Novel maltotriose-hydrolyzing thermoacidophilic type III pullulan hydrolase from Thermococcus kodakarensis

Appl Environ Microbiol. 2014 Feb;80(3):1108-15. doi: 10.1128/AEM.03139-13. Epub 2013 Dec 2.

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.

MeSH terms

  • Cloning, Molecular
  • Enzyme Stability
  • Glucans / metabolism*
  • Glycoside Hydrolases / genetics
  • Glycoside Hydrolases / isolation & purification
  • Glycoside Hydrolases / metabolism
  • Hydrogen-Ion Concentration
  • Hydrolases / genetics
  • Hydrolases / isolation & purification*
  • Hydrolases / metabolism*
  • Hydrolysis
  • Kinetics
  • Substrate Specificity
  • Temperature
  • Thermococcus / enzymology*
  • Thermococcus / genetics
  • Trisaccharides / metabolism*
  • alpha-Amylases / genetics
  • alpha-Amylases / isolation & purification
  • alpha-Amylases / metabolism
  • gamma-Cyclodextrins / metabolism

Substances

  • Glucans
  • Trisaccharides
  • gamma-Cyclodextrins
  • maltotriose
  • pullulan
  • Hydrolases
  • Glycoside Hydrolases
  • alpha-Amylases
  • pullulanase