Hyperproduction and Thermal Characterization of a Novel Invertase from a Double Mutant Derivative of Kluyveromyces marxianus 

Shaheen Aziz^1,2, Fatima Jalal², Muhammad Nawaz^2,3*, Bushra Niaz^2,3,Farman Ali Shah^1,2, Muhammad Hafeez-ur-Rahman Memon¹, Farooq Latif ², Shahid Nadeem³ and Muhammad Ibrahim Rajoka<sup>2,3

1</sup>Department of Chemical Engineering, Mehran University of Engineering and Technology (MUET), Jamshoro, Sindh, PK-76062 Pakistan
²Industrial Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, PK-38000 Pakistan
³Government College University, Allama Iqbal Road, Faisalabad, PK-38000 Pakistan

Article history:
Received March 16, 20100
Accepted December 8, 2010

Key words:
enthalpy/entropy, invertase, Kluyveromyces marxianus, medium optimization, purification, thermostability

Summary:
Kinetics of intracellular invertase production employing a double mutant derivative of Kluyveromyces marxianus was optimized by varying different process variables in a 23-litre fermentor. The maximum volumetric rate (Q_P) and invertase yield (Y_P/S) by M15 mutant were 1222 U/(L·h) and 160 U/g of substrate utilized, respectively (2-fold more than those of parental strain) at 50 °C on the molasses (150 g/L of total fermentable sugars) at pH=5.5. Glucose or sucrose (100, 150 or 170 g/L) did not repress invertase catabolically under the optimized fermentation conditions, contrary to the previous reports on other yeasts and filamentous fungi, where catabolite repression of sugars was predominant. Invertases derived by the wild (I_W) and mutant (I_M) strains were purified employing ammonium sulphate precipitation, and then characterized by column chromatographic techniques both kinetically and thermodynamically. The acidic limb of invertases was missing and collation of pK_a and the heat of ionization values indicated that carboxyl groups were involved in proton transfer during active catalysis. Ratios of K_cat/K_m and v_max/K_m indicated that I_M was significantly more specific for sucrose hydrolysis. The I_M exhibited stability in different buffers at pH=3.0–10.0 and temperature of 50–70 °C, as reflected by long half-lives. I_M showed significantly lower values of enthalpy of activation (ΔH*) and entropy of activation (ΔS*), while Gibbs free energy (ΔG*) was significantly increased at higher temperatures, making the I_M thermodynamically more thermostable. Thus I_M could be used as a catabolite-resistant invertase for the production of fructose syrup or high gravity ethanol.

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