Acta Phys. -Chim. Sin. ›› 1996, Vol. 12 ›› Issue (07): 629-634.doi: 10.3866/PKU.WHXB19960710

• ARTICLE • Previous Articles     Next Articles

Purification and Characterization of β-galactosidases from Cicer Arietinum

Li Xu-Yuan,Zhao Ke-Hao,Meng Yan-fa,Tu Wei-Xia   

  1. Department of Chemistry and Department of Bilelgy,Lanzhou University,Lanzhou 730000
  • Received:1995-11-13 Revised:1996-03-15 Published:1996-07-15
  • Contact: Li Xu-Yuan

Abstract:

 Gram chicken bean exhibits a high level of β-galactosidase activity, and the three components contained in it are responsible for this activity. β-galastosidase Ⅰ, Ⅱ and Ⅲ were separated by ammonium sulphate fractionation (30%-70% saturation) and by ion-exchange chromatography on DEAE-cellulose-32. β-galactosidase Ⅰ and Ⅱ,in particular, were purified by subsequent, chromatography on CM-cellulose-52. Specific activities of them were improved by 19 times and 48 times, meanwhile, recovery activities were 16% and 18% respectively. The two enzymes were homogeneous as judged by polyacrylamide gel electrophoresis and Sephadex G-200 molecular sieve chromatography. Their molecular weights were determined to be 24 000 and 58 000 respectively. β-galactosidase Ⅰhad an apparent KM of 6.0 ×10-3 mol•dm -3 for p-nitrophenyl-β-D-galactoside (PNPG) and 3.3 ×10-2mol•dm-3 for o-nitrophenyl-β-D-galactoside (ONPG). β-galactosidase Ⅱ had an apparent KM of 6.0 × 10-4 mol•dm-3 for PNPG and 1.0 × 10-3 mol•dm- 3 for ONPG. Galactose and lactose both competitively inhibited the activity of enzymes. Raffinose uncompetitively inhibited the activity of enzymes. lons Mg2+, Zn2+ and Ca2+ stimulated the activity. The two enzymes were markedly inhibited by Hg2+, PCMB and NEM, which suggested that tryptophan (-SH) was necessary for enzyme function. 

Key words: β-galactosidase, Cicer arietinum, Purification, Characterization, Enzyme catalytic function