Production and biochemical properties of proteases secreted by Aspergillus niger under solid state fermentation in response to different agroindustrial substrates

doi:10.1016/j.bcab.2014.06.001

Biocatalysis and Agricultural Biotechnology

Volume 3, Issue 4, October 2014, Pages 236-245

https://doi.org/10.1016/j.bcab.2014.06.001 Get rights and content

Abstract

This study reports the proteases production by Aspergillus niger LBA02 under solid state fermentation (SSF) using different agroindustrial by-products as substrates and the correlation between the protease production and some physical–chemical parameters. The biochemical properties of the proteases produced in each substrate were further investigated. The highest protease production was obtained using wheat bran as the substrate at 96 h fermentation. The results for chemical composition showed that the substrates with higher protein content induced the protease production at the first 48 h of fermentation. The crude extracts of proteases from A. niger LBA02 produced in wheat bran (PWB), soybean meal (PSM) and cottonseed meal (PCM) showed different biochemical properties when produced in different agroindustrial by-products. The biochemical characterization showed that the enzymes were most active over the pH range 3.0–4.0 and was stable from pH 2.5–4.5. The optimum temperature for activity was approximately 50.0 °C, and the enzymes were stable at 40.0–50.0 °C. In the study of the substrate specificity, the best substrate was hemoglobin from bovine blood with the highest relative activities of 183.84, 147.06 and 186.81% for PWB, PSM and PCM, respectively. The PWB showed higher ratio milk-clotting/protease activities (15.24) compared to PSM (0.38) and PCM (6.28).

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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Original Research PaperProduction and biochemical properties of proteases secreted by Aspergillus niger under solid state fermentation in response to different agroindustrial substrates

Abstract

Introduction

Section snippets

Chemical composition of the agroindustrial by-products

Determination of the water absorption index (WAI) of the agroindustrial by-products

Calculations and statistics

Chemical composition of the agroindustrial by-products

Conclusion

Acknowledgments

Food Chem.

Protein Expr. Purif.

Bioresour. Technol.

J. Biol. Chem.

Fungal. Biol.

Ind. Crops Prod.

Process Biochem.

J. Mol. Catal. B: Enzym.

Gelatinization of corn grits by roll and extrusion cooking

Cereal Sci. Today

Official Methods of Analysis of the Association of Official Agriculture Chemistry

Production and characterization of extracellular protease of mutant Aspergillus niger AB100 grown on fish scale

World J. Microbiol. Biotechnol.

Production and biochemical characterization of protease from Aspergillus oryzae: an evaluation of the physical-chemical parameters using agroindustrial wastes as supports

Biocat. Agric. Biotechnol.

Synergistic effects of agroindustrial wastes on simultaneous production of protease and α-amylase under solid state fermentation using a simplex centroid mixture design

Ind. Crops Prod

Protease production by Aspergillus oryzae in solid-state fermentation using agroindustrial substrates

J. Chem. Technol. Biotechnol.

Optimization of process conditions for the production of a prolylendopeptidase by Aspergillus niger ATCC 11414 in solid state fermentation

Food Sci. Biotechnol.

Characterization and cheese-making properties of rennet-like enzyme produced by a local algerian isolate of Aspergillus niger

Food Biotechnol.

Original Research Paper
Production and biochemical properties of proteases secreted by Aspergillus niger under solid state fermentation in response to different agroindustrial substrates