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

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 A degrees C and pH 5.0 and a half-life of 50 min at 100 A degrees 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, gamma-CD, and 6-O-maltooligosyl-beta-CD were [6)-alpha-d-Glcp-(1 -> aEuro parts per thousand 4)-alpha-d-Glcp-(1 -> aEuro parts per thousand 4)-alpha-d-Glcp-(1 ->](n), maltooctaose, and single maltooligosaccharide plus beta-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.