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

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 Ca 2+ 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.

Man/Cel5B, a Bifunctional Enzyme Having the Highest Mannanase Activity in the Hyperthermic Environment

“…These mannan and glucomannan polysaccharides may also be linked to α-1,6-linked galactose residues and the corresponding polysaccharides are called as galactomannan and galactoglucomannan, respectively. Generally, 1,4-β-D-Mannan mannanohydrolase or endo-1,4-β-mannosidase (EC 3.2.1.78) is called as βmannanase, which causes random cleavage of β-mannosidic linkages in mannan backbone (Zhu et al, 2020). Depending on sequence similarity, mannanases from various sources, such as bacteria, fungi, and plants, have been categorized within glycoside hydrolase families 5, 26, 113, and 134 within carbohydrate-active database (Ghosh et al, 2013).…”

β‐Mannanase production by Penicillium citrinum through solid‐state fermentation using açaí residual biomass (Euterpe oleracea)

“…In this article, mannanase remained active in the presence of Cu 2+ and Zn 2+ ; in contrast, ⊎-mannanase from Thermoanaerobacterium aotearoense SCUT27 was strongly suppressed by these cations. 14 The stability of the enzyme produced by P. citrinum isolated in this work in the presence of NaCl indicates that it could be applied to the bleaching of kraft pulp (in which Na + and Cl − concentrations can be high). 51 This result agrees with the mannanases reported by Liao et al 52 and Wang et al 53 produced by the genus Penicillium.…”

“…Mannanases are important biocatalysts for industrial processes; 10 for example, the random action of endo‐β‐mannanase against mannans generates mannooligosaccharides (MOS), which are used as prebiotics to promote healthy gut microbiota 11,12 . β‐Mannanase has received significant attention because of its widespread application in various industries, including food and animal feed production, the textile industry, bleaching of kraft pulp, detergents and pharmaceuticals 9,10,13,14 …”

“…11,12 ⊎-Mannanase has received significant attention because of its widespread application in various industries, including food and animal feed production, the textile industry, bleaching of kraft pulp, detergents and pharmaceuticals. 9,10,13,14 Solid-state fermentation (SSF) is a viable technique for producing industrial enzymes and provides a massive opportunity to convert low-cost agricultural waste into valuable products. 15,16 The SSF process is of particular economic interest for countries with an abundance of biomass and agro-industrial residues, which can be used as cheap raw materials.…”

Applications of Microbial β-Mannanases

“…Therefore, enzyme activity needs to be preserved while improving its thermostability. Zhu et al (2019) report that directed evolution based on iterative saturation mutagenesis applied to Man25 (a Thermoanaerobacterium aotearoense SCUT27 derived β-mannanase) at the five potential Ca 2+ binding sites lead to improvements in thermostability of the enzyme without decreasing its activity. Out of 7000 clones produced, the best mutant ManM3-3 (D143A) shows enhanced thermostability and satisfactory catalytic activity.…”