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

Qingping Liang,Zhemin Liu,Haijin Mou,Yuming Zhan,Mingxue Yuan,Changliang Zhu,Linyuan Cao

doi:10.3389/fmicb.2021.722347

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.

Highlights

Consensus sequence design (CSD) strategy offers a promising solution for designing enzymes of evaluated catalytic efficiency, while retaining other original properties (Sternke et al, 2019)
Analysis of the conserved domains of the gene sequences of ManAK and ManCbs showed that they both belonged to the glycoside hydrolase family 5 (GH5) family, and that the proportion of amino acid sequence homology in the catalytic region was as high as 86.4%
Two key amino acids (Glu188 and Glu296) in the catalytic sites of ManAK exist as proton donors and nucleophiles, respectively, which play an important role in hydrolysis (Zhang et al, 2019)

Summary

Introduction

Consensus sequence design (CSD) strategy offers a promising solution for designing enzymes of evaluated catalytic efficiency, while retaining other original properties (Sternke et al, 2019). Three mutants of dalcochinase were constructed (F196H, S251V, and M369E), and the catalytic efficiency was shown to have been significantly improved. This CSD strategy has rarely been used to improve the catalytic efficiency of β-mannanases. Strain HY-13, is a neutral-type mannanase in the same family, but has much higher catalytic activity than ManAK. Considering that ManAK and ManCbs share high sequence similarity (86.4%) in the catalytic region, it is theoretically feasible to enhance the catalytic efficiency of ManAK according to CSD strategy. Figure rendering was performed by PyMOL software (version 1.7.2; Delano Scientific, United States)

Methods

Results

Discussion

Conclusion