Characterization of the bga1‐encoded glycoside hydrolase family 35 β‐galactosidase of Hypocrea jecorina with galacto‐β‐d‐galactanase activity

Abstract

The enzyme b-galactosidase (EC 3.2.1.23) catalyses the hydrolysis of terminal nonreducing b-d-galactose residues in b-d-galactosides as, for example, lactose (1,4-O-b-d-galactopyranosyl-d-glucose) and structurally related compounds. It is found in plants and animals, as well as in a wide variety of microorganisms including yeasts, fungi, bacteria and Archaea. According to the Carbohydrate Active Enzymes database (http://www.cazy.org/)[1], b-galactosidases are members of four different glycoside hydrolase (GH) families (GH1, GH2, GH35 and GH42), indicating their structural diversity. In biotechnology, they are mainly applied for the hydrolysis of lactose to d-glucose and d-galactose in various products of the dairy industry [2,3]. This results in improved quality of the end product (softer texture of ice cream, faster ripening of cheese, etc.) and also lessens the problem of lactose intolerance, which is prevalent in more than half of the world's population [4]. In addition, b-galactosidases catalyse transgalactosylation reactions of various b-d-galactosides including lactose. Recently, these galacto-oligosaccharides have attracted considerable interest because of a proposed beneficial effects on human health [5,6].The filamentous fungus Hypocrea jecorina (anamorph: Trichoderma reesei) is a potent producer of The extracellular bga1-encoded b-galactosidase of Hypocrea jecorina (Trichoderma reesei) was overexpressed under the pyruvat kinase (pki1) promoter region and purified to apparent homogeneity. The monomeric enzyme is a glycoprotein with a molecular mass of 118.8 ± 0.5 kDa (MALDI-MS) and an isoelectric point of 6.6. Bga1 is active with several disaccharides, e.g. lactose, lactulose and galactobiose, as well as with aryland alkyl-b-d-galactosides. Based on the catalytic efficiencies, lactitol and lactobionic acid are the poorest substrates and o-nitrophenyl-b-d-galactoside and lactulose are the best. The pH optimum for the hydrolysis of galactosides is 5.0, and the optimum temperature was found to be 60°C. Bga1 is also capable of releasing d-galactose from b-galactans and is thus actually a galacto-b-d-galactanase. b-Galactosidase is inhibited by its reaction product d-galactose and the enzyme also shows a significant transferase activity which results in the formation of galacto-oligosaccharides.Abbreviations

Crystal structures of Trichoderma reesei β-galactosidase reveal conformational changes in the active site

“…Tr-b-gal is reported to be a glycoprotein that contains 11 putative N-linked glycosylation places (Gamauf et al, 2007). According to the structure, only one of them, Asn810 is located inside the protein, the remaining 10 putative sites are located on the surface of the protein.…”

“…The sequence and the enzymatic properties of this industrially potent enzyme have previously been reported (Seiboth et al, 2005) and (Gamauf et al, 2007). Tr-b-gal is active with several disaccharides, like lactose, lactulose, galactobiose, aryl-and alkyl-b-D-galactosides.…”

“…Tr-b-gal is active at the pH range 3-7 (maximum at pH 5). There is a substantial decrease in the activity at temperatures higher than 60°C (Gamauf et al, 2007). The highresolution structures that have been determined create an excellent starting point for an analysis of the function Tr-b-gal and the design of mutants in order to improve the enzymatic properties for industrial applications.…”

Secretomic Analysis Reveals Multi-Enzymatic Complexes in Trichoderma reesei Grown in Media Containing Lactose or Galactose

“…Thus, the protein concentration of lactose-induced secretome was higher (0.3 mg/mL) than galactose-induced sample (0.1 mg/mL). The higher protein concentration induced by lactose could be at least partially due to the secretion of the lactose-degrading enzyme β-galactosidase 1 [15].…”

“…Since lactose is not present in the natural environment of any filamentous fungus, the galactosidase activity present in GH 36 (complex I) could have other role in fungal metabolism, such as the glycidyl residue cleavage on glycoproteins or polysaccharide structures found in different plant or fungal cell walls [15].…”