No abstract

No abstract

The extremely thermophilic anaerobic archaeon strain B1001 was isolated from a hot-spring environment in Japan. The cells were irregular cocci, 0.5 to 1.0 μm in diameter. The new isolate grew at temperatures between 60 and 95°C (optimum, 85°C), from pH 5.0 to 9.0 (optimum, pH 7.0), and from 1.0 to 6.0% NaCl (optimum, 2.0%). The G+C content of the genomic DNA was 43.0 mol%. The 16S rRNA gene sequencing of strain B1001 indicated that it belongs to the genusThermococcus. During growth on starch, the strain produced a thermostable cyclomaltodextrin glucanotransferase (CGTase). The enzyme was purified 1,750-fold, and the molecular mass was determined to be 83 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Incubation at 120°C with SDS and 2-mercaptoethanol was required for complete unfolding. The optimum temperatures for starch-degrading activity and cyclodextrin synthesis activity were 110 and 90 to 100°C, respectively. The optimum pH for enzyme activity was pH 5.0 to 5.5. At pH 5.0, the half-life of the enzyme was 40 min at 110°C. The enzyme formed mainly α-cyclodextrin with small amounts of β- and γ-cyclodextrins from starch. This is the first report on the presence of the extremely thermostable CGTase from hyperthermophilic archaea.

No abstract

Chiral, racemic esters, ethyl (()-tetrahydrofuran-2-carboxylate 4c, methyl (()-r-phenylpropionate 9b, methyl (()-5,5-dimethyl-1,3-thiazolidine-4-carboxylate 12a, 2-methoxyethyl (()-1-(4tert-butylphenyl)-2-oxopyrrolidine-4-carboxylate 15a, (()-1benzyloxy-3-chloropropan-2-yl hydrogen succinate 18c, and (()-3-butyryloxyquinuclidinium butyrate [(()-20b‚n-PrCO 2 H], are resolved kinetically by enantioselective hydrolysis catalyzed by an Aspergillus melleus protease [E ) 60; 93.9% ee and 35% yield for (R)-tetrahydrofuran-2-carboxylic acid 4a], a Klebsiella oxytoca hydrolase [E > 200; 99.5% ee and 36% yield for (S)r-phenylpropionic acid 9a], a K. oxytoca hydrolase [E ) 145; 97.7% ee and 34% yield for (R)-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid 12b], a Bacillus breWis protease [E ) 77; 99% ee and 45% yield for (S)-15a], a Serratia marcescence esterase [E ) 49; 99% ee and 43% yield for (S)-18c], and an A. melleus protease [E ) 96; 96% ee and 42% yield for (R)-20b], respectively. Each enzymatic process is discussed with focus on the following tactical issues: (1) identification of a hydrolase with high enantioselectivity, (2) substrate concentrations not less than 1 M that allow for industrially viable volume efficiency (spacetime yield), (3) product separation by partition between organic and aqueous phases, and (4) alleviation of a hydrolysate inhibiting the enzymatic activity.