物理化学学报 >> 1996, Vol. 12 >> Issue (07): 629-634.doi: 10.3866/PKU.WHXB19960710

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鹰嘴豆β-半乳糖苷酶的分离纯化与表征

李绪渊,赵克浩,孟延发,涂伟霞   

  1. 兰州大学化学系，生物系，兰州 730000
  • 收稿日期:1995-11-13 修回日期:1996-03-15 发布日期:1996-07-15
  • 通讯作者:

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
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摘要：

从鹰嘴豆中分离得到了三种β－半乳糖苷酶（酶Ⅰ、酶Ⅱ和酶Ⅲ）. 将酶Ⅰ和酶Ⅱ进一步纯化，其比活力分别提高了19倍和48倍．酶活力回收率分别为16％和18％，测得它们的表观分子里分别为2.4×104和5.8×104, 最适pH分别5.9和5.0，最适温度分别为55℃和45℃．酶Ⅰ水解ONPG和PNPG的KM分别为33×10-2mol•dm-3和60×10-3mol•dm－3；酶Ⅱ水解ONPG和PNPG的KM分别为30×10－3mol•dm－3和60×10－4mol•dm－3.乳糖和半乳糖为该酶的竞争性可逆抑制剂，棉子糖为非竞争性可逆抑制剂．该酶受Hg2＋和PCMB强烈抑制和NEM明显抑制，而Mg2＋、Zn2＋和Ca2＋具有激活作用，推知巯基（－SH)是酶活性中心必须基团．

关键词: β-半乳糖苷酶, 鹰嘴豆, 分离纯化, 表征, 酶催化作用

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