Effect of C-terminal truncation on enzyme properties of recombinant amylopullulanase from Thermoanaerobacter pseudoethanolicus

• Published in: Extremophiles : life under extreme conditions Vol. 16; no. 3; pp. 395 - 403
• Main Authors: Lin, Fu-Pang, Ho, Yi-Hsuan, Lin, Hsu-Yang, Lin, Hui-Ju
• Format: Journal Article
• Language: English
• Published: Japan Springer Japan 01.05.2012; Springer; Springer Nature B.V
• Subjects: Amino Acid Sequence; Amino acids; Animal, plant and microbial ecology; Bacteria; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biochemistry; Biological and medical sciences; Biomedical and Life Sciences; Biotechnology; Catalysis; Enzymatic activity; Enzyme Stability - genetics; Enzymes; Fundamental and applied biological sciences. Psychology; Genetic recombination; Glycoside Hydrolases - chemistry; Glycoside Hydrolases - genetics; Glycoside Hydrolases - metabolism; Gram-Positive Endospore-Forming Rods - enzymology; Gram-Positive Endospore-Forming Rods - genetics; Hot Temperature; Life Sciences; Microbial Ecology; Microbiology; Mutagenesis; Original Paper; Protein Structure, Tertiary; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Deletion; Starch; Substrates; Thermoanaerobacter; Amylopullulanase; C-terminal truncation mutagenesis; Circular dichroism; Mutagenesis; Thermoanaerobacter pseudoethanolicus; Enzyme; Circular dichroism spectrometry; Bacteria; Extreme environment; Thermoanaerobacter
• ISSN: 1431-0651; 1433-4909
• DOI: 10.1007/s00792-012-0438-z

The smallest and enzymatically active molecule, TetApuQ818, was localized within the C-terminal Q818 amino acid residue after serial C-terminal truncation analysis of the recombinant amylopullulanase molecule (TetApuM955) from Thermoanaerobacter pseudoethanolicus . Kinetic analyses indicated that the overall catalytic efficiency, k cat / K m , of TetApuQ818 was 8–32% decreased for the pullulan and the soluble starch substrate, respectively. Changes to the substrate affinity, K m , and the turnover rate, k cat , were decreased significantly in both enzymatic activities of TetApuQ818. TetApuQ818 exhibited less thermostability than TetApuM955 when the temperature was raised above 85°C, but it had similar substrate-binding ability and hydrolysis products toward various substrates as TetApuM955 did. Both enzymes showed similar spectroscopies of fluorescence and circular dichroism, suggesting the active folding conformation was maintained after this C-terminal Q818 deletion. This study suggested that the binding ability of insoluble starch by TetApuM955 did not rely on the putative C-terminal carbohydrate binding module family 20 (CBM20) and two FnIII regions of TetApu, though the integrity of the AamyC module of TetApuQ818 was required for the enzyme activity.