Abstract
Abstract
Background
The interest for finding novel β-glucosidases that can improve the yields to produce second-generation (2G) biofuels is still very high. One of the most desired features for these enzymes is glucose tolerance, which enables their optimal activity under high-glucose concentrations. Besides, there is an additional focus of attention on finding novel enzymatic alternatives for glycoside synthesis, for which a mutated version of glycosidases, named glycosynthases, has gained much interest in recent years.
Results
In this work, a glucotolerant β-glucosidase (BGL-1) from the ascomycete fungus Talaromyces amestolkiae has been heterologously expressed in Pichia pastoris, purified, and characterized. The enzyme showed good efficiency on p-nitrophenyl glucopyranoside (pNPG) (Km= 3.36 ± 0.7 mM, kcat= 898.31 s−1), but its activity on cellooligosaccharides, the natural substrates of these enzymes, was much lower, which could limit its exploitation in lignocellulose degradation applications. Interestingly, when examining the substrate specificity of BGL-1, it showed to be more active on sophorose, the β-1,2 disaccharide of glucose, than on cellobiose. Besides, the transglycosylation profile of BGL-1 was examined, and, for expanding its synthetic capacities, it was converted into a glycosynthase. The mutant enzyme, named BGL-1-E521G, was able to use α-d-glucosyl-fluoride as donor in glycosylation reactions, and synthesized glucosylated derivatives of different pNP-sugars in a regioselective manner, as well as of some phenolic compounds of industrial interest, such as epigallocatechin gallate (EGCG).
Conclusions
In this work, we report the characterization of a novel glucotolerant 1,2-β-glucosidase, which also has a considerable activity on 1,4-β-glucosyl bonds, that has been cloned in P. pastoris, produced, purified and characterized. In addition, the enzyme was converted into an efficient glycosynthase, able to transfer glucose molecules to a diversity of acceptors for obtaining compounds of interest. The remarkable capacities of BGL-1 and its glycosynthase mutant, both in hydrolysis and synthesis, suggest that it could be an interesting tool for biotechnological applications.
Publisher
Springer Science and Business Media LLC
Subject
Applied Microbiology and Biotechnology,Bioengineering,Biotechnology
Reference56 articles.
1. Martínez AT, Speranza M, Ruiz-Dueñas FJ, Ferreira P, Camarero S, Guillén F, et al. Biodegradation of lignocellulosics: microbial, chemical, and enzymatic aspects of the fungal attack of lignin. Int Microbiol. 2005;8:194–205.
2. Martínez AT. How to break down crystalline cellulose. Science. 2016;352:1050.
3. Sørensen A, Lübeck M, Lübeck PS, Ahring BK. Fungal Beta-glucosidases: a bottleneck in industrial use of lignocellulosic materials. Biomolecules. 2013;3:612–31.
4. Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B. The carbohydrate-active enzymes database (CAZy) in. Nucleic Acids Res. 2013;2014:D490–5.
5. Ramani G, Meera B, Vanitha C, Rajendhran J, Gunasekaran P. Molecular cloning and expression of thermostable glucose-tolerant β-glucosidase of Penicillium funiculosum NCL1 in Pichia pastoris and its characterization. J Ind Microbiol Biotechnol. 2015;42:553–65.