Abstract
β-Mannanase was expressed in Thermoanaerobacterium aotearoense SCUT27 induced by locust bean gum (LBG). The open reading frame encoding a GH26 β-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 β-mannanase activity screening was established based on agar plates. The optimum temperature for the purified Man25 using LBG as a substrate was 55 °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 °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.
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