A glucotolerant β-glucosidase from the fungus Talaromyces amestolkiae and its conversion into a glycosynthase for glycosylation of phenolic compounds

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.

New Species of Talaromyces (Fungi) Isolated from Soil in Southwestern China

“…Its beneficial and harmful effects on humans have been well documented. Enzymes and pigments produced by some species of the genus are of industrial importance, such as β-glucosidase produced by T. amestolkiae N. Yilmaz et al [ 1 ] and T. cellulolyticus T. Fujii et al [ 2 ], and red pigments by T. atroroseus N. Yilmaz et al [ 3 , 4 ]. Talaromycosis caused by several species were also reported [ 5 , 6 ].…”

Fungal glycosyl hydrolases for sustainable plant biomass valorization: Talaromyces amestolkiae as a model fungus

“…BGL-1 is a monomeric protein, with the lowest molecular mass among the three BGLs studied. The extracellular activity secreted in P. pastoris was outstanding (75 U/mL), indicating the overexpression of bgl1 in this system (Méndez-Líter et al 2020). The monomeric units of BGL-2 and 3 have similar molecular mass, but BGL-3 showed to be a dimer when analyzed under non-denaturing conditions (Méndez-Líter et al 2017.…”

“…The assessment of the activity of the three BGLs on cellooligosacharides from 2 to 6 glucose units confirmed that they all acted on cellobiose and their efficiency varied with the chain length (Table 3). Despite the low hydrolytic efficiency of BGL-1 on the substrates assayed (Table 3), this enzyme showed an outstanding glucotolerance, and its inhibition constant against glucose (Ki = 3.78 M) is among the highest described to date (Méndez-Líter et al 2020). This work also revealed that BGL-1 is a versatile enzyme, since it had higher hydrolytic activity on sophorose (β-1,2 disaccharide of glucose) than on cellobiose (β-1,4 disaccharide of glucose).…”

“…In this occasion, the test included 70 potential acceptors that covered a wide variety of alcohols (sugars, sterols, phenolic compounds, or amino acids), finding that around half of the compounds tested were potentially glycosylable with BGL-2 and BGL-3, which represents an unusually high number of positive hits reactions (Méndez-Líter et al 2019). However, for BGL-1, the transglycosylation capacity was restricted to p-nitrophenol sugar derivatives of glucose (pNPG), galactose (pNPGal), and xylose (pNPX) (Méndez-Líter et al 2020).…”

Recent advances in enzymatic synthesis of β-glucan and cellulose

“…Protein engineering enabled the production of the β-1,2 transglycosylating BGL-1-E521G that was able to utilise phenolic acceptors such as epigallocatechin gallate (EGCC) in addition to pNP-sugars, whilst using α-glucosyl-fluoride as the donor. Consequently a variety of β-1,2-glucosylated phenolic compounds were produced in a regioselective manner [ 70 ].…”