科研之友
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摘要
A beta-galactosidase from Cicer arietinum seeds has been purified to apparent electrophoretic homogeneity using a combination of various fractionation and chromatographic techniques, giving a final specific activity of 220 units mg(-1), with approximately 1840 fold purification. Analysis of the protein by SDS-PAGE revealed two subunits with molecular masses of 48 and 38 kDa, respectively. These bands were further confirmed with LC-MS/MS, indicating that Chick pea beta-galactosidase (CpGAL) is a heterodimer. Molecular mass was determined to be 85 kDa by Superose-12 FPLC column, which is in agreement with the molecular mass suggested by mass spectroscopy to be 83 kDa. The optimum pH of the enzyme was 2.8 and it hydrolysed o-nitrophenyl beta-D galactopyranoside (ONPG) with a K-m value of 1.73 mM at 37 degrees C. The energy of activation (E-a) calculated in the range of 35 to 60 degrees C, using Arrhenius equation, was determined to be 11.32 kcal mol(-1). The enzyme could also hydrolyse lactose, with an optimum pH of 4.0 at 40 degrees C. K-m and E-a for lactose hydrolysis was found to be 10 mM and 10.57 kcal mol(-1), respectively. The enzyme was found to be comparatively thermostable showing maximum activity at 60 degrees C for both ONPG and lactose. Galactose was found to be the competitive inhibitor. beta-Galactosidase also exhibited glycoproteineous properties when applied on Con-A Sepharose column. The enzyme was localised in germinated seeds with X-gal activity staining and shown to be expressed prominently at grown radical tip and seed coat. Sequence alignment of CpGAL with other known plant beta-galactosidase showed high amino acid sequence homology.
- 出版日期2012-9-15
