Preparation Of Galacto-Oligosaccharides By Biocatalysis

Galacto-oligosaccharides as a functional oligosaccharide is widely used in infant formula milk powder,fermented milk,confectionery products,baked food,livestock feed,pet food and other fields due to its excellent physical and chemical properties and physiological effects.GOS mainly produced by?-galactosidase which is an important type of glycoside hydrolase,with hydrolytic activity and transglycosylation activity.Developing new?-galactosidase,exploring its enzymatic properties,and applying it to the preparation of galactooligosaccharides have very important research value and practical significance.In this thesis,on the basis of a new?-galactosidase-producing strain Klebsiella oxytoca ZJUH1705 obtained in the laboratory,two?-galactosidase genes bga1 and bga2 from Klebsiella oxytoca ZJUH1705 were cloned and expressed in E.coli BL21?DE3?.The amino acid sequence analysis of?-gal 1 and?-gal 2 indicated that they belong to different glycoside hydrolase families GH42 and GH2.Secondly,the expressed?-gal 1 and?-gal 2 were separated by Ni-chelating affinity chromatography,the enzyme activities of?-gal 1 and?-gal 2 were 217.6 U/mg and 57.9U/mg,respectively.SDS-PAGE and Native-PAGE analysis showed that?-gal 1 and?-gal 2 were homotrimers and dimers with monomer molecular weights of about 78 k Da and 118 k Da.The?-gal 1 and?-gal 2 are all mesophilic and neutral p H?-galactosidase with K_m of 5.62 m M and 3.91 m M,and V_max of 167.0?mol?mg^-1?min^-1and 14.6?mol?mg^-1?min^-1.In addition,?-gal 1 can only hydrolyze lactose and lactulose,while?-gal 2 can also hydrolyze lactose and lactulose and have transglycosylation activity.Finally,the process of GOS preparation by?-gal 2 was studied.The results showed that the optimal reaction condition was 37?,400 g/L initial lactose concentration,p H7.5,10 U/g _lactose enzyme,reaction time 48 h.The GOS yield is 45%,and the GOS concentration is 178 g/L.The reaction mixture mainly contains monosaccharides?glucose,galactose?,unreacted lactose and galacto-oligosaccharides?disaccharide,trisaccharide and a small amount of tetrasaccharide?.