Hung M-N, Lee B H
Department of Food Science and Agricultural Chemistry, McGill University, Ste-Anne-de-Bellevue, Quebec, Canada.
Appl Microbiol Biotechnol. 2002 Mar;58(4):439-45. doi: 10.1007/s00253-001-0911-6. Epub 2002 Feb 1.
A beta-galactosidase isoenzyme, beta-Gall, from Bifidobacterium infantis HL96, was expressed in Escherichia coli and purified to homogeneity. The molecular mass of the beta-Gall subunit was estimated to be 115 kDa by SDS-PAGE. The enzyme appeared to be a tetramer, with a molecular weight of about 470 kDa by native PAGE. The optimum temperature and pH for o-nitrophenyl-beta-D-galactopyranoside (ONPG) and lactose were 60 degrees C, pH 7.5, and 50 degrees C, pH 7.5, respectively. The enzyme was stable over a pH range of 5.0-8.5, and remained active for more than 80 min at pH 7.0, 50 degrees C. The enzyme activity was significantly increased by reducing agents. Maximum activity required the presence of both Na+ and K+, at a concentration of 10 mM. The enzyme was strongly inhibited by p-chloromercuribenzoic acid, divalent metal cations, and Cr3+, and to a lesser extent by EDTA and urea. The hydrolytic activity using lactose as a substrate was significantly inhibited by galactose. The Km, and Vmax values for ONPG and lactose were 2.6 mM, 262 U/mg, and 73.8 mM, 1.28 U/mg, respectively. beta-Gall possesses strong transgalactosylation activity. The production rate of galactooligosaccharides from 20% lactose at 30 and 60 degrees C was 120 mg/ml, and this rate increased to 190 mg/ml when 30% lactose was used.
来自婴儿双歧杆菌HL96的一种β-半乳糖苷酶同工酶β-Gal1在大肠杆菌中表达并纯化至同质。通过SDS-PAGE估计β-Gal1亚基的分子量为115 kDa。该酶似乎是一种四聚体，通过非变性PAGE测定其分子量约为470 kDa。对硝基苯基-β-D-吡喃半乳糖苷（ONPG）和乳糖的最佳温度和pH分别为60℃、pH 7.5以及50℃、pH 7.5。该酶在pH 5.0 - 8.5范围内稳定，在pH 7.0、50℃下80多分钟仍保持活性。还原剂可显著提高该酶的活性。最大活性需要同时存在浓度为10 mM的Na⁺和K⁺。该酶受到对氯汞苯甲酸、二价金属阳离子和Cr³⁺的强烈抑制，受EDTA和尿素的抑制程度较小。以乳糖为底物的水解活性受到半乳糖的显著抑制。ONPG和乳糖的Km值及Vmax值分别为2.6 mM、262 U/mg以及73.8 mM、1.28 U/mg。β-Gal1具有很强的转半乳糖基化活性。在30℃和60℃下，由20%乳糖生成低聚半乳糖的速率为120 mg/ml，当使用30%乳糖时，该速率增至190 mg/ml。
