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Thermostable amylopullulanase (TAPU) is valuable in starch saccharification industry for its capability to catalyze both α-1,4 and α-1,6 glucosidic bonds under the industrial starch liquefication condition. The majority of TAPUs belong to glycoside hydrolase family 57 (GH57). In this study, we performed a phylogenetic analysis of GH57 amylopullulanase (APU) based on the highly conserved DOMON_glucodextranase_like (DDL) domain and classified APUs according to their multidomain architectures, phylogenetic analysis and enzymatic characters. This study revealed that amylopullulanase, pullulanase, andα-amylase had passed through a long joint evolution process, in which DDL played an important role. The phylogenetic analysis of DDL domain showed that the GH57 APU is directly sharing a common ancestor with pullulanase, and the DDL domains in some species undergo evolution scenarios such as domain duplication and recombination., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.