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

Abstract

A gene encoding an amylopullulanase of the glycosyl hydrolase (GH) family 57 from Staphylothermus marinus (SMApu) was heterologously expressed in Escherichia coli. SMApu consisted of 639 amino acids with a molecular mass of 75.3 kDa. It only showed maximal amino acid identity of 17.1 % with that of Pyrococcus furiosus amylopullulanase in all identified amylases. Not like previously reported amylopullulanases, SMApu has no signal peptide but contains a continuous GH57N_Apu domain. It had the highest catalytic efficiency toward pullulan (k cat/K m , 342.34 s(-1) mL mg(-1)) and was extremely thermostable with maximal pullulan-degrading activity (42.1 U/mg) at 105 °C and pH 5.0 and a half-life of 50 min at 100 °C. Its activity increased to 116 % in the presence of 5 mM CaCl2. SMApu could also degrade cyclodextrins, which are resistant to the other amylopullulanases. The initial hydrolytic products from pullulan, γ-CD, and 6-O-maltooligosyl-β-CD were [6)-α-D-Glcp-(1 → 4)-α-D-Glcp-(1 → 4)-α-D-Glcp-(1→]n, maltooctaose, and single maltooligosaccharide plus β-CD, respectively. The final hydrolytic products from above-mentioned substrates were maltose and glucose. These results confirm that SMApu is a novel amylopullulanase of the family GH57 possessing the cyclodextrin-degrading activity of cyclomaltodextrinase.

Two potentially novel amylolytic enzyme specificities in the prokaryotic glycoside hydrolase α-amylase family GH57

“…It is worth mentioning that the tree shown in Fig. 4b represents currently the most complete evolutionary picture of the GH57 family, since the recently described dualspecificity amylopullulanase-cyclomaltodextrinase from S. marinus (Li et al, 2013), closely related to typical GH57 amylopullulanases, was included in the comparison.…”

“…a-Helices and b-strands (predicted by the Phyre-2 server; Kelley & Sternberg, 2009) are coloured in red and green, respectively. Abbreviations: AMY_Uncba, non-specified amylase from uncultured bacterium ; MGA_Pycfu, maltogenic amylase from P. furiosus (Comfort et al, 2008); AAMY_Mccja, a-amylase from M. jannaschii (Kim et al, 2001); PAMY_Bctth, putative aamylase-like protein from B. thetaiotaomicron (Janeč ek & Blesá k, 2011); APU_Pycfu, APU_Thchy, APU_Thcli and APU_Thcsi, amylopullulanases from P. furiosus (Dong et al, 1997), Thermococcus hydrothermalis (Erra-Pujada et al, 1999), Thermococcus litoralis and Thermococcus siculi (Jiao et al, 2011), respectively; APCD_Sttma, amylopullulanase-cyclomaltodextrinase from S. marinus (Li et al, 2013); BE_Thcko, BE_Thtma and BE_Theth, branching enzymes from Thermococcus kodakaraensis (Murakami et al, 2006), Thermotoga maritima and Thermus thermophilus (Palomo et al, 2011), respectively;4AGT_Arcfu, 4AGT_Dicth, 4AGT_Pycfu, 4AGT_Thcko, 4AGT_Thcli, 4-a-glucanotransferases from Archaeoglobus fulgidus (Labes & Schö nheit, 2007), D. thermophilum (Fukusumi et al, 1988), P. furiosus (Laderman et al, 1993a), Thermococcus kodakaraensis (Tachibana et al, 1997) and Thermococcus litoralis (Jeon et al, 1997), respectively; AGAL_Pycfu, a-galactosidase from P. furiosus (van Lieshout et al, 2003). CSRs are emphasized by rectangles and catalytic residues (CSR-3 glutamate, catalytic nucleophile; CSR-4 aspartate, proton donor) are indicated by asterisks.…”

“…Although it was described as an a-amylase , its sequence-structural features clearly suggest branching enzyme activity (Blesák & Janeček, 2012). Another example is a recently described amylopullulanase from Staphylothermus marinus that also exhibits cyclodextrinase activity (Li et al, 2013). It is worth mentioning that, of the current more than 900 members of family GH57, fewer than 20 proteins have thus far been biochemically characterized (Cantarel et al, 2009).…”

Properties and Applications of Extremozymes from Deep-Sea Extremophilic Microorganisms: A Mini Review

“…Functional screening of fosmid expression libraries derived from deep-sea hydrothermal vents identified an extreme cellulase that was active and thermostable at 92 • C. This enzyme showed endolytic activities against a variety of linear 1,4-β-glucans, such as phosphoric acid swollen cellulose, carboxymethyl cellulose, lichenan and β-glucan. Other industrial important thermostable polymer-degrading enzymes have also been identified and characterised from deep-sea environments, such as amylopullulanases, arylsulphatases and xylanases [53][54][55].…”

Marine Extremophiles: A Source of Hydrolases for Biotechnological Applications

“…The hyperthermophilic archaea Staphylothermus marinus , an isolate from the deep-sea associated with geothermal activity or hydrothermal vents, presents an optimum growth at 98 °C. A new thermostable amylopullulanase of the glycosyl hydrolase family from S. marinus , has recently been described with degradation activity towards pullulan and cyclodextrin at 105 °C [ 168 ]. One of the most thermostable and thermo-active pullulanases type II was described for Pyrococcus furiosus and Thermococcus litoralis , with activity ranging from 130 °C to 140 °C in the presence of 5 mM Ca ++ [ 169 ].…”