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

The gene for a new type of pullulan hydrolase from the hyperthermophilic ar chaeon Thermococcus aggregans was cloned and expressed in Escherichia coli. The 2181-bp open reading frame encodes a protein of 727 amino acids. A hyp othetical 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 a t 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 hal f-life of 3.5 h at 100 degrees C. Unlike all pullulan-hydrolysing enzymes d escribed 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 o f maltotriose, panose, maltose and glucose. The enzyme is also able to degr ade starch, amylose and amylopectin forming maltotriose and maltose as main products. (C) 2000 Federation of European Microbiological Societies. Publi shed by Elsevier Science B.V. All rights reserved.