Original Research PaperProduction and biochemical properties of proteases secreted by Aspergillus niger under solid state fermentation in response to different agroindustrial substrates
Introduction
Proteases are multifunctional enzymes accounting for nearly 60% of the whole enzyme market and are frequently used in detergent, leather, pharmaceuticals, food and biotechnology industries (Ramakrishna et al., 2010, Yin et al., 2013). They can be isolated from plants, animals and microorganisms. Of these sources, the microorganisms show great potential for protease production due to their broad biochemical diversity and their susceptibility to genetic manipulation. It has been estimated that microbial proteases represent approximately 40% of the total worldwide enzyme sales (Rao et al., 1998).
Several species of filamentous fungi have been exploited in industrial processes for the production of metabolites and industrial enzymes. Aspergillus niger has a long tradition of safe use in the production of enzymes and organic acids. Many of these products have listed as a “Generally Recognized as Safe (GRAS)” by the US Food and Drug Administration (Schuster et al., 2002). A. niger is one of the most important sources of fungal proteases. According Pel et al., (2007), genome sequencing shows that A. niger has 198 proteins involved in proteolytic degradation process.
Proteolytic enzymes can be produced by submerged and solid state fermentation. For the growth of fungi, solid state fermentation is most appropriate method because the solid substrates resemble the natural habitat of the fungi and improving their growth and the secretion of a wide range of extracellular enzymes. Some characteristics make solid state fermentation more attractive than submerged fermentation: simplicity, low cost, high yields and concentrations of the enzymes and the use of inexpensive and widely available agricultural residues as substrates (Chutmanop et al., 2008).
The biochemical characterization of enzymes is important to evaluate their biotechnological potential. The study of the protease properties, such as the substrate specificity, the optimum catalytic pH conditions and the temperature and stability profiles, can be used to predict the successful application of the enzyme to particular industries or processes (Castro and Sato, 2013a).
Previous work has shown that the expression and secretion of different sets of proteases and other enzymes, as well as their biochemical properties, can be regulated by the type of substrate used as carbon and nitrogen source (Speranza et al., 2011, Farnell et al., 2012). In this context, the main objectives of the present study were to evaluate the production of the protease by A. niger LBA02 under solid state fermentation using different agroindustrial by-products and to verify the correlation between some physical–chemical parameters, including chemical composition, water absorption index, particle size and packing density with the protease production. In addition, the biochemical properties of the proteases produced in each agroindustrial by-product, including the optimum pH and temperature for activity and stability, kinetic parameters Km and Vmax, substrate specificity and milk-clotting activities were investigated.
Section snippets
Chemical composition of the agroindustrial by-products
Moisture, protein content, lipids and ash of the agroindustrial by-products were determined by AOAC methods (AOAC, 2010). The carbohydrate content was determined by difference between the total value of 100% and the sum of the other components. The tests were performed in triplicate and the results were expressed as the mean±standard deviation.
Determination of the water absorption index (WAI) of the agroindustrial by-products
Their water absorption index (WAI) was determined using the method of Anderson et al. (1969) with slight modifications. Briefly, the sample (1.25 g) was
Calculations and statistics
Values are expressed as the arithmetic mean. The Tukey test was used to check the significant differences between the groups analyzed. The differences were considered significant when p-Value≤0.05.
Pearson correlation coefficient was used to measure the strength of linear dependence between two responses. The correlation coefficient ranges from –1 to 1. A value of 1 implies that a linear equation describes the relationship between the responses was perfectly and positive, while a value of −1
Chemical composition of the agroindustrial by-products
The centesimal compositions of the agroindustrial by-products used as fermentation substrates for production of protease by A. niger LBA02 under solid state fermentation are showed in Table 1. The enzymes production under solid state fermentation can be affected by the composition of the substrates and various cultivation factors. On protease production, for example, the presence of protein sources can induce the enzyme secretion by the microorganism. On the other hand, the substrate must have
Conclusion
The results obtained in our study showed that the protease production by Aspergillus niger can be affected by some physical chemical parameters, as the particle size and the water absorption index. Higher levels of protein in the agroindustrial by-products showed as important factor, inducing the on protease production in the first 48 h of fermentation. The biochemical characterization showed that the proteases produced in different agroindustrial by-products exhibited different properties. The
Acknowledgments
The work described in this paper was substantially supported by grants from São Paulo Research Foundation – FAPESP (Project no. 2011/10429-9), and the Department of Food Science, School of Food Engineering, University of Campinas, which are gratefully acknowledged.
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