title: 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.
authors.affiliation: Key Laboratory of Agricultural Products Processing in Jilin Provincial Universities, Changchun University, Satellite Road 6543, Changchun 130022, People's Republic of China.
authors.forename: Xiaolei
authors.initials: X
authors.lastname: Li
authors.forename: Dan
authors.initials: D
authors.lastname: Li
authors.forename: Kwan-Hwa
authors.initials: KH
authors.lastname: Park
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002122:Calcium Chloride / Q000378:metabolism; D003001:Cloning, Molecular; D003505:Cyclodextrins / Q000378:metabolism*; D019620:Desulfurococcaceae / Q000201:enzymology* / Q000235:genetics; D020536:Enzyme Activators / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005936:Glucans / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D003505:Cyclodextrins; D020536:Enzyme Activators; D005936:Glucans; D011994:Recombinant Proteins; C009109:pullulan; D006026:Glycoside Hydrolases; C082507:amylopullulanase; D002122:Calcium Chloride