A novel hyperthermophilic strictly chemolithoautotrophic member of the genus Methanococcus was isolated from a shallow (depth: 106 m) submarine vent system at the Kolbeinsey ridge, Iceland. The ...isolate grew between 45 and 91 degrees C with an optimum around 88 degrees C (doubling time: 25 min). It differs from Methanococcus jannaschii in its 16S rRNA sequence, its non-hybridizing DNA, and its selenium-independent growth. Therefore, the isolate represents a new species which we name Methanococcus igneus. Type strain is isolate "Kol 5" (DSM 5666).
A gene library of the thermophilic eubacterium, Rhodothermus marinus, strain 21, was prepared in pUC18 and used to transform Escherichia coli. Of 5400 transformants, two produced halos on lichenan ...plates after Congo‐red staining. Restriction mapping showed that the two clones shared an overlapping 1200‐bp DNA fragment, which was used for DNA sequencing. Five potential methionine (Met) translational‐initiation codons were identified. A putative signal peptide of 30 amino acids was identified with a hydrophobic core of nine hydrophobic amino acids. The molecular mass of the mature enzyme was estimated to be 29.7 kDa. A comparison of the primary protein sequence of β‐glucanase of Rhodothermus marinus with other glycosyl hydrolases showed 38.5% identity to the C‐terminal part of the β‐1,3‐glucanase of Bacillus circulars and limited identity to bacterial endo‐β‐1,3–1,4‐glucanases. The amino acid sequence showed high similarity to regions surrounding the catalytic Glu residue of bacterial β‐glucanases. A gene fragment of 889 bp containing the catalytic domain was overexpressed in E. coli using the pET23, T7‐phage RNA polymerase system. The enzyme showed activity on lichenan, β‐glucan and laminarin but not on CMC cellulose or xylan. The expressed enzyme was purified by heat treatment of the host. The enzyme had a temperature and pH optima of 85°C and pH 7.0, respectively, and was shown to retain full activity after incubation for 16 h at 80°C and have a half life of 3 h at 85°C.
