A GH57 Family Amylopullulanase from Deep-Sea Thermococcus siculi: Expression of the Gene and Characterization of the Recombinant Enzyme

Abstract

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.

Sequence fingerprints of enzyme specificities from the glycoside hydrolase family GH57

“…Sequences were collected based on basic protein BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) (Altschul et al 1990) searches using the complete sequences of 14 experimentally characterized GH57 enzymes: a-amylase from Methanocaldococcus jannaschii (Bult et al 1996;Kim et al 2001;Li and Peeples 2004), amylopullulanases from Pyrococcus furiosus (Dong et al 1997;Kang et al 2005), Thermococcus hydrothermalis (Erra-Pujada et al 1999), Thermococcus litoralis ) and Thermococcus siculi (Jiao et al 2011), branching enzymes from Thermococcus kodakaraensis (Murakami et al 2006;Santos et al 2011), Thermotoga maritima Dickmanns et al 2006) and Thermus thermophilus (Palomo et al 2011), 4-a-glucanotransferases from Archaeoglobus fulgidus (Labes and Schonheit 2007), Dictyoglomus thermophilum (Fukusumi et al 1988;Nakajima et al 2004), T. kodakaraensis (Tachibana et al 1997(Tachibana et al , 2000, T. litoralis (Jeon et al 1997) and P. furiosus (Laderman et al 1993a, b), and a-galactosidase from P. furiosus (van Lieshout et al 2003).…”

An extremely thermostable amylopullulanase from Staphylothermus marinus displays both pullulan- and cyclodextrin-degrading activities

“…The predicted amino acid sequence of SMApu showed only 17.1 % identity with that of PFApu (P. furiosus DSM 3638 amylopullulanase) (Dong et al 1997), and 14.8-10.4 % much lower identities with those of MJAmy (Methanocaldococcus jannaschii DSM 2661 α-amylase) (Kim et al 2001), TLApu (T. litoralis amylopullulanase) ), DT4αGT(Dictyoglomus thermophilum 4-α-glucanotransferase) (Nakajima et al 2004;Fukusumi et al 1988), TMAmyC (Termotoga maritima MSB8 α-amylase C) , THApu (T. hydrothermalis AL622 amylopullulanase) (Erra-Pujada et al 1999), and TSApu (T. siculi amylopullulanase) (Jiao et al 2011).…”

“…The archaeal amylopullulanases of the family GH57 from Thermococcus hydrothermalis (Erra-Pujada et al 1999), Thermococcus litoralis , Thermococcus siculi (Jiao et al 2011), and Pyrococcus furiosus (Rüdiger et al 1995;Dong et al 1997) and the archaeal pullulanases of the family GH13 from Thermococcus aggregans (Niehaus et al 2000) and Desulfurococcus mucosus (Duffner et al 2000) have been cloned and expressed in mesophilic Escherichia coli or Bacillus subtilis successfully. All these recombinant enzymes are thermostable with optimal temperature for the activity of between 85 and 105°C.…”

Effect of C-terminal truncation on enzyme properties of recombinant amylopullulanase from Thermoanaerobacter pseudoethanolicus

“…Apus isolated from a wide variety of microorganisms, especially from thermophiles, have great potential for industrial applications (Vieille and Zeikus 2001). Currently, six archael Apus such as Pyrococcus furiosus (Dong et al 1997), Thermococcus litoralis (Brown and Kelly 1993), P. woesei (Rudiger et al 1995), T. celer (Canganella et al 1994), T. hydrothermalis (Erra-Pujada et al 1999;Zona et al 2004), and T. siculi (Jiao et al 2011) were belonged to the glycoside hydrolase family 57 (GH57 Apus) (Janecek 2005). The remaining Apus, either mesophilic or thermophilic, including those from Thermoanaerobacter pseudoethanolicus (formerly, Thermoanaerobacter ethanolicus 39E, TetApu) , Thermoanaerobacter thermohydrosulfuricum (Melasniemi et al1990), Thermoanaerobacterium saccharolyticum B6ARI (Ramesh Matur et al 1994), Thermoanaerobacterium saccharolyticum NTOU1 (Lin et al 2011), and Thermoanaerobacterium thermosulfurigenes EM1 (Spreinat and Antranikian 1990) have motif structures, catalytic sites, and a general acid-base catalytic mechanism that are similar to those of the GHase family 13 (GH13 Apus).…”