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The gene encoding a new extracellular amylopullulanase (type II pullulanase) was cloned from an extremely thermophilic anaerobic archaeon Thermococcus siculi strain HJ21 isolated previously from a deep-sea hydrothermal vent. The functional hydrolytic domain of the amylopullulanase (TsiApuN) and its MalE fusion protein (MTsiApuN) were expressed heterologously. The complete amylopullulanase (TsiApu) was also purified from fermentation broth of the strain. The pullulanase and amylase activities of the three enzymes were characterized. TsiApu had optimum temperature of 95°C for the both activities, while MTsiApuN and TsiApuN had a higher optimum temperature of 100°C. The residual total activities of MTsiApuN and TsiApuN were both 89% after incubation at 100°C for 1 h, while that of TsiApu was 70%. For all the three enzymes the optimum pHs for amylase and pullulanase activities were 5.0 and 6.0, respectively. By analyzing enzymatic properties of the three enzymes, this study suggests that the carboxy terminal region of TsiApu might interfere with the thermoactivity. The acidic thermoactive amylopullulanases MTsiApuN and TsiApuN could be further employed for industrial saccharification of starch.

A Gram-negative, yellowish bacterial strain, designated Gsoil 1519 T , was isolated from soil of a ginseng field in Pocheon province (South Korea) and characterized using a polyphasic approach to determine its taxonomic position. Comparative 16S rRNA gene sequence analysis showed that strain Gsoil 1519 T belongs to the family 'Flexibacteraceae' and is related to Spirosoma rigui KCTC 12531 T (91.8 % similarity) and Spirosoma linguale LMG 10896 T (91.5 % similarity).Phylogenetic distances from any other recognized species within the family 'Flexibacteraceae' were greater than 14.7 %. The G+C content of the genomic DNA of strain Gsoil 1519 T was 50.1 %. The detection of MK-7 as the predominant menaquinone and a fatty acid profile with C 16 : 1 v5c, summed feature 4 (C 16 : 1 v7c and/or iso-C 15 : 0 2-OH), iso-C 15 : 0 and C 16 : 0 as the major acids supported the affiliation of strain Gsoil 1519 T to the genus Spirosoma. On the basis of its phenotypic properties and phylogenetic distinctiveness, strain Gsoil 1519 T should be classified in the genus Spirosoma as a representative of a novel species, for which the name Spirosoma panaciterrae sp. nov. is proposed. The type strain is Gsoil 1519 T (5KCTC 22263 T 5DSM 21099 T ).

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