Optimization and partial purification of beta-galactosidase production by Aspergillus niger isolated from Brazilian soils using soybean residue

(2019) 9:81 Martarello et al. AMB Expr https://doi.org/10.1186/s13568-019-0805-6 ORIGINAL ARTICLE Open Access Optimization and partial purification of beta‑galactosidase production by Aspergillus niger isolated from Brazilian soils using soybean residue Raquel Dall’Agnol Martarello1, Luana Cunha1, Samuel Leite Cardoso1, Marcela Medeiros de Freitas1, Damaris Silveira1, Yris Maria Fonseca‑Bazzo1, Mauricio Homem‑de‑Mello1, Edivaldo Ximenes Ferreira Filho2 and Pérola Oliveira Magalhães1* Abstract β-Galactosidases are widely used for industrial applications. These enzymes could be used in reactions of lactose hydrolysis and transgalactosylation. The objective of this study was the production, purification, and characterization of an extracellular β-galactosidase from a filamentous fungus, Aspergillus niger. The enzyme production was opti‑ mized by a factorial design. Maximal β-galactosidase activity (24.64 U/mL) was found in the system containing 2% of a soybean residue (w/v) at initial pH 7.0, 28 °C, 120 rpm in 7 days. ANOVA of the optimization study indicated that the response data on temperature and pH were significant (p < 0.05). The regression equation indicated that the R2 is 0.973. Ultrafiltration at a 100 and 30 kDa cutoff followed by gel filtration and anion exchange chromatography were carried out to purify the fungal β-galactosidase. SDS-PAGE revealed a protein with molecular weight of approximately 76 kDa. The partially purified enzyme showed an optimum temperature of 50 °C and optimum pH of 5.0, being stable under these conditions for 15 h. The enzyme was exposed to conditions approaching gastric pH and in pepsin’s presence, 80% of activity was preserved after 2 h. These results reveal a A. niger β-galactosidase obtained from residue with favorable characteristics for food industries. Keywords: Fungi, β-Galactosidase, Purification, Optimization, Agroindustrial residues Introduction Fermentation of agroindustrial residues received a great deal of attention in recent years. Many byproducts and raw materials from the food industry and agriculture, e.g., soybean residues, sugarcane bagasse, cotton stalk, corn cob, and mango peel have been used to produce biotechnological products owing to their high availability. They are also an alternative source of nutrients with low commercial cost (Moreira et al. 2012). *Correspondence: 1 Laboratory of Natural Products, Health Sciences School, Department of Pharmaceutical Sciences, University of Brasília, Brasília, DF CEP 7910‑900, Brazil Full list of author information is available at the end of the article The agroindustrial residues are mainly composed of lignocellulosic material. Considering that 90% of agroindustrial residues are discarded into the environment, the use of these residues as raw materials should reduce environmental pollution and may also increase the economic value of the residues (Moreira et al. 2012). Brazil is the second biggest producer of soybeans (Glycine max) worldwide. In the 2014–2015 harvest, soybean planting area reached 30.1 million hectares, producing a crop of 95 million tons of soybeans (Embrapa 2016). One application for soybean byproducts is fermentation by microorganisms including bacteria and fungi that are able to degrade the lignocellulosic material of the agricultural residues. These residues could be utilized by filamentous fungi as a carbon source for the production © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Martarello et al. AMB Expr (2019) 9:81 of enzymes, in particular hydrolytic ones (Moreira et al. 2012). Aspergillus fungi have been chosen for large-scale processes because they can produce large quantities and varieties of enzymes in a low-cost medium (Bergquist et al. 2002). Moreira et al. (2012) studied the degradation of lignocellulosic residues for production of enzymes of industrial significance such as xylanases, mannanases, pectinases, β-glucosidases, avicelases, phosphatases, and carboxymethyl cellulases by different species of fungi isolated from soil, including Aspergillusterreus, Aspergillusoryzae, and Aspergillusniger (Moreira et al. 2012). A large number of microorganisms have been assessed as potential sources of β-d-galactosidase (β-d-galactoside galactohydrolase, EC 3.2.1.23, most commonly known as lactase) to hydrolyze lactose into glucose and galactose for lactose-free milk production and products intended for lactose-intolerant consumers (Isobe et al. 2013a). Traditionally, the β-galactosidases most widely used in industry were obtained from Aspergillus spp. and Kluyveromyces spp. (Panesar et al. 2006), because these could be readily obtained with acceptable productivities and yields from cultivations of these microorganisms. Additionally, products obtained from these organisms are generally recognized as safe (GRAS status) for human consumption, which is critical for food related applications (Panesar et al. 2006). In Aspergillus niger the β-galactosidase enzymes are secreted to the extracellular medium, increasing the interest in finding new culture source for the production of this enzyme (Panesar et al. 2006). Besides, lactose is a hygroscopic sugar that has low solubility; it could induce crystallization and may cause technological problems for certain products in the dairy industry. The solubility and sweetness can be increased by the lactose hydrolysis. Many problems in refrigerated foods such as crystallization in dairy foods, precipitate formation in frozen foods, and development of a gritty texture may be reduced with lactose hydrolysis (Klein et al. 2010; Panesar et al. 2006). β-Galactosidases also participate in the synthesis of galactooligosaccharides (GOSs) and can be applied to functional foods such a slow-calorie foods or as an additive in fermented dairy products, breads, and drinks. Moreover, in the pharmaceutical industry, β-galactosidase is produced as a food supplement for lactose-intolerant people. Many symptoms of lactose intolerance are minimized by the use of exogenous β– galactosidase before ingestion of milk or dairy products (O’Connell and Walsh 2008; Oliveira et al. 2011). Optimization of the fermentation process can reduce the production costs for β-galactosidase and is important for enabling an industrial application and to obtain “green processes.” For this reason, the selection of a low-cost culture is fundamental (Liu et al. 2007). Optimization of Page 2 of 13 media components by the traditional “one-variable-at-atime” strategy is time-consuming and expensive when a large number of variables are considered. This method is incapable of detecting t (...truncated)