Produção e caracterização de B-galactosidase de Kluyveromyces marxianus ATCC 46537

Abstract

β-galactosidase has presented increased use in the dairy industry, since it can be used to produce a isomolecular mixture of glucose and galactose and also has wide application in biotechnological production of milk with a low-lactose content, in production of galacto-oligosaccharides from whey lactose and as a preliminary step to d-tagatose synthesis. Exist still a great interest in the optimization of fermentation conditions and of medium composition to increase the production of β-galactosidase in order to achieve a high yield of low cost production. The yeast Kluyveromyces marxianus is an important industrial yeast for fermentation of lactose, having classic applications in the production of biomass, ethanol and enzymes. In this work the production and characterization of β-galactosidase from K. marxianus ATCC 46537 produced by fermentation of whey permeate were studied. The fermentation medium used was based in permeate whey and analysis of sugars and ethanol were made by liquid chromatography. Enzymatic activity was determined by the initial rates and glucose formed was measured by glucose oxidase. The enzyme activity unit (U mL-1) was defined as μmol of glucose produced per minute per mL enzyme suspension at a temperature of 30 °C, pH 6.5, with an initial lactose concentration of 50 g L-1. The medium to obtain inoculum was the medium composed of yeast extract (6.0 g L-1), (NH4)2SO4 (6.0 g L-1), KH2PO4 (5,0 g L-1), MgSO4.7H2O (0.6 g L-1) permeate whey (lactose concentration of 50 g L-1) prepared in phosphate buffer 10-1 M pH 5.5. Was studied the growth kinetic of the yeast and was obtained the μmax of 0, 288 h-1 and generation time of 2.41 hours in this stage. Different methods of β-galactosidase extraction were evaluated and the extraction by vortex agitator and ultrasonic processor were who showed the best results. A Fractional Factorial Design was performed to study the influence of pH and medium composition on the production of β-galactosidase for which it was obtained that the maximum production of β-galactosidase was affected more significantly by lactose, followed by (NH4)2SO4, yeast extract and pH. The fermentative medium composition was evaluated using a Central Composite Design, the best results were obtained for lactose concentrations ranging from 100 to 150 g L-1, yeast extract lower than 6 g L-1 and (NH4)2SO4 between 2 and 8 g L-1. The simultaneous influence of agitation speed and aeration rate was evaluated by performing a Central Composite Design that resulted in enzymatic activity of 139.58 U mLfermented broth-1 at the central point (250 rpm and 0.75 vvm). Was evaluated the co-production of bioethanol by fermentation of whey permeate by using K. marxianus. The highest value observed for bioethanol concentration (36.94 g L-1) was obtained in the fermentation in which was used agitation speed of 427 rpm and aeration rate of 0.24 vvm, which implies a yield of 72.3 %. Was verified, by simultaneous analysis of the optimum regions for the answers of enzyme activity and bioethanol co-production, regions that imply high values for enzymatic activity and bioethanol, simultaneously, as follows: agitation speed 200-400 rpm with rate aeration of 0.4 to 1.2 vvm. These results indicate a promising alternative for the valorization of whey permeate in the simultaneous production of β-galactosidase and bioethanol. The enzyme extract produced were characterized and the kinetic constants obtained for Vmax and Km were respectively 11.01 U mL-1 and 3.62 g L-1. The produced enzyme was most active at a temperature of 45 °C and obtained high values of relative activity in lactic acid buffer at pH 5.5 to 7.3. The polyacrylamide gel electrophoresis the enzyme extract showed a subunit molecular mass of 52.3 kDa coincident with the reference sample (Lactozyme® 2600) indicating the presence of β-galactosidase subunits produced in the extract.