Study On The Efficient Production Of Tagatose By Biotransformation

D-tagatose,a natural rare ketohexose,has been one of the hot research topics in recent years.As a novel functional sweetener,D-tagatose has attracted extensive attention in food,cosmetics,and medicine industries due to its low calories,minimal glycemic effect,and dental care functions.In my laboratory,we obtained a strain of Lactobacillus brevis which was able to produce L-arabinose isomerase.Lactobacillus brevis is an ideal food grade strain to product D-tagatose.In this paper,the cultivation and enzyme production conditions were studied,the key enzyme in the production process was purified,and the high efficiency of D-tagatose production of L.brevis was explored.The fermentation conditions of L.brevis were studied by single factor experiment.And the optimal fermentation medium was determined as?g/L?:L-arabinose 5,corn steep liquor20,tripotassium phosphate 0.2,magnesium sulfate 0.05,sodium acetate 10.The optimum fermentation conditions were:initial pH 6.0-6.2,culture temperature 37°C,and culture time24 h.The enzyme activity increased about 3 folds after optimization.L-AI is a kind of endoenzyme of L.brevis.To obtain the crude enzyme,the cells disrupted by sonication and the supernatant was crude enzyme solution.Thereafter,the crude protein was further purified by ammonium sulfate fractionation?60%-80%?,dialysis desalting,DEAE-Sepharose CL-6B,and Native-PAGE gel extraction to obtain pure L-AI.The enzyme purity and molecular weight were estimated by the SDS-PAGE method and the oligomerization state of the enzyme was examined by the Native-PAGE.The L-AI was found to be stable at 45-75^°C,and at pH 7.0-9.0.Its optimum temperature and pH was determined as 65^°C and 7.0,respectively.Besides,we found that Ca²⁺,Cu²⁺,and Ba²⁺ions inhibited the enzyme activity,whereas the enzyme activity was significantly enhanced in the presence of Mg²⁺,Mn²⁺,or Co²⁺ions.Pure enzyme with molecular weight of 60.1 kDa was a monomer in solution.Furthermore,we characterized the kinetic parameters for L-AI and determined the K_m?129 mM?and the V_max(0.045 mM min^-1)values.The kinetic equation is V=0.045[s]/?129+[s]?.In this paper,the process conditions for the production of D-tagatose by whole-cell were optimized,and D-tagatose was produced by using whole-cell and L-AI as catalysts,D-galactose as the substrate.After the reaction of whole-cell for about 48 h or the reaction of L-AI for about 23 h,the conversion rate reached 45.1%,and the conversion was higer.And the cells can be used repeatedly for 3 times,L-AI can only be used repeatedly for 2 times.