Gene Cloning And Enzymological Characterizations Of Aspergillus Oryzae ?-galactosidases

?-Galactosidases(?-D-galactohydrolases,EC 3.2.1.23)are ubiquitous enzymes capable of hydrolyzing terminal ?-D-galactosyl moieties from substrates such as disaccharides,diverse glycoconjugates,and polysaccharides.They belong to the glycoside hydrolase.Under distinct reaction conditions,?-galactosidases can also catalyze transglycosylation reactions using various acceptor molecules,a capability used for the synthesis of galactooligosaccharides(GOS).?-Galactosidases have been widely used to hydrolyze lactose and render dairy products consumable for lactose-intolerant individuals.Further applications of these enzymes range from analytical studies to glycan remodeling and include various processes of biotechnological and medical importance.In 2015,the publish of aspergillus oryzae RIB40 genome make the study of?-galactosidases under whole genome possible.Through the analysis of aspergillus oryzae genome sequence information and find its genome have four encoding?-galactosidase gene sequence at least,but the biochemical vitality and function diversity between them are unknown.Therefore aspergillus oryzae ?-galactosidases are cloning expressed through the molecular cloning technology,and this research analysis comparatively the biochemical characteristics of them,and the aspergillus niger?-galactosidases system for comparative analysis with its biological characteristic,in order to compare systematically the similarities and differences of aspergillus oryzae?-galactosidases,laying the foundation of the development of a new generation of?-galactosidase products and realize the large-scale industrial production and application.The main results are as follows:The three aspergillus oryzae F1005F ?-galactosidases coding gene sequences(O158,AO,076)were cloned by PCR and then overexpressed recombinant Pichia pastoris strains(GS-0158,GS-AO,GS-076)were developed followed by the expressing plasmids(pPIC-O158,pPIC-AO,pPIC-076)constructed and screeing by MD medium and G418-YPD.Strains(pPIC-0158,pPIC-AO,pPIC-076)secretorily produced?-galactosidases induetively in 0.5%(v/v)Methanol for 120 hours in shaking flask fermentation.The biochemical properties of the three recombinant ?-galactosidases are examined.0158 perform the maximum activity under the condition of pH 4.0 and 50?;AO perform the maximum activity under the condition of pH 5.5 and 50?;076 perform the maximum activity under the condition of pH 7.0 and 50?.They have a good stability when the temperature no higher than 40? and pH range 4.5-7.5.Their activity are enhanced by Mn2+,whereas Fe2+and Cu2+inhibite them.The Km of 0158,AO and 076 towards lactose are determined to be 5.48 mmol/L,5.27 mmol/L and 6.21 mmol/L.0158,AO and 076 perform a specificity towards lactose as sole substrate with the activities of hydrolysis and transglycosylation.In addition,?-galactosidase 042,which is exist in the reference genome,is not exist in the aspergillus oryzae strain of this research.This study has cleared and compared the enzymatic properties of 0158,AO and 076,The similarities and differences between them are studied systematically,It will provide theoretical basis for further research and application of aspergillus oryzae P-galactosidases.