Improvement of the Catalytic Ability of a Thermostable and Acidophilic β-Mannanase Using a Consensus Sequence Design Strategy

Abstract

In order to improve the catalytic efficiency of a thermostable and acidophilic β-mannanase (ManAK; derived from marine Aspergillus kawachii IFO 4308), three mutants were designed by amino acid sequence consensus analysis with a second β-mannanase (ManCbs), which also belongs to the glycoside hydrolase family 5 (GH5) and has excellent catalytic efficiency. Three mutants were constructed and their biochemical characteristics were measured after heterologous expression in Pichia pastoris. The results revealed that the kcat/Km values of the three recombinant mannanases ManAKC292V, ManAKL293V, and ManAKL294H were enhanced by 303.0, 280.4, and 210.1%, respectively. Furthermore, ManAKL293V showed greater thermostability than ManAK, retaining 36.5% of the initial enzyme activity after incubation at 80°C for 5min. This study therefore provides a rational design strategy based on consensus sequence analysis to develop industrially valuable β-mannanase for future applications in marine aquafeed.

Structural‐guided design to improve the catalytic performance of aldo‐keto reductase KdAKR

“…For example, Gomez-Fernandez et al (2020) employed an in-house consensus method to derive variants of the high-redox potential laccase, which exhibited enhanced thermostability. Liang et al (2021) improved catalytic efficiency and thermostability of the β-mannanase using the consensus sequence design strategy. The thermostability and activity of the nitrilase were also improved through the consensus-guided mutagenesis (Xiong et al, 2022).…”