Cloning, overexpression, purification, and characterization of a polyextremophilic β-galactosidase from the Antarctic haloarchaeon Halorubrum lacusprofundi
Cloning, overexpression, purification, and characterization of a polyextremophilic β-galactosidase from the Antarctic haloarchaeon Halorubrum lacusprofundi
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DOI：
10.1186/1472-6750-13-3
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发表时间：
2013-01-16
影响因子：
3.5
通讯作者：
DasSarma, Shiladitya
中科院分区：
文献类型：
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作者：
Karan, Ram;Capes, Melinda D.;DasSarma, Shiladitya
工作背景：Halorubrum lacusprofundi是一种冷适应嗜盐古菌，分离自南极洲的深湖，一个常年寒冷和高盐度的湖泊。其基因组测序计划最近完成，提供了许多基因的预测编码多极端酶活性在极高的盐度和寒冷的温度。Lacusprofundi显示了用于碳水化合物利用的基因簇，其包含糖苷水解酶家族42 β-半乳糖苷酶基因，命名为BGA。为了研究β-半乳糖苷酶的生化特性，PCR扩增，克隆，并在遗传上易处理的盐古菌盐杆菌属NRC-1中表达的冷休克蛋白（cspD 2）基因启动子的控制下的bga基因。重组β-半乳糖苷酶蛋白的产量是H.采用凝胶过滤和疏水层析法纯化了lacusprofundi，并通过SDS-PAGE、LC-MS/MS和ONPG水解活性进行了鉴定。发现纯化的酶在宽的温度范围内（-5至60 ℃）具有活性，最适温度为50 ℃，在4 ℃时其最大活性的10%。该酶也表现出极端的嗜盐性，在4 M NaCl或KCl中具有最大活性。该酶在10-20%的乙醇水溶液中，包括甲醇、乙醇、正丁醇、异戊醇等，均具有较好的稳定性和活性。lacusprofundi β-半乳糖苷酶是一种在高盐浓度和低温和高温下具有活性的多极端酶。该酶在水-有机混合溶剂中也有活性，在合成化学中具有潜在的应用。H. lacuprofundi蛋白代表了重要的生物技术资源，并用于开发对水限制条件下的酶催化的了解。本研究为更好地理解H. lacusprofundi在常年寒冷的高盐环境中取得了成功，这与天体生物学有关。
Background: Halorubrum lacusprofundi is a cold-adapted halophilic archaeon isolated from Deep Lake, a perennially cold and hypersaline lake in Antarctica. Its genome sequencing project was recently completed, providing access to many genes predicted to encode polyextremophilic enzymes active in both extremely high salinity and cold temperatures.Results: Analysis of the genome sequence of H. lacusprofundi showed a gene cluster for carbohydrate utilization containing a glycoside hydrolase family 42 beta-galactosidase gene, named bga. In order to study the biochemical properties of the beta-galactosidase enzyme, the bga gene was PCR amplified, cloned, and expressed in the genetically tractable haloarchaeon Halobacterium sp. NRC-1 under the control of a cold shock protein (cspD2) gene promoter. The recombinant beta-galactosidase protein was produced at 20-fold higher levels compared to H. lacusprofundi, purified using gel filtration and hydrophobic interaction chromatography, and identified by SDS-PAGE, LC-MS/MS, and ONPG hydrolysis activity. The purified enzyme was found to be active over a wide temperature range (-5 to 60 degrees C) with an optimum of 50 degrees C, and 10% of its maximum activity at 4 degrees C. The enzyme also exhibited extremely halophilic character, with maximal activity in either 4 M NaCl or KCl. The polyextremophilic beta-galactosidase was also stable and active in 10-20% alcohol-aqueous solutions, containing methanol, ethanol, n-butanol, or isoamyl alcohol.Conclusion: The H. lacusprofundi beta-galactosidase is a polyextremophilic enzyme active in high salt concentrations and low and high temperature. The enzyme is also active in aqueous-organic mixed solvents, with potential applications in synthetic chemistry. H. lacuprofundi proteins represent a significant biotechnology resource and for developing insights into enzyme catalysis under water limiting conditions. This study provides a system for better understanding how H. lacusprofundi is successful in a perennially cold, hypersaline environment, with relevance to astrobiology.