Purification and properties of recombinant ?-glucosidase of the hyperthermophilic bacterium Thermotoga maritima

Gabelsberger, Josef; Liebl, Wolfgang; Schleifer, Karl‐Heinz

doi:10.1007/bf00170427

Cited by 48 publications

(41 citation statements)

References 28 publications

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“…The archaeal glycosidases contain, unlike most other glycosidases, no essential ϪSH groups for catalysis and form tetramers instead of monomers (21). One other multimeric member of the family is the bacterial glucosidase from the hyperthermophile Thermotoga maritima, which is a dimeric enzyme (14). This multimerization in enzymes from extreme thermophiles may be a way to protect groups that are susceptible to modification at high temperatures or to prevent local unfolding and thereby increasing their stability.…”

Section: Discussionmentioning

confidence: 99%

Characterization of the celB gene coding for beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus and its expression and site-directed mutation in Escherichia coli

et al. 1995

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The celB gene encoding the cellobiose-hydrolyzing enzyme ␤-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus has been identified, cloned, and sequenced. The transcription and translation initiation sites of the celB gene have been determined, and archaeal control sequences were identified. The celB gene was overexpressed in Escherichia coli, resulting in high-level (up to 20% of total protein) production of ␤-glucosidase that could be purified by a two-step purification procedure. The ␤-glucosidase produced by E. coli had kinetic and stability properties similar to those of the ␤-glucosidase purified from P. furiosus. The deduced amino acid sequence of CelB showed high similarity with those of ␤-glycosidases that belong to glycosyl hydrolase family 1, implicating a conserved structure. Replacement of the conserved glutamate 372 in the P. furiosus ␤-glucosidase by an aspartate or a glutamine led to a high reduction in specific activity (200-or 1,000-fold, respectively), indicating that this residue is the active site nucleophile involved in catalysis above 100؇C.The most extensively studied representative of the hyperthermophilic organisms that have an optimum growth temperature above 85ЊC is Pyrococcus furiosus (12). P. furiosus is able to grow on a wide range of substrates, including complex polymers such as starch, glycogen, peptone, and casein or simple carbon compounds like cellobiose, maltose, and pyruvate (12,20,33). The main fermentation products are CO 2 and H 2 or alanine, the latter acting as an alternative electron sink (22). The disaccharides cellobiose and maltose are hydrolyzed by intracellular glucosidases (5, 20). The generated glucose was proposed to be further metabolized via a nonphosphorylated Entner-Doudoroff pathway (27, 33). However, recently it was discovered that sugars are fermented by P. furiosus via an Embden-Meyerhof pathway that involves two ADP-dependent kinases (19).Characterization of proteins from hyperthermophiles revealed that they are extremely thermostable and may have an optimum temperature of catalysis that exceeds the maximum growth temperature of their host (1,18). In addition to their remarkable thermostability, proteins from hyperthermophiles are often found to be highly resistant to chemical denaturation and to degradation by proteases (13). One of the most thermostable enzymes identified up to now is the ␤-glucosidase from P. furiosus, with a half-life of 85 h at 100ЊC (20). During growth on cellobiose, ␤-glucosidase can make up to 5% of the total cell protein of P. furiosus and is involved in the hydrolysis of the ␤-1,4-glycosidic bond between the two glucose moieties of the disaccharide (20). In addition, ␤-glucosidases constitute a group of well-studied enzymes among members of all three domains of life, Eucarya, Bacteria, and Archaea (16,17,35). Therefore, the pyrococcal ␤-glucosidase is a suitable model enzyme for the molecular characterization of structure-function relations of hyperthermostable enzymes. To study these relations by protein eng...

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Section: Discussionmentioning

confidence: 99%

Characterization of the celB gene coding for beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus and its expression and site-directed mutation in Escherichia coli

et al. 1995

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“…Oligomerisation is a common characteristic of family-1 b-glycohydrolases from hyperthermophiles with those from the archae Sulfolobus solfataricus and Pyrococcus furiosus (Kengen & Stams, 1994) being tetrameric, and the enzyme from the hyperthermophilic bacterium Thermotoga maritima being a dimer (Gabelsberger et al, 1993). However the cyanogenic b-glycosidase from white clover is also a dimer in solution (Barrett et al, 1995), so no simple relationship can be derived.…”

Section: Factors Producing Thermostabilitymentioning

confidence: 98%

Crystal structure of the β-glycosidase from the hyperthermophilic archeon Sulfolobus solfataricus: resilience as a key factor in thermostability

Aguilar¹,

Sanderson²,

Moracci

et al. 1997

Journal of Molecular Biology

237

198

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“…Because of the exceptional number of possible combinations between carbohydrates, there are a large number of glycosidases of varying substrate speciWcity [9]. A -glycosidase with a remarkable -glucosidase activity and wide substrate speciWcity was puriWed and characterized from the hyperthermophile Thermotoga maritima [10]. The gene coding for this enzyme in our laboratory was overexpressed in Escherichia coli.…”

Section: Introductionmentioning

confidence: 99%

Hydrolysis of soy isoflavone glycosides by recombinant β-glucosidase from hyperthermophile Thermotoga maritima

Xue

Song

2009

J Ind Microbiol Biotechnol

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A recombinant Thermotoga maritima beta-glucosidase A (BglA) was purified to homogeneity for performing enzymatic hydrolysis of isoflavone glycosides from soy flour. The kinetic properties K(m), k(cat), and k(cat)/K(m) of BglA towards isoflavone glycosides, determined using high-performance liquid chromatography, confirmed the higher efficiency of BglA in hydrolyzing malonylglycosides than non-conjugated glycosides (daidzin and genistin). During hydrolysis of soy flour by BglA at 80 degrees C, the isoflavone glycosides (soluble form) were extracted from soy flour (solid state) into the solution (liquid state) in thermal condition and converted to their aglycones (insoluble form), which mostly existed in the pellet to be separated from BglA in the reaction solution. The enzymatic hydrolysis in one-step and two-step approaches yielded 0.38 and 0.35 mg genistein and daidzein per gram of soy flour, respectively. The optimum conditions for conversion of isoflavone aglycones were 100 U per gram of soy flour, substrate concentration 25% (w/v), and incubation time 3 h for 80 degrees C.

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Purification and properties of recombinant ?-glucosidase of the hyperthermophilic bacterium Thermotoga maritima

Cited by 48 publications

References 28 publications

Characterization of the celB gene coding for beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus and its expression and site-directed mutation in Escherichia coli

Characterization of the celB gene coding for beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus and its expression and site-directed mutation in Escherichia coli

Crystal structure of the β-glycosidase from the hyperthermophilic archeon Sulfolobus solfataricus: resilience as a key factor in thermostability

Hydrolysis of soy isoflavone glycosides by recombinant β-glucosidase from hyperthermophile Thermotoga maritima

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