Properties Of L-Arabinose Isomerase And Enzymatic Preparation Of D-Tagatose

D-tagatose, a rare natural hexoketose, is one of the rare sugars, which are defined as monosaccharides and their derivatives that rarely exist in nature. On a molecular level, D-tagatose is structurally identical to D-fructose, except for an inversion of the hydroxyl and hydrogen groups at the fourth carbon atom. The sweetness of D-tagatose is 92% that of sucrose with a similar taste. It has been tested that D-tagatose can be used in food industry GRAS. D-tagatose has low caloric value, in addition, it can prevent tooth decay, has no effect on plasma glucose levels, improve healthy bacterium. For these reasons, D-tagatose has received interest recently as a non-caloric sweetener substitute for sucrose.It is very important to establish a fast, convenient and effective method for data processing. According to the cysteine-carbazole test introduced by Dische, we studied test conditions and set up a simple and convenient, reliable mothord.A strain of Lactobacillus SK1.002 which can produce L-arabinose isomerase was stored in our laboratory. The effect of nutrients and other cultural conditions on the L-arabinose isomerase were studied. The optimal medium compositions and cultural conditions were(g/L): maltodextrin 28, yeast extract 10, corn steep liquor 22, CH3COONa 10, K3PO4 0.2, NaCl 0.01, FeSO4·7H2O 0.01, MgSO4·7H2O 0.2, MnSO4·2H2O 0.05, L-arabinose. 2.5 The optimum fermentation conditions were initial pH 8.4, cultivate temperature 37℃, inoculation volume 3%, fermentation time 12h. The enzyme activity reached 7.28 U/mL.The cells were disrupted by sonication and centrifuged, the supermatant was further purified by ammonium sulfate fractionation, dialysis. Properties of the L-arabinose isomerase were also studied. The optimal reaction temperature and pH of L-arabinose isomerase were 60℃and 7.0, respectively. The enzyme was stable within pH 4.0-8.0 under 30-60℃. The addition of Mn2+, Mg2+, Fe2+ ions enhanced the enzyme activity, whereas the addition of Ba2+, Zn2+ions inhibited it completely. With D-galactose as substrate, Km=1193.93mmol/L,Vmax=13.51μg/min.D-tagatose was isomerized from D-galactose using L-arabinose isomerase in three forms: cell-free enzyme, 10 and 40% (w/w) wet weight whole-cell. Effects of temperature, enzyme, substrate concentration and reaction time on the D-galactose conversion ratio were determined. The substrate concentration was found to be optimal at 0.833mol/L. A higher conversion yield of 38.9% (w/w) was obtained after about 72 hours by using 40%(w/w) wet weight whole-cell. Finally, by FT-IR Spectrometer test, we confirmed that L-arabinose isomerase mediated the isomerization of D-galactose into D-tagatose. The production of the bioconversion was D-tagatose.