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

• Published in: FEMS microbiology letters Vol. 190; no. 2; pp. 223 - 229
• Main Authors: Niehaus, Frank, Peters, Anke, Groudieva, Tatiana, Antranikian, Garabed
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 15.09.2000
• Subjects: amylopectin; amylose; Archaeon; Escherichia coli; Pullulan hydrolase; Starch; Thermococcales; Thermococcus aggregans; Archaeon; Thermococcales; Pullulan hydrolase; Starch
• ISSN: 0378-1097; 1574-6968
• DOI: 10.1016/S0378-1097(00)00339-6

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°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°C. The enzyme was stable at 90°C for several hours and exhibited a half-life of 2.5 h at 100°C. Unlike all pullulan-hydrolysing enzymes described to date, the enzyme is able to attack α-1,6- as well as α-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.