鹰嘴豆β-半乳糖苷酶的分离纯化与表征

Purification and Characterization of β-galactosidases from Cicer Arietinum

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．

Gram chicken bean exhibits a high level of β－-galactosidase activity, and the three components contairled 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 cnzymes 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 fo 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.