A Recombinant β-Mannanase from Thermoanaerobacterium aotearoense SCUT27: Biochemical Characterization and Its Thermostability Improvement

作者：Zhu, Muzi; Zhang, Ling; Yang, Fang; Cha, Yaping; Li, Shuang^*; Zhuo, Min; Huang, Shaobin; Li, Jianjun

来源：Journal of Agricultural and Food Chemistry, 2020, 68(3): 818-825.

DOI：10.1021/acs.jafc.9b06246

• beta-mannanase
• Thermoanaerobacterium aotearoense
• plate assay
• thermostability improvement

beta-Mannanase was expressed in Thermoanaerobacterium aotearoense SCUT27 induced by locust bean gum (LBG). The open reading frame encoding a GH26 beta-mannanase was identified and encoded a preprotein of 515 amino acids with a putative signal peptide. The enzyme without a signal sequence (Man25) was overexpressed in Escherichia coli with a specific activity of 1286.2 U/mg. Moreover, a facile method for beta-mannanase activity screening was established based on agar plates. The optimum temperature for the purified Man25 using LBG as a substrate was 55 degrees C. The catalytic activity and thermostability of Man25 displayed a strong dependence on calcium ions. Through saturation mutagenesis at the putative Ca2+ binding sites in Man25, the best mutant ManM3-3 (D143A) presented improvements in thermostability with 3.6-fold extended half-life at 55 degrees C compared with that of the wild-type. The results suggest that mutagenesis at metal binding sites could be an efficient approach to increase enzyme thermostability.

• 单位
  华南农业大学; 广东省微生物研究所