Molecular Characterization of the α-Glucosidase Gene (malA) from the Hyperthermophilic ArchaeonSulfolobus solfataricus

Abstract

Acidic hot springs are colonized by a diversity of hyperthermophilic organisms requiring extremes of temperature and pH for growth. To clarify how carbohydrates are consumed in such locations, the structural gene (malA) encoding the major soluble α-glucosidase (maltase) and flanking sequences fromSulfolobus solfataricus were cloned and characterized. This is the first report of an α-glucosidase gene from the archaeal domain. malA is 2,083 bp and encodes a protein of 693 amino acids with a calculated mass of 80.5 kDa. It is flanked on the 5′ side by an unusual 1-kb intergenic region. Northern blot analysis of themalA region identified transcripts for malA and an upstream open reading frame located 5′ to the 1-kb intergenic region. The malA transcription start site was located by primer extension analysis to a guanine residue 8 bp 5′ of themalA start codon. Gel mobility shift analysis of themalA promoter region suggests that sequences 3′ to position −33, including a consensus archaeal TATA box, play an essential role in malA expression. malA homologs were detected by Southern blot analysis in other S. solfataricus strains and in Sulfolobus shibatae, while no homologs were evident in Sulfolobus acidocaldarius, lending further support to the proposed revision of the genus Sulfolobus. Phylogenetic analyses indicate that the closest S. solfataricusα-glucosidase homologs are of mammalian origin. Characterization of the recombinant enzyme purified from Escherichia colirevealed differences from the natural enzyme in thermostability and electrophoretic behavior. Glycogen is a substrate for the recombinant enzyme. Unlike maltose hydrolysis, glycogen hydrolysis is optimal at the intracellular pH of the organism. These results indicate a unique role for the S. solfataricus α-glucosidase in carbohydrate metabolism.

Sugar transport in Sulfolobus solfataricus is mediated by two families of binding protein‐dependent ABC transporters

“…7C). The malA gene (contig 025 ORF 031 in the S. solfataricus P2 database) and adjacent regions have been characterized by Rolfsmeier et al (1998). Interestingly, a 2.4 kb transcript upstream of malA, termed ORF1, was found to be transcribed upon growth on maltose.…”

Effective production of a thermostable α-glucosidase fromSulfolobus solfataricus inEscherichia coli exploiting a microfiltration bioreactor

“…This en-zyme was purified from the thermoacidophilic archeon Sulfolobus solfataricus and it is the only one that has been cloned together with the ␣-glucosidase purified from S. solfataricus 98/2 (Sunna et al, 1997;Rolfmaier and Blum, 1995). Enzymes that hydrolyze glycosidic bonds play a very important role in the biodegradation of natural polysaccharides (Di Lernia et al, 1998;Rolfmaier et al, 1998), and the extreme thermostability of SsGA was especially interesting for biotechnological applications in the glucose syrup production industry (Godfrey and West, 1996). In fact, this is a saccharifying enzyme and its optimal reaction conditions are very similar to operational parameters typically used in proximate steps of the starch process industry.…”

Purification and Characterization of a Thermostable Starch‐Saccharifying Alpha‐1,4‐Glucan‐Glucohydrolase Produced by Bacillus licheniformis

“…The N‐terminal protein sequences obtained from B. licheniformis KIBGE‐IB3 and B. licheniformis KIBGE‐IB3M67 were NH 2 ‐Ser‐Ser‐Asn‐Lys‐Leu‐Thr‐Thr‐Ser‐Trp‐Gly and NH 2 ‐Met‐Asp‐Asn‐Lys‐Leu‐Thr‐Thr‐Ser‐Trp‐Gly, respectively. N‐Terminal sequences of both enzymes showed no strong homology with previously characterized glucoamylases ( Table ) . It was also observed that the first two amino acid residues from the N‐Terminal end of KIBGE‐IB3 were different from that of the KIBGE‐IB3M67.…”