This study reports the protease production from Aspergillus tamarii using agroindustrial residues as substrate for solid-state fermentation (SSF) and biochemical characterization. The highest protease production was obtained using wheat bran as substrate at 72 h fermentation with maximum proteolytic activity of 401.42 U/mL, collagenase of 243.0 U/mL and keratinase of 19.1 U/mL. The protease exhibited K M = 18.7 mg/mL and Vmax = 28.5 mg/mL/min. The optimal pH was 8.0 and stable in a wide pH range (5.0 - 11.0) during 24 h. The optimum temperature was 40 °C. The proteolytic activity was inhibited by Cu 2+ (33.98%) and Hg 2+ (22.69%). The enzyme was also inhibited by PMSF (65.11%), indicating that is a Serine Protease. These properties suggest that alkaline protease from A. tamarii URM4634 is suitable for application in food industries and leather processing. Additionally, the present findings opened new vistas in the utilization of wheat bran and other effective agroindustrial wastes as substrates for SSF.
Proteases catalyze the cleavage of peptide bonds in proteins, are the class of enzymes having applications in both physiological and commercial fields. Among all the different commercial enzymes, microbial protease in particular, consisting of more than 25% of biomolecules produced for industrial application and 65% of all the industrial enzymes sales in the world due to their applications in several industrial sectors like in the detergent, food, pharmaceuticals, chemicals, leather, paper and pulp and silk industries [
Proteases can be obtained from several sources, including plants, animals, and microorganisms. Even though a wide variety of microbial proteases are available, the use of these enzymes on industrial scale is still limited by their high production costs and the fact that their activity is often limited to a restricted range of biochemical characteristics [
The Solid State Fermentation (SSF) is especially suitable for the fungi growth because their moisture requirements are lower compared to the bacteria. In this technique, the enzymes produced are more concentrated than those in submerged fermentation. SSF is an inexpensive technique and can be widely applied to agricultural products or by-products as substrates. Furthermore, the substrate must be easy to handle, inexpensive and easy to purchase. The overall cost of enzyme production is very high (due to high cost of substrates and mediums used). Therefore, development of novel processes to increase the yield of proteases with respect to their industrial requirements coupled with lowering down the production cost is highly appreciable from the commercial point of view [
SSF is also a cost-effective process as it uses agroindustrial wastes, like seeds, peels, husks, bark, and bran to produce valuable bioactive molecules. Another important advantage of SSF is the higher growth rate exhibited by fungi on solid substrate as compared to submerged fermentation; the morphology of filamentous fungi allows them to colonize the substrate surface and matrix in search of nutrients, consequently secreting higher levels of metabolites and enzymes [
Filamentous fungi, such as Aspergillus spp., are explored for the production of industrial enzymes due to their ability to grow on solid or liquid substrates and having a large production of extracellular enzymes. Some strains from Aspergillus genus are considered non-toxic, recognized as a safe microorganism by the Food and Drug Administration (FDA), denominated Generally Recognized as Safe (GRAS), and used for human and animal nutrition [
In the Brazilian scenario, which is heavily focused on agriculture economy; especially soy, wheat, corn and sugarcane, the technological potential for efficient reuse of agro industrial waste can contribute to the development of high added value products such as enzymes, organic acids, flavors and fragrances, pigments, polysaccharides and hormones, adding value to this residue produced in large quantities [
In this study, we evaluated the inexpensive production of proteases from several GRAS fungal strains, using agroindustrial waste: wheat grains, Canadian lentils, amaranth flakes, soybean grains, nuggets sunflower, oat bran and wheat bran. The different residues used as substrate can modify fungi metabolic expression and produce enzymes with distinct characteristics, which can be applied in the biotechnological industry in many ways. The combination of the microbial cell and different substrates comprises metabolic and biotransformation process that can generate several cellular products. Additionally, the microorganism and substrate selection plus the biochemical characteristic of the enzyme produced are important factors to evaluate its biotechnological potential and target the possible applications for industrial processes [
The thirty-four Aspergillus strains used for screening were provided by the “Micoteca- URM” of Mycology Department, Centre of Biological Sciences of Federal University of Pernambuco (UFPE), Recife-PE, Brazil. The strain was preserved in mineral oil [
Inoculum spores were produced in Czapek Dox Agar tubes inclined containing a culture seven-days-old culture grown at 30˚C and suspended in a 3.0 mL of a solution consisting of 0.9% NaCl and 0.01% Tween 80, which was previously sterilized at 121˚C for 20 min. Aspergillus strains were inoculated (104 spores/mL) in soybean flour medium MS-2 [
After fermentation, the culture medium was centrifuged at 3000 g for 15 min at 4˚C to obtain the supernatant (enzyme crude extract). The screening of the microorganisms was performed in two steps. The first one was performed in submerged fermentation (SmF) and the second step was performed in solid-state fermentation (SSF), using different substrates.
The substrates used for screening by SSF were: Wheat grains, Wheat bran, Oat bran, Soybeans, Canadian Lentils, Flakes amaranth, Quinoa flakes and Nuggets Sunflower, obtained in the local market in the city of Garanhuns, Pernambuco, Brazil.
SSF was performed in 125mL-Erlenmeyer flasks containing 5 g of each agroindustrial substrate, nutrition solution and 107 spores/mL, corresponding to 40% moisture content. SSF was run for 72 h at 30˚C and the protease extract was obtained by addition of 7.5 mL of 0.1 M sodium phosphate (pH 7.0) per gram of fermented material and homogenized in shaker for 2 h. Solids were removed by centrifugation at 3000 g for 15 min at 4°C, and the supernatant was used as enzyme crude extract.
The experiments for protease production were performed according to a 23-full factorial design. The analyzed variables were: Substrate (3, 5 and 7 g); Moisture (30%, 40% and 50%); Temperature (25˚C, 30˚C and 35˚C). The central point runs were performed in quadruplicate to allow for pure error estimation. All graphic statistical analyses were made using the software Statistica 8.0 [
The protease activity was measured using azocasein as substrate described by Ginther [
The collagenase activity was performed using the Azocoll method described by Chavira et al. [
The Keratinase activity was assayed by Anbu et al. [
Fungal biomass estimation was carried out according to Castro et al. [
Coconut milk agar medium (CMA) were based on Lin and Dianese [
Briefly, 25 mL of YES agar was inoculated as single colonies in the center of Petri-dishes and incubated in the dark at 28˚C [
The kinetic parameters of the enzyme were determined using different concentrations of azocasein (2 ≤ S0 ≤ 100 mg/mL) for protease activity (section 2.4). All tests were carried out in triplicate and results were expressed as average values. Statistical analysis was performed using standard deviations of experimental data from the average values.
For biochemical characterization, the optimal activity and stability of enzymes at different pH and temperature, effects of metal ions and inhibitors were tested using enzymes produced in the best fermentation condition from Aspergillus tamarii URM4634 by SSF, according to 2.4 section.
The optimum pH for protease activity was determined using different buffers 0.2 M: citrate-phosphate (pH 5.0 - 7.0), Tris-HCl (pH 7.0 - 8.5) and glycine-NaOH (pH 8.5 - 11.0). The effect of pH on stability of the enzyme was verified by a previous incubation of the enzyme crude extract with above buffers at 5˚C. Aliquots were analyzed to determine residual protease activity at time intervals 0 h, 4 h, 8 h and 24 h.
The temperature effect was determined by performing the protease activity at temperatures from 5˚C to 90˚C. The temperature stability was measured by keeping the enzyme extract in the absence of substrate at temperatures from 5˚C to 90˚C. Aliquots were rapidly cooled ±25˚C, to ensure efficient refolding of the molecules of the enzyme optionally reversibly inactivated, were withdraw every 60 min to determine the residual activity at different times (0, 60, 120 and 180 min).
Protease activity was assessed in the presence of ions, as inhibitors or activators of activity. The effect of ionic solutions was evaluated at concentrations of 5 mM and 10 mM in 0.2 M Tris-HCl pH 7.2. The following ions were used: ZnSO4∙7H2O, MgSO4∙7H2O, CuSO4∙5H2O, FeSO4∙7H2O, CaCl2, HgCl2∙4H2O, KCl and NaCl, and incubated at 28˚C for 30 min. The enzyme activity without ions was considered as control (100%) and the Protease activity was determined by the method described in 2.4 Section.
To evaluate the effect of inhibitors on enzyme activity, the crude extract was exposed to the following protease inhibitors: Phenylmethylsulfonyl fluoride (10 mM), 2-merca- ptoethanol (10 mM)), Ethylenediaminetetraacetic acid (10 mM), Pepstatin A (1 mM) and Iodoacetic acid (10 mM) and were performed at 25˚C for 30 min. The enzyme activity without inhibitor was considered as control (100%).
SDS-PAGE was carried out using a 12% polyacrylamide running gel according to the method of Laemmli [
The proteolytic activity of the enzyme band was confirmed by zymogram analysis. To prepare a gelatin zymogram with 0.1% gelatin as substrate incorporated in the gel. Gels were loaded with 10 µL of concentrated supernatant, subject to electrophoresis at a constant current of 100 V at 25˚C and incubated for 30 min at room temperature with 2% (v/v) Triton X-100 and for 48h at 37˚C in 50 mMTris?HCl buffer and 15 mM CaCl2, pH 7.5. To azocasein zymogram with 0.1% azocasein as substrate incorporated in the gel. The azocasein was dissolved in Tris-HCl pH 8.8, keeping the same concentrations for performing the gelatin zymogram. Gels were finally stained and destained as described in the previous section.
Thirty-four strains of Aspergillus were evaluated among soy-based medium (MS-2) showed that all activities between 0.43 and 34.13 U/mL (
URM | Microorganisms | PA* (U/mL) | URM | Microorganisms | PA* (U/mL) |
---|---|---|---|---|---|
224 | Aspergillus terreus | 3.27 | 5741 | Aspergillus niger | 1.37 |
269 | Aspergillus heteromorphus | 15.77 | 5756 | Aspergillus niger | 2.27 |
1546 | Aspergillus carbonarius | 1.43 | 5774 | Aspergillus sydowii | 3.63 |
3266 | Aspergillus tamarii | 3.77 | 5778 | Aspergillus parasiticus | 12.87 |
3818 | Aspergillus carbonarius | 0.97 | 5787 | Aspergillus parasiticus | 21.07 |
3856 | Aspergillus niger | 2.70 | 5791 | Aspergillus flavus | 19.27 |
3916 | Aspergillus japonicas | 1.93 | 5792 | Aspergillus sclerotiorum | 14.30 |
4634 | Aspergillus tamarii | 29.90 | 5793 | Aspergillus flavus | 15.17 |
4658 | Aspergillus terreus | 7.77 | 5794 | Aspergillus flavus | 17.33 |
4924 | Aspergillus phoenicis | 10.13 | 5827 | Aspergillus melleus | 3.73 |
4953 | Aspergillus aculeatus | 1.43 | 5837 | Aspergillus niger | 17.47 |
5093 | Aspergillus terreus | 1.37 | 5838 | Aspergillus niger | 2.37 |
5182 | Aspergillus caespitosus | 0.43 | 5860 | Aspergillus sydowii | 7.43 |
5218 | Aspergillus niger | 0.73 | 5863 | Aspergillus niger | 2.17 |
5242 | Aspergillus japonicas | 3.73 | 5864 | Aspergillus terreus | 0.90 |
5701 | Aspergillus versicolor | 8.67 | 5870 | Aspergillus niveus | 0.60 |
5740 | Aspergillus flavus | 34.13 | 5895 | Aspergillus terreus var. aureus | 5.43 |
*PA-Protease activity.
Aspergillus flavus showed protease activity of 34.13 U/mL. However, this microorganism is known as mycotoxin producer such as aflatoxin B1 and B2, G1 and G2 and cyclopiazonic acid, being unfeasible its application in some biotechnological processes such as the food industry [
Aspergillus tamarii URM4634 was also selected because the high protease production compared to other microorganisms and the absence of release of aflatoxins. The constituents of CMA have an effect on fluorescent pigment production in coconut culture [
The study findings corroborate with the literature, emphasizing the potential of Aspergillus genus as a protease producer. Boer and Peralta [
In another study performed by Dhandapani et al. [
In the second step of screening, wheat grains, Canadian lentils, amaranth flakes, soybean grains, nuggets sunflower, oat bran and wheat bran were used as substrates for solid-state fermentation and evaluation of A. tamarii URM4634 growth into the agroindustrial by-products. Filamentous fungi are the most widely microorganisms used in SSF because of their ability to grow in solid substrates even in the absence of free water [
The best agroindustrial substrate for protease production in SSF was wheat bran and showed protease activity of 340 U/mL (
The best value of the protease activity was 404.67 U/mL, obtained from the average of the results of the central points (5 g wheat bran, 40% moisture at 30˚C) (
The analysis of the effects (
Nascimento et al. [
The best condition was also tested for specific proteolytic activities such as collagenase (243.0 U/mL) and keratinase (19.1 U/mL), due to their biotechnological potential. Lima et al., [
Runs | Substrate (g) | Moisture (%) | Temperature (°C) | PA* (U/mL) |
---|---|---|---|---|
1 | 3 | 30 | 25 | 353.83 |
2 | 7 | 30 | 25 | 337.00 |
3 | 3 | 50 | 25 | 353.67 |
4 | 7 | 50 | 25 | 383.17 |
5 | 3 | 30 | 35 | 312.50 |
6 | 7 | 30 | 35 | 235.17 |
7 | 3 | 50 | 35 | 251.17 |
8 | 7 | 50 | 35 | 263.67 |
9 (C) | 5 | 40 | 30 | 399.17 |
10(C) | 5 | 40 | 30 | 404.67 |
11(C) | 5 | 40 | 30 | 397.67 |
12(C) | 5 | 40 | 30 | 404.17 |
*PA―Protease Activity. (C)―Central points.
Factors | Effects |
---|---|
Substrate | −0.78 |
Moisture | 0.19 |
Temperature | −5.52* |
1 × 2 | 2.06 |
1 × 3 | −1.17 |
2 × 3 | −1.19 |
1 × 2 × 3 | 0.65 |
*Statistically significant at 95% confidence level (p < 0.05).
The evolution of fungal cellular growth in wheat bran was estimated by glucosamine level during 196 h of cultivation. A. tamarii URM4634 exhibited a maximum glucosamine level of 119.33 mg/g ± 4.8, after 96 h cultivation (
Similar values found in this study was also reported by Castro et al. [
luated different agroindustrial by-products such as wheat bran, soybean meal and cotton seed meal, showed glucosamine levels of 90.33 mg/g when grown in wheat bran, after 96 h of cultivation. Ramachandran et al. [
The KM and Vmax values were 18.7 mg/mL and 28.57 mg/mL/min, respectively. KM was related to the affinity of the enzyme toward azocasein substrate. Lineweaver-Burk plot of initial velocity of the extracellular protease from Aspergillus tamarii URM4634 is shown in
Mushtaq et al. [
Results of pH effect on enzyme activity and stability can be observed in
Myceliophthora sp., showed optimum pH 9.0 in SSF. On the other hand, the same protease and microorganism grown in submerged fermentation had its optimum pH 7.0 [
The enzyme showed 91.66% and 91.18% of its activity relative to the pH 7.0 and 8.5 in same buffer. The protease retained its activity of 82.88% and 50.28% in 0.2M Glycine-NaOH buffer pH 9.0 and 10.0, respectively. The results show that the enzyme was stable over a wide pH range (6.0 ? 11.0).
The pH stability of the enzyme is important for enzymatic characterization, before being marketed. The protease produced by Aspergillus tamarii URM4634 was stable at all pH values tested, showing 70% residual activity at pH 5.0 and 86.7% in pH 11.0 until 24 h. This pH stability of the protease of A. tamarii URM4634 showed potential for possible industrial applications. Protease with alkaline properties can be used in leather, detergents and pharmaceutical industry [
The temperature is one of the most critical parameters to be controlled in bioprocesses [
Yadav et al. [
Influences of ionic solutions were evaluated at concentrations of 5 mM and 10 mM (
Metal ions | 5 mM | 10 mM |
---|---|---|
Residual activity (%) | Residual activity (%) | |
Control | 100.0 ± 2.12 | 100.0 ± 2.12 |
K+ | 100.0 ± 2.45 | 100.0 ± 1.19 |
Ca2+ | 100.0 ± 2.01 | 112.88 ± 0.24 |
Zn2+ | 100.0 ± 1.13 | 111.36 ± 0.40 |
Mg2+ | 106.89 ± 1.89 | 116.39 ± 1.67 |
Na+ | 115.86 ± 1.90 | 114.98 ± 1.66 |
Fe2+ | 115.82 ± 1.86 | 93.95 ± 0.23 |
Hg2+ | 91.21 ± 1.27 | 77.31 ± 0.83 |
Cu2+ | 78.34 ± 0.65 | 66.02 ± 3.31 |
10 mM. However, the protease was inhibited by Hg2+ (22.69%) and Cu2+ (33.98%). Similarly results were obtained for alkaline protease produced by Aspergillus flavus [
Enzyme activity was inhibited 65.11% by 1mM phenylmethylsulfonyl fluoride (PMSF), classifying the enzyme as a serine protease. It was also slightly inhibited by 10 mM Ethylenediaminetetraacetic acid (EDTA)-metalloprotease inhibitor (71.15%), 10 mM 2- mercaptoethanol-cysteine protease inhibitor (81.62%), but was not inhibited when subjected to 1 µM Pepstatin A―the aspartic protease inhibitor (
The results of this study corroborate with results of the effect of the inhibitor, PMSF, on enzyme activity found by Bhunia et al. [
Inhibitors | Residual activity (%) |
---|---|
Control | 100.0 ± 2.58 |
EDTA | 71.15 ± 0.94 |
PMSF | 34.89 ± 2.00 |
2-Mercaptaethanol | 81.62 ± 0.76 |
Idoacetic acid | 94.33 ± 2.00 |
Pepstatin A | 100.0 ± 0.16 |
Microorganisms | Optimum temperature | Optimum pH | Type catalytic | References |
---|---|---|---|---|
Aspergillus tamarii | 40˚C | 8.0 | Serine protease | This work |
Aspergillus tamarii | NA | 9.0 | Serine protease | [ |
Aspergillus parasiticus | 50˚C | 7.0 | Serine protease | [ |
Aspergillus flavus | 45˚C | 7.5 | Serine protease | [ |
Aspergillus niger | NA | 9.0 | Metalloproteases | [ |
Aspergillus niger | 60˚C | 8.0 | Metalloproteases | [ |
Aspergillus niger | 40˚C | 3.5 | Aspartic protease | [ |
Aspergillus carbonarius | 40˚C | 3.0 | Cysteine protease | [ |
NA = Not Available.
SDS-PAGE and Zymogram were applied to verify the electrophoretic profile of the enzyme extract by A. tamarii URM4634 (
The results obtained in this study showed that the high levels of protease production by Aspergillus tamarii URM4634 could be achieved by adjusting the parameters, as the amount of substrate (5 g wheat bran), moisture (40%) and temperature (30˚C). The effect of temperature on the fermentation process showed a negative effect as an important factor, inducing the best protease production at 72 h. The biochemical characterization showed the optimum pH 8.0 and 40˚C and pH stability (5.0 - 11.0) and thermostability (10˚C - 40˚C). The enzyme is a serine protease and showed collagenolytic and keratinolytic activities. The protease produced by A. tamarii URM4634 showed enzymatic characteristics that are suitable for using in industrial applications as leather
processing and food industries, with low cost using wheat bran agroindustrial wastes as substrate.
The authors are greateful to FACEPE (Foundation for Science and Technology of the State of Pernambuco, Brazil) process IBPG-0257-5.07/14 for the scholarship to the first author, CNPq (National Council for Scientific and Technological Development, Brazil) and CAPES (National Council for the Improvement of Higher Education, Brazil) for the financial support.
da Silva, O.S., de Oliveira, R.L., Souza-Motta, C.M., Porto, A.L.F. and Porto, T.S. (2016) Novel Protease from Aspergillus tamari URM4634: Production and Characterization Using Inexpensive Agroindustrial Substrates by Solid-State Fermentation. Advances in Enzyme Research, 4, 125-143. http://dx.doi.org/10.4236/aer.2016.44012