br Acknowledgements This work was supported by grants of

Acknowledgements This work was supported by grants of the Argentine National Research Council (CONICET, PIP 0662). The authors express their appreciation to the Transmission Electron Microscopy Service (MET) of the Faculty of Veterinary Medicine of the National University of La Plata (Argentina) for their valuable collaboration. I am grateful to the late Rubén Oltolina (†Deceased 24 August 2016) for his invaluable technical assistance.
Introduction β-Galactosidase (β-d-galactohydrolases, EC 3.2.1.23) is an abundant glycoside hydrolase enzyme that catalyzes the hydrolysis of terminal β-d-galactosyl moieties to monosaccharides by breaking the glycosidic bond of substrates such as disaccharides, diverse glycoconjugates, and polysaccharides. Under defined reaction conditions, many β-galactosidases can catalyze transglycosylation reactions using various acceptor molecules. Depending on its biochemical activities, β-galactosidase activity has been used for (1) hydrolysis of lactose (thereby rendering dairy products consumable for lactose-intolerant individuals), analytical studies, glycan remodeling, and various processes of biotechnological and medical importance [1], [2], [3] and (2) synthesis of galactooligosaccharides (GOS) from lactose by transglycosylation [4]. β-Galactosidases can be derived from microbial sources including bacteria, yeasts, and filamentous fungi [4], [5], [6], [7]. Aspergillus-sourced β-galactosidases, especially those from Aspergillus oryzae, are most widely used, and detailed data on their characteristics and many applications have been previously reported [8], [9], [10]. The A. niger β-galactosidase was identified, purified, and characterized more than four decades ago [11], [12]. The lacA gene encoding A. niger β-galactosidase was first cloned and expressed in the yeast Saccharomyces cerevisiae in 1992 [13], [14] and later overexpressed in yeast [7] and Eremothecium gossypii (formerly Ashbya gossypii) [15]. Experimental evidence, especially from transcriptomic data, indicates that the β-galactosidase activity of A. niger is contributed by different isoenzymes [12], [16], [17], [18]. However, gene cloning, overexpression, structural analysis [19], and applications [20] have mainly been focused on A. niger LacA (or lactase A encoded by lacA). Advances in the LCQ-908 sequencing of A. niger[21] opened possibilities to further exploit this fungus to identify additional β-galactosidase-like enzymes, and a total of five putative β-galactosidases have been identified at genome-level [16], [17], [21]. In this study, in addition to the known LacA, genes encoding four new lactose-hydrolyzing enzymes were cloned, sequenced, and heterologously expressed, and their enzyme properties were established.
Materials and methods
Results
Discussion In this study, we identified five ORFs from A. niger F0215 representing putative β-galactosidases. The ORFs represented a previously described β-galactosidase (LacA) [7], [13], [15] and three other β-galactosidases (LacB, LacC, and LacE) that had been identified [16] but not previously biochemically characterized. In the present study, we cloned and expressed the four genes and characterized the biochemical properties and structures of the gene products. A sequence similar to the β-galactosidase ORF (XP_001391524.2) previously found in the genome of A. niger CBS 513.88 and designated as LacD was failed to be obtained and expressed. This ORF appears to represent a pseudogene. These results suggest that the β-galactosidase family of A. niger F0215 consists of four functional members (LacA, LacB, LacC, and LacE) belonging to family 35 of the glycoside hydrolases (www.cazy.org). Evidence reported by Widmer and Leuba [12] pointed to the possibility of the A. niger β-galactosidase family consisting of more than one member. They purified three β-galactosidases and identified them as glycoproteins with molecular weights of 124,000, 150,000, and 173,000; isoelectric points of about 4.6; pH optima between 2.5 and 4.0; and heat stability up to 60°C. These properties are similar to those established in our detailed investigations. However, the variation in the properties of the three glycoproteins was proposed to be mainly due to dissimilar carbohydrate contents and related to culture conditions [12]. Subsequently, a β-d-galactosidase from A. niger was purified and characterized [28]. Gel-filtration chromatography and SDS-PAGE analysis showed that its molecular weight was 300,000 and 130,000, respectively, suggesting that it was heavily glycosylated. This β-galactosidase was designated lacA, and the gene was cloned and expressed in S. cerevisiae[13], [14], yeast [7], and E. gossypii[15]. The transcription of lacA was induced by arabinose, xylose, xylan, and pectin [29].