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首页> 外文期刊>Applied and Environmental Microbiology >Biochemical and Phylogenetic Analyses of a Cold-Active β-Galactosidase from the Lactic Acid Bacterium Carnobacterium piscicola BA
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Biochemical and Phylogenetic Analyses of a Cold-Active β-Galactosidase from the Lactic Acid Bacterium Carnobacterium piscicola BA

机译：乳酸菌肉食杆菌BA的冷活性β-半乳糖苷酶的生化和系统发育分析

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摘要

We are investigating glycosyl hydrolases from new psychrophilic isolates to examine the adaptations of enzymes to low temperatures. A β-galactosidase from isolate BA, which we have classified as a strain of the lactic acid bacterium Carnobacterium piscicola, was capable of hydrolyzing the chromogen 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside (X-Gal) at 4°C and possessed higher activity in crude cell lysates at 25 than at 37°C. Sequence analysis of a cloned DNA fragment encoding this activity revealed a gene cluster containing three glycosyl hydrolases with homology to an α-galactosidase and two β-galactosidases. The larger of the two β-galactosidase genes, bgaB, encoded the 76.8-kDa cold-active enzyme. This gene was homologous to family 42 glycosyl hydrolases, a group which contains several thermophilic enzymes but none from lactic acid bacteria. The bgaB gene from isolate BA was subcloned in Escherichia coli, and its enzyme, BgaB, was purified. The purified enzyme was highly unstable and required 10% glycerol to maintain activity. Its optimal temperature for activity was 30°C, and it was inactivated at 40°C in 10 min. TheKm of freshly purified enzyme at 30°C was 1.7 mM, and the V max was 450 μmol · min?1 · mg?1 with o-nitrophenyl β-d-galactopyranoside. This cold-active enzyme is interesting because it is homologous to a thermophilic enzyme fromBacillus stearothermophilus, and comparisons could provide information about structural features important for activity at low temperatures.
机译：我们正在研究新的嗜冷菌中的糖基水解酶，以研究酶对低温的适应性。来自分离株BA的β-半乳糖苷酶（我们已将其分类为Piscicola乳酸菌）能够水解发色团5-溴-4-氯-3-吲哚基β-d-吡喃半乳糖苷（X-Gal）在4°C时，其在25°C的粗细胞裂解液中的活性要高于37°C。对编码这种活性的克隆DNA片段的序列分析揭示了一个基因簇，该簇含有三个与α-半乳糖苷酶和两个β-半乳糖苷酶同源的糖基水解酶。两个β-半乳糖苷酶基因中较大的一个bgaB编码76.8 kDa的冷活性酶。该基因与42族糖基水解酶同源，该家族包含几种嗜热酶，但不含乳酸菌。将来自分离株BA的bgaB基因亚克隆到大肠杆菌中，并纯化其酶BgaB。纯化的酶高度不稳定，需要10％的甘油来维持活性。其最佳活性温度为30°C，并在40°C下在10分钟内失活。用邻硝基苯基β-d-吡喃半乳糖苷在30℃下新鲜纯化的酶的Km为1.7mM，V max为450μmol·min·1·mg·1。这种冷活性酶很有趣，因为它与来自嗜热脂肪芽孢杆菌的嗜热酶同源，并且比较可以提供有关对于低温活性重要的结构特征的信息。

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