Cloning, expression and biochemical characterisation of a unique thermostable pullulan-hydrolysing enzyme from the hyperthermophilic archaeon Thermococcus aggregans

FEMS Microbiol Lett. 2000 Sep 15;190(2):223-9. doi: 10.1111/j.1574-6968.2000.tb09290.x.

Abstract

The gene for a new type of pullulan hydrolase from the hyperthermophilic archaeon Thermococcus aggregans was cloned and expressed in Escherichia coli. The 2181-bp open reading frame encodes a protein of 727 amino acids. A hypothetical membrane linker region was found to be cleaved during processing in E. coli. The recombinant enzyme was purified 70-fold by heat treatment, affinity and anion exchange chromatography. Optimal activity was detected at 95 degrees C at a broad pH range from 3.5 to 8.5 with an optimum at pH 6.5. More than 35% of enzymatic activity was detected even at 120 degrees C. The enzyme was stable at 90 degrees C for several hours and exhibited a half-life of 2.5 h at 100 degrees C. Unlike all pullulan-hydrolysing enzymes described to date, the enzyme is able to attack alpha-1,6- as well as alpha-1,4-glycosidic linkages in pullulan leading to the formation of a mixture of maltotriose, panose, maltose and glucose. The enzyme is also able to degrade starch, amylose and amylopectin forming maltotriose and maltose as main products.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Composition
  • Base Sequence
  • Cloning, Molecular*
  • DNA, Archaeal / chemistry
  • DNA, Archaeal / genetics
  • Glycoside Hydrolases / chemistry
  • Glycoside Hydrolases / genetics*
  • Glycoside Hydrolases / isolation & purification
  • Glycoside Hydrolases / metabolism*
  • Hydrogen-Ion Concentration
  • Molecular Sequence Data
  • Open Reading Frames
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Analysis, DNA
  • Starch / metabolism
  • Temperature
  • Thermococcus / enzymology*
  • Thermococcus / genetics

Substances

  • DNA, Archaeal
  • Recombinant Proteins
  • Starch
  • Glycoside Hydrolases
  • pullulanase

Associated data

  • GENBANK/UNKNOWN