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Modelling and optimisation of inulinase production by Kluyveromyces bulgaricus

In the work are presented surface response and physiological based modelling of production of microbial enzyme inulinase by Kluyveromyces bulgaricus (Kluyveromyces marxianus). Applied is the experimental design and surface response method for optimi-sation of temperature and pH on inulinase production cultivated on saccharose in shaking flasks. The process of inulinase synthesis is also modelled based on assumption of growth related inulinase production rate. The yeast growth rate is expressed by a balance with a paseudo rate limiting enzyme. Reversible temperature deactivation is modelled as a thermodynamic equilibrium with Arrhenius activation specific rates. Reversible pH inhibition is modelled by acidity dependent equilibrium between active enzyme and inhibited protonated and hydroxylated enzyme. The data set obtained by the experimental desin is applied for nonlinear regression of the physiological model. The results indicate that pH value of the cultivation medium showed is the most significant variable and it should be maintained at optimum value, 3, 5. The effect of temperature was slightly lower and optimal values are between 30 and 35&ordm ; ; C. 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 figures were: 100, 16-124, 36 U/ml (with sucrose as substrate for determination of enzyme activity) or 8, 6 -11, 6 U/ml (with inulin as substrate for determination of enzyme activity), respectively. The two modelling and optimisation methods are compared by ANOVA.

Kluyveromyces bulgaricus; inulin; inulinase; optimisation; response surface method; physiological modelling

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