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.
Artolozaga
M J
MJ
CDB, Centro de Desenvolvimento Biotecnológico, Joinville, Brazil.
Jonas
R
R
Schneider
A L
AL
Furlan
S A
SA
Carvalho-Jonas
M de F
Mde F
D001709:Biotechnology; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004983:Ethanolamines; D005524:Food Technology; D007202:Indicators and Reagents; D007716:Kluyveromyces / Q000201:enzymology*; D043322:Lactase; D012965:Sodium Chloride; D001616:beta-Galactosidase / Q000302:isolation & purification*
D004983:Ethanolamines; D007202:Indicators and Reagents; D012965:Sodium Chloride; D043322:Lactase; D001616:beta-Galactosidase; C007369:2-diethylaminoethanol