Biochemical characterization of engineered amylopullulanase from Thermoanaerobacter ethanolicus 39E-implicating the non-necessity of its 100 C-terminal amino acid residues.
The functional and structural significance of the C-terminal region of Thermoanaerobacter ethanolicus 39E amylopullulanase (TetApu) was explored using C-terminal truncation mutagenesis. Comparative studies between the engineered full-length (TetApuM955) and its truncated mutant (TetApuR855) included initial rate kinetics, fluorescence and CD spectrometric properties, substrate-binding and hydrolysis abilities, thermostability, and thermodenaturation kinetics. Kinetic analyses revealed that the overall catalytic efficiency, k (cat)/K (m), was slightly decreased for the truncated enzymes toward the soluble starch or pullulan substrate. Changes to the substrate affinity, K (m), and turnover rate, k (cat), varied in different directions for both types of substrates between TetApuM955 and TetApuR855. TetApuR855 exhibited a higher thermostability than TetApuM955, and retained similar substrate-binding ability and hydrolyzing efficiency against the raw starch substrate as TetApuM955 did. Fluorescence spectroscopy indicated that TetApuR855 retained an active folding conformation similar to TetApuM955. A CD-melting unfolding study was able to distinguish between TetApuM955 and TetApuR855 by the higher apparent transition temperature in TetApuR855. These results indicate that up to 100 amino acid residues, including most of the C-terminal fibronectin typeIII (FnIII) motif of TetApuM955, could be further removed without causing a seriously aberrant change in structure and a dramatic decrease in soluble starch and pullulan hydrolysis.
Lin
Hsu-Yang
HY
Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan.
Chuang
Hsu-Han
HH
Lin
Fu-Pang
FP
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D017931:DNA Primers; D004795:Enzyme Stability; D005818:Genetic Engineering; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013053:Spectrophotometry; D013213:Starch / Q000378:metabolism; D046968:Thermoanaerobacter / Q000201:enzymology*; D013816:Thermodynamics
D001426:Bacterial Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D013213:Starch; D006026:Glycoside Hydrolases; C082507:amylopullulanase