Tang Shuang-Yan, Le Quang-Tri, Shim Jae-Hoon, Yang Sung-Jae, Auh Joong-Huck, Park Cheonseok, Park Kwan-Hwa
Center for Agricultural Biomaterials, and Department of Food Science and Biotechnology, School of Agricultural Biotechnology, Seoul National University, South Korea.
FEBS J. 2006 Jul;273(14):3335-45. doi: 10.1111/j.1742-4658.2006.05337.x.
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 degrees C, which was 10 degrees 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 degrees 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 degrees C. The melting temperatures of III-1 and III-2, as determined by differential scanning calorimetry, increased by 6.1 degrees C and 11.4 degrees 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.
利用DNA改组技术提高嗜热嗜碱芽孢杆菌ET2麦芽ogenic淀粉酶的热稳定性。经过三轮改组和突变重组后，产生了两个高度耐热的突变体III-1和III-2。它们的最佳反应温度均为80℃，比野生型高10℃。突变酶III-1有七个突变：N147D、F195L、N263S、D311G、A344V、F397S和N508D。在78℃时，III-1的半衰期比野生型大约长20倍。突变酶III-2除了具有III-1中的突变外，还携带M375T，这导致了比活性的降低。在80℃时，III-2的半衰期为568分钟，而野生型的半衰期小于1分钟。通过差示扫描量热法测定，III-1和III-2的解链温度分别升高了6.1℃和11.4℃。氢键、疏水相互作用、静电相互作用、正确堆积和脱酰胺作用被预测为这些具有突变的酶热稳定性增强的机制。
