title: A protease-resistant α-galactosidase from Pleurotus djamor with broad pH stability and good hydrolytic activity toward raffinose family oligosaccharides.
abstract: An acidic α-galactosidase designated as PDGI (Pleurotus djamor α-galactosidase) was purified to homogeneity with 290-fold purification and a specific activity of 52.18 units/mg by means of ion exchange chromatography and gel filtration chromatography. PDGI is a monomeric protein exhibiting a molecular mass of 60kDa in SDS-PAGE and gel filtration. The optimum pH and temperature of the enzyme with pNPGal as substrate were 5.0 and 53.5°C, respectively. It displayed great pH stability within the pH range 3.0-10.0. Besides, the enzyme harbored remarkable resistance to acid protease and varying degrees of tolerance to other proteases: trypsin>collagenase Type-I>α-chymotrypsin neutral protease>proteinaseK. It was strongly inhibited by K+, Cd2+, Cu2+, Hg2+, Al3+, Fe3+and Ag+ions. The chemical modification reagents diethypyrocarbonate (DEPC), 2,3-butanedione (DIC) and trinitrophenol (TNBS) increased the activity of PDGI 1.5-fold whereas N-bromosuccinimide (NBS) and parachloro-mercuri-benzoate (PCMB) drastically suppressed its activity. PDGI displayed activity toward stachyose and raffinose. The Km values for hydrolysis of pNPGal, stachyose and raffinose were 0.76, 7.63 and 6.29mM, respectively. Furthermore, PDGI degraded raffinose and sthachyose. These results suggest that PDGI has great potential for elimination of the non-digestible and flatulence-causing oligosaccharides stachyose and raffinose from legumes.
authors.affiliation: State Key Laboratory for Agrobiotechnology and Department of Microbiology, China Agricultural University, Beijing 100193, China. Electronic address: huyujing1216@126.com.
authors.forename: Yujing
authors.initials: Y
authors.lastname: Hu
authors.affiliation: Institute of Biotechnology and Germplasmic Resource, Yunnan Academy of Agricultural Science, Kunming 650223, China. Electronic address: tiangt@aliyun.com.
authors.forename: Guoting
authors.initials: G
authors.lastname: Tian
authors.affiliation: College of Food Science and Technology, Nanjing Agricultural University, Weigang, Nanjing 210095, China. Electronic address: zhlychen@njau.edu.cn.
authors.forename: Liyan
authors.initials: L
authors.lastname: Zhao
authors.affiliation: State Key Laboratory for Agrobiotechnology and Department of Microbiology, China Agricultural University, Beijing 100193, China. Electronic address: hxwang@cau.edu.cn.
authors.forename: Hexiang
authors.initials: H
authors.lastname: Wang
authors.affiliation: School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China. Electronic address: b021770@mailserv.cuhk.edu.hk.
authors.forename: Tzi Bun
authors.initials: TB
authors.lastname: Ng
mesh_terms: D000595:Amino Acid Sequence; D002918:Chymotrypsin / Q000737:chemistry; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008670:Metals / Q000737:chemistry; D008970:Molecular Weight; D009844:Oligosaccharides / Q000737:chemistry; D020076:Pleurotus / Q000201:enzymology*; D011887:Raffinose / Q000737:chemistry*; D013379:Substrate Specificity; D000519:alpha-Galactosidase / Q000037:antagonists & inhibitors / Q000737:chemistry*
chemical_list: D005656:Fungal Proteins; D008670:Metals; D009844:Oligosaccharides; C005695:stachyose; D000519:alpha-Galactosidase; D002918:Chymotrypsin; C117608:alpha-chymotrypsin; D011887:Raffinose
keywords: Pleurotus djamor; Protease-resistant; Raffinose family oligosaccharides; α-Galactosidase