Yue Shan,Weili Yu,Lijuan Shen,Xuan Guo,He Zheng,Jinyi Zhong,Tao Hu,Yinglun Han
Haloalkane dehalogenase DhaA catalyzes the hydrolytic cleavage of carbon-halogen bonds and produces alcohol, a proton and a halide. However, DhaA suffers from the poor environmental stability, such as sensitivity to high temperature, low pH, hypersaline and organic solvent. In order to improve the environmental stability of DhaA, DhaA was covalently conjugated with inulin, a hydrophilic polysaccharide in the present study. Each DhaA was averagely conjugated with 7∼8 inulin molecules. The conjugated inulin could form a hydration layer around DhaA, which increased the conformational rigidity and decreased the entropy of the enzyme. Conjugation of inulin maintained 75.5 % of the enzymatic activity of DhaA and slightly altered the structure of DhaA. As compared with DhaA, the conjugate (inu-DhaA) showed slightly different kinetic parameters (K m of 2.9 μmol/L and K cat of 1.0 s −1 ). Inulin conjugation could delay the structural unfolding and/or slow the protonation process of DhaA under undesirable environment, including the long-term storage, low pH, hypersaline and organic solvent stability. As a result, the environmental stability of DhaA was markedly increased upon conjugation with inulin. Thus, inulin conjugation was an effective approach to enhance the environmental stability of DhaA.
Meiqi Wang,Weili Yu,Lijuan Shen,He Zheng,Xuan Guo,Jinyi Zhong,Tao Hu
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).
In this work, a facile fluorescence Eu3+-based metal organic frameworks (Eu MOF) sensor for ascorbic acid (AA) and ascorbate oxidase (AAO) detection was developed. The fluorescence of Eu MOF could be effectively quenched by Ce3+ but not Ce4+ at an appropriate concentration, thus, when the reductant AA was added into the solution containing Ce4+, Ce4+ was chemically reduced to Ce3+, which induced the decreased fluorescence signal of Eu MOF. However, when AAO was introduced, AA was effectively oxidized to dehydroascorbic acid (DHAA) under the catalysis of AAO, thus, Ce4+ could not be reduced, resulting in the fluorescence restore of Eu MOF. Hence, the concentration of AA and AAO could be determined by the fluorescence decrease and restore of Eu MOF. The fluorescent platform showed high sensitivity with the limit of detection of 0.32 µM for AA and 1.18 U/L for AAO, respectively. Moreover, the proposed method was successfully applied for AA and AAO determination in real samples, indicating great potential for biomedical application in complex matrices.
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