Enhancing thermostability of maltogenic amylase from Bacillus thermoalkalophilus ET2 by DNA shuffling

Abstract

DNA shuffling was used to improve the thermostability of maltogenic amylase from Bacillus thermoalkalophilus ET2. Two highly thermostable mutants, III-1 and III-2, were generated after three rounds of shuffling and recombination of mutations. Their optimal reaction temperatures were all 80°C, which was 10°C higher than that of the wild-type. The mutant enzyme III-1 carried seven mutations: N147D, F195L, N263S, D311G, A344V, F397S, and N508D. The half-life of III-1 was about 20 times greater than that of the wild-type at 78°C. The mutant enzyme III-2 carried M375T in addition to the mutations in III-1, which was responsible for the decrease in specific activity. The half-life of III-2 was 568 min while that of the wild-type was < 1 min at 80°C. The melting temperatures of III-1 and III-2, as determined by differential scanning calorimetry, increased by 6.1°C and 11.4°C, respectively. Hydrogen bonding, hydrophobic interaction, electrostatic interaction, proper packing, and deamidation were predicted as the mechanisms for the enhancement of thermostability in the enzymes with the mutations. © 2006 The Authors.