Purification and some properties of a β-galactosidase from the thermoacidophilic Alicyclobacillus acidocaldarius subsp. rittmannii isolated from Antarctica

Abstract

An intracellular β-galactosidase from a thermoacidophilic Alicyclobacillus acidocaldarius subsp. rittmannii was purified by procedures including precipitation with ammonium sulphate, gel permeation, ion-exchange and affinity chromatography and finally by preparative electrophoresis and some properties of the purified enzyme were determined. The homogenous enzyme had a specific activity of 113 U/mg protein, with a fold purification of 163 and a yield of 8%. The Km and kcat values for ONPG were determined as 8.9 mM and 1074 min −1, respectively in the purified β-galactosidase from A. acidocaldarius subspecies rittmannii. The bacteria produce thermostable β-galactosidase (EC 3.2.1.23) activity, which exhibits its optimum at the neutral pH region. The pH and temperature optima for the purified enzyme are 6.0 and 65 °C, respectively (10 min assay). β-Galactosidase specific activities of crude extracts obtained from bacterial cells grown in the presence and absence of lactose over a period of time (6–40 h) showed that β-galactosidase synthesis seems to be constitutive and increases by increasing time up to 40 h of cultivation. β-Galactosidase activity in the bacteria growing on the medium without lactose was 0.4 U/mg protein and increased up to 0.6 U/mg protein in the cells growing on the medium with lactose at 24 h (an increase by about 33% of its constitutive value), while it was 0.072 and 0.48 U/mg protein, respectively at 12 h (an increase by about 85% of its constitutive value). IPTG was also found to increase β-galactosidase activity over a short period of time. The molecular mass of the purified enzyme determined by gel filtration on FPLC and SDS-PAGE was 165 and 76 kDa, respectively. The β-galactosidase from Alicyclobacillus acidocaldarius subsp. rittmannii is most likely to belong to the glycoside hydrolyse family 42.