초고온성 아케아 써모코커스 NA1의 녹말분해효소의 과발현 및 특성

Abstract

Genomic analysis of a hyperthermophilic archaeon Thermococcus NA1. revealed the presence of an open reading frame consisting of 1,377 bp similar to GH13 a-amylases from the genus Thermococcus and Pyrococcus. This amylolytic enzyme, designated TNA1_amyl (Thermococcus sp. NA1 amylase), was cloned and expressed in Escherichia coli. The full-length protein was detected in the culture supernatant, indicating that the signal peptide was processed in E. coli and thus the mature form of the protein was reconstructed based on N-terminal sequencing data of the secreted recombinant protein. The purified protein by His-tagged affinity chromatography was highly thermostable with an optimum temperature of 80°C, and an optimum pH of 5.5. More than 50% of the maximum activity was retained in the range between pH 5.0 and pH 9.5. The enzyme displayed thermostability with half-life (t1/2) of 10 min at 90°C, and the thermostability of TNA1_amyl was significantly enhanced in the presence of calcium with half-life (t1/2) of 153 min at 90°C. TNA1_amyl hydrolyzed maltooligosaccharides, amylopectin and starch, not pullulan to produce mainly G2 to G7.