Purification and characterization of a thermostable β-Galactosidase from kidney beans (Phaseolus vulgaris L.) cv. PDR14

Using five different steps, beta-Galactosidase has been purified from kidney beans to apparent electrophoretic homogeniety with approximately 90-fold purification with a specific activity of 281 units mg(-1) protein. A single band was observed in native PAGE. Activity staining of the native gel with 5-bromo 4-chloro 3-indoxyl beta-D-galactopyranoside (X-Gal) at pH 4.0 also produced a single band. Analytical gel filtration in Superdex G-75 revealed the molecular mass of the native protein to be approximately 75 kD. 10% SDS-PAGE under reducing conditions showed two subunits of molecular masses, 45 and 30 kD, respectively. Hence, beta-galactosidase from kidney beans is a heterodimer, A typical protein profile with lambda(max) at 280 nm was observed and A(280)/A(260) ratio was 1.52. The N-terminal sequence of the 45 kD band showed 86 % sequence homology with an Arabidopsis thaliana and 85 % with Lycopersicon esculentum putative beta-galactosidase sequences. The Electrospray Mass Spectrometric analysis of this band also revealed a peptide fragment that had 90 % sequence homology with an Arabidopsis thaliana putative beta-galactosidase sequence. The N-terminal sequencing of the 30 kD band as well as mass spectrometric analysis both by MALDI-TOF and ES MS revealed certain sequences that matched with phytohemagglutinin of kidney beans. The optimum pH of the enzyme was 4.0 and it hydrolysed o- and p-nitrophenyl beta-D galactopyranoside with a K-m value of 0.63 mmol/L and 0.74 mmol/L, respectively. The energy of activation calculated from the Arrhenius equation was 14.8 kcal/mol enzyme site. The enzyme was found to be comparatively thermostable showing maximum activity at 67degreesC. Thermal denaturation of the enzyme at 65degreesC obeys single exponential decay with first order-rate constant 0.105 min(-1). Galactose, a hydrolytic product of this enzyme was a competitive inhibitor with a K-i of 2.7 mmol/L.