title: Functional characterization of a special thermophilic multifunctional amylase OPMA-N and its N-terminal domain.
abstract: A gene encoding a special thermophilic multifunctional amylase OPMA-N was cloned from Bacillus sp. ZW2531-1. OPMA-N has an additional 124-residue N-terminal domain compared with typical amylases and forms a relatively independent domain with a β-pleated sheet and random coil structure. Here we reported an unusual substrate and product specificities of OPMA-N and the impact of the additional N-terminal domain (1-124 aa) on the function and properties of OPMA-N. Both OPMA-N (12.82 U/mg) and its N-terminal domain-truncated ΔOPMA-N (12.55 U/mg) only degraded starch to produce oligosaccharides including maltose, maltotriose, isomaltotriose, and isomaltotetraose, but not to produce glucose. Therefore, the N-terminal domain did not determine its substrate and product specificities that were probably regulated by its C-terminal β-pleated sheet structure. However, the N-terminal domain of OPMA-N seemed to modulate its catalytic feature, leading to the production of more isomaltotriose and less maltose, and it seemed to contribute to OPMA-N's thermostability since OPMA-N showed higher activity than ΔOPMA-N in a temperature range from 40 to 80°C and the half-life (t(1/2)) was 5 h for OPMA-N and 2 h for ΔOPMA-N at 60°C. Both OPMA-N and ΔOPMA-N were Ca(2+)-independent, but their activities could be influenced by Cu(2+), Ni(2+), Zn(2+), EDTA, SDS (1 mM), or Triton-X100 (1%). Kinetic analysis and starch-adsorption assay indicated that the N-terminal domain of OPMA-N could increase the OPMA-N-starch binding and subsequently increase the catalytic efficiency of OPMA-N for starch. In particular, the N-terminal domain of OPMA-N did not determine its oligomerization, because both OPMA-N and ΔOPMA-N could exist in the forms of monomer, homodimer, and homooligomer at the same time.
authors.affiliation: Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, College of Life Science, Jilin University, Changchun, China.
authors.forename: Fan
authors.initials: F
authors.lastname: Li
authors.forename: Xuejun
authors.initials: X
authors.lastname: Zhu
authors.forename: Yanfei
authors.initials: Y
authors.lastname: Li
authors.forename: Hao
authors.initials: H
authors.lastname: Cao
authors.forename: Yingjiu
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000681:Amylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis / Q000187:drug effects; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D008670:Metals / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009844:Oligosaccharides / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013213:Starch / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014312:Trisaccharides / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008670:Metals; D009844:Oligosaccharides; D014312:Trisaccharides; C045441:isomaltotriose; C047512:isomaltotetraose; C008317:maltotriose; D008320:Maltose; D013213:Starch; D000681:Amylases