| Popis: |
Background: The interest for finding novel b-glucosidases that can improve the yields for the production of second-generation (2G) biofuels is still very high. One of the most desired features for these enzymes is the glucose tolerance, which allows them to act optimally under elevated 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, have 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 production of the enzyme in the yeast was very high, reaching 75 U/mL, and it allowed purification in just one step with a yield of 80%. Although the enzyme showed good efficiency on p NPG ( K m =3.36 ± 0.7 mM, k cat =898.31 s -1 ), the activity detected on cellooligosaccharides, the natural substrates of the enzyme, was much lower, which could limit its exploitation in lignocellulose degradation processes. Interestingly, when examining the substrate specificity of BGL-1, it showed to be more active on sophorose, the b-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 p NP-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, which can transfer glucose molecules to glucose to a diversity of acceptors for obtaining compounds of interest. The remarkable capacities of BGL-1 and its glycosynthase mutant, both in hydrolysis or in synthesis, suggest that it could be an enzyme with applications for industrial processes. |
