Purification Of β-Fructosyltransferase And β-Fructofuransidase From Aspergillus Oryzaea GX0011 And Their Application In Production Of Fructooligosaccharides

Fructooligosaccharides(FOS) have received particular attention in recent years because of their favorable biological and functional properties such as low calorie, noncariogenic sweeteners, consecutive improvement of the intestinal microbial flora, relief of constipation, decrease of total cholesterol and lipid in the serum. They could be produced by transferring one to three molecules of fructose to the fructose residue in sucrose byβ-fructosyltransferase(FTS).β- fructofuranosidase (FFS) catalyzed the hydrolysis of sucrose and fructooligosaccharides by recognizing the fructose moiety of their molecules. In the present paper, FTS and FFS from Aspergillus oryzae GX0011 were studied. The experiments were shown just as follows:The optimum culture medium of GX0011 on the base of 200 ml tomato culture broth in a 500-ml Erlenmeyer flask was determined by liquid submerged fermentation(LSF). Through orthogonal test design and single factor experiment, influence of carbon resource, nitrogen resource, trace element, temperature, pH and rotating speed on the production of FTS and FFS has been investigated. The optimum condition of LSF was sucrose 2.4%, yeast extract 0.5%, K⁺ 0.1%, 29℃, pH6.5, 32h and 170rpm. The maximum productivity of FTS(16.928U/ml) was 5-fold higher than that with the medium before optimization.FTS and FFS were isolated and purified from crude extract of GX1100 by the procedures of heat-denaturing, ammonium sulphate fractionation, ion column chromatography on DEAE-Sephadex A-50 (1st), Octyl-Sepharose Cl-4B, Sephacryl S-300 and DEAE-Sephadex A-50(2nd). The purified enzymes were homogeneous on SDS-PAGE with molecular weight of 51 kDa and 35 kDa. The final purified fold, specific activity and the yield of the purified FTS were 121.7, 1975U/mg and 17%. And those of FFS were 396.4, 983U/mg and 7% respectively. On SDS-PAGE, both enzymes gave clear, not diffused glycoprotein bands with different electrophoretic mobility. This result and lower molecular weight indicated that FTS was not glycoproteins. Furthermore, FFS exhibited only hydrolytic activity, while FTS catalyzed exclusively fructosyltransfer reaction.The two enzymes exhibited distinctly different pH and temperature profiles. The optimum reaction pH and temperature for FTS were found to be 5.8 and 40℃, while FFS showed the optimum activity at pH 4.0 and 55℃. FTS was stable below 45℃and pH5.5～7.5, while FFS stable condition were less than 60℃and pH 3.0～5.0. Furthermore, the two enzymes exhibited obviously different kinetic constants confirming their different nature. The K_m and V_max values were calculated to be 240.530g/L and 2.217g/L/min for FTS and 0.2585 g/L/min and 10.5719g/L for FFS , respectively. Fructose didn't affect FTS activation and glucose was a competitive inhibitors for FTS, vice versa. The K_i of glucose to FTS was 173.472 g/L and fructose to FFS was 3.664g/L. At 10% and 50% sucrose concentration purified FTS produced FOS at the yield of 57.47% and 62.85%, respectively. In addition, Metal ions and other chemicals had different effects on the activities of two enzymes, but no actived obviously reagent was found during the work.The effects of chemical modification such as PMSF, NBS, EDC and TNBS on FTS were measured when sucrose was added or not. The decrease of activity has linear correlation with the concentration of modify reagent. So it could be concluded that the active center of FOS may include serine group, tryptophan group and aspartic acid group (glutamic acid). The lysine group also had a certain effect on the enzyme, which may effect the configuration of the enzyme. And their IC₅₀ were 27.08mmol/L, 5.50 mmol/L, 25.86 mmol/L and 18.59 mmol/L, respectively.Ethanol and other multiple inhibitors(A and B) had obviously different effects on the activity of two enzymes. FFS was almost completely abolished with ethanol, A and B, while FTS maintained or intensively enhanced its original activity. The effects of ultrasonic on the enzyme were also studied. FFS was residual 43% of its original activity on (A+300)W, 20 min or (A+700)W, 5 min on the given frequency, while FTS almost maintained 100% activity. At 10% and 50% sucrose solutions FTS after ultrasonic disposal produced FOS at the yield of 57.47% and 62.85 % respectly against 47.3 % and 53.5 % with the crude extract.