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. 2021 Oct 21;11(11):467. doi: 10.1007/s13205-021-03027-9

Functional properties and potential application of ethanol tolerant β-glucosidases from Pichia ofunaensis and Trichosporon multisporum yeasts

Jaqueline Elaine Vaz 1, Lacan Rabelo 1, Mohammed Anas Zaiter 1, Waldir Eduardo Simioni Pereira 1, Gustavo Metzker 1, Maurício Boscolo 1, Roberto da Silva 1, Eleni Gomes 1, Ronivaldo Rodrigues da Silva 1,✉
PMCID: PMC8531188  PMID: 34745818

Abstract

β-Glucosidases have been extensively investigated to integrate the enzyme complex for cellulose fiber saccharification and for improving the aroma of wine. To produce these enzymes, greater attention has been given to filamentous fungi and bacteria, and few investigations have targeted the potential applications of enzymes secreted by yeasts. Addressing this issue, in this study, β-glucosidases were produced by the Pichia ofunaensis and Trichosporon multisporum yeasts, via solid state fermentation with wheat bran as a substrate. When using p-Nitrophenyl β-d-glucopyranoside (pNPG) as an enzyme substrate, maximum β-glucosidase activities were detected at pH 5.5–6.0 and 50–60 °C for P. ofunaensis, and pH 5–6 and 55 °C for T. multisporum. Both enzymes were able to hydrolyze cellobiose and exhibited stability over a wide range of pH (3.5–9.0) for 24 h at 4 °C, thermostability up to 50 °C for 1 h and tolerance to 10 mM phenolic compounds. Negative modulation on enzyme activity was observed in the presence of Cu2+, Fe3+, Zn2+, Al3+ and Hg2+, while both β-glucosidases were tolerant to 30% methanol, isopropanol and acetone. In the presence of ethanol and glucose, enzymes from P. ofunaensis were the more active and stable of the two. These enzymes, especially the P. ofunaensis β-glucosidases, could be tested in enology for improving the aroma of wine and for integrating a cellulolytic complex to produce 2G ethanol.

Keywords: Agro-industrial residues, Cellobiose, Cellulolytic complex, Fungi, 2G ethanol, Yeast

Introduction

β-Glucosidases, β-d-glucoside glucohydrolase (EC 3.2.1.21), are enzymes capable of hydrolyzing glycosidic bonds involving d-glucose residues into alkyl-, amino- or aryl-β-d-glycosides, cyanogenic glycosides, disaccharides and short oligosaccharides. Some enzymes, via transglycosylation, can also synthesize glycosyl links between different molecules (Salgado et al. 2018). The cellobiose hydrolysis is a key step for the enzymatic depolymerization of cellulose, which means β-glucosidases have been extensively investigated to compose the cellulolytic complex, especially for 2G ethanol production (Singh et al. 2016; Srivastava et al. 2018). Cellobiose is a final product of other cellulolytic enzymes and its accumulation in the reaction medium may promote cellulase inhibition. It makes β-glucosidases essential enzymes to hydrolyze cellobiose and release free glucose for fermentation (Yeoman et al. 2010; Singh et al. 2016).

Apart from their use for producing biofuels, β-glucosidases are important in the beverage industry, especially for improving the aroma of wine (González-Pombo et al. 2011; Zang et al. 2018) and the flavor of tea and fruit juice (Keerti et al. 2014). Enzyme tolerance to glucose, acidic pH and ethanol are essential requirements to use β-glucosidases in enology and in the simultaneous saccharification–fermentation of cellulose (Villena et al. 2007; Kuusk and Väljamäe 2017). β-Glucosidases with these attributes and that can be obtained at low cost are required for these applications.

In this scenario, agricultural residues, such as wheat bran, can be used as a cheap and alternative substrate for prospecting different microbial enzymes, since they are widely available (Bhatia et al. 2002; Sadh et al. 2018). Due to their rich carbohydrate and protein content (Wieser et al. 2020), residues, such as straw and wheat bran, are correlated with increased enzyme productivity when used as substrates in microbial fermentation (Kalogeris et al. 2003; Leite et al. 2007; Silva et al. 2019).

In general, prospecting for microbial enzymes from bacteria and filamentous fungi has been explored; yet, there has been very little focus on yeast enzymes. Thus, this was the motivation of our study, which can offer new enzymes for application in different industrial segments. Here, β-glucosidases produced by the Pichia ofunaensis and Trichosporon multisporum yeasts have been investigated. They were biochemically characterized to evaluate their potential for use in both situations; enology and for integrating the cellulolytic complex to produce 2G ethanol.

Materials and methods

Microorganisms

In this study, the P. ofunaensis 1A-14 and T. multisporum 1A-10 yeast strains, isolated from ant nests and stored in the working collection of the Biochemistry and Applied Microbiology Laboratory, Ibilce/Unesp, S. J. Rio Preto/SP, Brazil, were used. The conservation of pure cultures was done by storage at − 80 °C. Glass beads, washed with nitric acid and sterilized, were added to the yeast suspensions, which were kept overnight in glycerol (15%) in the freezer at − 20 °C and then transferred to a freezer at − 80 °C.

Solid-state fermentaion (SSF)

First, P. ofunaensis 1A-14 and T. multisporum 1A-10 cells were inoculated in slant tubes containing YEPD medium (2% peptone, 1% yeast extract, 2% glucose and 2% agar), and incubated for 48 h at 28 °C. Subsequently, the cells were resuspended in sterile water and inoculated to 100 mL YEPD medium without agar in a 250 mL Erlenmeyer flasks, for 24 h at 28 °C under 200 rpm agitation. After yeast growth, the culture media were centrifuged at 10,000×g for 10 min at 4 °C. Then the supernatant was discarded and the biomass resuspended in sterile saline solution composed of K2HPO4 (0.1%), KH2PO4 (0.1%), (NH4)2SO4 (0.1%) and MgSO4·7H2O (0.02%) and used to inoculate 1 mg of cells for each SSF flasks.

For SSF, polypropylene bags (15 × 25 cm) containing 5 g of wheat bran as substrate were used. The wheat bran was hydrated with 10 mL of sterile saline solution. After yeast inoculum with 1 mg of cells per fermentation flask, the yeast growth was carried out at 28 °C for 9 days. Next, the fermentation flasks were removed by adding 50 mL of distilled water and homogenized by stirring for 1 h at 160 rpm at 25 °C. Then, the material was filtered through muslin fabric and centrifuged at 10,000×g for 10 min at 4 °C. The supernatant was used as an enzyme extract, verifying the activity for β-glucosidase using the substrate p-nitrophenyl β-d-glucopyranoside (pNPG).

For the functional characterization of the β-glucosidases, the extract obtained from the cultivation of each yeast was concentrated and partially purified using a 10 kDa cut-off filter membrane, surface area 420 cm2 (GE Healthcare, USA) coupled to a QuixStand system (GE Healthcare, Little Chalfont, UK) with a PRP-09WM peristaltic recirculation pump (Watson-Marlow, Marlow, UK).

Determination of β-glucosidase activity

β-Glucosidase activities were determined using 25 µL of partially purified enzyme extracts (1.5 mg/mL), 125 µL of 0.1 M acetate buffer pH 5.0, and 125 µL of pNPG substrate at 4 mM diluted in this same buffer. The reactions were carried out at 40 °C for 15 min and stopped by the addition of 1 mL of 2 M sodium carbonate (Na2CO3) (Silva et al. 2019). Then, the absorbance was monitored in a spectrophotometer at 410 nm. Enzyme activity was expressed in U/mL, considering one unit (U) as the amount of enzyme needed to release 1 µmol of p-nitrophenol per min, using a standard curve for p-nitrophenol as reference. The test was performed in triplicate.

Functional characterization of β-glucosidase activities from partially purified enzyme extracts

Influence of pH and temperature on the activity and stability of β-glucosidases

Using pNPG as substrate, the effects of pH and temperature on β-glucosidase activities were evaluated. For optimal pH testing, the reaction mixture was composed as mentioned in the previous section with variations in the buffering reagents, ranging from pH 3.0 to 10.0: glycine (pH 3.0 and 3.5), acetate (pH 4.0, 4.5, and 5.0), Mes (pH 5.5, 6.0, and 6.5), Hepes (pH 7.0 and 7.5), Bicine (pH 8.0, 8.5, and 9.0), and Caps (pH 9.5 and 10.0), all at 0.1 M, at 40 °C. To see the effect of temperature on the enzyme activity, the reaction mixtures were incubated from 30 to 70 °C, with variations of 5 °C. The assay was performed using the optimum pH previously determined. Maximum activities for pH and temperature were expressed in relative activity, where the maximum enzyme activities were used as 100%. All tests were performed in triplicate.

To investigate the stability at pH, the partially purified enzyme extracts were initially incubated with each buffer mentioned above for 24 h at 4 °C, without substrate. For thermal stability, the enzyme extracts were incubated at 40 °C, 50 °C, 55 °C and 60 °C for 15, 30 and 60 min. In both enzyme stability assays, after the incubation time, the enzyme reaction was carried out using the predetermined optimum pH and temperature. The tests were carried out in triplicate. The results obtained were expressed in residual activity, considering the enzyme activity without treatment as 100%.

Using d-cellobiose as a substrate, the effects of pH and temperature on enzyme activity were also evaluated. For pH, the reaction mixture was composed of 50 µL of partially purified enzyme extracts and 250 µL of 1% cellobiose solution diluted in each different pH buffer, using the same buffer solutions mentioned previously in the pH range 3.0–8.0. Reactions were carried out for 3 h at 50 °C and interrupted by incubation at 96 °C for 10 min. Afterward, 1 mL of the Katal glucose kit reagent (Katal Biotecnologica Ind.e Com., Belo Horizonte, Brazil) was added to the reaction, followed by incubation at 37 °C for 10 min and the absorbance was monitored in a spectrophotometer at 505 nm. To determine the influence of temperature on enzyme activity, the reactions were carried out in the range of 30–70 °C in a previously determined optimal pH buffer. All tests were performed in triplicate, and activities were expressed in relative activity.

Effect of cations on β-glucosidase activity

The effects of cations derived from chloride salts: Li+, Na+, K+, Ba2+, Ca2+, Mg2+, Mn2+, Ni2+, Cu2+, Co2+, Hg2+, Zn2+, Fe3+ and Al3+, at concentrations of 5 and 10 mM, were evaluated. Initially, the partially purified enzyme extracts were incubated with each of these ions for 5 min at 25 °C (de Amo et al. 2019; Pereira et al. 2020). Then, the enzyme reaction was carried out at optimum temperature and pH using 4 mM pNPG as a substrate. All experiments were performed in triplicate and expressed as residual activity, considering the enzyme activity without ions as 100%.

Effect of organic solvents on β-glucosidase activity

The influence of ethanol, methanol, isopropanol and acetone solvents was evaluated at concentrations of 2, 10, 20, 30 and 50% (v/v) on the activities of the β-glucosidases from P. ofunaensis and T. multisporum. Initially, the partially purified enzyme extracts were incubated with each of these solvents for 5 min at 25 °C (de Amo et al. 2019; Pereira et al. 2020). Then, the enzyme reaction was carried out at optimum temperature and pH using 4 mM pNPG as a substrate. All experiments were performed in triplicate and expressed as residual activity, considering the enzyme activity without solvents as 100%.

Additionally, to assess the suitability of applying these enzymes to improve the aroma of alcoholic beverages, such as wine, or their use in the simultaneous saccharification and fermentation of lignocellulosic material, the effects of stability and activity of the enzyme extracts in the presence of ethanol at pH 3.5 and 5.5 (pH consistent with wine and optimum pH for enzyme activity, respectively), were also evaluated for 120 h at 25 °C.

The enzymes were incubated with 0.1 M glycine buffer pH 3.5 or 0.1 M Mes buffer pH 5.5 containing 15% ethanol, and a corresponding experiment with enzymes incubated with the buffers without ethanol. Aliquots were collected at these times: 2, 4, 6, 12, 24, 48, 72, 96 and 120 h; and subjected to an enzymatic reaction with pNPG (4 mM)—samples from each group of experiments were submitted to a reaction using the same buffer solution of the incubation step (at pH 3.5 or 5.5), with or without ethanol 15% at the final concentration.

Reactions were carried out at 50 °C and in triplicate. The results obtained were expressed as residual activity, considering the enzyme activities at time zero as 100% for each experiment group.

Effect of glucose and phenolic compounds on β-glucosidase activity

Due to their ability to inhibit the activity of β-glucosidases, glucose and phenolic compounds were incubated, separately, with the partially purified enzyme extracts for 5 min at 25 °C (de Amo et al. 2019; Pereira et al. 2020). Next, the enzyme activity was evaluated using 4 mM pNPG as a substrate at optmum pH and temperature. Glucose was evaluated at concentrations from 1.25 to 290 mg/mL, and phenolic compounds were evaluated at 10 mM, using vanillin, syringaldehyde, and syringic, 4-hydroxybenzoic, vanillic, ferulic, p-coumaric, gallic and tannic acids. The tests were performed in triplicate and expressed as a residual activity, considering the enzyme activity without treatment as 100%.

Total protein concentration

To determine the total protein concentration, we used Bradford's reagent (Sigma-Aldrich) and bovine serum albumin (BSA) as a standard (Bradford 1976).

Statistical analyses

We used the GraphPad Prism software (version 5.0) to calculate statistical variance (ANOVA). Differences between samples were considered statistically significant if p < 0.05.

Results and discussion

Effect of pH and temperature on the activity and stability of β-glucosidases

Figure 1A and B shows the effect of pH and temperature on the β-glucosidase activity of both yeasts, using pNPG at 4 mM and cellobiose 1% as substrates. The highest hydrolytic activity on pNPG substrate was found at pH 5.5–6.0 (p = 0.09) and 50–60 °C (p = 0.1) for P. ofunaensis enzyme extract; and pH 5–6 (p = 0.1) and 55 °C for T. multisporum. In these assays, the enzymes showed abrupt decreases in their activities after the optimal temperatures, maintaining around 60% at 60 ºC (T. multisporum), and 20% at 65 °C (P. ofunaensis). Both β-glucosidases did not show any activity at 70 °C.

Fig. 1.

Fig. 1

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Effect of pH (A) and temperature (B) on β-glucosidase activities of P. ofunaensis and T. multisporum, using 4 mM pNPG and 1% cellobiose as substrates. For pH assay, the reactions were performed using 0.1 M buffers in the pH range of 3–10 at 40 °C, and in 0.1 M Mes buffer at pH 5.5, for temperature curve (30–70 °C). Values are expressed as mean (n = 3) ± standard deviation

The activity of the β-glucosidases from P. ofunaensis on cellobiose was higher at pH 4.5–5.5 (p = 0.06) and 55 °C; and for T. multisporum at pH 5.0 and 50 °C. The conditions for the highest enzyme activities using pNPG and cellobiose showed slight differences, which is explained by the strict relationship of the enzyme with each substrate, in this case a different structure between (1) a synthetic substrate containing one glycone and aglycone group, and (2) a disaccharide. The pH and temperature influence the ionization state and molecular kinetics, respectively, of the reaction components (substrate and enzyme). This is crucial for the best interaction of an enzyme with each substrate and the conversion of a substrate into a reaction product. Bonfá et al. (2018) reported a similar result, where the characterization of β-glucosidase from Myceliophthora thermophila showed optimal activity at 60 °C and a pH of 5.0 on pNPG as a substrate but when using cellobiose, the highest activity was observed at 50 °C and pH 4.5.

The conditions of maximum activity observed in this study were similar to those reported by Silva et al. (2019) for a β-glucosidase from P. guilliermondii with optimal activity at pH 3.5–5.5 and 50–55 °C, and different from those observed for some fungal β-glucosidases, such as BglF and BglJ β-glucosidases from Aspergillus oryzae, where the maximum activities were at pH 6.0 and 45 °C, and pH 4.5 and 40 °C, respectively (Kudo et al. 2015). Leite et al. (2007) obtained maximum activity for the β-glucosidase of A. pullulans at pH 4.0–4.5 and 80 °C, and for the Thermoascus aurantiacus fungus at pH 4.5 and 75 °C.

The stability of the enzymes at different pH was investigated after being kept for 24 h at 4 °C (Fig. 2A). Enzymes from P. ofunaensis and T. multisporum maintained more than 70% and 80% of their initial activity in the pH range 3–10. Leite et al. (2007) reported high pH stability (more than 90% activity) for 24 h at room temperature for A. pullulans β-glucosidase when incubated in the pH range of 4.0–9.5, and pH range of 4.5–6.5 for T. aurantiacus β-glucosidase. Silva et al. (2019) obtained a similar result with β-glucosidase from P. guilliermondii, whose stability in the pH range 3–10 was around 80% for 24 h at 4 °C. Other fungal β-glucosidases also exhibited stability over a wide range of pH (Almeida et al. 2015; Kudo et al. 2015; Bonfá et al. 2018).

Fig. 2.

Fig. 2

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Effect of pH (A) on the stability of β-glucosidases incubated at 4 °C for 24 h, in the absence of substrate. Thermal stability of β-glucosidases from P. ofunaensis (B) and T. multisporum (C). Reactions were performed using 4 mM pNPG as a substrate, 0.1 M Mes buffer, pH 5.5, at 55 °C. Values were expressed as mean (n = 3) ± standard deviation

The thermal stability of the β-glucosidases from P. ofunaensis (Fig. 2B) and T. multisporum (Fig. 2C) was evaluated at temperatures of 40, 50, 55 and 60 °C for 15, 30 and 60 min. The enzymes of both species were stable up to 50 °C for 60 min, maintaining more than 75% of activity. The enzyme from P. ofunaensis maintained 44% activity when incubated for 60 min at 55 °C, while the enzyme from T. multisporum maintained only 13% activity. From 60 °C up, there was no enzyme activity in any of the enzyme extracts. The thermostability observed for the β-glucosidases of these yeasts was higher than that reported for some fungal β-glucosidases. BglJ β-glucosidase from A. oryzae (Kudo et al. 2015) and β-glucosidase from P. guilliermondii (So et al. 2010) were totally inactivated when incubated for 30 min at 45 °C and 50 °C, respectively.

The characteristics of the β-glucosidases of these two yeasts, with activity and stability at acidic pH and at temperatures up to 50 °C, and also capable of hydrolyzing cellobiose, point to the possibility of using these enzymes as part of the cellulolytic complex, which may be useful in the final stages of saccharification of cellulose fiber, reducing the cellulase inhibition effect by cellobiose, and favoring the release of free glucose for anaerobic fermentation.

Effect of cations on the activity of β-glucosidases

Cations can interact with amino acids in different portions of the protein and at the catalytic site, consequently, it may cause positive or negative modulation on enzyme activity. Among the metal ions evaluated, none of them caused an increase in enzyme activity at both concentrations (5 and 10 mM), while negative effects on β-glucosidases were mainly observed for Cu2+, Fe3+, Zn2+, Al3+ and Hg2+ ions, Cu2+ being the ion that caused the largest reduction in enzyme activity (Table 1). At a concentration of 10 mM of this ion, decreases of 50% and 43% were noted for the enzymes from P. ofunaensis and T. multisporum, respectively.

Table 1.

Influence of ions derived from chloride salts, at 5 and 10 mM, on the β-glucosidase activities of P. ofunaensis and T. multisporum

Chloride salts Relative activity (%)
Pichia ofunaensis Trichosporon multisporum
5 mM 10 mM 5 mM 10 mM
CoCl2·6H2O 86.00 ± 0.20 85.00 ± 2.30 90.50 ± 2.30 84.80 ± 2.20
CuCl2·2H2O 65.70 ± 1.50 49.50 ± 0.80 61.50 ± 1.65 57.25 ± 0.50
NiCl2 90.00 ± 0.60 83.50 ± 1.00 88.00 ± 0.60 89.50 ± 1.70
FeCl3·6H2O 81.00 ± 0.85 62.00 ± 0.20 88.00 ± 1.70 79.50 ± 1.20
ZnCl2 75.50 ± 0.80 65.00 ± 1.70 74.50 ± 2.50 64.80 ± 0.80
HgCl2 77.00 ± 1.80 77.00 ± 0.85 89.50 ± 0.90 84.50 ± 0.40
MnCl2·4H2O 88.00 ± 0.50 82.50 ± 1.25 94.50 ± 0.56 90.00 ± 0.65
KCl 84.00 ± 1.00 82.50 ± 1.40 89.00 ± 2.50 85.00 ± 0.90
LiCl 89.50 ± 1.60 85.00 ± 0.50 91.00 ± 1.80 86.50 ± 1.10
MgCl2·6H2O 88.00 ± 0.50 84.00 ± 0.70 90.50 ± 1.50 84.50 ± 1.50
CaCl2 85.00 ± 0.25 83.50 ± 1.70 90.00 ± 1.70 80.50 ± 2.11
BaCl2 82.50 ± 0.25 80.50 ± 1.20 90.50 ± 1.50 81.80 ± 0.10
AlCl3 73.00 ± 0.85 66.00 ± 0.50 95.25 ± 2.70 77.50 ± 1.10
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Reactions were performed using 4 mM pNPG as a substrate, 0.1 M Mes buffer, pH 5.5, at 55 °C. Values are expressed as mean (n = 3) ± standard deviation

Transition metals, such as Cu2+, exhibit affinity for thiol, amine and carboxylic groups present in amino acids (Shriver and Atkins 2017). These ions generally oxidize the functional thiol groups of cysteine residues and can reduce enzyme activity. In addition to the redox reaction, the loss of enzyme activity can also occur through covalent bonding of copper with the functional groups above mentioned. The β-glucosidases produced in this study showed a greater tolerance to this ion than that from Penicillium simplicissimum (Bai et al. 2013), which maintained only 29.08% of its initial activity, in the presence of 10 mM of Cu2+. In another study, a β-glucosidase from Aspergillus oryzae only maintained 34% of its initial activity in the presence of 1 mM Cu2+ (Tang et al. 2014). Kaur et al. (2007), studying the β-glucosidase from Melanocarpus sp., also reported a reduction of more than 30% in enzyme activity caused by the addition of 10 mM of this ion.

Also, Fe3+ and Hg2+ ions may be involved in a redox reaction or covalent bonding with the above-mentioned groups, while Zn2+, which is a redox inactive ion, may be involved only in covalent bonding of the β-glucosidases (Tejirian and Xu 2010). The Fe3+ suppression of β-glucosidase activity from A. oryzae was also reported by Tang et al. (2014), where 1 mM of this ion reduced 45% of the initial enzyme activity; while for β-glucosidase from P. guilliermondii, about 60% activity was maintained at 5 mM of Fe3+ (Silva et al. 2019). The β-glucosidase from Dekkera bruxellensis maintained 78% of its catalytic performance with 10 mM of this ion, a result very similar to that obtained for T. multisporum (Kuo et al. 2018).

In the presence of Hg2+, at 5 and 10 mM, the β-glucosidase activities of P. ofunaensis fermentative extract were about 77%, while for T. multisporum it was above 84%. Olajuyigbe et al. (2016) reported about 80% and just over 50% residual activity of the Fusarium oxysporum enzyme at concentrations of 5 and 10 mM of this ion, respectively, while Almeida et al. (2015) reported about a 60% reduction in Penicillim verruculosum β-glucosidase activity in the presence of 10 mM Hg2+. Furthermore, the results obtained in this study differ from those found by Riou et al. (1998) who reported a complete loss of activity of the β-glucosidase from A. oryzae at 5 mM Hg2+.

The Zn2+ ion, at 10 mM, reduced the activities of the β-glucosidases of both yeasts, maintaining about 64% of enzyme performance. For this ion, the enzyme extracts presented here were more tolerant than the β-glucosidase from M. thermophila reported by Bonfá et al. (2018), which maintained 63% activity at 2.5 mM Zn2+, as well as for the β-glucosidase from A. oryzae that kept only 17% of its initial activity at 5 mM Zn2+ (Riou et al. 1998).

For the Al3+ ion, at 10 mM, enzyme extract from P. ofunaensis exhibited a reduction of 34% in its activity, while there was a 23% reduction in the activity of the T. multisporum enzymes. The effect on enzyme activity caused by the presence of this ion is associated with the formation of covalent bonds with non-metals, such as sulfur, oxygen and nitrogen (Shriver and Atkins 2017). In the presence of 5 mM Al3+, Silva et al. (2019) observed a 39% decrease in the activity of β-glucosidase from P. guilliermondii, and at 2.5 mM Al3+, Bonfá et al. (2018) reported a decrease of around 17% in the activity of M. thermophila β-glucosidase.

The alkali metal ions only caused a slight change in the activities of both β-glucosidase extracts, which may be related to ion pairing, since alkaline-earth metals and alkali metals can perform strong ion-pair interactions with proteins.

Effect of organic solvents on the activity of β-glucosidases

The effect of methanol, ethanol, isopropanol and acetone on the enzyme activity was evaluated at concentrations of 2, 10, 20, 30 and 50% (v/v), as shown in Fig. 3. Ethanol distinctly affected the two enzyme extracts (Fig. 3A), while the other solvents similarly influenced the β-glucosidases of the two species. For enzymes from P. ofunaensis, in the presence of 2–10% ethanol, there was a slight increase, up to 6%, in the activity of β-glucosidases. At 20% ethanol, P. ofunaensis enzymes maintained 100% activity, while for β-glucosidases from T. multisporum remained between 70 and 80%. The increase in the concentration of ethanol resulted in a reduction in the enzyme activity of both yeasts. However, none of the β-glucosidases had their activities totally compromised, even at the highest concentration (50% ethanol), the catalytic performance was still above 60%.

Fig. 3.

Fig. 3

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Effect of organic solvents: ethanol (A), methanol (B), isopropanol (C) and acetone (D) at concentrations of 2, 10, 20, 30 and 50% on the β-glucosidase activities of P. ofunaensis and T. multisporum. Reactions were performed using 4 mM pNPG as a substrate, 0.1 M Mes buffer, pH 5.5, at 55 °C. Values were expressed as mean (n = 3) ± standard deviation

Kudo et al. (2015) reported the repressive action of ethanol on the activity of two β-glucosidases from A. oryzae. In the presence of 20% ethanol, less than 40% of the initial BglA activity and the total loss of BglJ activity was observed. In another study, β-glucosidase from P. guilliermondii had approximately 50% of its activity reduced in the presence of 50% ethanol (Silva et al. 2019).

The increase in β-glucosidase activity in the presence of ethanol has already been observed by other authors. Barbagallo et al. (2004) reported an increase in the activity of β-glucosidases from Pichia anomala and Saccharomyces cerevisiae in the presence of 10–20% ethanol. Silva et al. (2019) also reported a 10–15% increase in the β-glucosidase activity of P. guilliermondii at a concentration of 10–30% of ethanol. A. niger β-glucosidase also had a 7% increase in its activity at 10% ethanol (Zhao et al. 2013). In the study of β-glucosidase from M. thermophila, Karnaouri et al. (2013) observed that short-chain alcohols (ethanol, methanol and isopropanol) caused an increase in β-glucosidase activity. It is possible that the alcohol-induced change in the polarity of the medium can facilitate the enzyme access to the substrate, as well as stabilize the conformation of the enzyme (Pemberton et al. 1980; Mateo and Di Stefano 1997). However, increasing the concentration of these solvents can result in protein precipitation or denaturation.

Methanol and isopropanol (Fig. 3B, C), at concentrations of 2–30%, did not affect the enzyme activities. In the highest concentration (50%), for methanol, the β-glucosidase of P. ofunaensis maintained around 57% of activity, while T. multisporum around 62%; for isopropanol, activity above 75% was seen for both enzyme extracts. Finally, acetone (Fig. 3D) was the organic solvent with the lowest denaturing effect among all those evaluated in this work. Over all the acetone concentrations used, the activities of both β-glucosidases remained above 80%.

The β-glucosidase from P. guilliermondii (Silva et al. 2019) was tolerant to up to 50% of isopropanol and up to 30% of methanol and acetone. At 50% concentration, isopropanol did not affect P. guilliermondii enzyme activity, unlike that observed for P. ofunaensis and T. multisporum, whose 50% isopropanol caused a 25% reduction in the enzyme activities. The results obtained for P. guilliermondii in the presence of 50% methanol were similar to those obtained here, causing about a 40% reduction in enzyme activity. For 50% acetone, β-glucosidase from P. guilliermondii maintained only 9% of its activity, while enzymes from P. ofunaensis and T. multisporum maintained activities above 80%.

In contrast to the β-glucosidases in this study, Zhao et al. (2013) reported that 10%, methanol caused a 51% increase in activity of the β-glucosidase from A. niger expressed in Pichia pastoris. Acetone, on the other hand, caused a greater reduction in the activity of this β-glucosidase, different from that which was seen for P. ofunaensis and T. multisporum enzyme extracts. In 40% acetone, the A. niger enzyme maintained 39% of its catalytic performance.

Organic solvents are widely used in protein concentration steps; enzyme tolerance to these solvents indicates they can be tested to this proposal with reduced enzyme denaturation, or, in the case of ethanol, the additional use of these enzymes in alcoholic beverages may be evaluated. Thus, we investigated the stability and activity of β-glucosidases from both yeasts at acidic pH and ethanol 15% (conditions similar to those present in wine), as well as in a medium with optimum pH activity (pH 5.5), for 120 h at 25 °C.

After 120 h at pH 3.5 and 15% ethanol, the β-glucosidase from P. ofunaensis maintained more than 85% of its activity, a result very similar to incubation under these same conditions without the addition of ethanol (Fig. 4A). For incubations at pH 5.5 up to 48 h, high stability, with above 90% activity was seen in both experiments, with and without ethanol. However, in incubation with ethanol, the activity remained practically constant up to 96 h, unlike that demonstrated in the absence of this alcohol, which showed a reduction in activity to 70%.

Fig. 4.

Fig. 4

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Activities of β-glucosidases from P. ofunaensis (A) and T. multisporum (B) after incubation for 120 h at 25 °C with or without 15% ethanol at pH 3.5 (0.1 M glycine buffer) or pH 5.5 (0.1 M Mes buffer). pNPG at 4 mM was used as a substrate, diluted in 0.1 M buffer, pH 3.5 or 5.5, and with or without ethanol depending on the incubation conditions. Values were expressed as mean (n = 3) ± standard deviation

For the enzyme extract of T. multisporum incubated at pH 3.5 and 15% ethanol, a lower enzyme activity compared to P. ofunaensis β-glucosidases was seen, maintaining approximately 65% of activity after incubation for 4 h and 45% at the end of 120 h. Under these same conditions, without ethanol, more than 90% of its catalytic performance was detected (Fig. 4B). At pH 5.5, a sample incubated without ethanol for 24 h exhibited higher activity, above 90%, compared to the experiment with ethanol (approximately 70%). At the end of 120 h, the enzyme extracts from both experiments showed similar results, activity between 65 and 70%.

The enzyme incubation for 120 h with 15% ethanol at acidic pH indicated a similar response to that previously detected (Fig. 3A), with enzymes from P. ofunaensis being more tolerant to ethanol than enzymes from T. multisporum, which reinforces the possibility of using these β-glucosidases, especially those from P. ofunaensis, for application in the wine industry. Silva et al. (2019) performed a similar assay that resulted in more than 80% activity incubating the P. guilliermondii enzyme in the presence of 10% ethanol at pH 3.5 for 72 h at 25 °C.

The food and drink industries have shown a growing interest in enzymes, in particular the winemaking and juice sectors, which have focused their attention on β-glucosidases (Singh et al. 2016). These enzymes can improve the aroma of juices and wines through the hydrolysis of glycosidic aroma precursors, especially terpene glycosides (Winterhalter and Skouroumounis 1997; Maicas and Mateo 2016; Bonciani et al. 2018).

Acid or enzymatic pathways can hydrolyze odorless non-volatile glycosides. Acid hydrolysis can cause rearrangements in the aglycone structure along with the formation of undesirable odors (Hernandez-Orte et al. 2009). However, enzymatic hydrolysis only cleaves the glycosidic bond without changing the aglycone, which makes this procedure more efficient. In this case, an ideal β-glucosidase should be stable at low pH (pH between 2.5 and 3.8), tolerant to glucose and active in the presence of 10–15% of ethanol (Riou et al. 1998). In this study, the enzymes presented have these biochemical characteristics and can be attractive for this application proposal.

Effect of glucose and phenolic compounds on β-glucosidase activity

The assessment of the effect of glucose on enzyme activity is important as β-glucosidases can be inhibited by the accumulation of their reaction product. Comparing the enzyme extracts from the two yeasts in the presence of glucose (Fig. 5), the β-glucosidases produced by P. ofunaensis lost only 12% activity in 1.25 mg/mL of this sugar, while the β-glucosidase extract from T. multisporum was more affected, reducing about 43% activity. At the highest concentration (290 mg/mL glucose), both enzyme extracts still maintained activity, around 25% for P. ofunaensis and 20% for T. multisporum.

Fig. 5.

Fig. 5

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Effect of glucose on the β-glucosidase activities of P. ofunaensis and T. multisporum. Reactions were performed using 4 mM pNPG as a substrate, 0.1 M Mes buffer, pH 5.5, at 55 °C. Values were expressed as mean (n = 3) ± standard deviation

Unlike our study, Silva et al. (2019) observed a 13% increase in the activity of the β-glucosidase of P. guilliermondii in the presence of 25–100 mM glucose (approximately 4.5–18 mg/mL). For concentrations above 18 mg/mL of this sugar, Silva et al. (2019) noted a reduction in enzyme activity, similar to those observed for P. ofunaensis and T. multisporum. At a concentration of 180 mg/mL (1 M) glucose, the enzyme from P. guilliermondii maintained 40% of initial activity, while in a concentration range close to this, of 170–200 mg/mL in our study, residual activities were between 42–36% and 25–22% for P. ofunaensis and T. multisporum, respectively. This shows similarities in glucose tolerance to those for β-glucosidases from P. guilliermondii and P. ofunaensis.

The enzymes described here were more glucose tolerant than the enzymes from T. aurantiacus e A. pullulans (Leite et al. 2008), which exhibited complete inhibition in the presence of 5% glucose (50 mg/mL). In contrast, at this same glucose concentration, enzyme extracts from P. ofuanensis and T. multisporum maintained 75% and 42% of their activities, respectively.

Additional to glucose, the effects of phenolic compounds on β-glucosidases activities were also evaluated (Table 2). For all compounds, the residual activity was maintained above 88%, except for gallic and tannic acids. Residual activities of 77% were noted for both enzyme extracts in the presence of gallic acid, and 48% and 63% activities for P. ofunaensis and T. multisporum extracts, respectively, in the presence of tannic acid.

Table 2.

Effect of phenolic compounds (10 mM) on the β-glucosidase activities of P. ofunaensis and T. multisporum

Phenolic compounds (10 mM) Relative activity (%)
Pichia ofunaensis Trichosporon multisporum
Syringic acid 96.60 ± 1.90 104.00 ± 1.60
Vanillin 96.10 ± 2.90 99.80 ± 1.80
4-Hydroxybenzoic acid 94.70 ± 4.20 98.20 ± 3.10
Vanillic acid 89.00 ± 2.00 97.20 ± 2.20
Ferulic acid 92.90 ± 2.50 98.60 ± 1.40
Syringaldehyde 90.90 ± 2.30 90.40 ± 2.70
p-coumaric acid 92.70 ± 2.60 88.10 ± 1.40
Gallic acid 77.70 ± 1.00 77.40 ± 1.70
Tannic acid 48.20 ± 0.80 63.10 ± 2.50
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Reactions were performed using 4 mM pNPG as a substrate, 0.1 M Mes buffer, pH 5.5, at 55 °C. Values are expressed as mean (n = 3) ± standard deviation

The same was observed by Ximenes et al. (2011), where the commercial β-glucosidase from Trichoderma reesei (Spezyme CP®) showed a significant inhibition by tannic and gallic acids after short and long times of incubation. The enzyme extracts described in this work were more tolerant than that reported by Bonfá et al. (2018), whose results obtained for M. thermophila β-glucosidase, in reactions using pNPG as a substrate, indicated a greater inhibition of enzyme activity in the presence of 2.5 mM p-coumaric acid (approximately 70% residual activity) and no detected activity at 0.0625 mM tannic acid.

In general, enzyme extract from P. ofunaensis was less sensitive to glucose compared to T. multisporum, a characteristic that, added to greater tolerance to ethanol, makes enzymes from P. ofunaensis more suitable for future use in wines. In addition, the results indicated that these enzyme extracts might be used in the hydrolysis of lignocellulosic materials, and do not require the pure enzyme. In situations where chromatographic steps are not necessary, it is possible to highlight the advantage of a cheaper process, since purification steps decrease the final yield of the enzyme.

Conclusion

Yeast enzymes have been little explored considering the diverse species of these microorganisms in nature. Studies like the one conducted here can offer new enzymes for application in different industrial segments and this research is driven by the worldwide demand for the use of sustainable technologies. In this regard, using wheat bran as a substrate for fermentation, the production and characterization of β-glucosidases by P. ofunaensis and T. multisporum were described. The study highlighted their tolerance to different physicochemical agents, especially the P. ofunaensis enzymes, which were stable to glucose and ethanol at pH 3.5 and 5.5. β-Glucosidases with these characteristics attract interest for use in improving wine aroma and degrading cellobiose during cellulose fiber saccharification. These findings support future studies to evaluate the application of these enzymes in the aforementioned industrial segments.

Acknowledgements

The authors would like to acknowledge the financial support provided by Fundação de Amparo à Pesquisa do Estado de São Paulo-FAPESP (processes 2017/06399-3, 2018/07036-4 and 2017/06066-4).

Declarations

Conflict of interest

The authors declare no financial or commercial conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Consent for publication

Not applicable.

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