Hyperproduction and thermal characterization of a novel invertase from a double mutant derivative of Kluyveromyces marxianus

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.sub.P]) and invertase yield ([Y.sub.P/S]) by M15 mutant were 1222 U/(L x 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.sub.W]) and mutant ([I.sub.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.sub.a] and the heat of ionization values indicated that carboxyl groups were involved in proton transfer during active catalysis. Ratios of [K.sub.cat]/[K.sub.m] and [v.sub.max]/[K.sub.m] indicated that [I.sub.M] was significantly more specific for sucrose hydrolysis. The [I.sub.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.sub.M] showed significantly lower values of enthalpy of activation (Δ[H.sup.*]) and entropy of activation (Δ[S.sup.*]), while Gibbs free energy (Δ[G.sup.*]) was significantly increased at higher temperatures, making the [I.sub.m] thermodynamically more thermostable. Thus [I.sub.m] could be used as a catabolite-resistant invertase for the production of fructose syrup or high gravity ethanol. Key words: enthalpy/entropy, invertase, Kluyveromyces marxianus, medium optimization, purification, thermostability