Insolubilization of inulinase on magnetite chitosan microparticles, an easily recoverable and reusable support

Abstract

Inulinase (E.C. 3.2.1.7), a highly productive enzyme could provide a solution to industries looking forward to a robust biocatalyst which is stable and recyclable for the production of high fructose syrup. This objective can be realized by an amalgamation of magnetic nanobiotechnology and enzyme engineering. The present work explores the ramifications of this concept through the insolubilization of inulinase on to magnetite nanoparticles entrapped in chitosan (cMNPs). Statistical methods were used to identify the optimum concentration of EDAC (N-(3-dimethylaminopropyl)-N′-ethyl carbodiimide hydrochloride), cMNPs, and cross-linking time which play a pivotal role in deciding the overall efficiency of the biocatalyst. The optimum concentrations for preparation of the magnetic biocatalyst were found to be 15.29mg and 23.76mg for EDAC and cMNPs respectively, for which the maximum activity recovery obtained, was 81.40% with an optimum cross linking time of 1.73h. The optimum pH and temperature for soluble inulinase (SI) was found to be 6.0 and 70°C whereas that of magnetically insolubilized inulinase (MII) shifted to 4.6 and 50°C. The insolubilized inulinase could be recycled for up to 15 cycles with substantial activity at least for a year at 4°C. This feature coupled with ease separation of biocatalyst and a fivefold increase in the t1/2 value at temperature of 80°C are evidence of enhanced thermal and operational stability inherited by the enzyme after insolubilization, indicating a high stability in practical operation.