Biochemical characterization of three Aspergillus niger ß-galactosidases

Background: ß-Galactosidases catalyze both hydrolytic and transgalactosylation reactions and therefore have many applications in food, medical, and biotechnological fields. Aspergillus niger has been a main source of ß-galactosidase, but the properties of this enzyme are incompletely studied. Results: Three new ß-galactosidases belonging to glycosyl hydrolase family 35 from A. niger F0215 were cloned, expressed, and biochemically characterized. In addition to the known activity of LacA encoded by lacA, three putative ß-galactosidases, designated as LacB, LacC, and LacE encoded by the genes lacB, lacC, and lacE, respectively, were successfully cloned, sequenced, and expressed and secreted by Pichia pastoris. These three proteins and LacA have N-terminal signal sequences and are therefore predicted to be extracellular enzymes. They have the typical structure of fungal ß-galactosidases with defined hydrolytic and transgalactosylation activities on lactose. However, their activity properties differed. In particular, LacB and lacE displayed maximum hydrolytic activity at pH 4­5 and 50°C, while LacC exhibited maximum activity at pH 3.5 and 60°C. All ß-galactosidases performed transgalactosylation activity optimally in an acidic environment. Conclusions: Three new ß-galactosidases belonging to glycosyl hydrolase family 35 from A. niger F0215 were cloned and biochemically characterized. In addition to the known LacA, A. niger has at least three ß-galactosidase family members with remarkably different biochemical properties.

Producción y caracterización parcial de beta-galactosidasa de Kluyveromyces lactis propagada en suero de leche desproteinizado / Production and partial characterization of beta-galactosidase from Kluyveromyces lactis grown in deproteinized whey

The purpose of this work was to optimize the beta-galactosidase production by Kluyveromyces lactis, applying the Surface Response Methodology (SRM) and using deproteinized whey as fermentation medium. An Orthogonal Central Compound Design (OCCD) was used without repetition, with four factors: temperature, pH, agitation speed and fermentation time. Then, enzyme activity (U/ml) as response variable was used. Thirty trials in twenty-five treatments, with six repetitions at the central point, were carried out, in a New Brunswick Bioflo 2000 fermentor with a volume of 2 liters. The deproteinized whey obtained by thermocoagulation was chemically analyzed. The results were: moisture 93.83 per cent, total solids 6.17 per cent, protein 0.44 per cent, lactose 4.85 per cent, acidity 0.43 per cent and pH 4.58. The best conditions in the enzyme production were: temperature 30.3 degrees C, pH 4.68, agitation speed 191 r.p.m. and fermentation time 18.5 h. with an enzyme production of 8.3 U/ml. The degree of purification obtained was 7.4 times and the yield was 50.8 per cent. The purified enzyme had an optimum temperature of 60 degrees C and a pH of 6.2. This work shows that the yeast Kluyveromyces lactis grown in deproteinized whey is able to produce the enzyme beta-galactosidase and SRM can be used in the fermentology processes, specifically in determining the best suitable operation conditions.

Properties of a new fungal ß-Galactosidase with potential application in the dairy industry

ß-Galactosidase or ß-D-galactohydrolase (EC.3.2.1.23) is an important enzyme industrially used for the hydrolysis of lactose from milk and milk whey for several applications. Lately, the importance of this enzyme was enhanced by its galactosyltransferase activity, which is responsible for the synthesis of transgalctosylated oligosaccharides (TOS) that act as functional foods, with several beneficial effects on consumers. Penicillium simplicissimum, a strain isolated from soil, when grown in semi-solid medium showed good productivity of ß-galactosidase with galactosyltransferase activity. The optimum pH for hydrolysis was in 4.0-4.6 range and the optimum pH for galactosyltransferase activity was in the 6.0-7.0 range. The optimum temperature for hydrolysis and transferase activity was 55-60§C and 50§C, respectively, and the enzyme showed high thermostability for the hydrolytic activity. The enzyme showed a potential for several industrial applications such as removal of 67 (per cent) of the lactose from milk and 84 (per cent) of the lactose from milk whey when incubated at their original pH (4.5 and 6.34, respectively) under optimum temperature conditions. When incubated with a 40 (per cent) lactose solution in 150 mM McIlvaine buffer, pH 4.5, at 55§C the enzyme converted 86.5 (per cent) of the lactose to its component monosaccharides. When incubated with a 60 (per cent) lactose solution in the same buffer but at pH 6.5 and 50§C, the enzyme can synthetize up to 30.5 (per cent) TOS, with 39.5 (per cent) lactose and 30 (per cent) monosaccharides remaining in the preparation.

Studies on beta-galactosidase from Candida pseudotropicalis III- Purification and some properties of the pure enzyme

Beta-galactosidase from Candida pseudotropicalis was partially purified by fractional precipitation with ammonium sulfate. Purification was achieved by sephadex G-75 and the cellulose DE-52 column chromatography, the purification fold was 5.2, 20.6 and 104.2, respectively. The purity of enzyme was checked on polyacrylamide gel Disc electrophoresis. The properties of the purified enzyme have been studied. The Km and V max were 4 x 10-5 M and 27 x 106 M/min-1, respectively. The maximum enzyme activity was obtained in 0.2 M potassium phosphate buffer at pH 7.0 and incubation temperature 30C for 25 minutes. The effect of temperature, pH on enzyme stability and activity was studied. The enzyme was strongly activated by Mg+2, while as Mn+2, Ca+2, Na+2 and Zn+2 had an activating effect at low concentrations and Cu+2 showed complete inhibition