Recovery of ß-galactosidase from the yeast Kluyveromyces lactis by cell permeabilization with sarkosyl

Abstract

We present here a novel method for recovering ß-galactosidase (ß-gal) from the yeast Kluyveromyces lactis (NRRL 1118) by means of sarkosyl permeabilization. The yeast was permeabilized with 0.2% (w/v) sarkosyl in 0.1 M potassium phosphate buffer for 30 min at 25 °C. After centrifuging and washing, the permeabilized cells were incubated in buffer at pH 7.2, 35 °C for 6–7 h resulting in a nearly soluble extract containing 80% of the ß-galactosidase and about 40% of the cell protein. Transmission electron microscopy showed that permeabilized cells underwent gross structural changes during enzyme release but did not exhibit significant physical breakage. An extract obtained from a 40 mg mL–1 suspension of permeabilized cells was partially purified and concentrated 10-fold through diafiltration and ultrafiltration. The concentrated enzyme solution mixed with 50% (v/v) glycerol proved to be stable for at least 10 months at 5 °C. The kinetic properties of the ß-galactosidase preparation including the hydrolysis of milk, were similar to those exhibited by a commonly used commercial ß-galactosidase derived from the same yeast species. Sarkosyl is a biodegradable detergent with extremely low toxicity, thus making this compound an attractive permeabilizing agent for the downstream -processing of enzymes used in the food industry.