One step partial purification of beta-D-galactosidase from Kluyveromyces marxianus CDB 002 using STREAMLINE-DEAE.

The intracellular enzyme beta-D-galactosidase provides interesting applications in the dairy industry, which are able to solve problems related to product processing, or can alleviate lactose intolerance in some populations. In order to obtain a technical enzyme, yeast cells of Kluyveromyces marxianus CDB 002 were disrupted by high pressure homogenization and an innovative chromatographic technique was tested for the recovery of beta-D-galactosidase. A STREAMLINE 25 column, containing 65 ml STREAMLINE-DEAE was equilibrated with 50 mM potassium phosphate buffer pH 7.5 at an upward flow of 250 cmh-1. 100-200 ml cell homogenate were applied onto the expanded gel. After unbound proteins and cellular debris were washed out, the bed was allowed to sediment and beta-D-galactosidase was eluted with a downward flow of 0.2 M NaCl in the same buffer. A 6-fold purification factor was achieved with 63% activity recovery, while removing cell debris at a single step, thus avoiding a centrifugation step. Concentration and volume of the applied sample affected purification and gel performance. The results presented show STREAMLINE-DEAE chromatography to be an interesting method for the production of beta-D-galactosidase as a technical enzyme, since it can also be applied on a large scale without much modification.

Pyranose 2-oxidase from Phanerochaete chrysosporium--further biochemical characterisation.

Pyranose 2-oxidase (P2O) was purified 43-fold to apparent homogeneity from the basidiomycete Phanerochaete chrysosporium using liquid chromatography on phenyl Sepharose, Mono Q (twice) and phenyl Superose. The native enzyme has a molecular mass of about 250 kDa (based on native PAGE) and is composed of four identical subunits of 65 kDa. It contains three isoforms of isoelectric point (pI) 5.0, 5.05 and 5.15 and does not appear to be a glycoprotein. P2O is optimally stable at pH 8.0 and up to 60 degrees C. It is active over a broad pH range (5.0-9.0) with maximum activity at pH 8.0-8.5 and at 55 degrees C, and a broad substrate specificity. D-Glucose is the preferred substrate, but 1-beta-aurothioglucose, 6-deoxy-D-glucose, L-sorbose, D-xylose, 5-thioglucose, D-glucono-1,5-lactone, maltose and 2-deoxy-D-glucose are also oxidised at relatively high rates. A Ping Pong Bi Bi mechanism was demonstrated for the P2O reaction at pH 8.0, with a catalytic constant (kcat) of 111.0 s-1 and an affinity constant (Km) of 1.43 mM for D-glucose and 83.2 microM for oxygen. Whereas the steady-state kinetics for glucose oxidation were unaffected by the medium at pH > or = 7.0, at low pH both pH and buffer composition affected the P2O kinetics with the kcat/K(m) value decreasing with decreasing pH. The greatest effect was observed in acetate buffer (0.1 M, pH 4.5), where the kcat decreased to 60.9 s-1 and the K(m) increased to 240 mM. The activity of P2O was completely inhibited by 10 mM HgCl2, AgNO3 and ZnCl2, and 50% by lead acetate, CuCl2 and MnCl2.