Novel Maltotriose-Hydrolyzing Thermoacidophilic Type III Pullulan Hydrolase from Thermococcus kodakarensis

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 alpha-amylase activities. The highest activities were observed at 95 to 100 degrees 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 degrees C. Its half-life at 100 degrees C was 45 min when incubated either at pH 6.5 or 8.5. The K-m value toward pullulan was 2 mg ml(-1), with a V-max 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 alpha-1,6 and alpha-1,4 glycosidic linkages in pullulan. The most preferred substrate, after pullulan, was gamma-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 (alpha, beta, and gamma), mainly into maltose. A unique feature of TK-PUL was the ability to hydrolyze maltotriose into maltose and glucose.