Purification and characterization of an extremely thermostable β‐glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus

Abstract

Cell-free extracts of cellobiose-grown cells of the hyperthermophile Pyrococcus furiosus contain very high activities (19.8 U/mg) of a P-glucosidase. The cytoplasmic enzyme was purified 22-fold to apparent homogeneity, indicating that the enzyme comprises nearly 5% of the total cell protein.The native P-glucosidase has a molecular mass of 230 -+ 20 m a , composed of 58 ? 2-kDa subunits. The enzyme has a pl of 4.40. Thiol groups are not essential for activity, nor is the enzyme dependent on divalent cations or a high ionic strength. The enzyme shows optimum activity at pH5.0 and 102-105°C. From Lineweaver-Burk plots, V,,, values of 470U/mg and 700U/mg were found for cellobiose (K, = 20 mM) andp-nitrophenyl-P-D-glucopyranoside ( K , = 0.15 mM), respectively. The purified enzyme also exhibits high j?-galactosidase activity and P-xylosidase activity, but shows no activity towards a-linked disaccharides or P-linked polymers, like cellulose.The purified P-glucosidase shows a remarkable thermostability with a half life of 85 h at 100°C and 13 h at 110°C.Hyperthermophilic microorganisms have their optimal growth temperature at 80-110°C [l]. With a few exceptions, they all belong to the domain of the Archaea [2]. The majority of these extreme thermophiles are strictly anaerobic and depend on the reduction of elemental sulfur (So) for growth. Due to its rapid growth and relatively high cell yields, Pyrococcusfuriosus is one of the best studied representatives of this group. It grows by a fermentative type of metabolism, using yeast extract, peptone, casein, starch, maltose or pyruvate as substrate. Acetate, H, and CO, are reported as the major end products [3,4]. In the presence of s", H,S is formed instead of H,. This reduction of So to H,S is apparently required only as a means to eliminate inhibition In order to utilize the different polymers, P furiosus harbours several hydrolytic enzyme activities. As many as 13 active proteinases have been demonstrated, both extracellular and intracellular [5-71. Furthermore, depending on the type of carbohydrates present in the growth medium, a-amylase, pullulanase or a-glucosidase activity has been detected [ 8,

The effects of temperature on the kinetics and stability of mesophilic and thermophilic 3-phosphoglycerate kinases

“…These enzymes follow closely the predicted rise in chemical reaction velocity with temperature and give linear Arrhenius plots. Other enzymes, such as trout-brain acetylcholinesterase [29], Thermotoga maritima enolase [30], Pyrococcus furiosus β-glucosidase [31] and the phosphoglycerate kinases studied in this paper, have much slower and non-exponential increases in reaction velocity. Indeed, the increase in k cat with temperature was practically linear for the PGKs.…”

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

“…-Glucosidase activity was measured according to the procedure of Kengen et al 5) Citrate buffer (0.1 M, pH 5.0 at 90 C, 4.0 cm 3 ) containing 10 mM pNPG was incubated with native or immobilized Pfu--glucosidase in a screwcapped 10 cm 3 vial for 5 min at 90 C. In the case of the immobilized enzyme, the reaction mixture was stirred magnetically. The reaction solution (0.5 cm 3 ) was pipetted into 4.5 cm 3 of 10 wt % sodium carbonate solution for quenching.…”

“…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.…”

“…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.…”

Occurrence, Biological Activities and Synthesis of Kaurane Diterpenes and their Glycosides

“…In industry, glycosidases are used for glycosidation reactions and these enzymes can be extracted from diverse natural sources, such as Prunus amygdalus (almond tree) and Pyrococcus furiosus (microorganism) [102,139]. The first and foremost natural function of these enzymes is to promote the hydrolysis of the glycosidic bonds.…”