2National Institute of Biotechnology and Genetic Engineering, Jang Road, Faisalabad, Pakistan
3Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore 54590, Pakistan
* To whom correspondence should be addressed.
Received August 25, 2009; Revision received October 3, 2009
A putative β-glucosidase gene from the genome of Bacillus halodurans C-125 was expressed in E. coli under the regulation of T7lac promoter. On induction with isopropyl-β-D-1-thiogalactopyranoside, the enzyme expressed at ~40% of the cell protein producing 238 mg/liter culture. With increase in culture cell density to A600 12 in auto-inducing M9NG medium, β-glucosidase production increased 3-fold. Approximately 70% of the expressed enzyme was in a soluble form, while the rest was in an insoluble fraction of the cell lysate. The soluble and active form of the expressed enzyme was purified by ammonium sulfate precipitation followed by ion-exchange chromatography to a purity >98%. The mass of the enzyme as determined by MALDI-TOF mass spectrometry was 51,601 Da, which is nearly the same as the calculated value. Phylogenetic analysis of the β-glucosidase of B. halodurans was found to cluster with members of the genus Bacillus. Temperature and pH optima of the enzyme were found to be 45°C and 8.0, respectively, under the assay conditions. Km and kcat against p-nitrophenyl-β-D-glucopyranoside were 4 mM and 0.75 sec–1, respectively. To our knowledge, this is the first report of high-level expression and characterization of a β-glucosidase from B. halodurans.
KEY WORDS: expression, auto-inducing medium, MALDI-TOF analysis, β-glucosidase, Bacillus halodurans, E. coli