A native-feather-degrading thermophilic anaerobe was isolated from a geothermal hot stream in Indonesia. Isolate AW-1, identified as a member of the species Fervidobacterium islandicum, was shown to degrade native feathers (0.8%, w/v) completely at 70 degrees C and pH 7 with a maximum specific growth rate (0.14 h(-1)) in Thermotoga- Fervidobacterium(TF) medium. After 24 h of culture, feather degradation led to an increase in free amino acids such as histidine, cysteine and lysine. Moreover, nutritionally essential amino acids such as tryptophan and methionine, which are rare in feather keratin, were also produced as microbial metabolites. A homomultimeric membrane-bound keratinolytic protease (>200 kDa; 97 kDa subunits) was purified from a cell extract of F. islandicum AW-1. The enzyme exhibited activity toward casein and soluble keratin optimally at 100 degrees C and pH 9, and had a half-life of 90 min at 100 degrees C. The enzyme showed higher specific activity for the keratinous substrates than other proteases and catalyzed the cleavage of peptide bonds more rapidly following the reduction of disulfide bridges in feather keratin by 10 mM dithiothreitol. Therefore, the enzyme from F. islandicum AW-1 is a novel, thermostable keratinolytic serine protease.

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Gene araA encoding an l‐arabinose isomerase (AraA) from the hyperthermophile, Thermotoga neapolitana 5068 was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a polypeptide of 496 residues with a calculated molecular mass of 56 677 Da. The deduced amino acid sequence has 94.8% identical amino acids compared with the residues in a putative l‐arabinose isomerase of Thermotoga maritima. The recombinant enzyme expressed in E. coli was purified to homogeneity by heat treatment, ion exchange chromatography and gel filtration. The thermophilic enzyme had a maximum activity of l‐arabinose isomerization and d‐galactose isomerization at 85°C, and required divalent cations such as Co2+ and Mn2+ for its activity and thermostability. The apparent Km values of the enzyme for l‐arabinose and d‐galactose were 116 mM (vmax, 119 μmol min−1 mg−1) and 250 mM (vmax, 14.3 μmol min−1 mg−1), respectively, that were determined in the presence of both 1 mM Co2+ and 1 mM Mn2+. A 68% conversion of d‐galactose to d‐tagatose was obtained using the recombinant enzyme at the isomerization temperature of 80°C.

A gene, tayI, encoding a novel subtilisin-like protease, designated thermicin, from the extremely thermophilic bacterium Thermoanaerobacter yonseiensis KB-1 (DSM 13777) was cloned by using a sequence tag containing the consensus sequence of proteases. The gene consisted of 1,239 nucleotides, and the deduced amino acid sequence indicated that it is a preproenzyme with a 311-residue mature protein composed of canonical catalytic residues (Asp29, His64, and Ser252). Thermicin was overproduced in E. coli as a fusion protein with a histidine tag and purified by nickel nitrilotriacetic acid affinity chromatography. Thermicin from E. coli showed maximum proteolytic activity at 92.5 degrees C and pH 9.0, and its half-life was 30 h at 80 degrees C. In order to determine cleavage specificity, thermicin was incubated with insulin beta chain, and the resulting peptides were analyzed by matrix assisted laser desorption/ionization-time of flight mass spectrometry. The carboxyl group side of the Val12, Leu15,17, Gly23, and Pro28 residues was cleaved. Thermicin is well known to hydrolyze Gly- and Pro-rich collagens. The K (m) and k (cat)/ K (m) values of thermicin for the hydrolysis of the synthetic substrate L-Gly-Pro- p-nitroaniline were 54.16 microM and 142.05 (10(5) s(-1) M(-1)), respectively, at 92.5 degrees C and pH 9.0. Amino acid sequence comparison and phylogenetic analysis indicated that this enzyme belongs to a new subgroup with respect to its molecular evolution, when compared with previously characterized subtilisins. This result indicates that thermicin is a novel enzyme different from other thermostable proteases.