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首页> 外文期刊>Journal of bacteriology >Biochemical Characterization of a β-Galactosidase with a Low Temperature Optimum Obtained from an Antarctic Arthrobacter Isolate
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Biochemical Characterization of a β-Galactosidase with a Low Temperature Optimum Obtained from an Antarctic Arthrobacter Isolate

机译：从南极节杆菌分离物获得的低温最佳的β-半乳糖苷酶的生化特性

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A psychrophilic gram-positive isolate was obtained from Antarctic Dry Valley soil. It utilized lactose, had a rod-coccus cycle, and contained lysine as the diamino acid in its cell wall. Consistent with these physiological traits, the 16S ribosomal DNA sequence showed that it was phylogenetically related to other Arthrobacter species. A gene (bgaS) encoding a family 2 β-galactosidase was cloned from this organism into an Escherichia coli host. Preliminary results showed that the enzyme was cold active (optimal activity at 18°C and 50% activity remaining at 0°C) and heat labile (inactivated within 10 min at 37°C). To enable rapid purification, vectors were constructed adding histidine residues to the BgaS enzyme and its E. coli LacZ counterpart, which was purified for comparison. The His tag additions reduced the specific activities of both β-galactosidases but did not alter the other characteristics of the enzymes. Kinetic studies using o-nitrophenyl-β-d-galactopyranoside showed that BgaS with and without a His tag had greater catalytic activity at and below 20°C than the comparable LacZ β-galactosidases. The BgaS heat lability was investigated by ultracentrifugation, where the active enzyme was a homotetramer at 4°C but dissociated into inactive monomers at 25°C. Comparisons of family 2 β-galactosidase amino acid compositions and modeling studies with the LacZ structure did not mimic suggested trends for conferring enzyme flexibility at low temperatures, consistent with the changes affecting thermal adaptation being localized and subtle. Mutation studies of the BgaS enzyme should aid our understanding of such specific, localized changes affecting enzyme thermal properties.
机译：从南极干旱谷土壤中获得了嗜冷革兰氏阳性分离株。它利用乳糖，具有棒球菌循环，并在其细胞壁中含有赖氨酸作为二氨基酸。与这些生理特征一致，16S核糖体DNA序列显示它与其他 Arthrobacter 物种在系统发育上相关。从该生物体中克隆了一个编码家族2β-半乳糖苷酶的基因（ bgaS ）到大肠杆菌宿主中。初步结果显示该酶具有冷活性（在18°C时具有最佳活性，在0°C时仍具有50％的活性）和热不稳定（在37°C时10分钟内失活）。为了能够快速纯化，构建了将组氨酸残基添加至BgaS酶及其 E的载体。大肠杆菌LacZ对应物，经过纯化用于比较。 His标签的添加降低了两种β-半乳糖苷酶的比活性，但没有改变酶的其他特性。使用 o -硝基苯基-β-d-吡喃半乳糖苷的动力学研究表明，带有和不带有His标签的BgaS在20°C和低于20°C的催化活性均高于可比的LacZβ-半乳糖苷酶。通过超速离心研究BgaS的热不稳定性，其中活性酶在4°C下为均四聚体，但在25°C时解离为无活性单体。 2族β-半乳糖苷酶氨基酸组成的比较和具有LacZ结构的模型研究并未模仿暗示在低温下赋予酶柔性的趋势，这与影响热适应性的变化是局部的和微妙的一致。 BgaS酶的突变研究应有助于我们了解影响酶热特性的这种特定的局部变化。

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