BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS Cloning, purification, and characterization of β-galactosidase from Bacillus licheniformis DSM 13 Onladda Juajun & Thu-Ha Nguyen & Thomas Maischberger & Sanaullah Iqbal & Dietmar Haltrich & Montarop Yamabhai Received: 16 June 2010 / Revised: 21 August 2010 / Accepted: 23 August 2010 # Springer-Verlag 2010 Abstract The gene encoding homodimeric β-galactosidase (lacA) from Bacillus licheniformis DSM 13 was cloned and overexpressed in Escherichia coli, and the resulting recombinant enzyme was characterized in detail. The optimum temperature and pH of the enzyme, for both o-nitrophenyl-β-D-galactoside (oNPG) and lactose hydro- lysis, were 50°C and 6.5, respectively. The recombinant enzyme is stable in the range of pH 5 to 9 at 37°C and over a wide range of temperatures (4–42°C) at pH 6.5 for up to 1 month. The K m values of LacA for lactose and oNPG are 169 and 13.7 mM, respectively, and it is strongly inhibited by the hydrolysis products, i.e., glucose and galactose. The monovalent ions Na + and K + in the concentration range of 1–100 mM as well as the divalent metal cations Mg 2+ , Mn 2+ , and Ca 2+ at a concentration of 1 mM slightly activate enzyme activity. This enzyme can be beneficial for applica- tion in lactose hydrolysis especially at elevated temperatures due to its pronounced temperature stability; however, the transgalactosylation potential of this enzyme for the produc- tion of galacto-oligosaccharides (GOS) from lactose was low, with only 12% GOS (w/w) of total sugars obtained when the initial lactose concentration was 200 g/L. Keywords β-Galactosidase . Bacillus licheniformis . Lactose hydrolysis . Transglycosylation . Recombinant Introduction β-Galactosidases (β-gal, lactase, EC 3.2.1.23) catalyze the hydrolysis and transgalactosylation of β-D-galactopyranosides (such as lactose) (Nakayama and Amachi 1999) and are found widespread in nature. β-Galactosidases have been isolated and characterized from many different sources including microorganisms, plants, and animals. At present, more than a hundred putative β-galactosidase sequences can be deduced from various databases, and these can be classified into four different glycoside hydrolase (GH) families GH-1, GH-2, GH-35, and GH-42, based on functional similarities (Cantarel et al. 2009). Microbial β- galactosidases have attracted considerable attention for biotechnological applications, and hence numerous reports (Halbmayr et al. 2008; Nguyen et al. 2006; Rahim and Lee 1991; Rajakala and Karthigai 2006) and reviews (Husain 2010; Nakayama and Amachi 1999; Park and Oh 2010a) on the characterization of these enzymes from various organisms have been published. The hydrolysis of lactose Electronic supplementary material The online version of this article (doi:10.1007/s00253-010-2862-2) contains supplementary material, which is available to authorized users. O. Juajun : M. Yamabhai (*) School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, 111 University Avenue, Nakhon Ratchasima 30000, Thailand e-mail: montarop@g.sut.ac.th O. Juajun Department of Food Science and Technology, Faculty of Agricultural and Technology, Rajamangala University of Technology Isan, Surin Campus, Surin 32000, Thailand T.-H. Nguyen : T. Maischberger Applied Biocatalysis Research Center, 8010 Graz, Austria T.-H. Nguyen : T. Maischberger : S. Iqbal : D. Haltrich Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU—University of Natural Resources and Life Sciences Vienna, 1190 Vienna, Austria Appl Microbiol Biotechnol DOI 10.1007/s00253-010-2862-2