Molecular Cloning And Biochemical Charaterization Of Beta-galactanase Agha From Aspergillus Niger

?-galactamase(EC 3.2.1.89)is a class of enzymes that hydrolyze 1,4-?-D-galactoside bonds in galactan.It promotes deep hydrolysis of galactan to prepare galacto-oligosaccharides.Galacto-oligosaccharides are probiotics with good probiotic functions in various functional oligosaccharides and have a wide range of applications in food and pharmaceutical industries.In the present study,an endo-?-1,4-galactamase from Aspergillus niger was cloned,expressed and biochemically characterized for its potential application for galacto-oligosacharides preparation,which may provide reference values for future studies on preparation methods of galacto-oligosaccharides.The main results are as follows:Gene aghA encoding endo-?-1,4-galactanase from Aspergillus niger gene aghA was successfully cloned and expressed in Pichia pastoris,and the recombinant strain GS115(pPIC-aghA)was subsequently constructed.In the shaking flask experiments,the activity of the recombinant enzyme AghA was 130.01 U/mL.This recombinant enzyme was then purified to homogeneity by ammonium sulfate precipitation,desalting and gel chromatography,SDS-PAGE analysis,the molecular weight of ?-galactanase AghA was 38 kDa and the specific activity was 7440.55 U/mg.and the purification fold and yield of were 24.701 and 16.79%,respectively.The optimal reaction temperature and pH of AghA was determined to be 45? and 4.5,respectively.After incubation at 30-50? or 4.0-5.0 for 1 h,this enzyme still retained more than 80%of its initial activity.AghA was slightly inhibted by Zn2+ and Co2+,but was strongly inhibted by Fe3+.The Km of the endo-?-1,4-galactanase AghA to galactan was 0.08±0.005 mg/mL,and the Vmax was 400±24 mg·mL-1·min-1.As compared with ONPG,AghA preferred to hydrolyze pectin.As analyzed by thin layer and high performance liquid chromatographies,AghA can hydrolyze potato pulp into galactooligosaccharides which mainly consists of galactose,galactobiose and galactotriose,with a small amount of galactatinose.The conditions for the preparartion of galacto-oligosaccharides from potato pulp were then optimized by the"change one factor at a time" method and orthogonal experimental design,and the optimal conditions obtained were as follows:the amount of AghA,hydrolysis temperature and pH were 86.6 U/g substrate,45? and 4.0,respectively.After hydrolysis under this optimized condition,the relative concentration of galacto-oligosaccharides was 6.24mg/mL.