A protease-resistant alpha-galactosidase from Pleurotus citrinopileatus with broad substrate specificity and good hydrolytic activity on raffinose family oligosaccharides
文献类型: 外文期刊
作者: Hu, Yujing 1 ; Tian, Guoting 3 ; Geng, Xueran 1 ; Zhang, Weiwei 1 ; Zhao, Liyan 4 ; Wang, Hexiang 1 ; Ng, Tzi Bun 5 ;
作者机构: 1.China Agr Univ, State Key Lab Agrobiotechnol, Beijing 100193, Peoples R China
2.China Agr Univ, Dept Microbiol, Beijing 100193, Peoples R China
3.Yunnan Acad Agr Sci, Inst Biotechnol & Germplasm Resource, Kunming 650223, Peoples R China
4.Nanjing Agr Univ, Coll Food Sci & Technol, Nanjing 210095, Jiangsu, Peoples R China
5.Chinese Univ Hong Kong, Fac Med, Sch Biomed Sci, Shatin, Hong Kong, Peoples R China
关键词: alpha-Galactosidase;Pleurotus citrinopileatus;Protease-resistant;Raffinose family oligosaccharides
期刊名称:PROCESS BIOCHEMISTRY ( 影响因子:3.757; 五年影响因子:3.665 )
ISSN: 1359-5113
年卷期: 2016 年 51 卷 4 期
页码:
收录情况: SCI
摘要: An acidic alpha-galactosidase designated as PCGI was isolated from the fruiting bodies of Pleurotus citrinopileatus with 264-fold purification and a specific activity of 7.92 units/mg. It was purified to homogeneity by ion exchange chromatography and gel filtration chromatography. PCGI is a heterodimeric protein consisting of a 33 kDa and a 27 kDa subunit in SDS-PAGE. The purified enzyme was identified by MALDI-TOF-MS. It belongs to the GH27 family. The optimum pH and temperature of the enzyme with pNPGaI as substrate were 4.4 and 50 degrees C, respectively. Besides, it displayed remarkable resistance to acid protease, neutral protease, alpha-chymotrypsin, and trypsin. It was strongly inhibited by Cd2+, Cu2+, Hg2+,Al3+, Fe3+ and Ag+ ions. Diethypyrocarbonate (DEPC) doubled the activity of PCGI whereas N-bromosuccinimide (NBS) drastically decreased it. PCGI displayed wide substrate diversity with activity toward substrates such as stachyose, raffinose, melibiose. The Km values for hydrolysis of pNPGaI, stachyose, raffinose, and melibiose were 0.2, 16.7, 18.9, and 6.3 mM, respectively. Galactose (Ki =0.92 mM) and melibiose (Ki = 7.13 mM) competitively inhibited the enzymes. Futhermore, it completely degraded raffinose and sthachyose. These results suggest that PCGI has great potential for removal of the non-digestible and flatulence-causing oligosaccharides stachyose and raffinose from legumes. (C) 2016 The Authors. Published by Elsevier Ltd.
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