Sequential cultivation method for β-fructofuranosidase production from URM4634, evaluation of their biochemical and kinetic/thermodynamic characteristics, and application on sucrose hydrolysis.

The present study focused on evaluating the sequential fermentation (SF) method for FFase production from URM4634 using soybean bran as substrate. The SF was performed using soybean bran as substrate at 72 h and 30 °C and the maximum hydrolytic activity (44.00 U mL), corresponding to an increase of 2.98-fold to about SmF using sucrose as substrate. Already the maximum transfructosylating activity was 26.10 U mL. The FFase presents maximum hydrolytic activity at pH 5.0-6.0 and transfructosylating at pH 6.0 and 60 °C for both enzyme activities. The enzyme showed a typical hydrolytic kinetic profile evidenced by more affinity by sucrose hydrolysis reaction than the fructosyl transfer one. From kinetic and thermodynamic data of thermal denaturation, it was observed that the enzyme presents suitable at 55 °C, evidenced by the large half-life (990.21 min) and values (3289.41 min). The maximum release of reducing sugars (8.45 g L) was obtained in hydrolysis of 20% sucrose during 180 min. The results obtained for FFase production by SF proved that this method can be used satisfactorily for sucrose-degrading enzymes and can contribute to the development of the SF technique to produce different industrial-interest enzymes.