PRODUCTION, PURIFICATION AND IMMOBILIZATION OF INULINASE FROM KLUYVEROMYCES-FRAGILIS

Kluyveromyces fragilis (NCIM 3217), Kluyveromyces marxianus (NCIM 3231), Hansenula polymorpha (NCIM 3377). Pichia fermentan (NCIM 3408), Pichia polymorpha (NCIM 3419) and Debaryomyces castellii (NCIM 3446) were grown on an inulin-based growth medium. Only K. fragilis produced extracellular inulinase with a maximum after 36 h of growth at 25-27-degrees-C. Sucrose and fructose were weak inducers of inulinase as compared to inulin whereas with glucose the inulinase level was minimal. An aqueous extract of chicory roots containing 1%, fructan was a better carbon source than inulin and peptone was the best nitrogen source for the production of inulinase. The maximum yield of inulinase was about 7 units cm-3 of medium. The invertase to inulinase ratio of 10 in the culture filtrate was reduced to 1.6 on purifying inulinase by ethanol precipitation followed by chromatography on Sephadex G-200, DEAE-cellulose and CM-cellulose columns. Using this purification procedure. inulinase was purified 26-fold. With inulin as substrate, the shape of the velocity curve was nearer to a sigmoidal pattern whereas with sucrose the curve was hyperbolic. The molecular weight of inulinase was determined as 250 +/- 10 kDa. The crude and purified inulinase preparations did not release sucrose or oligosaccharides from inulin, indicating that the enzyme has primarily exo-inulinase activity. Using the metal-link chelation method, 40% of inulinase was immobilised on cellulose. Maximum activity of crude, purified and immobilised inulinase preparations was observed at 55-degrees-C. The half-life of immobilised inulinase at 25-degrees-C was 5 days.