Identification of a Novel α-Galactosidase from the Hyperthermophilic ArchaeonSulfolobus solfataricus

Abstract

Sulfolobus solfataricus is an aerobic crenarchaeon that thrives in acidic volcanic pools. In this study, we have purified and characterized a thermostable ␣-galactosidase from cell extracts of S. solfataricus P2 grown on the trisaccharide raffinose. The enzyme, designated GalS, is highly specific for ␣-linked galactosides, which are optimally hydrolyzed at pH 5 and 90°C. The protein consists of 74.7-kDa subunits and has been identified as the gene product of open reading frame Sso3127. Its primary sequence is most related to plant enzymes of glycoside hydrolase family 36, which are involved in the synthesis and degradation of raffinose and stachyose. Both the galS gene from S. solfataricus P2 and an orthologous gene from Sulfolobus tokodaii have been cloned and functionally expressed in Escherichia coli, and their activity was confirmed. At present, these Sulfolobus enzymes not only constitute a distinct type of thermostable ␣-galactosidases within glycoside hydrolase clan D but also represent the first members from the Archaea.␣-Galactosidases (␣-Gals) (EC 3.2.1.22) are a widespread class of enzymes that liberate galactose from the nonreducing end of sugars. In bacteria, yeasts, and fungi, these enzymes are usually involved in the degradation of various plant saccharides, which can then serve as a carbon and energy source for growth. Plants synthesize ␣-galactosides such as raffinose and stachyose as the major energy storage molecules in leaves, roots, and tubers. Moreover, these oligosaccharides have been associated with cold and desiccation tolerance of seeds (28). Both raffinose and stachyose are produced by two specialized synthases (raffinose synthase [EC 2.4.1.82] and stachyose synthase [EC 2.4.1.67]) that use galactinol as a galactosyl donor (39). The oligosaccharides are degraded during seed germination by the action of two distinct types of ␣-Gals that differ in their optimal pH of catalysis. While the acid ␣-Gal type is most likely active in the acidic environment of the vacuole and the apoplasm, the alkaline ␣-Gal type probably catalyzes galactose release in the more neutral or alkaline cytoplasm (4,17,31). Mammals express an ␣-Gal and an ␣-N-acetylgalactosaminidase (␣-NAGal) in lysosomal bodies to degrade glycolipids, glycoproteins, and oligosaccharides. In humans, mutations in

Molecular Characterization of an NADPH-Dependent Acetoin Reductase/2,3-Butanediol Dehydrogenase from Clostridium beijerinckii NCIMB 8052

“…This preculture was used to inoculate 50 ml LB medium in conical flasks and incubated for 8 h (37°C, 120 rpm). After 8 h of growth, the cultures were placed on ice for 1 h to induce host chaperones (18) and subsequently induced by adding 0.5 mM isopropyl-␤-D-1-thiogalactopyranoside (IPTG). E. coli cells were harvested after 15 h of growth at 20°C and resuspended in 20 mM Tris-HCl buffer, pH 7.5, containing 1 mM tris(2-carboxyethyl)phosphine (TCEP) as a reducing agent (19).…”

Identification of a GH110 Subfamily of α1,3-Galactosidases

“…A continuous substrate depletion assay, coupled with a NADP ϩ -mediated oxidation of released galactose by glucose dehydrogenase, was used to determine the k cat /K m values of the oligosaccharide substrate, which was carried out in 400-l reaction volumes containing 0.05 mM substrate, 0.5 mM NADP ϩ , and 2 units of Thermoplasma acidophilum glucose dehydrogenase (Sigma-Aldrich), and absorption at 340 nm was monitored over a period of 5-60 min (20).…”

Nanorobotics : A Newer Platform for Molecular Diagnose

“…The research and development of nanorobots for common application in fields such as medicine and defense technology should lead as for a safer and healthier future [11], nanorobots are also candidates for industrial applications. The advent of molecular nanotechnology will again expand and enormously the effectiveness, comfort and speed of future medical treatments while at the same time significantly reducing risk, cost and invasiveness [35]. The higher dedications for the development of nanorobotics will leads to improvisation of personalized medicines within the time frame.…”

“…Integrated nanosensors can be utilized for such a task in order to find intensity of E-cadherin signals. Thus they can be employed effectively for treating cancer [35].…”