Purification of thermostable α‐galactosidase from Irpex lacteus and its use for hydrolysis of oligosaccharides

Abstract

A monomeric α-galactosidase (ILGI) from the mushroom Irpex lacteus was purified 94.19-fold to electrophoretic homogeneity. ILGI exhibited a specific activity of 18.36 U mg(-1) and demonstrated a molecular mass of 60 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). ILGI was optimally active at 80 °C and pH 5.0, and it was stable over a temperature range of 4-70 °C and a wide pH range of 2.0-12.0. ILGI was completely inactivated by Ag(+) and Hg(2+) ions and N-bromosuccinimide (NBS). Moreover, ILGI exhibited good resistance to proteases. Galactose acted as a noncompetitive inhibitor with Ki and Kis of 3.34 and 0.29 mM, respectively. The α-galactosidase presented a broad substrate specificity, which included p-nitrophenyl α-D-galactopyranoside (pNPGal), melibiose, stachyose, and raffinose with Km values of 1.27, 3.24, 7.1, and 22.12 mM, correspondingly. ILGI exhibited efficient and complete hydrolysis to raffinose and stachyose. The aforementioned features of this enzyme suggest its potential value in food and feed industries.

Cloning, expression, and characterization of thermal‐stable and pH‐stable agarase from mangrove sediments

“…Agarose, which is extracted from marine red macroalgae, is considered as an ideal resource for producing fermentable monosaccharides. Fermentable monosaccharides are important precursors in the bioenergy field because of the great yields of agarose and a lack of lignocellulose that facilitates the extraction of agarose . Alcohols and H 2 have been produced after agarose saccharification in previous studies .…”

Optimization of Saccharomyces cerevisiae α-galactosidase production and application in the degradation of raffinose family oligosaccharides

“…Substrate specificity seems to be determined more by molecular state of the protein than similarities in their amino acid sequence. Monomeric enzymes belonging to different families of GH can act on small oligosaccharides and polymeric galactomannans [35, 70, 71]; however, those that are organized as high MW multimeric complexes only can hydrolyse small oligosaccharides [28, 72]. ScAGal is a tetrameric enzyme and is a further example that the inability to act on polymeric substrates might be due to its multimeric structure restricting access to the active site of the enzyme.…”

“…There are many references to α-Gals isolated from different organisms; many of them have been identified in fungi [22, 23, 32–36], bacteria [29, 37–40], plants [41], and even in the gut of insects [42], and have been characterized because of the big industrial potential for the hydrolysis of RFOs and/or galactomannans. However, there are more current works that have informed α-Gals from original sources [22, 23, 29, 33–36, 41] than the use of expression hosts [32, 37–40, 42] for the enhanced production of enzyme. Moreover, in most cases, the enzyme purification process requires many steps, but there are few studies that have optimized overexpression α-Gals production conditions for cost-effective use on an industrial scale [43–45].…”

Improved Foods Using Enzymes from Basidiomycetes

“…It may be of interest for high-temperature processes (Figure 3). Fungal α-galactosidases were discovered in Irpex lacteus [51,52], Leucopaxillus tricolor [53] and in Tremella aurantialba [54]. These enzymes, some of which are available in recombinant form, were generally very active, thermostable and working within a broad pH range.…”