The one-pot immobilization of halophilic phenylalanine dehydrogenase from marine microorganism with metal ions modified reduced graphene oxide (CRGO) material was studied. Phenylalanine dehydrogenase was from Bacillus nanhaiensi and expressed with a C-terminal His-tag. Investigation of CRGO, CRGO-PEI, CRCO-Mn, and CRGO-PEI-Mn for one-pot purification and immobilization of phenylalanine dehydrogenase from crude enzyme solution was carried out. Enzyme activity yield rate achieved 80.0% by immobilization with CRCO-Mn, and the loading capacity was 6.7 mg/mg. Manganese ion coordination greatly improved the selectivity of the CRGO for the target His-tagged enzyme. Furthermore, the effect of NaCl concentration on the immobilization was investigated, which the loading capacity of CRGO-PEI and CRGO-Mn-PEI was increased by 10.7% and 30.6% with 1 M NaCl, respectively. The adsorption curves of crude enzyme one-pot immobilized by CRGO-Mn and purified enzyme immobilized by CRGO-Mn were similar. Therefore, one-pot immobilization strategy is promising for industrial application with advantages such as high efficiency and low cost, which shorten the pipelines for enzyme discovery towards industrial applications through the establishing of marine enzyme collections.
Keywords: Amino acid dehydrogenase; Functionalized carbon materials; One-pot immobilization; Organic solvent tolerance; Phenylalanine dehydrogenase.
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