Optimization Of Arthrobacter.sp SYBC-La1 Fermentation For β-galactosidase Production And Enzyme Characterization

Lactulose (4-O-β-D-galactopyranosyl-D-fructose) is a synthetic ketose disaccharide, It can be obtained from lactose by chemical synthesis. Lactulose specifically promotes the inte- stinal proliferation of Bifidobacterium, Lactulose is used in pharmaceuticals as a gentle la -xative and to treat hyperammonemia. Lactulose is currently manufactured by chemical synthesis involving the alkaline isomerization of lactose. The process includes expensive separation and purification steps to remove byproducts. Biocatalysis process with clean production and easy purification byβ-galactosidase can overcome the disadvantages of lactulose production by chemical synthesis.10 strains of microorganisms producingβ-galactosidase were screened from soil by mark of 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside. One bacterium strain producingβ-ga- lactosidase with transgalactosylation activity was screened from it by testing lactulose In 10 mM phosphate buffer (pH 7.0) containing 40 % lactose, 20 % fructose, 400 U/L enzyme activity, reacted at 35℃with shaking for 8h, the products were analyzed by high-performance liquid chromatography (HPLC). The result show that the reacted product from sample containsβ-galactosidase from strain (2-1) contains lactulose. Clone and analyze the 16S rDNA sequence . By making a comprehensive view on all the results of taxonomy, the strain was identified as Arthrobacter sp SYBC La1.The optimized fermentation medium contained (g/L): glycerol 20, yeast extract 14, Fe3+ 1.5 mmoL/L, pH 6.5. The optimized fermentation condition was: 10 h of seed age, 30 mL of the suitable medium volume size in 250 mL flasks, 12 % of the inoculum size, 16 h of the cultiva tion time and 25℃of the fermentation temperature. Theβ-galactosidase activity was increased 1.07times from 0.322 U/mL to 0.688 U/mL after optimization.The optimum pH ofβ-galactosidase was 6.5, with relative activities were all above 50 % at the rang of pH 5.5 to pH 8. The optimum temperature ofβ-galactosidase was 30℃, It was prove to have instability on height teampreture,β-galactosidase could be activated by Mn2+ K+,Zn2+and Mg2+. It was strongly inhibited by Cu2+. With theprocedure of ammonium sulfate precipitation,ion exchange chromatography and gel filtration .The purification and yield were 22.34-fold and 20.5 % respectively.