PRODUCTION OF LEVAN-TYPE FRUCTOOLIGOSACCHARIDE BY IMMOBILIZED LEVANSUCRASE

Abstract

Levansucrase produces levan-type fructooligosaccharide (L-FOS) by hydrolyzing sucrose, and then transfers the fructofuranosyl residue to sucrose or the growing L-FOS chain, liberating glucose. Levansucrase gene (lsRN) of Bacillus licheniformis RN-01 was expressed in Escherichia coli Top-10, using in 3X LB medium. The efficiency of levansucrase (LsRN) when covalently bound on chitosan bead, partially deacetylated chitin/chitosan, fine particle chitosan, epoxide Sepabeads EC-EP bead, and amino epoxide Sepabeads EC-HFA bead was 96%, 94%, 11%, 38%, and 30%, respectively. Levansucrase immobilized on CTS beads lost less than 25% of its activity after 10 cycles of repeated use. In contrast, levansucrase immobilized on Sepabead EC-EP or Sepabead EC-HFA lost over 60% after only 5 cycles of repeated use. The optimum pH and temperature of the immobilized enzyme on CTS beads were broader than that of free enzyme, pH 6.0 at 40 oC compared to pH 4.0-6.0, 40-50 oC, respectively. Levansucrase immobilized on CTS beads could tolerate sucrose concentration up to 50% (w/v) while retaining over 95% of its activity, which is significantly better than levansucrase immobilized on Sepabead EC-EP and EC-HFA and free enzyme. Immobilization of levansucrase on CTS bead could slightly protect levansucrase from the inactivation by Cu2+, Zn2+ and Fe3+, as well as SDS and EDTA. Interestingly, immobilizing levansucrase on CTS beads further elevate the enhancement of levansucrase activity by Mn2+ more than 2 folds. The stability of immobilized levansucrase activity was found to be highest on CTS beads, retaining approximately 70% activity after 12 h incubation in 50 mM citrate buffer, pH 6.0, at 50 oC. L-FOS products were produced by LsRN-N251Y for 7.83 g at the first cycle and decreased to 5.22 g at fifth cycle. LsRN-Y246S produced L-FOS for 8.36 g and 6.26 g for the first and fifth cycle, respectively. The 8.14 g and 4.56 g of L-FOS at the first and fifth cycle respectively were produced from LsRN-Y246W. Scale up production of L-FOS was performed, using packed-bed immobilized levansucrase reactor. The packed-bed column contained 1,890 U of immobilized levansucrase. A 250 g (50% (w/v)) of sucrose solution was feed into the column with an upward flow direction with a flow rate of 10 mL/min, at 40 oC. The medium chain and long chain of L-FOS products produced at 12 h about 118.39 g and 130.45 g from LsRN-Y246S and Y246W, respectively. LsRN-N251Y produced short chain L-FOS products, 102.93 g at 24 h. L-FOS products were successfully fractionated into DP1 –DP9 by Biogel P2 column chromatography.