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), Pic hia polymorpha (NCIM 3419) and Debaryomyces castellii (NCIM 3446) were grown on an inulin-based growth medium. Only K. fragilis produced ext racellular inulinase with a maximum after 36 h of growth at 25-27-degr ees-C. Sucrose and fructose were weak inducers of inulinase as compare d to inulin whereas with glucose the inulinase level was minimal. An a queous extract of chicory roots containing 1%, fructan was a better ca rbon source than inulin and peptone was the best nitrogen source for t he 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 c ulture filtrate was reduced to 1.6 on purifying inulinase by ethanol p recipitation followed by chromatography on Sephadex G-200, DEAE-cellul ose and CM-cellulose columns. Using this purification procedure. inuli nase 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 determ ined as 250 +/- 10 kDa. The crude and purified inulinase preparations did not release sucrose or oligosaccharides from inulin, indicating th at the enzyme has primarily exo-inulinase activity. Using the metal-li nk chelation method, 40% of inulinase was immobilised on cellulose. Ma ximum activity of crude, purified and immobilised inulinase preparatio ns was observed at 55-degrees-C. The half-life of immobilised inulinas e at 25-degrees-C was 5 days.