title: Novel maltotriose-hydrolyzing thermoacidophilic type III pullulan hydrolase from Thermococcus kodakarensis.
abstract: A novel thermoacidophilic pullulan-hydrolyzing enzyme (PUL) from hyperthermophilic archaeon Thermococcus kodakarensis (TK-PUL) that efficiently hydrolyzes starch under industrial conditions in the absence of any additional metal ions was cloned and characterized. TK-PUL possessed both pullulanase and α-amylase activities. The highest activities were observed at 95 to 100°C. Although the enzyme was active over a broad pH range (3.0 to 8.5), the pH optima for both activities were 3.5 in acetate buffer and 4.2 in citrate buffer. TK-PUL was stable for several hours at 90°C. Its half-life at 100°C was 45 min when incubated either at pH 6.5 or 8.5. The Km value toward pullulan was 2 mg ml(-1), with a Vmax of 109 U mg(-1). Metal ions were not required for the activity and stability of recombinant TK-PUL. The enzyme was able to hydrolyze both α-1,6 and α-1,4 glycosidic linkages in pullulan. The most preferred substrate, after pullulan, was γ-cyclodextrin, which is a novel feature for this type of enzyme. Additionally, the enzyme hydrolyzed a variety of polysaccharides, including starch, glycogen, dextrin, amylose, amylopectin, and cyclodextrins (α, β, and γ), mainly into maltose. A unique feature of TK-PUL was the ability to hydrolyze maltotriose into maltose and glucose.
authors.affiliation: School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore, Pakistan.
authors.forename: Nasir
authors.initials: N
authors.lastname: Ahmad
authors.forename: Naeem
authors.initials: N
authors.lastname: Rashid
authors.forename: Muhammad Saleem
authors.initials: MS
authors.lastname: Haider
authors.forename: Mehwish
authors.initials: M
authors.lastname: Akram
authors.forename: Muhammad
authors.initials: M
authors.lastname: Akhtar
mesh_terms: D003001:Cloning, Molecular; D004795:Enzyme Stability; D005936:Glucans / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D013379:Substrate Specificity; D013696:Temperature; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics; D014312:Trisaccharides / Q000378:metabolism*; D000516:alpha-Amylases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D047408:gamma-Cyclodextrins / Q000378:metabolism
chemical_list: D005936:Glucans; D014312:Trisaccharides; D047408:gamma-Cyclodextrins; C008317:maltotriose; C009109:pullulan; D006867:Hydrolases; D006026:Glycoside Hydrolases; D000516:alpha-Amylases; C100181:pullulanase