title: Truncation of the unique N-terminal domain improved the thermos-stability and specific activity of alkaline α-amylase Amy703.
abstract: High pH condition is of special interest for the potential applications of alkaline α-amylase in textile and detergent industries. Thus, there is a continuous demand to improve the amylase's properties to meet the requirements set by specific applications. Here we reported the systematic study of modular domain engineering to improve the specific activity and stability of the alkaline α-amylase from Bacillus pseudofirmus 703. The specific activity of the N-terminal domain truncated mutant (N-Amy) increased by ~35-fold with a significantly improved thermo-stability. Kinetic analysis demonstrated that the Kcat and Kcat/Kmof N-Amy were enhanced by 1300-fold and 425.7-fold, respectively, representing the largest catalytic activity improvement of the engineered α-amylases through the methods of domain deletion, fusion or swapping. In addition, different from the wild-type Amy703, no exogenous Ca(2+) were required for N-Amy to maintain its full catalytic activity, implying its superior potential for many industrial processes. Circular dichroism analysis and structure modeling revealed that the increased compactness and α-helical content were the main contributors for the improved thermo-stability of N-Amy, while the improved catalytic efficiency was mainly attributed by the increased conformational flexibility around the active center.
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, The College of Life Science, Hubei University, Wuhan, 430062, People's Republic of China.
authors.forename: Zhenghui
authors.initials: Z
authors.lastname: Lu
authors.affiliation: Tianjin Institutes of Industrial Biotechnology, Chinese Academy of Science, Tianjin, 300308, China.
authors.forename: Qinhong
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, The College of Life Science, Hubei University, Wuhan, 430062, People's Republic of China.
authors.forename: Sijing
authors.initials: S
authors.lastname: Jiang
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, The College of Life Science, Hubei University, Wuhan, 430062, People's Republic of China.
authors.forename: Guimin
authors.initials: G
authors.lastname: Zhang
authors.affiliation: Tianjin Institutes of Industrial Biotechnology, Chinese Academy of Science, Tianjin, 300308, China.
authors.forename: Yanhe
authors.initials: Y
authors.lastname: Ma
mesh_terms: D001407:Bacillus / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics*; D002942:Circular Dichroism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D015202:Protein Engineering; D017434:Protein Structure, Tertiary / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000516:alpha-Amylases