Protease from Aspergillus oryzae: Biochemical Characterization and Application as a Potential Biocatalyst for Production of Protein Hydrolysates with Antioxidant Activities
This study reports the biochemical characterization of a protease from Aspergillus oryzae LBA 01 and the study of the antioxidant properties of protein hydrolysates produced with this protease. The biochemical characterization showed that the enzyme was most active over the pH range 5.0–5.5 and was stable from pH 4.5 to 5.5. The optimum temperature range for activity was 55–60°C, and the enzyme was stable at temperatures below 45°C. The activation energy () for azocasein hydrolysis and temperature quotient () were found to be 37.98？kJ？mol？1 and 1.64–1.53 at temperature range from 30 to 55°C, respectively. The enzyme exhibited of 97.63？min and a value of 324.31 at the optimum temperature for activity (57.2°C). Protease from A. oryzae LBA 01 was shown as a potentially useful biocatalyst for protein hydrolysis, increasing the antioxidant activities of soy protein isolate, bovine whey protein, and egg white protein from 2.0- to 10.0-fold. 1. Introduction Proteases are multifunctional enzymes and represent a fundamental group of enzymes due to diversity of their physiological roles and biotechnological applications [1]. These enzymes are extremely important in the pharmaceutical, medical, food, and biotechnology industries, accounting for nearly 60% of the whole enzyme market [2]. It has been estimated that microbial proteases represent approximately 40% of the total worldwide enzyme sales [3]. Fungi are a potential source of proteases due to their broad biochemical diversity, their susceptibility to genetic manipulation, high productivity, and being extracellular and are easily recoverable from the fermentation medium [1, 4]. Aspergillus oryzae (A. oryzae) is a filamentous fungus listed as a “Generally Recognized as Safe (GRAS)” organism by the US Food and Drug Administration. It has a long history of use in the food industry in the production of traditional fermented foods, due to its high proteolytic activity and its use of sugar [5, 6]. According to Machida et al. [7] the molecular history of the organism shows that A. oryzae has the largest expansion of hydrolytic genes (135 proteinase genes). The biochemical characterization of enzymes is important to evaluate their biotechnological potential. The study of the protease properties, such as the influence of inhibitors or activators, the substrate specificity, the optimum catalytic pH conditions and the temperature, and stability profiles and kinetic parameters, can be used to predict the successful application of the enzyme to particular industries or processes. The application of proteases to the
References
[1]
T. A. S. Silva, A. Knob, C. R. Tremacoldi, M. R. Brochetto-Braga, and E. C. Carmona, “Purification and some properties of an extracellular acid protease from Aspergillus clavatus,” World Journal of Microbiology and Biotechnology, vol. 27, no. 11, pp. 2491–2497, 2011.
[2]
V. Ramakrishna, S. Rajasekhar, and L. S. Reddy, “Identification and purification of metalloprotease from dry grass pea (Lathyrus sativus L.) seeds,” Applied Biochemistry and Biotechnology, vol. 160, no. 1, pp. 63–71, 2010.
[3]
M. B. Rao, A. M. Tanksale, M. S. Ghatge, and V. V. Deshpande, “Molecular and biotechnological aspects of microbial proteases,” Microbiology and Molecular Biology Reviews, vol. 62, no. 3, pp. 597–635, 1998.
[4]
K. S. Vishwanatha, A. G. Appu Rao, and S. A. Singh, “Characterisation of acid protease expressed from Aspergillus oryzae MTCC 5341,” Food Chemistry, vol. 114, no. 2, pp. 402–407, 2009.
[5]
T. Gotou, T. Shinoda, S. Mizuno, and N. Yamamoto, “Purification and identification of proteolytic enzymes from Aspergillus oryzae capable of producing the antihypertensive peptide Ile-Pro-Pro,” Journal of Bioscience and Bioengineering, vol. 107, no. 6, pp. 615–619, 2009.
[6]
M. Matsushita-Morita, S. Tada, S. Suzuki et al., “Overexpression and characterization of an extracellular Leucine Aminopeptidase from Aspergillus oryzae,” Current Microbiology, vol. 62, no. 2, pp. 557–564, 2011.
[7]
M. Machida, K. Asai, M. Sano, et al., “Genome sequencing and analysis of Aspergillus oryzae,” Nature, vol. 438, no. 7071, pp. 1157–1161, 2005.
[8]
D. Y. Zhou, B. W. Zhu, L. Qiao et al., “In vitro antioxidant activity of enzymatic hydrolysates prepared from abalone (Haliotis discushannai Ino) viscera,” Food and Bioproducts Processing, vol. 90, no. 2, pp. 148–154, 2012.
[9]
J. Charney and R. M. Tomarelli, “A colorimetric method for the determination of the proteolytic activity of duodenal juice,” The Journal of Biological Chemistry, vol. 171, pp. 501–505, 1947.
[10]
B. F. Chow and M. A. Peticolas, “A rapid method for the determination of proteolytic activities of enzyme preparations,” The Journal of General Physiology, vol. 32, no. 1, pp. 17–24, 1948.
[11]
Food Chemical Codex, General Tests and Apparatus, Proteolytic Activity, Fungal (SAP), National Academy Press, Washington, DC, USA, 1981.
[12]
K. S. Siddiqui, M. J. Azhar, M. H. Rashid, and M. I. Rajoka, “Activity and thermostability of carboxymethylcellulase from Aspergillus niger is strongly influenced by non-covalently attached polysaccharides,” World Journal of Microbiology and Biotechnology, vol. 12, no. 3, pp. 213–216, 1996.
[13]
A. Jakób, J. Bryjak, H. Wójtowicz, V. Illeová, J. Annus, and M. Polakovi？, “Inactivation kinetics of food enzymes during ohmic heating,” Food Chemistry, vol. 123, no. 2, pp. 369–376, 2010.
[14]
M. Dixon and E. C. Webb, Enzymes, Academic Press, New York, NY, USA, 1979.
[15]
H. Lineweaver and D. Burk, “The determination of enzyme dissociation constants,” Journal of the American Chemical Society, vol. 56, no. 3, pp. 658–666, 1934.
[16]
A. Dávalos, C. Gómez-Cordovés, and B. Bartolomé, “Extending applicability of the oxygen radical absorbance capacity (ORAC-fluorescein) assay,” Journal of Agricultural and Food Chemistry, vol. 52, no. 1, pp. 48–54, 2004.
[17]
A. Bougatef, M. Hajji, R. Balti, I. Lassoued, Y. Triki-Ellouz, and M. Nasri, “Antioxidant and free radical-scavenging activities of smooth hound (Mustelus mustelus) muscle protein hydrolysates obtained by gastrointestinal proteases,” Food Chemistry, vol. 114, no. 4, pp. 1198–1205, 2009.
[18]
S. Kumar, N. S. Sharma, M. R. Saharan, and R. Singh, “Extracellular acid protease from Rhizopus oryzae: purification and characterization,” Process Biochemistry, vol. 40, no. 5, pp. 1701–1705, 2005.
[19]
Y. Esparza, A. Huaiquil, L. Neira, et al., “Optimization of process conditions for the production of a prolylendopeptidase by Aspergillus niger ATCC 11414 in solid state fermentation,” Food Science and Biotechnology, vol. 20, no. 5, pp. 1323–1330, 2011.
[20]
M. J. García-Gómez, S. Huerta-Ochoa, O. Loera-Corral, and L. A. Prado-Barragán, “Advantages of a proteolytic extract by Aspergillus oryzae from fish flour over a commercial proteolytic preparation,” Food Chemistry, vol. 112, no. 3, pp. 604–608, 2009.
[21]
D. Wang, Z.-Y. Zheng, J. Feng et al., “Influence of sodium chloride on thermal denaturation of a high-salt-tolerant neutral protease from Aspergillus oryzae,” Food Science and Biotechnology, vol. 22, no. 5, pp. 1–7, 2013.
[22]
V. Sant'Anna, A. P. F. Corrêa, D. J. Daroit, and A. Brandelli, “Kinetic modeling of thermal inactivation of the Bacillus sp. protease P7,” Bioprocess and Biosystems Engineering, vol. 36, no. 7, pp. 993–998, 2013.
[23]
M. Melikoglu, C. S. K. Lin, and C. Webb, “Kinetic studies on the multi-enzyme solution produced via solid state fermentation of waste bread by Aspergillus awamori,” Biochemical Engineering Journal, vol. 80, pp. 76–82, 2013.
[24]
D. Huang, B. Ou, and R. L. Prior, “The chemistry behind antioxidant capacity assays,” Journal of Agricultural and Food Chemistry, vol. 53, no. 6, pp. 1841–1856, 2005.
[25]
R. L. Prior, X. Wu, and K. Schaich, “Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements,” Journal of Agricultural and Food Chemistry, vol. 53, no. 10, pp. 4290–4302, 2005.
[26]
T. G. Tavares, M. M. Contreras, M. Amorim, et al., “Optimisation, by response surface methodology, of degree of hydrolysis and antioxidant and ACE-inhibitory activities of whey protein hydrolysates obtained with cardoon extract,” International Dairy Journal, vol. 21, no. 12, pp. 926–933, 2011.
[27]
M. D. M. Contreras, B. Hernández-Ledesma, L. Amigo, P. J. Martín-álvarez, and I. Recio, “Production of antioxidant hydrolyzates from a whey protein concentrate with thermolysin: optimization by response surface methodology,” LWT: Food Science and Technology, vol. 44, no. 1, pp. 9–15, 2011.
[28]
G. Di Pierro, M. B. O'Keeffe, A. Poyarkov, G. Lomolino, and R. J. Fitzgerald, “Antioxidant activity of bovine casein hydrolysates produced by Ficus carica L.-derived proteinase,” Food Chemistry, vol. 156, pp. 305–311, 2014.
[29]
P. Yang, H. Ke, P. Hong, S. Zeng, and W. Cao, “Antioxidant activity of bigeye tuna (Thunnus obesus) head protein hydrolysate prepared with Alcalase,” International Journal of Food Science and Technology, vol. 46, no. 12, pp. 2460–2466, 2011.
[30]
Y. Zhuang and L. Sun, “Preparation of reactive oxygen scavenging peptides from tilapia (Oreochromis niloticus) skin gelatin: optimization using response surface methodology,” Journal of Food Science, vol. 76, no. 3, pp. 483–489, 2011.
[31]
L. Zhang, J. Li, and K. Zhou, “Chelating and radical scavenging activities of soy protein hydrolysates prepared from microbial proteases and their effect on meat lipid peroxidation,” Bioresource Technology, vol. 101, no. 7, pp. 2084–2089, 2010.
[32]
H. Tanzadehpanah, A. Asoodeh, and J. Chamani, “An antioxidant peptide derived from Ostrich (Struthio camelus) egg white protein hydrolysates,” Food Research International, vol. 49, no. 1, pp. 105–111, 2012.
