Purification and Characterization of a Protease Using Aspergillus oryzae Under Submerged Fermentation Using Dairy By-Products as a Substrate

Abstract

Whey, a large-scale dairy industry by-product, can be converted into whey protein concentrate (WPC), providing a cost-effective nutrient-rich substrate for microbial fermentation. We investigated protease production by Aspergillus oryzae using WPC as the sole substrate in submerged fermentation. Following fermentation, the protease was purified sequentially from the crude extract by salting-out, which yielded a substantial purification factor (~39), and subsequent ion-exchange chromatography. The non-adsorbed chromatographic fraction showed the highest protease activity (92.6 U/mL) and revealed one main protein band ~45 kDa via SDS-PAGE. Enzyme characterization demonstrated activity across neutral-to-alkaline conditions, optimal at pH 9.0 and 37 °C, with stability maintained between 30 °C and 37 °C. The enzyme was classified as a serine protease based on strong inhibition by PMSF and SDS; its activity was also inhibited by Zn²⁺, Mg²⁺, and K⁺, but enhanced by Ca²⁺. This work validates WPC as an efficient substrate for protease production by A. oryzae and presents a promising strategy for valorizing industrial by-products through sustainable biotechnology.

1. Introduction

The use of enzymes in industrial processes has become indispensable in recent years, mainly due to their sustainable nature, low energy demand, and high cost-effectiveness, making them a more efficient alternative to conventional chemical agents [1]. Among the main enzymes of industrial interest, proteases stand out, possessing a wide range of applications, including food processing, the pharmaceutical industry, and medicinal applications [2].

Currently, the demand for industrial enzymes is predominantly met by proteases from microbial sources, as they are considered more economical and efficient compared to plant and animal sources [3]. In this context, filamentous fungi, particularly those of the Aspergillus genus, which are recognized by the Food and Drug Administration (FDA) as “Generally Recognized as Safe” (GRAS) [4], have gained great prominence in protease production due to characteristics such as the ease of handling these organisms, rapid growth in simple and inexpensive culture media, and extracellular enzyme production, which facilitates their extraction [5].

Additionally, the efficiency of enzymatic production is also directly related to the chemical composition of the raw material used as a substrate and its accessibility, being crucial factors in selecting economically viable alternatives to reduce process costs [6]. Thus, the use of industrial residues for enzyme production has proven to be an economically sustainable alternative, aligning with the principles of the circular economy [7].

Whey, a by-product of cheese production, is rich in lactose, proteins, and minerals, making it an attractive substrate for microbial fermentation and enzyme production. Its composition not only provides essential nutrients for microbial growth but also offers an eco-friendly solution to valorize dairy waste, reducing environmental pollution and promoting resource efficiency [7]. Aspergillus niger and Aspergillus terreus have been extensively studied for protease production using whey. These fungi show significant proteolytic activity, with specific activities of 179.0 and 294.7 U mg⁻¹ protein, respectively, after purification. The production is influenced by nitrogen sources and cosupplements, with yeast extract and certain amino acids enhancing protease yield. Aspergillus ochraceus and Aspergillus flavus also demonstrate strong proteolytic activities, particularly in high-salt conditions, which is beneficial for certain industrial applications [8]. The utilization of whey as a substrate for protease production aligns with the growing interest in sustainable bioprocesses, as it transforms a waste product into a valuable resource. Furthermore, the application of whey in enzyme production can significantly reduce the overall costs of industrial processes, making it an economically viable option for large-scale applications. This approach not only addresses waste management challenges in the dairy industry but also contributes to the development of greener and more sustainable industrial practices [6].

Among industrial residues with high biotechnological potential, whey stands out as a by-product generated on a large scale by the dairy industry. This sector holds an important place in the food industry, and consequently, the generation of waste from its processes increases proportionally to its growth [9]. Therefore, one of the challenges of this industry is the proper management of its by-products, especially whey derived from cheese production, as a significant portion of this material is still improperly discarded, potentially causing significant environmental impacts [10].

This work presents an innovative approach by utilizing cheese production waste from an industry in Garanhuns, Agreste region of Pernambuco, Brazil, for the production of a protease enzyme of high biotechnological interest. The study not only explores an efficient method for valorising dairy by-products but also demonstrates how local industrial waste can be transformed into a valuable resource for enzyme production. For companies in the sector, this technique offers a dual advantage: it adds economic value to a previously discarded by-product while significantly reducing environmental impact, as improper whey disposal poses serious ecological risks, including water pollution and soil degradation. By integrating sustainable bioprocesses into dairy waste management, this research contributes to circular economic practices, providing industries with a cost-effective and eco-friendly alternative that aligns with global sustainability goals.

2. Results

2.1. Production and Purification of Proteases Using Whey Concentrate

Optimized production conditions (pH, temperature, substrate concentration), guided by previous studies, yielded a crude extract with a maximum protease activity of 144.64 U/mL. Due to this high activity, purification techniques were implemented to isolate a pure enzyme with high specific activity, as summarized in

Table 1. Purification steps of the protease produced by A. oryzae.

Purification Steps	Volume (mL)	TP (mg)	Pa (U/mL)	SPa (U/mg)	Y (%)	PF
Crude Extract	100	410.83	144.64	0.35	100.00	1.00
Acetone	10	16.80	141.31	8.40	4.09	23.88
Etanol	10	12.37	134.71	10.88	3.01	30.92
Salting out	10	9.44	132.28	14.00	2.29	39.77
HiTrap ANX FF	1.0	0.116	92.60	795.98	0.028	2260.8

TP—total protein; P_a—total protease activity; SP_a—specific protease activity; Y—yield; PF—purification factor.

. Each purification step was evaluated for yield and purification factor, confirming efficient protease separation. Comparing precipitation methods, organic solvents (acetone and ethanol) resulted in recovery percentages of approximately 3–4%, with ethanol achieving a higher purification factor (31) than acetone (24). Although the salting-out method yielded a lower recovery (around 2%), it demonstrated a superior purification factor (39.77) while maintaining protease activity comparable to the solvent precipitation methods.

The application of ion exchange chromatography in enzyme purification, particularly for proteases, is a well-documented process that leverages the differential adsorption of proteins based on their charge properties. In the given scenario, the protease activity was primarily concentrated in the unadsorbed fraction (P1) with 92.6 U/mL, while the adsorbed fraction (P2) showed significantly lower activity at 7.11 U/mL as demonstrated in Figure 1.

Polyacrylamide gel electrophoresis (SDS-PAGE) of the non-adsorbed fraction obtained from ion exchange chromatography revealed a distinct band with a molecular weight between approximately 43 and 45 kDa, as shown in Figure 2.

2.2. Evaluation of Enzyme Functional Properties

2.2.1. Effect of pH on Enzymatic Activity

The protease produced by A. oryzae demonstrated optimal enzymatic activity under alkaline conditions, with the highest activity observed at pH 9.0 (Figure 3). The enzyme’s activity significantly decreased outside the pH range of 7.0 to 9.0, suggesting that it is most stable and effective in neutral or basic environments. This behavior highlights the protease’s potential for industrial applications where alkaline conditions are prevalent.

2.2.2. Effect of Temperature on Enzymatic Activity and Stability

Figure 4 presents the results regarding the effect of temperature on the activity and stability of the enzyme produced by A. oryzae. The temperature of maximum protease activity was observed to be 37 °C (Figure 4A). Regarding thermal stability (Figure 4B), a decline in enzymatic activity was observed after the first 15 min of incubation at 60 °C and 70 °C, with enzymatic activity reducing by approximately 89% and 55%, respectively, compared to the initial activity, retaining less than 20% of its activity after 30 min of incubation at these temperatures. At lower temperatures, the enzyme remained stable, except at 25 °C, where a reduction of about 31% in relation to the initial activity was observed after 30 min. After 60 min of incubation at 40 °C and 50 °C, slight reductions of approximately 10% and 13% in protease activity, respectively, were noted, while at 30 °C and 37 °C, the enzymatic activity remained stable throughout the entire incubation period.

2.2.3. Effect of Inhibitors, Ions, and Surfactants on Enzymatic Activity

Table 2. Effect of ions, inhibitors, and surfactants on the proteolytic activity of the enzyme produced by A. oryzae.

Residual Activity (%)
Íons		Inhibitors		Surfactants (%, v/v)
	20 mM		10 mM		0.5%	1%	2%
Zn2+	66.8	EDTA	82.4	Tween 20	96.5	96.8	94.9
Ca2+	103.9	Pepstatin A	96.2	Tween 80	90.6	91.2	96.9
Mg2+	61.6	PMSF	36.4	Triton X100	91.8	96.2	97.7
Na+	96.4	Iodoacetic Acid	87.6	SDS	55.6	26.0	16.8
K+	62.5	β-Mercapto	100.1
Ba2+	64.6
Fe2+	61.3
Cu2+	68.2
Control	100%

summarizes the action of different substances on the proteolytic activity of the enzyme produced by A. oryzae. Regarding the action of inhibitors, PMSF induced a reduction of approximately 64% in enzymatic activity, while EDTA and iodoacetic acid promoted reductions of about 18% and 13%, respectively, compared to the control. On the other hand, in the presence of β-mercaptoethanol and pepstatin A, the enzyme fully maintained its activity relative to the control.

It is known that ions can modulate enzymatic activity by binding to amino acid residues present in the active site and in the enzyme’s structure, leading to conformational changes that may induce either an increase or inhibition of activity. Regarding the influence of different ions on proteolytic activity, in the presence of Zn²⁺, Mg²⁺, K⁺, Ba²⁺, Fe²⁺, and Cu²⁺, similar reductions were observed, ranging from 32% to 38% relative to the control. On the other hand, Na⁺ and Ca²⁺ induced a reduction and an increase in proteolytic activity of about 3%, respectively.

Regarding the effect of surfactants, the protease produced in this study demonstrated high stability in the presence of non-ionic surfactants such as Triton X and Tween, maintaining about 90% of its activity compared to the control. However, it showed pronounced enzymatic inhibition in the presence of SDS (sodium dodecyl sulfate), with this effect being directly proportional to the surfactant concentration. The enzyme’s activity was reduced by approximately 45%, 74%, and 83% at concentrations of 0.5%, 1%, and 2%, respectively. Conversely, the strong inhibitory effect of SDS, an anionic surfactant, likely stems from its ability to denature proteins by disrupting hydrophobic interactions and breaking disulfide bonds, leading to a loss of enzymatic activity. This sensitivity to SDS highlights the enzyme’s vulnerability to harsh chemical environments and underscores the need for careful optimization of conditions in practical applications.

3. Discussion

3.1. Assessment of Whey-Based Protease Production and Purification

Microbial enzyme production is strongly influenced by fermentation process conditions, such as medium pH, temperature, and agitation (Suberu et al., 2019) [11]. In the production of fungal proteolytic enzymes via submerged fermentation, it is common to use pH levels near neutrality, moderate temperatures, and low substrate concentrations in the fermentation medium [12]. Based on this, similar conditions to those reported in the literature were applied, leading to significant protease activity.

Our results demonstrate the potential of using whey protein concentrate as the sole substrate in the fermentation medium. However, other studies have used whey only as a supplement for enzyme production. Kumura et al. [13] reported protease activity from A. oryzae using whey-supplemented media, while Ahmad et al. [14] achieved enzyme production using Lactobacillus plantarum and Enterococcus faecium under optimized conditions. More recently, El-Gayar et al. [15] demonstrated the feasibility of using whey in combination with other components to enhance protease yield. These findings highlight the biotechnological potential of whey as a substrate for enzyme production, particularly for applications in dairy industries, where proteases play a crucial role in milk coagulation.

Given the high protease activity observed in the crude extract, various purification techniques were employed to obtain a pure enzyme with high specific activity. A comparison of different methods revealed variations in recovery rates and purification factors. Regarding organic solvent precipitation, both acetone and ethanol were tested, with ethanol exhibiting a slightly higher purification factor. The salting-out method, despite yielding a lower recovery percentage compared to organic solvents, resulted in a higher purification factor, maintaining protease activity levels comparable to other methods. The salt-precipitated fraction was further purified using ion-exchange chromatography, where protease activity was predominantly concentrated in the non-adsorbed fraction, while the adsorbed fraction retained lower enzymatic activity (Figure 1).

The degree of enzyme purification correlates with the requirements of various applications in which proteases can be utilized. Despite the lower recovery percentage, ion-exchange chromatography provided a highly purified enzyme, suitable for applications demanding high purity levels, such as healthcare and pharmaceutical industries. In contrast, precipitation, while achieving higher recovery rates, does not constitute an efficient method for obtaining analytically pure enzymes. Nevertheless, precipitation remains a useful step in sample preparation before high-resolution chromatography and for applications where extreme purity is not required [16].

From the non-adsorbed fraction obtained via ion-exchange chromatography, polyacrylamide gel electrophoresis (SDS-PAGE) revealed distinct protein bands within a molecular weight range comparable to those reported in previous studies, as shown in Figure 2. Santos et al. [17] described a broad molecular weight range for proteases from A. tamarii, while Silva et al. [18] identified a collagenolytic protease with a specific molecular weight from the same fungal strain.

3.2. Functional Characterization of Enzymatic Properties

3.2.1. pH-Dependent Activity Profile of the Enzyme

The pH of the medium can influence enzymatic functions in various ways by altering the protonation and deprotonation states of amino acid residues at the catalytic site, due to changes in the concentration of H⁺ ions in the solution. These alterations can lead to structural modifications of the enzyme [19]. Consequently, when the pH deviates from the optimal value, a decrease in enzymatic activity is observed, potentially indicating a denaturation process [19]. In this study, the protease produced by Aspergillus oryzae exhibited significant activity at neutral and alkaline pH values. Previous studies have also reported the production of slightly alkaline proteases by various fungal strains. El-Khonezy et al. [20] and Farooq et al. [21] demonstrated that proteases produced by A. ochraceus and A. oryzae exhibited maximum activity under alkaline conditions. In contrast, Mamo et al. [22] observed that proteases from A. oryzae displayed peak activity at acidic pH, suggesting that different Aspergillus species may produce enzymes with distinct optimal pH ranges.

3.2.2. Temperature-Dependent Catalytic Performance and Stability

According to the “classical model” that explains the influence of temperature on enzymatic activity, there is a directly proportional relationship between these two variables [23]. However, at high temperatures, enzymes may undergo denaturation and consequently lose their functionality, often irreversibly [24,25]. Thus, similar to pH, enzymes have a temperature range within which they can maintain their activity without structural alterations [24].

The optimal temperature for the protease produced by A. oryzae in this study was 37 °C, which aligns with the findings of Vinuthna and Raju [26], who observed an optimal temperature of 35 °C for A. oryzae NCIM 637 protease. However, Kamara et al. [27] working with A. niger and Mamo et al. [28] using a strain of A. oryzae reported optimal protease activity at 50 °C and 60 °C, respectively. The variations in both optimal pH and temperature may be related to the nature of the microorganism used, as well as the production conditions, highlighting that different strains of the same species can produce proteases with distinct characteristics.

3.2.3. Chemical Modulators of Enzyme Catalysis: Inhibitors, Ions, and Surfactants

The classification of a protease can be determined based on its response to inhibitory substances [28]. Since PMSF is a specific inhibitor of serine proteases, its action suggests that the produced protease belongs to this enzymatic class. The inhibition mechanism occurs through the sulfonation of the serine residue in the active site, preventing enzymatic catalysis [28]. This inhibitory effect has been widely reported for proteases produced by filamentous fungi [12,20,29]. The purified enzyme from Albatrellus confluens is confirmed as a serine protease due to its complete inhibition by PMSF, which sulfonates the serine residue in the active site, blocking enzymatic activity. No inhibition was observed with E-64, pepstatin, or EDTA, indicating that these substances do not affect this specific protease. The enzyme’s strong substrate specificity and optimal alkaline activity further support its classification as a serine protease, highlighting its potential for therapeutic applications [29]. However, although PMSF is a well-known marker for serine proteases, other substances can also modulate enzymatic activity [30]. Studies have shown that inhibitors such as EDTA and Pepstatin A may also interfere with the activity of certain serine proteases [12].

Ions and surfactants exert significant influence on enzymatic activity through distinct mechanisms. Ions can fine-tune protease function by directly engaging with amino acid residues within or near the active site or by inducing broader structural rearrangements. These interactions can either amplify catalytic efficiency by acting as essential cofactors that stabilize the enzyme’s active conformation or diminish it by competing for binding sites or triggering inhibitory conformational changes [19,31]. Research consistently demonstrates the diverse impact of metal ions on proteolytic activity, with some enhancing and others hindering enzyme function [12,20,28].

Surfactants, on the other hand, affect protease stability and activity primarily by interacting with the enzyme’s overall structure. The remarkable stability of this protease in the presence of non-ionic surfactants, such as Triton X and Tween, underscores its promising potential for integration into industrial applications, notably in detergent formulations. Conversely, anionic surfactants like SDS typically disrupt enzymatic activity by causing protein denaturation. This denaturation arises from the interaction of SDS molecules with the polypeptide chain, leading to structural alterations and the potential exposure or disruption of crucial active site residues [32]. The scientific literature indicates that the effect of surfactants is highly dependent on their specific chemical properties and concentration, with outcomes ranging from the preservation of activity to complete inhibition [12,28,33]. The observed high tolerance to non-ionic surfactants further bolsters the biotechnological appeal of this protease for enzymatic formulations, broadening its scope for industrial utilization.

4. Materials and Methods

4.1. Microorganism and Fermentation Medium

The Aspergillus oryzae (Ahlb.) Cohn strain (URM 8345), isolated from soil in the Caatinga region, was kindly provided by the URM-Micoteca Profa. Maria Auxiliadora Cavalcanti Collection at the Federal University of Pernambuco (UFPE). The culture was maintained at 4 °C in tubes with slanted Potato Dextrose Agar (PDA). The protease was produced by submerged fermentation using the protein concentrate obtained from whey, a by-product of cheese production. The whey used as a substrate in this study was derived from the production process of “Queijo Coalho” cheese. It was obtained from Polilac Industry, located in the city of Garanhuns, Pernambuco, Brazil. Upon receipt, the whey was stored at 4 °C until use and was employed without any prior chemical modification to maintain its natural composition for fermentation experiments. The substrate (1%, w/v) was solubilized in distilled water and subsequently autoclaved at 121 °C for 15 min for sterilization.

4.2. Submerged Fermentation

For the inoculum, fungal spores were produced in test tubes from the cultured culture, grown for 7 days at 30 °C, and then suspended in 3.0 mL of a previously sterilized solution of 0.9% NaCl (w/v) and 0.01% Tween 80 (v/v). Fermentations were carried out for 72 h at 30 °C under agitation at 150 rpm in 250 mL Erlenmeyer flasks containing 100 mL of fermentation medium, inoculated with 10⁴ spores/mL. After the process, the fermented medium was filtered through 0.45 μm nitrocellulose membranes to obtain the cell-free enzymatic extract.

4.3. Protein Assay and Proteolytic Activity

Protein content was determined using the bicinchoninic acid method with the Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, MA, USA), using bovine serum albumin (BSA) as a standard according to Smith et al. [34]. Proteolytic activity was determined by the Ginther [35] method using azocasein (1% p/v) as a substrate. An aliquot of the enzymatic extract (150 μL) was mixed with the azocasein substrate (250 μL) and incubated at 37 °C for 1 h. Afterward, the reaction was stopped by adding 1.0 mL of 10% trichloroacetic acid (TCA). Then, the reaction mixture was centrifuged (3000× g, 15 min), and the supernatant (800 μL) was homogenized with 200 μL of 1.8 M NaOH. One unit of protease activity was defined as the amount of enzyme that produces a 0.1 increase in absorbance per hour at 420 nm. Experiments were performed in triplicate (UV-1800 spectrophotometer (Shimadzu Corporation, Kyoto, Japan.

4.4. Purification

4.4.1. Precipitation

The enzymatic extract (10 mL) was subjected to pre-purification by salting out (0–60% ammonium sulfate) and organic solvents (70% acetone and ethanol) [22,36]. The protein concentrate was resuspended in 1.0 mL of 0.1 M Tris-HCl buffer (pH 7.0) for further analysis.

4.4.2. High-Resolution Chromatography Purification

Purification was carried out using the ÄKTA pure chromatography system (GE Healthcare, Chicago, IL, USA) coupled with a computerized data acquisition system (UNICORN). The precipitated extract was applied to a HiTrap ANX FF 1 mL ion exchange column (Sigma-Aldrich, St. Louis, MO, USA), equilibrated with 0.1 M Tris-HCl buffer (pH 7.0). Elution of the non-adsorbed enzymes was performed with the same buffer under a flow rate of 0.5 mL/min. The adsorbed enzymes were eluted with 1.0 M NaCl diluted with the same buffer at the same flow rate. The protein profile of the separation was assessed at a wavelength of 280 nm.

4.5. Polyacrylamide Gel Electrophoresis (SDS-PAGE)

SDS-PAGE was performed using a 10% polyacrylamide gel according to Laemmli [37]. Protein bands were detected by Coomassie Brilliant Blue staining, and destaining was performed in a solution containing water, acetic acid, and methanol (4.5:1:4.5).

4.6. Enzymatic Characterization

4.6.1. Effect of pH and Temperature on Enzymatic Activity and Stability

To investigate the influence of pH on enzymatic activity, a 1% (w/v) azocasein substrate was prepared using buffer solutions at various pH values: sodium citrate (pH 5.0 and 6.0), Tris-HCl (pH 7.0, 8.0, and 9.0), and carbonate-bicarbonate (pH 10.0). Proteolytic activity assays were conducted following the protocol outlined in Section 4.3. To determine the optimal temperature for enzymatic activity, the reaction was performed across a temperature gradient from 25 °C to 100 °C. For these assays, the 1% (w/v) azocasein substrate was solubilized in the buffer solution exhibiting the highest proteolytic activity, as determined in the optimal pH experiments. Regarding thermal stability, the enzyme was pre-incubated at temperatures ranging from 25 °C to 70 °C for specific durations (15, 30, and 60 min). Subsequently, the residual proteolytic activity was measured under the previously established optimal temperature conditions.

4.6.2. Effect of Ions, Inhibitors, and Surfactants on Proteolytic Activity

The effect of substances inhibiting enzymatic activity was evaluated using ethylenediaminetetraacetic acid (EDTA) as a metaloprotease inhibitor, phenylmethanesulfonyl fluoride (PMSF) for serine-proteases, and pepstatin A for aspartic-protease and iodoacetic acid and β-mercaptoethanol, which inhibit cysteine-protease, at a concentration of 10 mM each. The effect of different ions on enzymatic activity was evaluated using concentrations of 20 mM of the following ions: Ca²⁺, Cu²⁺, Fe²⁺, Mg²⁺, Na+, Zn²⁺, Ba²⁺, and K⁺. For surfactants, the effect of Triton X-100, Tween 20, Tween 80, and sodium dodecyl sulfate (SDS) at 0.5%, 1.0%, and 2.0% was analyzed. For all assays, each substance was kept in contact with the enzyme for 30 min at room temperature, then the proteolytic activity reaction was performed. The results were expressed in relative activity, with the control (100%) being without the addition of substances.

5. Conclusions

The production of proteases by Aspergillus oryzae using cheese whey, an industrial by-product, as the sole substrate presents a sustainable and innovative strategy for advancing industrial biotechnology. This study demonstrated the successful optimization of fermentation conditions to significantly enhance enzymatic production. The isolated protease was characterized as a serine protease with an approximate molecular weight of 45 kDa, an optimal temperature around 37 °C, and an optimal pH of 9.0, indicating its robustness and versatility. These properties make the enzyme highly attractive for industrial applications, particularly in processes requiring alkaline conditions. Furthermore, the use of cheese whey valorizes a major by-product of the cheese industry, contributing to waste reduction and adding economic value to dairy production chains. These findings open new perspectives for the development of environmentally responsible technologies and promote the sustainable use of natural resources in industrial practices.