Characterization of a protease-resistant alpha-galactosidase from Aspergillus oryzae YZ1 and its application in hydrolysis of raffinose family oligosaccharides from soymilk

摘要

The alpha-galactosidase gene (galC) was cloned fromAspergillus oryzae YZ1 and expressed in Pichia pastoris. The galC (2319 bp) containing two introns encoded a protein of 726 amino acids. The activity of the alpha-galactosidase (GalC) increased 1- fold after coding sequence optimization. Purified GalC exhibited a single protein band (100 kDa) in SDS-PAGE. The optimum pH and temperature of GalC were pH 4.66 and 50 degrees C, respectively. Like many GH36 family alpha-galactosidases, GalC displayed its activities towards raffinose and stachyose. The Km values for pNPG, raffinose and stachyosewere 2.16, 4.63 and 8.54mM, respectively. The GalC retained about 90% activity within the pH range 3.0-8.0. The activity of GalC was inhibited by Cu2+, while Ca2+ increased the enzyme activity. Different concentrations of glucose, mannose, galactose, xylose and sucrose slightly affected the activity of GalC. The GalC displayed strong resistance to trypsin, alpha-chymotrypsin, and proteinase K. Under simulated gastric conditions, GalC maintained most of its native activity after pepsin treatment for 3 h. The GalC could also effectively degrade raffinose and stachyose in soymilk. The GalC with high hydrolysis efficiency towards raffinose family oligosaccharides (RFOs) and strong resistance to proteases is considered to have great potential in food and feed industries. (c) 2020 Elsevier B.V. All rights reserved.