Laboratoire de Microorganismes et de Biomolécules, Centre de Biotechnologie de Sfax, BP 1177, Sfax 3018, Tunisia.
Bioresour Technol. 2011 Jan;102(2):1740-6. doi: 10.1016/j.biortech.2010.08.082. Epub 2010 Aug 26.
Based on sequence alignments and homology modeling, Gly 312 and Lys 436 of the maltogenic amylase from Bacillus sp. US149 (MAUS149) were selected as targets for site-directed mutagenesis to improve the thermostability of the enzyme. Variants of MAUS149 with amino acid substitutions G312A, K436R and G312A-K436R had substrate specificities, kinetic parameters and pH optima similar to those of the wild-type enzyme; however, the enzymes with substitutions K436R and G312A-K436R, had an optimal temperature of 45 °C instead of the 40 °C for the wild-type enzyme. The half-life time at 55 °C increased from 15 to 25 min for the double mutant. Molecular modeling suggests that the increase in thermostability was due to new hydrophobic interactions and the formation of a salt bridge and hydrogen bond in the G312A and K436R variants, respectively. The double mutant could be a potential candidate for application in the bread industry.
基于序列比对和同源建模，选择芽孢杆菌麦芽淀粉酶（MAUS149）中的 Gly312 和 Lys436 作为定点突变的靶标，以提高酶的热稳定性。具有氨基酸取代 G312A、K436R 和 G312A-K436R 的 MAUS149 变体的底物特异性、动力学参数和 pH 最佳值与野生型酶相似；然而，具有取代 K436R 和 G312A-K436R 的酶的最适温度为 45°C，而不是野生型酶的 40°C。双突变体在 55°C 下的半衰期从 15 分钟增加到 25 分钟。分子建模表明，热稳定性的提高是由于新的疏水相互作用以及 G312A 和 K436R 变体中盐桥和氢键的形成所致。该双突变体可能是在面包工业中应用的潜在候选者。
