A study of β-galactosidases from thermoacidophiles

Microorganisms that grow under multiple stress conditions, such as thermoacidophiles, are a rich source of novel glycosyl hydrolases. β-Galactosidases isolated from these extreme microorganisms would be of academic interest by extending the knowledge with regard to such novel enzymes that are active at elevated temperature and/or low pH. These biocatalysts may also find possible applications in various industrial processes where harsh processing conditions are used. It is the purpose of this study to identify novel β-galactosidases from thermoacidophiles for further investigation. This work focused on Picrophilus torridus DSM 9790, the most acidophilic thermophile discovered to date, in addition to selected thermoacidophilic alicyclobacilli. A β-galactosidase from P. torridus DSM 9790 was identified using bioinformatic analysis, while that from Alicyclobacillus vulcanalis DSM 16176 was selected after initial characterisation of crude β-galactosidases produced by a range of alicyclobacilli. Despite extensive cloning trials, recombinant production of these enzymes failed to yield a biologically active form of either protein. The β-galactosidases were purified to homogeneity from their native sources and LC-MS/MS analysis was used to confirm the identity of the purified proteins. Extensive characterisation of the enzymes was carried out and included investigations into the pH and temperature activity and thermostability of the target enzymes. The P. torridus DSM 9790 β-galactosidase displayed maximal activity at 70 °C and at acidic pH values of 5.0-5.5. Optimal temperature was identical for the A. vulcanalis DSM 16176 β-galactosidase but this enzyme had a higher pH optimum of 6.0. Both enzymes were found to be thermostable at a significant level indicating their potential use in high temperature industrial processes. Some preliminary application studies were carried out using the selected β- galactosidases as biocatalysts in the synthesis of the synthetic disaccharide lactulose at high temperature. A biocatalyst process characterised by environmentally ‘clean’ production and straight forward purification would be considered an environmentallyfriendly alternative strategy to the currently employed chemical alkaline isomerisation for production of lactulose. The β-galactosidase from P. torridus DSM 9790 showed some potential in this regard.