An Extremely Thermostable β-Glucosidase from the Hyperthermophilic Archaeon<i>Pyrococcus Furiosus</i>; A Comparison with Other Glycosidases

Kengen, Servé W. M.; Stams, Alfons Johannes Maria

doi:10.3109/10242429409034379

Cited by 30 publications

(17 citation statements)

References 33 publications

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“…Up to now, the highest optimal temperatures for activity have been reported for the amylomaltases from T. aquaticus (80°C) (50) and Aquifex aeolicus (90°C) (1), which makes PyAMase the most thermoactive amylomaltase described to date. The biphasic behavior of the Arrhenius plot indicates that the active site of PyAMase undergoes a conformational change upon an increasing in temperature, which has been observed for other extreme thermostable enzymes as well (28). The putative disulfide bonds in PyAMase could contribute to its stability by reducing the conformational entropy of the denatured state (15).…”

Section: Discussionmentioning

confidence: 61%

Amylomaltase ofPyrobaculum aerophilumIM2 Produces Thermoreversible Starch Gels

Kaper

Talik

Ettema

et al. 2005

Appl Environ Microbiol

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Amylomaltases are 4-␣-glucanotransferases (EC 2.4.1.25) of glycoside hydrolase family 77 that transfer ␣-1,4-linked glucans to another acceptor, which can be the 4-OH group of an ␣-1,4-linked glucan or glucose. The amylomaltase-encoding gene (PAE1209) from the hyperthermophilic archaeon Pyrobaculum aerophilum IM2 was cloned and expressed in Escherichia coli, and the gene product (PyAMase) was characterized. PyAMase displays optimal activity at pH 6.7 and 95°C and is the most thermostable amylomaltase described to date. The thermostability of PyAMase was reduced in the presence of 2 mM dithiothreitol, which agreed with the identification of two possible cysteine disulfide bridges in a three-dimensional model of PyAMase. The kinetics for the disproportionation of malto-oligosaccharides, inhibition by acarbose, and binding mode of the substrates in the active site were determined. Acting on gelatinized food-grade potato starch, PyAMase produced a thermoreversible starch product with gelatin-like properties. This thermoreversible gel has potential applications in the food industry. This is the first report on an archaeal amylomaltase.

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Section: Discussionmentioning

confidence: 61%

Amylomaltase ofPyrobaculum aerophilumIM2 Produces Thermoreversible Starch Gels

Kaper

Talik

Ettema

et al. 2005

Appl Environ Microbiol

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“…This subdivision is supported by differences in biochemical characteristics of the archaeal glycosidases and those of the other glycosidases. 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).…”

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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“…Synthesis of various glycosides has mainly been achieved using mesophilic glycosidases such as -glucosidase (EC 3.2.1.21) from almonds via reversed hydrolysis reaction or transglycosylation reaction. 1,2) On the other hand, thermostable glycosidases have been recently isolated from hyperthermophilic microorganisms [3][4][5][6][7][8][9] and utilized for synthesis of alkyl glucosides and oligosaccharides. 2,[10][11][12][13][14][15] Glycosylation using thermostable glycosidases was performed at an elevated temperature to compensate for the low solubility of glycosyl acceptor and/or donor.…”

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confidence: 99%

“…2) The -glucosidase form Pyrococcus furiosus (Pfu--glucosidase) is one of the most thermostable glycosidases and has respectable -galactosidase and -mannosidase activity. [5][6][7][8] Only two methods for immobilization of Pfu--glucosidase have been reported. Immobilized Pfu--glucosidase onto Eupergit C Ò has been used for synthesis of alkyl -glucosides 10,11) and oligosaccharides, 15) while the immobilized enzyme was considerably inactivated during both transglucosylation at 75 C 10) and condensation of glucose at 60 C. 15) Okahata and his coworkers have developed effective transglycosylation using lipid-coated glycosidases, including Pfu--glucosidase in aqueous-organic two-phase systems or in homogeneous organic solvents, [16][17][18][19][20][21][22] but recovery and reuse of lipid-coated glycosidases have not been attempted.…”

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confidence: 99%

Preparation and Properties of Gelatin-Immobilized β-Glucosidase fromPyrococcus furiosus

Nagatomo

Matsushita

Sugamoto

et al. 2005

Bioscience, Biotechnology, and Biochemistry

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Hyperthermostable -glucosidase from Pyrococcus furiosus was enclosed in gelatin gel by cross-linking with transglutaminase. Gelatin-immobilized -glucosidase was considerably more thermostable than the native enzyme. Lyophilized immobilisate was stored at 90 C for 1 month without loss of activity. The immobilized -glucosidase catalyzed transglucosylation of 5-phenylpentanol with 10.0 equivalent of cellobiose at pH 5.0 and 70 C for 12 h to afford 5-phenylpentyl -Dglucopyranoside in 41% yield. The immobilized enzyme was more effective than the native one in transglucosylation. The gelatin-immobilized Pfu--glucosidase recovered from the first run of the reaction was reusable on successive runs.

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An Extremely Thermostable β-Glucosidase from the Hyperthermophilic ArchaeonPyrococcus Furiosus; A Comparison with Other Glycosidases

Cited by 30 publications

References 33 publications

Amylomaltase ofPyrobaculum aerophilumIM2 Produces Thermoreversible Starch Gels

Amylomaltase ofPyrobaculum aerophilumIM2 Produces Thermoreversible Starch Gels

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

Preparation and Properties of Gelatin-Immobilized β-Glucosidase fromPyrococcus furiosus

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