title: Fusion of an oligopeptide to the N terminus of an alkaline α-amylase from Alkalimonas amylolytica simultaneously improves the enzyme's catalytic efficiency, thermal stability, and resistance to oxidation.
abstract: In this study, we constructed and expressed six fusion proteins composed of oligopeptides attached to the N terminus of the alkaline α-amylase (AmyK) from Alkalimonas amylolytica. The oligopeptides had various effects on the functional and structural characteristics of AmyK. AmyK-p1, the fusion protein containing peptide 1 (AEAEAKAKAEAEAKAK), exhibited improved specific activity, catalytic efficiency, alkaline stability, thermal stability, and oxidative stability compared with AmyK. Compared with AmyK, the specific activity and catalytic constant (kcat) of AmyK-p1 were increased by 4.1-fold and 3.5-fold, respectively. The following properties were also improved in AmyK-p1 compared with AmyK: kcat/Km increased from 1.8 liter/(g·min) to 9.7 liter/(g·min), stable pH range was extended from 7.0 to 11.0 to 7.0 to 12.0, optimal temperature increased from 50°C to 55°C, and the half-life at 60°C increased by ∼2-fold. Moreover, AmyK-p1 showed improved resistance to oxidation and retained 54% of its activity after incubation with H2O2, compared with 20% activity retained by AmyK. Finally, AmyK-p1 was more compatible than AmyK with the commercial solid detergents tested. The mechanisms responsible for these changes were analyzed by comparing the three-dimensional (3-D) structural models of AmyK and AmyK-p1. The significantly enhanced catalytic efficiency and stability of AmyK-p1 suggests its potential as a detergent ingredient. In addition, the oligopeptide fusion strategy described here may be useful for improving the catalytic efficiency and stability of other industrial enzymes.
authors: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China.
authors: Haiquan
authors: H
authors: Yang
authors: Xinyao
authors: X
authors: Lu
authors: Long
authors: L
authors: Liu
authors: Jianghua
authors: J
authors: Li
authors: Hyun-dong
authors: HD
authors: Shin
authors: Rachel R
authors: RR
authors: Chen
authors: Guocheng
authors: G
authors: Du
authors: Jian
authors: J
authors: Chen
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D055162:Biocatalysis; D003902:Detergents; D004795:Enzyme Stability; D020563:Gammaproteobacteria / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D006207:Half-Life; D006358:Hot Temperature; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008961:Models, Structural; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins; D000516:alpha-Amylases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D003902:Detergents; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006861:Hydrogen Peroxide; D000516:alpha-Amylases