Mutagenesis for improvement of activity and thermostability of amylomaltase from Corynebacterium glutamicum

作者：Nimpiboon Pitchanan; Kaulpiboon Jarunee; Krusong Kuakarun; Nakamura Shigeyoshi; Kidokoro Shun ichi; Pongsawasdi Piamsook^*

来源：International Journal of Biological Macromolecules, 2016, 86: 820-828.

DOI：10.1016/j.ijbiomac.2016.02.022

• Amylomaltase
• Random mutagenesis
• Site-directed mutagenesis
• Thermostability
• Large-ring cyclodextrin
• Corynebacterium glutamicum

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.

• 出版日期2016-5