag (44,45) and fused at the N terminus of the dockerin-bearing laccase. The resulting bifunctional chimera was successfully expressed and purified.…”

Section: Conversion Of the Selected Enzymes To The Cellulosomal Modementioning

confidence: 99%

Toward combined delignification and saccharification of wheat straw by a laccase-containing designer cellulosome

Davidi

Moraïs

Artzi

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Efficient breakdown of lignocellulose polymers into simple molecules is a key technological bottleneck limiting the production of plant-derived biofuels and chemicals. In nature, plant biomass degradation is achieved by the action of a wide range of microbial enzymes. In aerobic microorganisms, these enzymes are secreted as discrete elements in contrast to certain anaerobic bacteria, where they are assembled into large multienzyme complexes termed cellulosomes. These complexes allow for very efficient hydrolysis of cellulose and hemicellulose due to the spatial proximity of synergistically acting enzymes and to the limited diffusion of the enzymes and their products. Recently, designer cellulosomes have been developed to incorporate foreign enzymatic activities in cellulosomes so as to enhance lignocellulose hydrolysis further. In this study, we complemented a cellulosome active on cellulose and hemicellulose by addition of an enzyme active on lignin. To do so, we designed a dockerin-fused variant of a recently characterized laccase from the aerobic bacterium Thermobifida fusca. The resultant chimera exhibited activity levels similar to the wild-type enzyme and properly integrated into the designer cellulosome. The resulting complex yielded a twofold increase in the amount of reducing sugars released from wheat straw compared with the same system lacking the laccase. The unorthodox use of aerobic enzymes in designer cellulosome machinery effects simultaneous degradation of the three major components of the plant cell wall (cellulose, hemicellulose, and lignin), paving the way for more efficient lignocellulose conversion into soluble sugars en route to alternative fuels production.

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“…This family-10 xylanases retains high activity over a broad range of pH, and is most active in the neutral range. The native XT6 displays the highest initial reaction rate at 75°C and has been successfully used in a large-scale biobleaching mill trial (Khasin et al, 1993;Lapidot et al, 1996). After several rounds of screenings for both activity and stability, a series of mutants with enhanced thermal tolerance was obtained without compromising activity.…”

Section: Introductionmentioning

confidence: 99%

Improving the thermostability of Geobacillus stearothermophilus xylanase XT6 by directed evolution and site-directed mutagenesis

Zhang

Pei

et al. 2010

Bioresource Technology

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Overexpression and single-step purification of a thermostable xylanase from Bacillus stearothermophilus T-6

Cited by 51 publications

References 12 publications

Glycosynthase Activity of Geobacillus stearothermophilus GH52 β‐Xylosidase: Efficient Synthesis of Xylooligosaccharides from α‐D‐Xylopyranosyl Fluoride through a Conjugated Reaction

Glycosynthase Activity of Geobacillus stearothermophilus GH52 β‐Xylosidase: Efficient Synthesis of Xylooligosaccharides from α‐D‐Xylopyranosyl Fluoride through a Conjugated Reaction

Toward combined delignification and saccharification of wheat straw by a laccase-containing designer cellulosome

Improving the thermostability of Geobacillus stearothermophilus xylanase XT6 by directed evolution and site-directed mutagenesis

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