Mutagenesis for improvement of activity and thermostability of amylomaltase from Corynebacterium glutamicum
Mutagenesis for improvement of activity and thermostability of amylomaltase from Corynebacterium glutamicum
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DOI：
10.1016/j.ijbiomac.2016.02.022
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发表时间：
2016-05-01
影响因子：
8.2
通讯作者：
Pongsawasdi, Piamsook
中科院分区：
文献类型：
--
作者：
Nimpiboon, Pitchanan;Kaulpiboon, Jarunee;Pongsawasdi, Piamsook
本工作旨在通过随机和定点突变提高嗜温谷氨酸棒杆菌淀粉麦芽糖酶的热稳定性。从易错PCR中，选择与野生型相比在50 ° C具有更高热稳定性的突变CgAM并测序。结果表明，该突变体含有一个A406 V突变。然后进行定点诱变以构建A406 V和A406 L。两种突变的CgAMs均表现出更高的分子间转葡萄糖基化活性，最适温度向上移动，最适pH略有增加，以进行缩合和环化反应。当与野生型相比时，两种突变的CgAM在35-40 ° C下的热稳定性显著增加，其中来自DSC光谱的峰值温度更高。A406 V对活性和热稳定性的影响大于A406 L。A406 V-和A406 L-CgAM的催化效率值k（cat）/K-m分别是野生型的2.9和1.4倍，主要是由于k（cat）的显著增加。LR-CD产物分析表明，与野生型酶相比，A406 V具有更高的产物产率，特别是在更长的孵育时间和更高的温度下。(C)2016爱思唯尔B. V.保留所有权利。
This work aims to improve thermostability of amylomaltase from a mesophilic Corynebacterium glutamicum (CgAM) by random and site-directed mutagenesis. From error prone PCR, a mutated CgAM with higher thermostability at 50 degrees C compared to the wild-type was selected and sequenced. The result showed that the mutant contains a single mutation of A406V. Site-directed mutagenesis was then performed to construct A406V and A406L. Both mutated CgAMs showed higher intermolecular transglucosylation activity with an upward shift in the optimum temperature and a slight increase in the optimum pH for disproportionation and cyclization reactions. Thermostability of both mutated CgAMs at 35-40 degrees C was significantly increased with a higher peak temperature from DSC spectra when compared to the wild type. A406V had a greater effect on activity and thermostability than A406L. The catalytic efficiency values k(cat)/K-m of A406V- and A406L-CgAMs were 2.9 and 1.4 times higher than that of the wild-type, respectively, mainly due to a significant increase in k(cat). LR-CD product analysis demonstrated that A406V gave higher product yield, especially at longer incubation time and higher temperature, in comparison to the wild-type enzyme. (C) 2016 Elsevier B.V. All rights reserved.