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Brazilian Journal of Microbiology logoLink to Brazilian Journal of Microbiology
. 2022 Apr 11;53(3):1599–1611. doi: 10.1007/s42770-022-00750-0

Study of protease activity from Aspergillus awamori INCQS2B.361U2/1 extracellular fraction and modification of culture medium composition to isolate a novel aspartic protease

Raquel Elisa da Silva-López 1,✉, Thayane Aparecida Alves de Araujo 1, Hélvio José Jalles Monteiro 1,2, Érika Maria Gomes Ferreira Teixeira 1, Lucas Tupi 2, Elba Pinto da Silva Bon 2
PMCID: PMC9433587  PMID: 35411453

Abstract

Aspergillus awamori was cultivated in a modified Breccia medium, and the extracellular fraction was obtained, which presented 260 ± 15 µg of protein/mg and specific protease activity of 3.87 ± 0.52 mM.min−1.mg of protein−1 using Nα-p-tosyl-L-arginine methyl ester hydrochloride (L-TAME) as substrate. This fraction showed major proteins about 104 and 44 kDa and maximal protease activity at pH 5.5, 6.5, and 9.0, suggesting that A. awamori secretes acidic, neutral, and alkaline proteases with expressive thermal stability, however, aspartic protease was the most important activity. When yeast extract was supplemented to a modified Breccia medium, A. awamori protein secretion and protease activity were maximal and the affinity chromatography on pepstatin-agarose was employed to isolate the aspartic protease activity, which was called ASPA, with approximately 75 kDa. ASPA maximal activity was obtained at pH 4.5 and 6.5, and 50 °C. Pepstatin inhibited about 80% of ASPA activity, with IC50 and Ki values of 0.154 and 0.072 μM, respectively. ASPA cleaved protein and peptides substrates with the highest activity against gelatin (95 U/mg) and good peptidase activity with KM 0.0589 mM and Vmax 1.909 mM.min−1.mg protein−1, using L-TAME as substrate. A. awamori extracellular fraction is a source of proteases with important activity, and the supplementation of modified Breccia medium increased the aspartic protease production. This enzyme presented different biochemical characteristics from the previously reported A. awamori aspartic proteases. Therefore, ASPA is an excellent candidate for biotechnological application due to its important activity and thermostability.

Keywords: Aspergillus awamori, Extracellular fraction, Culture medium modification supplementation, Aspartic protease isolation, Enzyme characterization

Introduction

Many researchers have investigated enzyme production due to its wide range of physiological, analytical, and industrial applications [1, 2]. Among all biological resources, microorganisms are particularly important because of their extensive biochemical diversity and the possibility of large-scale culture through fermentative processes. Moreover, these organisms are known to play a key role in the production of both extracellular and intracellular enzymes at a commercial scale, and more than 3000 different microbial extracellular enzymes have been reported. [3]

Filamentous fungi from the Aspergillus genus consist of over 340 officially recognized species. In nature, they are the main agents of decomposition for various organic substances. These fungi reproduce primarily by forming asexual spores (conidia) produced in specialized multicellular structures called conidiophores. Although it causes aspergillosis, which is transmitted by inhalation of Aspergillus conidia and can be critical in patients with immunity and chronic respiratory disorders,[4] this fungus is an economically relevant and useful microorganism that is extensively used in the fermentation industry to produce enzymes, organic acids, vitamins, and antibiotics. [5]

Proteases are important enzymes and are nearly two-thirds of the total industrial enzymes currently used. Microbial production is the main source of proteases [6]. Proteases are used in a wide range of applications, including in food, beverage, meat, detergents, tannery, peptide synthesis, leather, and pharmaceutical industries [7]. Over one-quarter of commercial proteases from fungal sources are from Aspergillus species and are employed for a variety of purposes [8]. An acid proline-specific endoprotease from Aspergillus niger prevented the chill-haze formation during beer production that is induced by the interaction of polyphenol with proline-rich proteins [9]. The acid protease from A. niger is capable of degrading Ochratoxin A, which is a mycotoxin present in food commodities, reducing its toxicity [10]. Some types of proteases were identified in different A. niger strains and most of them belong to the A1-family aspartic proteases [11]. A. oryzae expressed basic,[12] neutrals [13], and acid proteases [12]; however, the most important activity was the aspartic type. Furthermore, an aspartic protease from A. oryzae exhibits, when consumed, a probiotic effect in rats. [14].

Aspergillus awamori is classified as black aspergilli as a member of the Aspergillus section Nigri. [15] This fungus presented a relevant production of xylanase, ferulic acid esterase, and β-xylosidase, which are important in the biomass saccharification process [16] and heterologous proteins because of their high secretion capacity [17]. This fungus has been used in pharmaceutical applications, including the expression of lactoferrin, a human milk protein [18]. It is also a source of an acid-resistant, thermal stable, non-mutagenic peptide that demonstrates diabetes management potential [19]. A. awamori also produces proteases, such as Aspergillopepsin A [20] and Aspergillopepsin B,[21] which are used in the food and biotechnological industries [22, 23]. These enzymes are closely related to aspartic proteases and are the most important proteases produced [24]. Aspergillopepsins genes have already been sequenced; however, little information is known about the biochemistry and kinetic characteristics of these aspartic proteases. The secretion of A. awamori proteases can cleave heterologous proteins expressed by this fungus, reducing its yield. [25].

Therefore, the aims of this work were to study the protease activity from A. awamori extracellular fraction cultivated in a modified Breccia medium to modify the culture conditions of A. awamori for increased production of aspartic protease as well as isolate and characterize secreted aspartic proteases.

Materials and methods

Culture conditions and extracellular fraction preparation

The A. awamori fungus strain was deposited in the fungi culture collection of the National Institute of Quality Control in Health (INCQS 2B.361 U2/1) of the Oswaldo Cruz Foundation. The fungus was propagated on potato dextrose agar (PDA) plates at 30 °C until dense black sporulation was observed. Spores were collected by adding 2 mL of sterilized distilled water to the plate, followed by gentle scraping. A standardized stock spore suspension presenting 106–107 spores/mL in 0.5% (v/v) glycerol was maintained at − 4 °C.

Breccia medium [26] and a modified Breccia medium were employed for A. awamori growth containing suitable nutrients for growth and propagation. Media compositions are provided in Table 1.

Table 1.

Composition of 1 L of Breccia media (

Adapted from Breccia et al. 1995)

Reagents Breccia medium Modified Breccia
medium
Ammonium sulfate [(NH4)2SO4] (g/L) *** 2.7
Sodium nitrate (NaNO3) (g/L) 1.2 ***
Potassium phosphate monobasic (KH2PO4)(g/L) 3.0 3.0
Potassium phosphate dibasic (K2HPO4) 6.0 g 6.0 g
Magnesium sulfate heptahydrate (MgSO4.7H2O) 0.2 g 0.2 g
Calcium chloride (CaCl2) (g/L) 0.05 g 0.05 g
Ferrous sulfate heptahydrate, 5 mg/mL (mL/L) 1 1

Cobalt chloride hexahydrate (CoCl2.6H2O)

2.0 mg/mL

 1 mL 1 mL

Manganese sulfate tetrahydrate (MnSO4.4H2O)

1.6 mg/mL

 1 mL 1 mL
Zinc sulfate heptahydrate (ZnSO4.7H2O) 1.4 mg/mL 1 mL 1 mL
Yeast extract (g/L) 12.0 g 14.0 g
Carbon source (wheat bran) (g/L) 30.0 g 30.0 g
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***Not added

The cultivation of A. awamori started with a pre-inoculum of 1% of the stock spore suspension in Brecccia medium (200 RPM at 30 °C). After 48 h, an inoculum was made using a modified Breccia medium and 10% of the total volume of the pre-inoculum (200 RPM at 30 °C). The culture supernatant was analyzed from day 0 to 8 of cultivation, and day 7 was the one chosen for the collection of the supernatant. On that day, the cells were harvested by centrifugation (2000 × g for 10 min at 4 °C), and the culture supernatant was collected and centrifuged (10,000 × g for 30 min at 4 °C) to obtain the extracellular fraction. The clear material was collected for further analysis and protease purification.

The yeast extract concentration and presence of other nitrogen sources (ammonium sulfate, sodium nitrate, and urea) were changed in the modified Breccia medium to increase the protease expression. Initially, four Ye-Breccia media were prepared that contained yeast extract concentrations of 8, 10, 12, and 16 g/L. Another four Osn-Breccia media were prepared using only one source of nitrogen: only yeast extract (14.0 g/L), only ammonium sulfate (2.7 g/L), and only sodium nitrate (1.2 g/L, and the last one with only urea (0.3 g/L). No buffers were used. In other words, the potassium phosphate monobasic (KH2PO4) and potassium phosphate dibasic (K2HPO4) were not added to all these media. The modified Breccia medium contains 14.0 g/L of yeast extract and 2.7 g/L of ammonium sulfate; however, it does not have sodium nitrate and urea (Table 1).

Ammonium sulfate precipitation and enzyme purification

Solid (NH4)2 SO4 was added to the culture supernatant at 45 and 60% saturation. After gently stirring at 4 °C overnight, the suspension was centrifuged (10,000 × g for 30 min at 4 °C). The pellet was collected and resuspended in 3 volumes of distilled water and dialyzed overnight in 2 L of 10 mM sodium citrate, pH 5.5 buffer at 4 °C. After removal of the insoluble material by centrifugation (10,000 × g for 30 min at 4 °C), the clear supernatant was loaded onto a pepstatin-agarose affinity column (5 mL, Sigma Co, St. Louis, MO, USA), previously equilibrated with 50 mM glycine–HCl, pH 3.0. After exhaustive washing (10 bed volumes), the active material was eluted (at a flow rate of 1 mL/min) with 50 mM Tris–HCl, pH 9.2 [27]. Tubes of 1 mL were collected, and the absorbance at 280 nm was monitored. The fractions with higher absorbance were pooled and denominated as aspartic protease from Aspergillus awamori extracellular fraction (ASPA), which was stored at − 4 °C for further analysis.

Protein measurement

The protein content of the fraction and the purified enzyme were measured by Bradford’s method [28] using bovine serum albumin as standard.

Polyacrylamide gel electrophoresis and substrate gel electrophoresis

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the method of Laemmli [29]. The gel solution was composed of acrylamide: running gel with 12% (v/v) and stacking gel with 4% (v/v). Samples from A. awamori were heated for 5 min at 100 °C with sample buffer (62.5 mM Tris–HCl pH 6.8, 2.5% SDS, 0.002% bromophenol blue, 5% β-mercaptoethanol, and 10% glycerol). About 20 μg of protein was applied into each well from the solidified gel, and 100 V was applied. Subsequently, the gels were stained with Coomassie Blue R-250, and the molecular weight was determined using Bio-Rad’s Precision Plus Protein™ standards as molecular mass standards. Gelatin substrate gel electrophoresis was conducted under reducing and nonreducing conditions, and the protocol was similar to the previously described protocol, however, the sample was not heated. After electrophoresis, the gel was not stained, but it was washed with 2.5% Triton X-100 for 1 h to remove SDS and incubated overnight at room temperature in 50 mM sodium citrate pH 5.5 to allow proteolysis. The next day, the gels were stained with 0.1% amide black and distained using methanol/acetic acid/distilled water (3:1:6) [30]. Gelatinolytic activities were detected by clear zones indicating the degradation of the substrate and quantified by measuring the area intensity of indicated band subtracted identical background area intensity of the same lane. The densitometric analysis was performed using Image J (Public Domain).

Protease activity assays

Enzyme assays and determination of kinetic parameters

Chromogenic substrate using Nα-p-Tosyl-L-arginine methyl ester hydrochloride (L-TAME) (125 µM), purchased from Sigma, which is a peptidomimetic that has arginine at the P1 site, was digested in different buffers at room temperature. After the addition of the enzyme, the extracellular fraction (10 μg) or the ASPA (1 μg) protein, the substrate digestion was followed by measuring the absorbance increase at 247 nm, and the specific activity was expressed as mM.min−1.mg of protein−1. [30] The steady-state parameter Michaelis constant (KM) was determined from initial velocity measurements at 8 different substrate concentrations (0.01 to 1.00 mM). Plots of velocity vs. substrate concentration were used to determine KM values through nonlinear regression (Prism, version 3.0 GraphPad Software, San Diego, CA, USA).

Influence of pH on enzyme activity

For the pH dependency assays, the extracellular fraction (10 μg) or the ASPA (1 μg) was incubated for 15 min at room temperature with 0.1 µg/mL casein and gelatin with different buffers. The buffers used were 50 mM sodium citrate (pH 4.0–6.5), 50 mM Tris/HCl (pH 7.0–8.5), and 50 mM carbonate/bicarbonate (pH 9.0–10.0). The hydrolysis of the substrate was measured by the increase of absorbance at 280 nm and expressed as described before [31]. All data represent the average and standard error of the mean of three independent experiments performed in quadruplicates.

Influence of temperature on enzyme activity

To determine the effect of the temperature on enzyme activity, the extracellular fraction (10 µg) was incubated in buffers at pH 5.5, 6.5, and 9.0, and the ASPA (1 µg) in 50 mM sodium citrate, pH 5.5, for 30 min at different temperatures ranging from 20 to 70 °C using casein (0.1 µg/mL) and L-TAME (125 µM) as substrates. Hydrolysis of the substrates was measured as described before, and all data represent the average and standard error of the mean of three independent experiments performed in quadruplicates.

For thermal stability assays, the extracellular fraction (10 µg) was previously incubated in buffers at pH 5.5, 6.5, and 9.0, and the ASPA (1 µg) in 50 mM sodium citrate pH 5.5 at 60 °C for up to 24 h. The reaction was triggered by adding L-TAME (125 µM). The residual activity was calculated by determining the protease activity at 25 °C without previous incubation as 100%. All data represent the average and standard error of the mean of three independent experiments performed in quadruplicates.

Protease assays for protein substrates

Protein substrates hemoglobin (100 µg/mL), bovine serum albumin (BSA) (100 µg/mL), gelatin (100 µg/mL), and casein (50 µg/mL) were dissolved in 50 mM sodium citrate at a final volume of 500 μL and incubated with gentle agitation for 30 min at room temperature with 1 μg of ASPA. The reactions were stopped by the addition of 500 μL of 10% (v/v) trichloroacetic acid. The tubes were centrifuged (10.000 × g for 10 min at 4 °C), and the absorbance of the supernatants was measured at 280 nm. One unit of enzymatic activity was defined as the amount of enzyme required to cause an increase of 0.1 in the absorbance under standard conditions [32]. All data represent the average and standard error of the mean of three independent experiments performed in quadruplicates.

Effect of protease inhibitors

The type of protease was studied using specific inhibitors for protease classes, using casein (0.1 µg/mL) and L-TAME (125 µM) as substrates. Different concentrations of inhibitors—ethylenediaminetetraacetic acid (EDTA), L-trans-epoxysuccinyleucylamido-(4-guanidino) butane (E-64), pepstatin, benzamidine, iodoacetamide, phenanthroline, and phenylmethysulfonyl fluoride (PMSF)—were incubated with 10 µg of the extracellular fraction proteins and 1 μg of ASPA for 30 min at room temperature. The reaction commenced upon the addition of the substrates in 50 mM sodium citrate at room temperature for 15 min, and the activity was measured as described in the previous section. Appropriate controls were carried out in parallel using the same enzyme solutions free of inhibitors. Inhibition was expressed as the percentage of the appropriate control activity [33]. All data represent the average and standard error of the mean of three independent experiments performed in quadruplicates.

IC50 is the inhibitor concentration required for 50% inhibition of enzyme activity. Inhibition constants (Ki) is the dissociation equilibrium constant of the enzyme-inhibitor complex, in other words, Ki is the inhibitor amount needed to tie up half the free enzyme, and both values can be used as quantitative indexes for the inhibitor potency and is important in the characterization of a protease. [30] To study these parameters, ASPA (1 μg) was incubated with pepstatin at 8 different concentrations (0.01 to 10 µM). Plots of inhibition activity (%) vs inhibitor concentrations (µM) were applied to determine IC50 and Ki values through nonlinear regression using the Lineweaver–Burk equation (Prism, version 5.0 GraphPad Software, San Diego, CA, USA).

Results and discussion

Characterization of protease activity from A. awamori extracellular fraction

Preliminary characterization of the extracellular fraction from A. awamori was performed using a sample from the 7th day of culture in a modified Breccia medium because the most pronounced activity of cellulosic enzymes by this fungus took place around that day [34]. Moreover, the viability of A. awamori decreases beginning on the 8th day of cultivation, and enzyme production dramatically reduces [35]. The expression of both cellulolytic and proteolytic enzymes may occur at the same time for nutritional reasons, to obtain glucose and amino acids from polysaccharides and proteins from the extracellular medium. Protein measurement showed that A. awamori extracellular fraction presented about 260 ± 15 µg of protein/mg of lyophilized fraction (212 mL) and peptidase activity about 3.87 ± 0.52 (µM.min−1.mg of protein−1) × 10−6 using L-TAME as substrate.

The extracellular fraction of A. awamori was analyzed by SDS-PAGE 12% under nonreducing (Fig. 1 A and D) and reducing (Fig. 1 B) conditions. As expected, the extracellular fraction exhibited many proteins with different molecular weights. The sample from the nonreducing condition gel (Fig. 1 A) had lower quality and homogeneity than the reducing gel because the reducing agent β-mercaptoethanol slowed down the dispersion of protein chains [29] (Fig. 1 B); however, both gels had similar protein profiles. Major proteins of 125 and 60 kDa were found only under nonreducing conditions; likely they are constituted of multiple polypeptide chains that were decomposed by β-mercaptoethanol generating proteins with lower molecular weights. The densitometry of gel proved, as visually observed, that the major proteins had about 104 and 44 kDa, which correspond to 20 and 27%, respectively, of the total of proteins present in the gel (Fig. 1 C). The secreted extracellular fraction of A. awamori presented expressive gelatinolytic activity during the electrophoresis, and the clear band at about 75 kDa refers to an estimated molecular weight of active protease (Fig. 1 D).

Fig. 1.

Fig. 1

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SDS-PAGE of A. awamori extracellular fraction. A 12% under nonreducing conditions; B 12% under reducing conditions and C respective densitometry analysis; D gelatin zymogram 10% under the nonreducing condition

The pH value may cause ionization of amino acid residues of the enzymes that are essential for catalysis, thus influencing the enzymatic activity [36]. This information about the protease classes is also valuable because these enzymes have maximal activity in a particular pH range [37]. In Fig. 2 A, using casein as substrate, the maximal protease activity was observed at 5.5, 6.5, and 9.0 pH values. When gelatin was used as the substrate (Fig. 2 B), maximal activity changed to 6.5, 7.0, and 9.0 pH values. These results could suggest the presence of aspartic, cysteine, and serine proteases in the extracellular fraction of A. awamori culture because they act in acid (5.5), slightly acid (6.5), and neutral to alkaline pH (7.0 and 9.0), respectively. [37] The activity of aspartic protease had already been reported in A. awamori, with maximal activity at pH 5.0 [38], and in other species of Aspergillus, such as A. fumigatus, [39] A. foetidus, [40] A. terreus, [41] A. niger, [42] and A. oryzae, [12] with optimum pH of 4.2 to 5.0, 5.0, 5.0, 3.5, and 5.0 to 5.5, respectively. However, the present work is the first report about the serine protease activity in the extracellular medium of A. awamori, suggesting that this type of protease is also secreted by this fungus.

Fig. 2.

Fig. 2

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The pH and temperature influence on protease activity of A. awamori extracellular fraction. pH effects using casein (A) and gelatin (B) as substrates; temperature effects using L-TAME (C) and casein (D) as substrates in distinct pH values; protease activity stability of A. awamori extracellular fraction after 24 h at 60 °C in 5.5, 6.5, and 9.0 pH values. All data represent the average and standard error of the mean of three independent experiments performed in quadruplicates

Temperature is another important factor that affects the velocity of the reactions catalyzed by enzymes. The reaction rate improves as temperature rises due to the increment of the kinetic energy of the molecules. However, if this energy increases, it can break the hydrogen and hydrophobic bonds that maintain protein tridimensional structure, leading to denaturation [43]. The effect of temperature on an extracellular fraction of A. awamori for peptidase and protease activity was evaluated using L-TAME and casein as substrates. When L-TAME was employed at pH 5.5, 6.5, and 9.0, peptidase activity was similar and independent of pH value, with maximal at 50 °C, followed by a decrease in activity at 60 °C, possibly because of enzyme denaturation (Fig. 2 C). With casein as substrate, different patterns were observed: at pH 5.5, the activity increased until 40 °C; at pH 6.5, the maximal activity was at 30 °C; and at pH 9.0, maximal activities were at 20 and 50 °C (Fig. 2 D). These results could be due to the heterogeneity of proteases present in A. awamori extracellular fraction. Acid proteases from other Aspergillus species exhibited similar temperature values in which the protease activity was maximally ranging from 30 to 60 °C. [12, 38, 40–42] Furthermore, protease activity of this A. awamori fraction was almost completely preserved (92 to 97%) in all pH values analyzed, after 24 h at 60 °C, without substrates, demonstrating important thermal stability of these enzymes (Fig. 2 E).

Specific inhibitors from different protease classes were employed to study the types of proteolytic enzymes in A. awamori extracellular fraction (Table 2). The most important inhibition was observed using pepstatin (45.2%) at pH 5.5. Pepstatin, produced by many species of Actinomyces, is a hexapeptide that contains statine (Iva-Val-Val-Sta-Ala-Sta) and is a very potent inhibitor of pepsin-like aspartic proteases [44]. These results suggest the presence of aspartic protease activity in this fraction. The inhibition of 16.1% at pH 9.0 using PMSF and using benzamidine (12.8%), both serine protease inhibitors, suggested the presence of serine protease activity secreted by A. awamori. Evidence of serine proteases has not been reported in A. awamori; however, these activities were found in A. fumigattus,[45] A. tamarii, [46] and A. oryzae. [12, 13].

Table 2.

Residual activity of proteases from A. awamori extracellular fraction using specific protease inhibitors in distinct pH values

Inhibition (%)
Inhibitor [] Inhibitor class pH 5.5 pH 6.5 pH 9.0
Benzamidine 1 mM Serine 0.4 ± 0.0 12.8 ± 0.2 4.0 ± 0.8
PMSF 1 mM Serine 0.0 ± 0.0 0.0 ± 0.0 16.1 ± 1.6
Iodocetamide 1 mM Serine/cystein 3.1 ± 0.1 4.6 ± 0.6 0.0 ± 0.0
E-64 1 μM Cystein 2.1 ± 0.1 4.9 ± 0.9 4.2 ± 0.2
Phenantroline 10 mM Metallo 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
Pepstatin 1 μM Aspartic 45.2 ± 2.78 27.9 ± 1.6 37.7 ± 3.7
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Study of protease activity during A. awamori cultivation

The next step of this work was to study the protease activity in the A. awamori extracellular fraction during fungus cultivation. Figure 3 A illustrates that the highest protein amount occurred on the 6th day, and the proteolytic activity against casein was maximal on the 4th and 7th days (Fig. 3 B). The progression of activity from secreted proteases against L-TAME demonstrated that the 7th day had the best activity because of the major activity variation (Fig. 3 C). In addition, SDS-PAGE 12% (Fig. 3 D) reveals major expression of the protein on the 7th day of cultivation, which is reinforced by densitometry (Fig. 3 E), confirming that the maximum protein secretion and protease activity by A. awamori occurred on the 7th day of cultivation.

Fig. 3.

Fig. 3

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Protein amount from day 0 to the 7th day of cultivation (A), proteolytic activity against casein (B), activity progression of A. awamori extracellular fraction using L-TAME as substrate (C), SDS-PAGE 12% under reducing condition (D), and each bar corresponding the sum of densitometric areas from all proteins on each day of culture (E)

Effect of yeast extract concentration and nitrogen sources of modified Breccia medium for protease production

The composition change of the modified Breccia medium was proposed to verify if the alterations would interfere with protease activity and protein expression of A. awamori extracellular fraction. Nitrogen is present in the composition of amino acids, proteins, and other substances for A. awamori growth, such as vitamins, purins, and pyrimidines [47]. Thus, the variation of the nitrogen source concentration (the yeast extract) could modify the culture supernatant characteristics, such as the pH, and as a consequence, vary the enzymatic activity of the culture supernatant. The pH analysis of culture supernatant was crucial because pH interferes with the activity of secreted enzymes. [48].

In the new versions of modified Breccia media, no buffers were used that prevent sudden pH change during the A. awamori cultivation. The pH on day 7 for each yeast extract concentration assayed was measured, and an increment of alkaline pH value near 8.75 was observed using 14.0 g/L of yeast extract; however, for the media with other yeast extract concentrations, the final pH value was about 6.5 to 7.0 (Fig. 4 A). Aspergillus species are generally more tolerant to alkaline pH than acidic pH [49]. In addition, the amount of protein was directly correlated to the concentration of yeast extract (Fig. 4 B). Peptidase activity against L-TAME (Fig. 4 C) and protease activity against casein (Fig. 4 D) was studied, and the maximum activity was noted at 14.0 and 12.0 g/L, respectively, which demonstrates the possible heterogeneity of proteases in the extracellular fraction of A. awamori depends on substrate. SDS-PAGE 10% containing gelatin demonstrated maximum proteolysis from A. awamori extracellular fraction using a new modified Breccia medium with 14.0 g/L of yeast extract (Fig. 5). Therefore, the yeast extract concentration chosen for medium preparation was 14.0 g/L because the highest protease activity was obtained.

Fig. 4.

Fig. 4

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pH variation (A), protein amount (B), activity against L-TAME (C), and casein (D) of cultivation medium according to yeast extract concentration. The samples were collected on the 7th day of cultivation. All data represent the average and standard error of the mean of two independent experiments performed in quadruplicates

Fig. 5.

Fig. 5

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Gelatin zymogram 10% of A. awamori extracellular fraction using different concentrations of yeast extract. The samples were collected on the 7th day of cultivation. Molecular weight markers (A); media yeast extract concentrations: 8 g/L (B), 10 g/L (C), 12 g/L (D), 14 g/L (E), and 16 g/L (F)

As nitrogen is essential for the growth and propagation of fungi,[50] changes in nitrogen sources were proposed to improve the protease production of A. awamori. Two organic (urea and yeast extract) and two inorganics (sodium nitrate and ammonium sulfate) sources were chosen according to the previous report [16]. Fig. 6 A shows that the protein secretion by A. awamori was maximal when organic sources were supplemented with a modified Breccia medium. The most expressive peptidase activity against L-TAME was observed using yeast extract and ammonium sulfate (Fig. 6 B); however, when casein was employed as the substrate, the maximal protease activity was obtained with supplementation of ammonium sulfate, followed by yeast extract (Fig. 6 C). Therefore, the YeAs-Breccia medium adopted for A. awamori cultivation had: 14.0 g/L of yeast extract and 2.7 g/L of ammonium sulfate, similar to the modified Breccia medium; however, it did not have potassium phosphate monobasic (KH2PO4) and potassium phosphate dibasic (K2HPO4), which work as a buffer.

Fig. 6.

Fig. 6

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Protein secretion by A. awamori using organic (yeast extract and urea) and inorganic (sodium nitrate and ammonium sulfate) sources to prepare modified Breccia medium (A), activity against L-TAME (B), and activity against casein (C). The samples were collected on the 7th day of cultivation. All data represent the average and standard error of the mean of two independent experiments performed in quadruplicates

Isolation and characterization of aspartic protease from A. awamori

The YeAs-Breccia medium was employed to isolate acidic proteases from the culture supernatant of A. awamori, which was performed using affinity chromatography. The overall purification of approximately 5.5-fold yielded 84% of aspartic protease obtained from 1.4 mg of proteins from A. awamori extracellular fraction (Table 3). Figure 7 illustrates the purification profile of aspartic protease using the pepstatin-agarose column, showing a single signal with maximal absorbance of about 1.0 in 280 nm, which is typical of affinity chromatography. Fraction 4, with maximal absorbance, was submitted to SDS-PAGE analysis under nonreducing conditions, and the one step of affinity chromatography produced homogeneous material with approximately 75 kDa (Fig. 7, insert B). These results indicate that aspartic protease isolated by pepstatin-agarose differed in molecular weight from the previous 45 kDa A. awamori aspartic protease,[38] and aspartic proteases reported from other Aspergillus species, such as the 39 kDa of A. fumigatus, [39] the 23.8 kDa of A. terreus, [41] and the 50 kDa of A. niger. [42] The novel aspartic protease from A. awamori with a high affinity for pepstatin was denominated ASPA.

Table 3.

Purification of aspartic proteases from Aspergillus awamori

Total protein (mg) Enzyme activity (µmol.min−1) Specific activity (µmol.min−1.mg protein−1) Purification (-fold) Yield (%)
Extracellular fraction 1.40 5.02 × 10−2 0.23 - 100.00%
Affinity column 0.13 4.23 × 10−2 1.26 5.48 84.26%
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Fig. 7.

Fig. 7

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Purification profile of the A. awamori aspartic protease in the Pepstatin-affinity column and the SDS-PAGE analysis. A Molecular weight; B ASPA

Effect of pH and temperature in ASPA

The pH dependence of ASPA activity, using L-TAME as substrate, demonstrated maximal enzyme activities around pH 4.5 and 6.5, which is a pH range typical of acidic proteases [51], such as aspartic type proteases (Fig. 8 A).

Fig. 8.

Fig. 8

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Effect of pH and temperature on ASPA activity. Maximal pH (A) and temperature (B) of ASPA using pH 4.5 and temperatures ranging from 20 to 80 °C. C Thermal stability using A. awamori extracellular fraction and ASPA at 60 °C for 24 h at pH 4.5. All data represent the average and standard error of the mean of three independent experiments performed in quadruplicates, using L-TAME as substrate

The optimum temperature profile of ASPA was analyzed using pH 4.5 and temperatures ranging from 20 to 80 °C. ASPA displayed maximal activity at 50 °C and preserved more than 70% of its activity at 60 °C (Fig. 8 B). As discussed before, aspartic proteases from other Aspergillus had maximum activity at 30 °C,[41] 55 °C, [12, 38, 40] and 60 °C for A. niger. [12, 42].

The thermal stability was studied using the extracellular fraction of A. awamori and ASPA for comparison, in which both were pre-incubated at 60 °C for 24 h with buffer at pH 5.5 and 4.5, respectively. The protease activity from the A. awamori fraction was almost completely preserved (~ 93%), whereas the isolated protease showed residual activity of 72%, in other words, ASPA lost about 28% of activity, and the extracellular fraction only lost ~ 7% (Fig. 8 C). A. awamori might produce and secrete to the extracellular environment some compounds, such as quercetin, ascorbic acid, and kaempferol, present in the extracellular fraction, and have some effects in the preservation of protein structure [52]. The acidic protease activity from extracellular fraction and ASPA showed high thermal stability crucial for their employment in some biotechnological procedures. [6]

Effect of commercial protease inhibitors in ASPA

The mechanism of enzymatic catalysis was studied using specific protease inhibitors, such as EDTA, benzamidine, pepstatin, and E-64. EDTA is a chelating agent that sequesters metal ions and interferes with metalloprotease activity;[53] benzamidine is a reversible inhibitor of trypsin, trypsin-like enzymes, and serine proteases; [54] pepstatin is a specific inhibitor to aspartic proteases; E-64 is a cysteine protease inhibitor that was isolated from the fungus Aspergillus japonicus. [55] EDTA and benzamidine did not affect the activity of ASPA. As expected, pepstatin inhibited about 80% of ASPA activity; however, E-64 decreased the ASPA activity by about 17% (Fig. 9 A). The inhibitory effect of pepstatin concentration on ASPA demonstrated IC50 and Ki values of 0.1540 and 0.072 μM, respectively, which were calculated according to the inhibition activity vs inhibitor concentrations curve (Fig. 9 B), and are in accordance with the range of Ki values observed for other aspartic proteases from Aspergillus species [12, 41, 42]. These results support that ASPA is an aspartic protease secreted by A. awamori that exhibited sensibility to E-64, possibly because this cysteine protease inhibitor irreversibly binds to a thiol group of cysteine residues near active sites of certain proteases [56]. Although cysteine protease activity has not yet been reported in any species of the genus Aspergillus, some aspartic proteases were insensitive to pepstatin. [57, 58].

Fig. 9.

Fig. 9

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Effect of protease inhibitors on ASPA activity (A) and Ki e IC50 determination of pepstatin on ASPA activity (B). All assays employed L-TAME as substrate. All data represent the average and standard error of the mean of three independent experiments performed in quadruplicates

Substrate specificity of ASPA

ASPA exhibited important protease activity when it was assayed using gelatin, BSA, hemoglobin, and casein as substrates. The highest enzymatic activity was observed using gelatin, followed by BSA, hemoglobin, and casein (Fig. 10 A), suggesting that ASPA can participate in processing important proteins for food and pharmaceutical industries [59]. The peptidase activity of ASPA was studied using the peptidomimetic substrate, L-TAME. Although it has an arginine residue at the P1 site and is more specific for serine proteases, L-TAME is an ester substrate and can be hydrolyzed by aspartic proteases because all proteases are able to break this kind of bond, as well as the peptide bonds. The Michaelis–Menten equation was used to evaluate the parameters, Michaelis constant (KM) and maximum velocity (Vmax), for L-TAME and ASPA. KM is the substrate concentration [S] responsible for half of the Vmax and is related to first-order kinetics and the affinity of the enzyme (E) for the S. On the other hand, when the reaction is at Vmax, [S] is much larger than [E], and the enzyme is saturated with the substrate [47]; thus, the reaction is zero-order kinetics [60]. According to Fig. 10 B, ASPA presented KM = 0.0589 mM and Vmax = 1.909 mM.min−1.mg of protein−1, using L-TAME as substrate. As expected, serine proteases presented lower KM values for this substrate because it is more specific for this type of protease. [32].

Fig. 10.

Fig. 10

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Substrate specificity of ASPA on proteins (A) and KM determination using L-TAME as substrate (B). All data represent the average and standard error of the mean of three independent experiments performed in quadruplicates, using L-TAME as substrate

Conclusion

The preliminary characterization of the extracellular fraction of A. awamori indicated mostly the aspartic protease activity with important thermal stability with maximal activity on the 7th day of cultivation. The supplementation of the culture medium with an organic source rich in protein increased the secretion of this acidic protease activity. The 75 kDa aspartic protease was isolated from an extracellular fraction of A. awamori, ASPA, which exhibited distinct biochemical and kinetic characteristics from the aspartic protease of A. awamori previously, reported. When the fungus was subjected to distinct cultivation conditions, it could have expressed an aspartic protease more adapted to the new conditions. In addition, the simple, inexpensive, and high yield isolation strategy employed in the present work was very different from that previously described, and as a result, a new protease was isolated with expressive biotechnology potential to be employed in many industrial processes, such as pharmaceutical preparations as an acidic therapeutic protease.

Acknowledgements

The authors are thankful to Sharon de Queiroz Silva from Bioethanol Laboratory for assistance with the fungus cultivation. This paper was revised by Ms. Patrícia Fernandes Ferreira and Dr. Maria Antonieta Ferrara, researchers at the Department of Natural Products from FIOCRUZ.

Funding

This study was supported by National Council for Scientific and Technological Development (CNPq-grant number 490029/2009–4) and the Oswaldo Cruz Foundation (FIOCRUZ, PROEP CNPq/ FARMANGUINHOS-407839/2017–8).

Declarations

Conflict of interest

The authors declare no competing interests.

Footnotes

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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