Journal
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
Volume 79, Issue 9, Pages 3049-3058Publisher
AMER SOC MICROBIOLOGY
DOI: 10.1128/AEM.03785-12
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Funding
- Priority Academic Program Development of Jiangsu Higher Education Institutions
- 111 Project [111-2-06]
- National High Technology Research and Development Program of China (863 Program) [2012AA022202, 2012CB720806]
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In this study, we constructed and expressed six fusion proteins composed of oligopeptides attached to the N terminus of the alkaline alpha-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 (k(cat)) 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: k(cat)/K-m 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 degrees C to 55 degrees C, and the half-life at 60 degrees C increased by similar to 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.
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