Recombinant Aspergillus β-galactosidases as a robust glycomic and biotechnological tool

Martin Dragosits 0 1 Stefan Pflgl 0 1 Simone Kurz 0 1 Ebrahim Razzazi-Fazeli 0 1 Iain B. H. Wilson 0 1 Dubravko Rendic 0 1 0 E. Razzazi-Fazeli VetCORE Facility for Research, University of Veterinary Medicine , Vienna, Austria 1 S. Pflgl Department of Biotechnology, University of Natural Resources and Life Sciences , Vienna, Austria 2 ) Department of Chemistry, University of Natural Resources and Life Sciences , Muthgasse 18, Vienna 1190, Austria Galactosidases are widespread enzymes that are used for manifold applications, including production of prebiotics, biosynthesis of different transgalactosylated products, improving lactose tolerance and in various analytical approaches. The nature of these applications often require galactosidases to be present in a purified form with clearly defined properties, including precisely determined substrate specificities, low sensitivity to inhibitors, and high efficiency and stability under distinct conditions. In this study, we present the recombinant expression and purification of two previously uncharacterized -galactosidases from Aspergillus nidulans as well as one -galactosidase from Aspergillus niger. All enzymes were active toward p -nitrophenyl--D - galactopyranoside as substrate and displayed similar temperature and pH optima. The purified recombinant galactosidases digested various complex substrates containing terminal galactose -1,4 linked to either N -acetylglucosamine or fucose, such as N -glycans derived from bovine fibrin and Caenorhabditis elegans . In our comparative study of the recombinant galactosidases with the commercially available galactosidase from Aspergillus oryzae , all enzymes also displayed various degrees of activity toward complex oligosaccharides containing -1,3-linked terminal galactose residues. All recombinant enzymes were found to be robust in the presence of various organic solvents, temperature variations, and freeze/thaw cycles and were also tested for their ability to synthesize galactooligosaccharides. Furthermore, the use of fermentors considerably increased the yield of recombinant galactosidases. Taken together, we demonstrate that purified recombinant galactosidases from A. niger and from A. nidulans are suitable for various glycobiological and biotechnological applications. - -Galactosidases (-D -galactohydrolases, EC 3.2.1.23) are ubiquitous enzymes capable of hydrolyzing terminal -D galactosyl moieties from substrates such as disaccharides, diverse glycoconjugates, and polysaccharides. They belong to the glycoside hydrolase clan A (GH-A), which encompasses, among others, -galactosidase families 35 and 42. Under distinct reaction conditions, -galactosidases can also catalyze transglycosylation reactions using various acceptor molecules, a capability used for the synthesis of, e.g., galactooligosaccharides (GOS) (Park and Oh 2010). Galactosidases have been widely used to hydrolyze lactose and render dairy products consumable for lactose-intolerant individuals. Further applications of these enzymes range from analytical studies (Titz et al. 2009) to glycan remodeling (Iskratsch et al. 2009) and include various processes of biotechnological and medical importance (Husain 2010). The -galactosidases in general use are mainly derived from microbial sources, which include both prokaryotic and eukaryotic organisms. They can be purified at high yields directly from fungal organisms (Tanaka et al. 1975; Husain 2010; Sen et al. 2012) or produced in a recombinant form at high levels in different expression systems (Wang et al. 2009; Oliveira et al. 2011). Other commonly used sources of galactosidases include bacteria (e.g., bifidobacteria (Hung et al. 2001; Hsu et al. 2005) and Streptococcus pneumonia (Hughes and Jeanloz 1964)), yeasts (e.g., Kluyveromyces lactis (Dickson and Markin 1978)), plants (e.g., from jack bean (Arakawa et al. 1974; Li et al. 1975)), and animals (e.g., bovine testes -galactosidase (Distler and Jourdian 1973)). One of the most widely used fungal -galactosidase originates from Aspergillus oryzae . As such, detailed data on its characteristics and many applications have been reported (Scheckermann et al. 1997; Zeleny et al. 1997; Torres et al. 2003; Vera et al. 2011). The -galactosidase from Aspergillus niger was previously used to overcome the Lac phenotype of Saccharomyces cerevisiae (Kumar et al. 1992) and its heterologous expression in various yeast hosts as well as its use in lactose hydrolysis and transgalactosylation reactions was reviewed recently (Oliveira et al. 2011). A closely related recombinant -galactosidase from A. niger van Tiegh has been recently described as a highly acid-stable enzyme with prospective use in the treatment of lactose intolerance (Hu et al. 2010) and a purified native -galactosidase from this strain possesses a rather low pH optimum (OConnell and Walsh 2010). In 2008, two -galactosidases secreted by Aspergillus carbonarius ATCC6276 were purified to homogeneity and were shown to be resilient to simulated gastric conditions (OConnell and Walsh 2008). Most recently, a galactosidase was purified from Aspergillus alliaceus and partially characterized; this enzyme, unlike various other fungal galactosidases, has a pH optimum in the neutral range, an enzymatic property suitable for the preparation of low-lactose milk (Sen et al. 2012). Since the Aspergillus spp. are obviously an established source of robust but different -galactosidases, we sought to thoroughly characterize pure forms of recombinant galactosidases using various substrates and conditions. In addition, as the A. oryzae -galactosidase only digests terminal -1,3-linked galactose residues at a very slow rate (Zeleny et al. 1997), we examined whether any of the galactosidases from other Aspergillus spp. display higher activity toward these residues, a feature interesting for glycobiological analyses. In this study, we report on the expression and characterization of two -galactosidases from Aspergillus nidulans and one -galactosidase from A. niger. We have expressed Cterminally His-tagged versions of these previously putative -galactosidases in Pichia pastoris as an expression host and purified the recombinant enzymes to apparent homogeneity. In-depth characterization of the recombinant proteins and comparison with the commercially available, native galactosidase from A. oryzae were performed. Additionally, we emphasized the evaluation of the recombinant enzymes as an analytical tool, such as for glycobiological analyses, which is an important application of -galactosidases rarely evaluated for novel enzymes. The obtained data show that these recombinant -galactosidases are promising as reagents for a variety of glycobiological and biotechnological applications. Materials and methods Open reading frames (ORF) from A. niger (lacA , gene ID 4977988) and A. nidulans (lacA, gene ID 2876531; lacB, gene ID 2876757) were polymerase chain reaction (PCR) amplified (Expand Polymerase) fr (...truncated)