Study On α-galactosidases And β-galactosidase From Bispora Sp. MEY-1

α-Galactosidase (EC3.2.1.22) andβ-galactosidase (EC 3.2.1.23) are two types of glycoside hydrolases widely existed in nature, which can cleaveα- orβ-linked galactose residues from a series of compounds. The modification of galactomannan byα-galactosidase extended its applications in food, pharmacy, and pulp industries.β-Galactosidase hydrolyze lactose in milk into galactose and gluctose. Supplementation of microbialβ-galactosidase could alleviate the symptoms caused by lactose intolerance. In this study, threeα-galactosidase genes and oneβ-galactosidase gene from the acidophilic fungus Bispore sp. MEY-1 were cloned, expressed and characterized.All the threeα-galactosidases AgalA,AgalB and AgalC from Bispore sp. MEY-1 belong to glycoside hydrolase family 27. Both of AgalA and AgalC exhibited the highest amino acid sequence identity (52%) with theα-galactosidase from Lachancea thermotolerans; however, the amino acid identity between AgalA and AgalC was only 57.6%. The deduced amino acid sequence of AgalB showed the highest identity (36%) with that of theα-galactosidase from Penicillium simplicissimum, and 24.9% and 27.2% to that of AgalA and AgalC, respectively. The optimal pH of AgalA,AgalB and AgalC were 5.5,3.5 and 7.0, respectively. The temperature optima of the threeα-galactosidases were 55°C,55°C and 45°C, respectively. AgalA,AgalB and AgalC exhibited different activity towards different substrates. AgalA only hydrolyzes synthetic substrate pNPG; AgalB and AgalC have ability to not only hydrolyze small galacto-oligosaccharides (melibiose, raffinose and stachyose), but also galactomannan polysaccharides (guar gum and locust bean gum). The specific activity of AgalA,AgalB and AgalC towards pNPG was 31.2 U/mg,581 U/mg and 128.8 U/mg, respectively. The synergistic action of AgalB and family 5β-mannanase Man5A from the same organism to degrade guar gum showed that AgalB not only degrade the side-chain of guar gum alone, but also acts synergisitically withβ-mannanase. When increased the incubation period of AgalB, the action of Man5A was enhanced significantly. Neither AgalA nor AgalC had ability to act synergisitically withβ-mannanase.Belonging to glycoside hydrolase family 35,β-galactosidase BgalA showed optimal activity at pH 1.5 and 60°C. BgalA was stable over the whole pH range and was strongly resistant to pepsin and trypsin digestion. Most of metal ions have no significant effects on its activity. The specific activity of r-BgalA towards ONPG was 99.5 U/mg. The Km and Vmax of BgalA for ONPG and lactose are 5.22 mM and 120.8μmol/ (min·mg), and 0.31 mM and 137.3μmol/ (min·mg), respectively. Compared with the low hydrolysis ratio (3.5%) of commercialβ-galactosidase from Aspergillus oryzae, BgalA was more stable and showed higher hydrolysis ability (85.8%) toward milk lactose under simulated gastric conditions.In summary,α-galactosidase AgalB andβ-galactosidase BgalA were resistant to acid and proteases, and have great potential in industrial applications. This study provides more information for development of future industrial enzymes.