节杆菌β-半乳糖苷酶的分离纯化及酶学性质研究

Purification and characterization of β-galactosidase from Arthrobacter sp.

分离纯化了由节杆菌所产的β-半乳糖苷酶(EC 3.2.1.23)并研究了其酶学性质。以乳糖发酵培养基培养,离心收集菌体,超声破碎细胞得到粗酶液;采用硫酸铵沉淀、Phenyl-Sepharose疏水、DEAE-Sepharose离子交换和p-aminobenzyl-1-thio-β-D-galactopyranoside(PABTG)亲和层析方法进行酶的分离纯化,纯化倍数达到31.4;经SDS-PAGE检测分子量为30 kD。酶学性质的研究表明:该酶最适pH值为6.6,最适反应温度为37℃,pH在5.5～8.0,温度低于30℃时,相对酶活在1 h内保持在50%以上,较为稳定。以邻硝基苯-β-D-吡喃半乳糖苷(ONPG)为底物,米氏常数为4.64 mmol/L。

The purification and characterization of β-galactosidase （EC 3.2.1.23） produced by Arthrobacter sp. were investigated. bacterial cells cultured were gathered by eentrifugation and disrupted by ultrasonication. The crude enzyme solution was precipi- tated by ammocium sulphate. Further enzyme purification was carried out by Phenyl- Sepharcrose, DEAGSephamse and p-amino- benzyl-l-thio-β-D-galactopymnoside （PAge） affinity chrcrnatogmphy. The purified β-galactosidase was illustrated as a single band in SDS-PAGE with a purification fold of 31.4 and an aplmrent rmlecular weight of 30 kD. The maximum activity of the enzyme was detemained at pH 6.6 and 37 12. Above 50 % of the macoimum activity of the enzyme was detected at pH 5.5-- 8.0 and 30 ℃. The Km for ONPG was 4.64 mmol/L.