Enhancing thermostability of maltogenic amylase from <i>Bacillus thermoalkalophilus</i> ET2 by DNA shuffling

다국어 초록 (Multilingual Abstract)

<P>DNA shuffling was used to improve the thermostability of maltogenic amylase from <I>Bacillus thermoalkalophilus</I> 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.</P>