Scholarly article on topic 'Production of β-glucosidase on solid-state fermentation by Lichtheimia ramosa in agroindustrial residues: Characterization and catalytic properties of the enzymatic extract'

Production of β-glucosidase on solid-state fermentation by Lichtheimia ramosa in agroindustrial residues: Characterization and catalytic properties of the enzymatic extract Academic research paper on "Agricultural biotechnology"

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Electronic Journal of Biotechnology
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{Cellobiase / "Cellulases and hemicellulases" / "Industrial enzymes" / "Microbial enzymes"}

Abstract of research paper on Agricultural biotechnology, author of scientific article — Nayara Fernanda Lisboa Garcia, Flávia Regina da Silva Santos, Fabiano Avelino Gonçalves, Marcelo Fossa da Paz, Gustavo Graciano Fonseca, et al.

Abstract Background β-Glucosidases catalyze the hydrolysis of cellobiose and cellodextrins, releasing glucose as the main product. This enzyme is used in the food, pharmaceutical, and biofuel industries. The aim of this work is to improve the β-glucosidase production by the fungus Lichtheimia ramosa by solid-state fermentation (SSF) using various agroindustrial residues and to evaluate the catalytic properties of this enzyme. Results A high production of β-glucosidase, about 274U/g of dry substrate (or 27.4U/mL), was obtained by cultivating the fungus on wheat bran with 65% of initial substrate moisture, at 96h of incubation at 35°C. The enzymatic extract also exhibited carboxymethylcellulase (CMCase), xylanase, and β-xylosidase activities. The optimal activity of β-glucosidase was observed at pH5.5 and 65°C and was stable over a pH range of 3.5–10.5. The enzyme maintained its activity (about 98% residual activity) after 1h at 55°C. The enzyme was subject to reversible competitive inhibition with glucose and showed high catalytic activity in solutions containing up to 10% of ethanol. Conclusions β-Glucosidase characteristics associated with its ability to hydrolyze cellobiose, underscore the utility of this enzyme in diverse industrial processes.

Academic research paper on topic "Production of β-glucosidase on solid-state fermentation by Lichtheimia ramosa in agroindustrial residues: Characterization and catalytic properties of the enzymatic extract"


EJBT-00106; No of Pages 6

Electronic Journal of Biotechnology xxx (2015) xxx-xxx



Contents lists available at ScienceDirect

Electronic Journal of Biotechnology

Production of ^-glucosidase on solid-state fermentation by Lichtheimia ramosa in agroindustrial residues: Characterization and catalytic properties of the enzymatic extract

Nayara Fernanda Lisboa Garcia a, Flávia Regina da Silva Santos a, Fabiano Avelino Gon^alves b, Marcelo Fossa da Paz a, Gustavo Graciano Fonseca b, Rodrigo Simoes Ribeiro Leite a *

a Laboratório de Enzimologia e Processos Fermentativos, Faculdade de Ciencias Biológicas e Ambientáis, Universidade Federal da Grande Dourados, Dourados, MS, Brazil b Laboratório de Bioengenharia, Faculdade de Engenharia, Universidade Federal da Grande Dourados, Dourados, MS, Brazil



Article history: Received 18 February 2015 Accepted 15 May 2015 Available online xxxx

Keywords: Cellobiase

Cellulases and hemicellulases Industrial enzymes Microbial enzymes

Background: p-Glucosidases catalyze the hydrolysis of cellobiose and cellodextrins, releasing glucose as the main 18 product. This enzyme is used in the food, pharmaceutical, and biofuel industries. The aim of this work is to 19 improve the p-glucosidase production by the fungus Lichtheimia ramosa by solid-state fermentation (SSF) 20 using various agroindustrial residues and to evaluate the catalytic properties of this enzyme. 21

Results: A high production of p-glucosidase, about 274 U/g of dry substrate (or 27.4 U/mL), was obtained by 22 cultivating the fungus on wheat bran with 65% of initial substrate moisture, at 96 h of incubation at 35°C. The 23 enzymatic extract also exhibited carboxymethylcellulase (CMCase), xylanase, and p-xylosidase activities. The 24 optimal activity of p-glucosidase was observed at pH 5.5 and 65°C and was stable over a pH range of 3.5-10.5. 25 The enzyme maintained its activity (about 98% residual activity) after 1 h at 55°C. The enzyme was subject to 26 reversible competitive inhibition with glucose and showed high catalytic activity in solutions containing up to 27 10% of ethanol. 28

Conclusions: p-Glucosidase characteristics associated with its ability to hydrolyze cellobiose, underscore the 29 utility of this enzyme in diverse industrial processes. 30

) 2015 Pontificia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V. All rights reserved.

1. Introduction

The pronounced scarcity of fossil fuels related to environmental problems resulting from their processing and consumption has prompted the search for alternative sources of biofuels and renewable energy. This in turn, has generated significant interest in the use of cellulases and other enzymes to convert vegetal biomass into fermentable sugars [1].

Enzymatic hydrolysis of cellulose to glucose requires at least three different enzymes including endo-glucanases (EC, that internally hydrolyze cellulose chains, reducing its degree of polymerization; exo-glucanases (EC that attack the non-reducing and reducing extremities of cellulose, releasing cellobiose; and ß-glucosidases (EC that hydrolyze cellobiose and oligosaccharides, thereby releasing glucose [2].

The ability of ß-glucosidase to utilize different glycosidic substrates renders it suitable for several industrial processes, including the

* Corresponding author. E-mail address: (R.S.R. Leite). Peer review under responsibility of Pontificia Universidad Católica de Valparaíso.

enzymatic hydrolysis of cellulose in order to obtain fermentable 53 sugars and the production of functional foods derived from soy. The 54 enzyme is also used in the juice and beverage industry, where it can 55 improve the aromatic quality of wine and other grape derivatives [3]. 56 The obtainment of industrial enzymes in a sustainable and 57 economically viable manner requires the pursuit of renewable 58 raw materials and processes at low cost. The use of solid-state 59 fermentation (SSF) can reduce the environmental impact and 60 add value to the by-products of agroindustry [4]. The iterative 61 improvement and advantages of SSF have been described in several 62 reports, which studied the influence of different cultivation 63 parameters on the production of microbial enzymes [5,6]. The 64 advantages of SSF include the simplicity of growth conditions, because 65 they are very similar to the environmental systems where many 66 microorganisms develop (especially filamentous fungi); the reduced 67 energy consumption, and that complex equipment or sophisticated 68 control systems are not required. The method also results in higher 69 levels of productivity and low catabolite repression, and favors 70 increased stability of the secreted enzymes [7]. 71

In general, the industrial applicability of an enzyme is closely related 72 to the cost of its production and physicochemical characteristics. The 73 016/j.ejbt.2015.05.007

0717-3458/© 2015 Pontificia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V. All rights reserved.


N.F.L. Garcia et al. / Electronic Journal of Biotechnology xxx (2015) xxx-xxx

production costs can be reduced by screening hyper producer strains, associated with the cultivation process optimized in low-cost mediums [3]. Previous work conducted by our Research Group revealed high p-glucosidase production by the fungus Lichtheimia ramosa by SSF using several lignocellulosic materials [6,8]. This study aimed to optimize the p-glucosidase production by this fungus by SSF. The p-glucosidase produced was biochemically characterized and the catalytic properties of the enzymatic extract were evaluated.

2. Material and methods

2.1. Microorganism

The filamentous fungus L. ramosa was isolated from sugarcane bagasse provided by Sao Fernando A^ucar e Alcool Ltda., Dourados, MS, Brazil [8]. The microorganism was maintained on Sabouraud Dextrose Agar medium; after growth at 28°C for 48 h, the strain was stored at 4°C.

22. Inoculum

The organism was cultivated in inclined 250 mL Erlenmeyer flasks containing 40 mL of Sabouraud Dextrose Agar and maintained for 48°C h at 28°C. A fungal suspension was obtained by adding 25 mL of nutrient solution and gently scraping the surface of the culture. The nutrient solution was composed of 0.1% ammonium sulfate, 0.1% magnesium sulfate heptahydrate, and 0.1% ammonium nitrate (w/v) [9]. As inoculum, 5 mL of this suspension was transferred to each 250 mL Erlenmeyer flask containing lignocellulosic material as substrates.

2.6. Characterization of fi-glucosidase produced by the fungus L. ramosa 126

2.6.1. Effect of pH 127 The optimum pH for p-glucosidase activity was determined by 128

measuring the activity at 50°C at different pH values (3.0-8.0), with 129 increments of 0.5, using 0.1 M citrate-phosphate buffer solution. The 130 pH stability was determined incubating the enzyme for 24 h at 25°C at 131 different pH values, appropriately diluted with buffer solutions: 0.1 M 132 citrate-phosphate (pH 3.0-8.0), 0.1 M Tris-HCl (pH 8.0-8.5), and 133 0.1 M glycine NaOH (pH 8.5-10.5), with increments of 0.5, adopting 134 as 100% the highest value of residual activity obtained after the 135 samples treatment. The residual activity was determined under 136 optimal conditions of pH and temperature. 137

2.6.2. Effect oftemperature 138 The optimum temperature for p-glucosidase activity was obtained 139

by determining the enzymatic activity over a temperature range of 140 30°C-75°C, with increments of 5°C, at the respective optimum pH. 141 Thermostability was determined by incubating the enzyme for 1 h at 142 different temperatures (30°C-70°C), with increments of 5°C, adopting 143 as 100% the highest value of residual activity obtained after the 144 samples treatment. The residual activities were measured under 145 optimal conditions of pH and temperature. 146

2.6.3. Effect of glucose and ethanol on fi-glucosidase activity 147 The enzymatic activity was quantified with the addition of glucose or 148

ethanol at different concentrations in the reaction mixture (0-200 mM 149 glucose or 0-30% of ethanol). The activities were measured under 150 optimal conditions of pH and temperature. 151

2.7. Catalytic potential of the enzymatic extract 152

2.3. Solid-state fermentation (SSF)

The enzyme was produced by cultivating the fungus in 250 mL Erlenmeyer flasks containing 5 g of substrate (wheat bran, soy bran, corn cob, corn straw, rice peel, or sugarcane bagasse), previously washed and dried at 60°C for 24 h. The optimal substrate for enzyme production was used in subsequent steps to evaluate the effects of varying the pH of cultivation medium, moisture content, temperature, and time of cultivation. The parameter selected in each step was used for further cultivation, in an iterative strategy designed to optimize the fermentation process for p-glucosidase production. All material was previously autoclaved for 20 min at 121°C, and the experiments were performed in duplicate.

2.4. Enzyme extraction

The extraction of the enzyme from the fermented substrate was carried out by adding 50 mL of distilled water, and constantly shaking at 100 rpm for 1 h. The sample was filtered and centrifuged at 3000 x g for 5 min. The supernatant was considered the enzymatic extract and was used in the following steps.

The CMCase and xylanase activities were quantified using 3% 153

carboxymethylcellulose (Sigma C5678) and 1% xylan (Sigma 154

Birch-Wood), respectively. The reducing sugar released was quantified 155

by the DNS method [11]. The p-xylosidase activity was measured with 156

the synthetic substrate p-nitrophenyl-p-D-xylopyranoside (4 mM, 157

Sigma), following the methodology described in Section 2.5. The 158

potential to hydrolyze cellobiose was evaluated with a glucose-oxidase 159

kit (Glucose-PP Analisa). Specifically, 100 |jL of the enzymatic extract 160

was added to 0.9 mL of 50 mM sodium acetate buffer containing 0.5% 161

cellobiose (Fluka). One unit of enzymatic activity was defined as the 162

amount of enzyme capable of producing 1 ^mol of product per min of 163

reaction. 164

3. Results and discussion 165

3.1. Production of fi-glucosidase by solid-state fermentation 166

3.1.1. Selection of substrates for fi-glucosidase production 167

Among the tested substrates, the cultivation of the fungus L. ramosa 168

in wheat bran provided higher p-glucosidase production (162.2 U/g 169

or 16.22 U/mL) (Table 1). The wheat bran has suitable nutritional 170

composition as a substrate for microbial growth; it contains 171

2.5. Determination of fi-glucosidase activity

The p-glucosidase activity was determined with 50 |jL of enzymatic extract, 250 |jL of sodium acetate buffer (0.1 M, pH 4.5), and 250 |jL of p-nitrophenyl-p-D-glucopyranoside (4 mM, pNPpG, Sigma) during a 10 min reaction at 50°C. The enzymatic reaction was stopped with 2 mL of sodium carbonate (2 M), and the liberated product was spectrophotometrically quantified at 410 nm. One unit of enzyme activity was defined as the amount of enzyme required to release 1 |jmol of nitrophenol per minute of reaction [10].

Table 1

p-Glucosidase production in different agroindustrial substrates (75% of initial moisture) by L. ramosa by solid state fermentation, at 96 h of incubation, under 28°C.

Substrate U/mL U/g

Wheat bran 16.22 ± 0.42 162.2 ±4.2

Soy bran 1.15 ±0.07 11.5 ±0.7

Corn cob 0.35 ± 0.04 3.5 ± 0.45

Corn straw 0.27 ± 0.02 2.7 ± 0.2

Rice peel 0.068 ± 0.00 0.68 ± 0.00

Sugarcane bagasse 1.11 ± 0.025 11.1 ± 0.25

t1.1 t1.2 t1.3

t1.5 t1.6 t1.7 t1.8 t1.9 t1.10


N.F.L. Garcia et al. / Electronic Journal of Biotechnology xxx (2015) xxx-xxx

appropriate quantities of carbohydrates, proteins, fats, fiber and ashes (Ca, Mg, P, K, S), favoring enzymes production [12].

High level (3-glucosidase production can be achieved during the culture of microorganisms by solid-state fermentation, using wheat bran as either the main substrate, or as a substantial component of the mixture [3,8,12,13]. Thus, wheat bran was selected for subsequent assays in order to optimize the cultivation process for (3-glucosidase production.

3.1.2. influence of fermentative parameters on fi-glucosidase production using wheat bran as substrate

The greatest (3-glucosidase production by the fungus was obtained from the cultivation where the initial pH of the nutrient solution was adjusted to 5.0 (Fig. 1a). However, the microorganism showed considerable enzyme production for all pH values evaluated. Previous reports indicate that filamentous fungi produce cellulases at pH values below neutrality [14]. Most filamentous fungi show optimal growth in slightly acidic pH. In general, values of pH higher than 7.0 reduce fungal growth and, thereby reducing the enzyme production [15].

The pH was not controlled during the cultivation process due to the heterogeneity of the process of solid-state fermentation. According to Pandey et al. [16], the difficulty of monitoring and controlling fermentation parameters in solid-state fermentation is perhaps, the main drawback of this process. Variations of pH during the fermentation process are due to the metabolic activity of the microorganisms, and may be increased or decreased according to the by-products released or the nutrients consumed during the process.

Among the moisture values evaluated, the highest enzyme production was obtained in wheat bran with 65% of initial moisture (Fig. 1b). Values between 60 and 70% of moisture are often used for cultivation of filamentous fungi when the aim is to produce (3-glucosidase. Leite et al. [3] reported the (3-glucosidase production (70 U/g of substrate) by the cultivation of Thermoascus aurantiacus in wheat bran with 60% of moisture. Brijwani et al. [13] obtained higher production of (3-glucosidase (10.71 U/g) using soybean peel and wheat bran with 70% of moisture, during co-cultivation of Trichoderma reesei and Aspergillus oryzae by solid-state fermentation.

The moisture in solid-state fermentation can influence the synthesis and secretion of extracellular enzymes. The presence of free water between the particles of the substrate reduces the porosity of the

medium, interfering with the gas transfer and temperature. On the 211 other hand, the low moisture content can decrease the solubility of 212 nutrients, disfavoring microbial metabolic activity [17]. 213

The ideal temperature for (3-glucosidase production by L. ramosa was 214 35°C, about 249.0 U/g (24.9 U/mL) (Fig. 1c). Fig. 1c reveals that a higher 215 amount of enzyme was produced in cultures carried out at 30°C-40°C. 216 This optimal temperature for enzyme production is higher than the 217 range most commonly considered optimal for the cultivation of 218 mesophilic microorganisms, which is usually between 28°C and 30°C 219 [13,18]. This characteristic favors the use of this strain in industrial 220 processes, where variations in process temperature are acceptable, 221 considering that the control of fermentation parameters on a large 222 scale is not as precise as in laboratory conditions. 223

A considerable reduction in enzyme production was evident 224 in cultures performed at 25°C and 45°C (Fig. 1c). Temperatures 225 that are significantly lower than the optimal for microbial growth 226 disfavor nutrient transport and the exchange of products between the 227 intracellular and extracellular environment. This is because low 228 temperatures reduce both the permeability of the plasma membrane 229 and the speed of the metabolic reactions. On the other hand, very high 230 temperatures cause the collapse of membranous structures and 231 denature structural proteins and enzymes. Accordingly, both cases 232 result in reduced enzyme production [12]. 233

Finally, the influence of cultivation time was investigated. The 234 highest enzyme production was obtained at 96 h of incubation at 35°C 235 using wheat bran as the substrate with 65% of moisture and pH 5.0 236 (Fig. 1d). The overall optimization of the process increased the 237 (3-glucosidase production from 162.2 U/g (16.22 U/mL) to 274.0 U/g 238 (27.4 U/mL) (Table 1 and Fig. 1d, respectively). In addition to 239 increased enzyme production, the optimization permitted a reduction 240 in cultivation time to less than the duration used in preliminary assays 241 carried out by our research group [4,8]. Gon^alves et al. [8] reported 242 the production of 17.26 U/mL of (3-glucosidase in cultivation of the 243 fungus L. ramosa for 120 h by solid-state fermentation. Our current 244 results reinforce the importance of optimizing the culture parameters, 245 as evidenced by a 68.9% increase in the (3-glucosidase production 246 when compared to the initial values. 247

The reduced cultivation time, achieved in the present work, is also a 248 key improvement for fermentation techniques that use L. ramosa, since 249 the cost of enzyme production is proportional to incubation time. A 250

P. 200

30 35 40 Temperature ( C)

60 65 70 Mttsture (%)

CJ 250 <5

£ 200 » 150

72 9« Trne (h)

Fig. 1. Influence of fermentation parameters on p-glucosidase production by the fungus L. ramosa, by SSF in wheat bran. (a) Influence of initial cultivation pH; (b) Influence of initial substrate moisture; (c) Influence of cultivation temperature; (d) Influence of cultivation time. Conditions: pH 5.0; moisture 65%; temperature 35°C.


4 N.F.L. Garcia et al. / Electronic Journal of Biotechnology xxx (2015) xxx-xxx

reduced amount of enzyme was found in extracts obtained after 96 h of cultivation (Fig. 1d). This is likely explained by the consumption of the culture medium nutrients and the excretion of by-products by the microorganism used for fermentation. Such by-products, which may interfere with protein synthesis as well as enzymatic activity, include proteases and substances that reduce macro and micronutrients, alter the pH, and decrease water availability [19].

The level of p-glucosidase production obtained during the current study is significantly higher than that described by other groups. Leite et al. [3] obtained 7.0 U/mL of p-glucosidase with 72 h of T. aurantiacus cultivation in wheat bran. Delabona et al. [20] obtained 105.82 U/g of substrate by cultivation of the fungus Aspergillus fumigatus in wheat bran for 96 h. Xin and Geng [21] obtained 61.6 U/g of substrate with T. reesei cultivated at 26°C for 192 h on woodchips. Ng et al. [2], reported the production of 159.1 U/g of substrate by the fungus Penicillium citrinum YS40-5 after cultivation in rice bran for 96 h. Zimbardi et al. [22] optimized the p-glucosidase production by the fungus Colletotrichum graminicola in wheat bran, with a maximum production substrate of 159.3 U/g, after 168 h. Silva et al. [6] reported the production of 0.061 U/mL of p-glucosidase by the fungus L. ramosa in pequi residue (typical fruit of the Cerrado vegetation), after 48 h in solid-state fermentation.

The enzymatic extract obtained, under optimal culture conditions, was used in subsequent steps for the biochemical characterization of p-glucosidase.

3.2. Characterization offi-glucosidase produced by the fungus L. ramosa

3.2.1. Effect ofpH and temperature

The p-glucosidase produced by the fungus L. ramosa showed higher catalytic activity at pH 5.5 and temperature of 65°C (Fig. 2a and Fig. 2b).

Surprisingly, the enzyme showed higher catalytic activity at temperatures above 50°C, not observed routinely in enzymes produced by mesophilic microorganisms. Belancic et al. [23] obtained the best activity at pH 5.0 and optimum temperature of 40°C for p-glucosidase produced by Debaryomyces vanrijiae. Most fungal p-glucosidases show optimum activity between 40°C and 50°C and at pH between 4.0 and 6.0 [24,25]. However, some studies have reported

the production of extremely stable p-glucosidase by mesophilic 288 strains [3]. 289

The p-glucosidase produced by the fungus L. ramosa showed 290 remarkable structural stability. The enzyme retained its original 291 activity after 24 h of incubation over a pH range of 3.5-10.5 (Fig. 2c). 292 Regarding the thermal stability of the enzyme, about 90% of the 293 catalytic activity was recovered after 1 h at 55°C (Fig. 2d). 294

The results are more significant when compared with previously 295 published data. p-glucosidase produced by different species of 296 Penicillium showed stability from pH 4.0 to 6.0 [26,27]. The enzyme 297 produced by the fungus Trichoderma harzianum was stable at 298 temperatures below 55°C for 15 min, maintaining only 36% of 299 initial activity after 15 min at 60°C [28]. Delabona et al. [5] describe 300 the stability of p-glucosidase produced by the fungus A. fumigatus 301 P40M2 at temperatures from 40°C to 60°C and at pH 3.0 to 5.5. 302 The p-glucosidase produced by the yeast Sporidiobolus pararoseus 303 maintained its catalytic activity for 1 h at40°C; at higher temperatures, 304 only 30% of the initial activity was recovered [24]. 305

3.2.2. Effect ofglucose and ethanol on fi-glucosidase activity 306

Evaluation of the effect of ethanol on enzymatic activity is essential 307 in studies with p-glucosidases, since these enzymes are frequently 308 exposed to substantial concentrations of alcohol in many industrial 309 applications [29]. 310

Fig. 3a shows the effect of different concentrations of ethanol (0- 311 30%) on the enzymatic activity. Ethanol concentrations up to 5% 312 potentiated the enzymatic activity, and elicited an increase of up to 313 20% compared to the initial activity. Increasing ethanol concentration 314 to 15% dramatically reduced enzymatic activity to only 22% of 315 the original level. However, at a concentration of 10% of ethanol, 316 p-glucosidase retained a level of catalytic activity similar to the 317 control. Considering that the final ethanol concentration in fermented 318 broths obtained by traditional processes is around 10% [30], we infer 319 that the enzyme is sufficiently stable to be applied in industrial 320 fermentation processes containing ethanol. 321

The increase in the catalytic potential of p-glucosidases observed by 322 the ethanol addition is related to the glucosyltransferase activity [31]. 323 Ethanol can increase the rate of reaction by acting as preferred 324 acceptor of glycosyl residues during enzymatic catalysis [32]. The 325

Fig. 2. Effect of pH (a), and temperature (b), on the activity (c), and stability (d) of p-glucosidase, produced by the fungus L. ramosa by SSF in wheat bran, with 65% of moisture and initial pH 5.0, incubated for 96 h at 35°C.


N.F.L. Garcia et al. / Electronic Journal of Biotechnology xxx (2015) xxx-xxx 5

Ethanol (%) Glucose (mM)

Fig. 3. (a) effect of ethanol, and (b ) glucose, on the activity of p-glucosidase produced by L. ramosa by SSF in wheat bran, with 65% of moisture and initial pH 5.0, incubated for 96 h at 35°C.

326 hydrolysis and transglycosylation occur through the same biochemical

327 pathway, differing only in the nature of the final acceptor [25].

328 Different concentrations of glucose were added to the reaction

329 mixture to evaluate the behavior of p-glucosidase in the presence

330 of this inhibitor. The enzyme was strongly inhibited by glucose,

331 maintaining approximately 30% of its original activity in the reaction

332 carried out with 100 mM glucose (Fig. 3b).

333 The majority of microbial p-glucosidases are inhibited by glucose,

334 which is a major limitation of the use of these enzymes in industrial

335 processes [3]. High glucose concentrations can interfere directly or

336 indirectly with substrate binding to the enzyme active site, reducing

337 the reaction rate [33]. The inhibition of p-glucosidase produced by

338 L. ramosa was completely reversed when the substrate concentration

339 was increased to the same glucose concentration, indicating that the

340 interaction of the enzyme with the inhibitor is competitive.

341 Competitive inhibition can be reversed by increasing substrate

342 concentration; the same fact is not observed in non-competitive

343 inhibition. In competitive inhibition, the inhibitor and the substrate

344 compete for the same binding site of the enzyme (the active site, in

345 this case). Thus, increasing the substrate concentration to equal or

346 greater values than those of the inhibitor, favors the binding of the

347 enzyme to the substrate, which is reflected in the reversibility of

348 enzymatic inhibition [3].

349 The reversibility of inhibition by glucose and stability to ethanol

350 confirm the potential of this p-glucosidase for applications that

351 require simultaneous saccharification and fermentation processes [3],

352 where the monosaccharides released by enzymatic hydrolysis are

353 simultaneously converted to ethanol by fermenting microorganisms

354 [34,35].

355 3.3. Catalytic potential of the enzymatic extract

356 The production of other cellulases and also hemicellulases by

357 L. ramosa, in optimized culture conditions, was evaluated in this work

358 (Table 2). The enzymatic extract exhibited CMCase (152.1 U/g or

359 15.21 U/mL), xylanase (28.5 U/g or 2.85 U/mL), and p-xylosidase

360 (115.7 U/g or 11.57 U/mL) activity.

361 The production of the CMCase and hemicellulases by L. ramosa is not

362 very impressive when compared with hyper-producing strains.

t2.1 Table 2

t2.2 Catalytic potential of the enzymatic extract obtained by solid-state fermentation by L. t2.3 ramosa in wheat bran, with 65% of moisture and initial pH 5.0, incubated for 96 h at 35°C.

t2.4 Enzyme Substrate U/mL U/g

t2.5 CMCase Carboxymethylcellulose 15.21 152.1

t2.6 ß-Glucosidase p-nitrophenyl-ß-D-glucopyranoside (pNPG) 23.47 237.7

t2.7 ß-Glucosidase Cellobiose 23.45 234.5

t2.8 Xylanase Xylan 2.85 28.5

t2.9 ß-Xylosidase Xylopyranoside p-nitrophenyl-ß-D (pNPX) 11.57 115.7

However, relatively little is known regarding the characteristics of 363

these enzymes, and thus further studies in this area are required. 364

Moreover, there is the possibility to improve these enzymes 365

production in new works of culture optimization. Silva et al. [36] 366

reported the production of 60 U/mL of CMCase and 107 U/mL by the 367

fungus T. aurantiacus by solid-state fermentation, using corncob as 368

substrate. Delabona et al. [20] report the production of 160.1 U/g of 369

CMCase and 1055.62 U/g of xylanase by the fungus A. fumigatus 370

cultivated in agroindustrial residue products. 371

Another interesting aspect of the current study, shown in Table 2, is 372

the impressive potential of the enzyme to hydrolyze cellobiose, as it 373

yields values similar to those obtained with a synthetic substrate 374

(pNPG). The microbial p-glucosidases can be classified into three 375

major groups: (1) Aryl p-glucosidases, which exhibit high specificity 376

to hydrolyze aryl-glycosides substrates, (2) true cellobiases, which are 377

enzymes with that hydrolyze cellobiose with high specificity and (3) 378

enzymes with low specificity: enzymes that act on different types of 379

glycosides substrates [25,31]. Apparently, p-glucosidase expressed 380

from the fungus L. ramosa has low specificity; that is, it has the 381

potential to hydrolyze different glycosides substrates. However, to 382

confirm this hypothesis, further studies should be performed with the 383

purified enzyme. According to Bhatia et al. [25], most of microbial 384

p-glucosidases are classified in the third group. 385

4. Conclusions 386

L. ramosa has proven to be a remarkable fungus for use in 387

p-glucosidase production when cultivated by solid-state fermentation 388

using wheat bran as the substrate. This fungus is capable of producing 389

several enzymes, including CMCase and p-xylosidase. p-Glucosidase 390

was highly stable across a range of pH and temperatures and retained 391

its original activity in solutions containing 10% of ethanol. 392

Furthermore, the inhibitory effects of glucose were completely 393

reversed at high substrate concentrations. The enzyme can hydrolyze 394

different glycoside substrates; due to these characteristics, the 395

p-glucosidase produced by L. ramosa can be used for the production of 396

second-generation ethanol as well as for the improvement of food and 397

beverage quality. Q2

Financial support 399

The authors gratefully acknowledge the financial support of the 400

Conselho Nacional de Desenvolvimento Científico e Tecnológico 401

(CNPq), the Fundaçao de Apoio ao Desenvolvimento do Ensino, 402

Ciência e Tecnologia do Estado de Mato Grosso do Sul (FUNDECT), and 403 the Coordenaçao de Aperfeiçoamento Pessoal de Nível Superior 404



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