将卤代烷脱卤酶 DhaA 与阿拉伯半乳聚糖缀合以提高其稳定性。
Conjugation of haloalkane dehalogenase DhaA with arabinogalactan to increase its stability.
机构信息
State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, China; University of Chinese Academy of Sciences, Beijing, 100190, China.
State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, China.
出版信息
J Biotechnol. 2021 Jul 20;335:47-54. doi: 10.1016/j.jbiotec.2021.06.002. Epub 2021 Jun 9.
Haloalkane dehalogenase DhaA can catalyze the hydrolytic cleavage of carbonhalogen bonds, along with production of the corresponding alcohol, a proton and a halide. However, DhaA suffers from poor environmental tolerance, such as sensitivity to high temperature, low pH and hypersaline. Arabinogalactan (AG) is a hydrophilic polysaccharide with highly branched long chains. DhaA was conjugated with AG to improve the environmental stability of DhaA in the present study. Each DhaA was averagely conjugated with 4∼5 AG molecules. Conjugation of AG essentially maintained the enzymatic activity of DhaA (91.4 %) without apparent structural alteration. The hydration layer formed by AG could reduce the solvent accessible area of DhaA and slow the protonation process, thereby improving the pH and high salt stability of DhaA. In particular, the remaining activities of the conjugate (AG-DhaA) were 35.3 % after treatment at pH4.0 for 1 h, and 80.8 % in 1 M NaCl after treatment for 16 h. As compared with DhaA, AG-DhaA showed slightly different kinetic parameters (K M of 1.90 μmol/L and k cat of 2.60 s -1).
卤代烷脱卤酶 DhaA 可以催化碳卤键的水解断裂，同时生成相应的醇、质子和卤化物。然而，DhaA 对环境的耐受性较差，如对高温、低 pH 值和高盐度敏感。阿拉伯半乳聚糖（AG）是一种具有高度支化长链的亲水性多糖。本研究将 DhaA 与 AG 偶联，以提高 DhaA 的环境稳定性。每个 DhaA 平均与 4∼5 个 AG 分子偶联。AG 的偶联基本上保持了 DhaA 的酶活性（91.4%），而没有明显的结构改变。AG 形成的水合层可以减少 DhaA 的溶剂可及面积并减缓质子化过程，从而提高 DhaA 的 pH 值和高盐稳定性。特别是，偶联物（AG-DhaA）在 pH4.0 下处理 1 小时后，剩余活性为 35.3%，在 1 M NaCl 下处理 16 小时后，剩余活性为 80.8%。与 DhaA 相比，AG-DhaA 表现出略有不同的动力学参数（K M 为 1.90 μmol/L，k cat 为 2.60 s -1）。
Zotero 插件