Optimisation of inulinase production by Kluyveromyces bulgaricus

Present work is based on observation of effects of pH and temperature of fermentation on the production of microbial enzyme inulinase by Kluyveromyces bulgaricus (former Kluyveromyces marxianus). Inulinase hydrolyses inulin, an oligosaccharide which can be isolated from plants such as Jerusalem artichoke, chicory or dahlia, into pure fructose (1). Fructooligosaccharides have great potential in food industry because they can be used as calorie-reduced and noncariogenic sweeteners. Fructose formation from inulin is a single step enzymatic reaction and yields are up to 95 % fructose. On contrary, conventional fructose production from starch needs at least three enzymatic steps, yielding only 45 % fructose (2). Process of inulinase production was optimised by using experimental design method. pH value of the cultivation medium showed to be the most significant variable and it should be maintained at optimum value, 3.6. The effect of temperature was slightly lower and optimal values are between 30 and 33 oC. At a low pH value of the cultivation medium, the microorganism was not able to produce enough enzyme and enzyme activities were low. Similar effect was caused by high temperature. Highest values of enzyme activities were achieved at optimal fermentation conditions and the values were: 100.16-124.36 IU/ml (with sucrose as substrate for determination of enzyme activity) or 8.6 -11.6 IU/ml (with inulin as substrate), respectively. The method of factorial design and response surface analysis makes it possible to study several factors simultaneously, to quantify the individual effect of each factor and to investigate their possible interactions (3). The model based on physiological assumptions is also applied. Assumed is a single enzyme rate determing growth (Monod kinetics) with proportional inulinase production rate. Applied are the models of reversible temperature and acidity inhibition based on thermodynamic equilibrium between active and inhibited enzyme states. Predictions by the two models are compared by ANOVA.