Gene Expression And Characterization Of The Recombinant Galactosidases From Rhizomucor Miehei And Paenibacillus Barengoltzii

Galactosidases from microorganisms have been attracted considerable research interest in the recent years because of their potential applications in food,feed,pharmaceutical,environmental and other fields.In this study,the gene cloning,expression and characterization of a novel ?-galactosidase from Rhizomucor miehei CAU432 and two ?-galactosidases from Paenibacillus barengoltzii CAU904 were investigated,and the main results were as follows:The a-galactosidase gene(RmgalB)was cloned from the thermophilic fungus R.miehei and expressed in Pichia pastoris.The gene belonging to glycoside hydrolase(GH)family 36 has an open reading fram(ORF)of 2241 bp encoding 746 amino acids with two introns.The recombinant a-galactosidase(RmgalB)was secreted up to 1953.9 U mL-1 by high cell density fermention in a 5 L fermentor,which is the highest yield obtained for a-galactosidase by far.The purified enzyme is a tetramer gave a single band corresponding to a molecular mass of 83.1 kDa on SDS-PAGE.The enzyme exhibited a high specific activity of 505.5 U mg-1.The optimal temperature and pH of RmgalB were determined to be 55 ? and pH 5.5,respectively.It was stable within pH 5.5-9.5 and up to 55 ?.RmgalB displayed specificity toward raffinose and stachyose,and completely hydrolyzed the anti-nutritive raffinose family oligosaccharides(RFOs).These properties make RmgalB useful in food and feed industries.A ?-galactosidase gene(PbBgal2A)was cloned for the first time from P.barengoltzii expressed in Escherichia coli.The coding region of 3045 bp encodes a protein of 1014 amino acids with a deduced molecular mass of 115.7 kDa The recombinant ?-galactosidase,belonging to GH family 2 had a specific activity of 45.1 U mg-1.PbBgal2A was optimally active at pH 7.5 and 45 ?,respectively.It was stable up to 45 ? and within pH 6.0-8.0.The recombinant ?-galactosidase was able to hydrolyze lactose efficiently at pH 7.5 and 40 ?.PbBgal2A also efficiently hydrolysed lactose from whey and milk.It also possessed transglycosylation activities at high concentrantions of lactose.These properties make PbBgal2A an ideal candidate for commercial use,in the production of lactose-free milk and galacto-oligosaccharides.A novel gene(PbBgal42A)encoding ?-galactosidase from P.barengoltzii was cloned and expressed in E.coli.The gene had an ORF of 2097 bp encoding 698 amino acids.The recombinant ?-galactosidase was purified with a molecular mass of 168.3 kDa.The purified enzyme had a specific activity of 623.9 U mg-1.The optimal temperature and pH of PbBgal42A were determined to be 45 ? and 7.0,respectively.It was stable up to 50 ? and within pH 6.5-10.5.PbBgal42A efficiently degraded' ?-galactan to yield galactose as end product,which was different from most of other ?-galactosidases from GH family 42.These properties make PbBgal42A useful in several industries.