Purification and characterization of a maltase from the extremely thermophilic crenarchaeote Sulfolobus solfataricus

Abstract

A soluble maltase (␣-glucosidase) with an apparent subunit mass of 80 kDa was purified to homogeneity from Sulfolobus solfataricus. The enzyme liberates glucose from maltose and malto-oligomers. Maximal activity was observed at 105؇C, with half-lives of 11 h (85؇C), 3.0 h (95؇C), and 2.75 h (100؇C). The enzyme was generally resistant to proteolysis and denaturants including aliphatic alcohols. n-Propanol treatment at 85؇C increased both K m and V max for maltose hydrolysis.Sulfolobus solfataricus (5) is an aerobic, extremely thermophilic microorganism found in acidic hotsprings. It has been assigned to the subgroup Crenarchaeota within the domain Archaea by 16S rDNA sequence analysis (17). S. solfataricus has the ability to grow lithoautotrophically utilizing sulfur as an energy source or chemoheterotrophically on a variety of sugars and polysaccharides, including starch, as sole carbon and energy sources (2, 5, 7). Starch catabolism frequently depends upon a secreted ␣-amylase which generates linear maltodextrins from starch as well as a cell-associated ␣-glucosidase (maltase) which converts maltose and maltodextrins to glucose (11). Extremely thermophilic ␣-amylases have been identified in members of the obligately anaerobic sulfur-reducing genus Pyrococcus (3, 13, 15), which is classified within the euryarchaeotal branch of the domain Archaea. A monomeric 125-kDa ␣-glucosidase has also been identified in this group (4, 12). Several lines of evidence suggested that an ␣-glucosidase was similarly present in the crenarchaeote S. solfactaricus. These included the ability of the organism to utilize maltose as the sole carbon and energy source (7) and the presence of a p-nitrophenyl-␣-D-glucopyranoside (PNPG) hydrolytic activity in crude cell extracts (1).S. solfataricus 98/2. S. solfataricus 98/2 was obtained from L. Hochstein (10). Cells were cultured at 80ЊC as described previously (2) at pH 3.0 with maltose at 0.2% (wt/vol) as the sole carbon and energy source. Cultures were grown in 500-ml volumes and were harvested in late exponential phase, which was equivalent to a cell density of 10 9 cells per ml and an A 540 of 1.0. Cells were recovered by centrifugation at 4ЊC, and the resulting cell pellet was frozen at Ϫ20ЊC. Species identification was determined by rDNA sequence analysis (9). Such information was important in light of the confusion regarding Sulfolobus isolates (14,18). DNA sequence analysis of a 487-bp 16S rDNA sequence indicated that strain 98/2 shared 100% homology with S. solfataricus P2, 98.8% homology with S. shibatae B12 (6), and 87.6% homology with S. acidocaldarius (9,14,18). In addition, S. solfataricus P2 and 98/2 utilize a range of sugars, including maltose, mannose, and trehalose, which cannot be utilized by S. acidocaldarius, as well as cellobiose, which is used either poorly or not at all by either S. acidocaldarius or S. shibatae (7).Purification of the S. solfataricus maltase. The colorimetric maltose analog PNPG was used as a substrate to monitor maltase activity during the purifica...

Identification of an archaeal mercury regulon by chromatin immunoprecipitation

“…Archaeal strains, plasmids and primers are indicated (Table S1, available in the online Supplementary Material). S. solfataricus strain PBL2025 and its derivatives were grown at 80 uC with aeration in batch culture as described previously (Allen, 1959;Rolfsmeier & Blum, 1995;Worthington et al, 2003) in Allen's basal salts (Allen, 1959) as modified by Brock et al (1972) at a pH of 3.0. Liquid media were supplemented with either 0.2 % lactose (w/v), 0.2 % sucrose (w/v) or 0.2 % (w/v) tryptone as carbon and energy sources as indicated.…”

Mercury Inactivates Transcription and the Generalized Transcription Factor TFB in the Archaeon Sulfolobus solfataricus

“…S. solfataricus (38) was cultivated in batch cultures, as described previously (19,39). Cells were grown at 80°C in the medium of Allen (1) as modified by Brock et al (9) at a pH of 3.0 in 250-ml screw-cap flasks.…”

Identification and Molecular Characterization of the First α-Xylosidase from an Archaeon

“…Although the substrate specificity of these gene products remains to be identified, they could be involved in the degradation and assimilation of plant polysaccharides. The only examples of glycosyl hydrolases characterized in this genus are a secreted ␣-amylase, an intracellular ␣-glucosidase from S. solfataricus strain 98/2 (17,18), and a ␤-glycosidase that has been purified from strains MT4 and P2 (19,20). The two ␣-glycosyl hydrolases are involved in the utilization of dextrins, whereas the function in vivo of the ␤-glycosidase (LacS), which is under active study in our laboratory, is still obscure (21)(22)(23).…”

“…In particular, the C terminus of XylS (roughly from positions 100 to 700), corresponding to a conserved domain in the ProDom data base (accession number PD001543), produced 35%, 30%, 29%, and 27% identity with the hypothetical protein of Erwinia herbicola (Erwglu), the ␣-glucosidase from S. solfataricus (MalA), the ␣-xylosidases from L. pentosus (XylQ), and Thermotoga maritima (Tmxyl), respectively. The Erwglu and Tmxyl enzymes have not been characterized biochemically, whereas MalA has been studied in detail and appears to be a typical ␣-glucosidase specific for maltose and malto-oligosaccharides (17). XylQ is an ␣-xylosidase with high specificity for isoprimeverose (␣-D-xylopyranosyl-1,6-D-glucopyranose); the xylQ gene is clustered with xylP, encoding an hypothetical membrane protein transporter, in an operon that is involved in the metabolism of this disaccharide (9).…”