Abstract
Formylglycine-generating enzymes can selectively recognize and oxidize cysteine residues within the sulfatase sub motif at the terminus of proteins to form aldehyde-bearing formylglycine (FGly) residues, and are normally used in protein labeling. In this study, an aldehyde tag was introduced to proteins using formylglycine-generating enzymes encoded by a reconstructed set of the pET28a plasmid system for enzyme immobilization. The haloacid dehalogenase ST2570 from Sulfolobus tokodaii was used as a model enzyme. The C-terminal aldehyde-tagged ST2570 (ST2570CQ) exhibited significant enzymological properties, such as new free aldehyde groups, a high level of protein expression and improved enzyme activity. SBA-15 has widely been used as an immobilization support for its large surface and excellent thermal and chemical stability. It was functionalized with amino groups by aminopropyltriethoxysilane. The C-terminal aldehyde-tagged ST2570 was immobilized to SBA-15 by covalent binding. The site-specific immobilization of ST2570 avoided the chemical denaturation that occurs in general covalent immobilization and resulted in better fastening compared to physical adsorption. The site-specific immobilized ST2570 showed 3-fold higher thermal stability, 1.2-fold higher catalytic ability and improved operational stability than free ST2570. The site-specific immobilized ST2570 retained 60% of its original activity after seven cycles of batch operation, and it was superior to the ST2570 immobilized to SBA-15 by physical adsorption, which loses 40% of its original activity when used for the second time. It is remarkable that the site-specific immobilized ST2570 still retained 100% of its original activity after 10 cycles of reuse in the semi-continuous flow reactor. Overall, these results provide support for the industrial-scale production and application of site-specific, covalently immobilized ST2570.
Highlights
Enzymes are biocatalysts that catalyze reactions under mild conditions with high efficiency, and stereospecificity or enantioselectivity, and they reduce or avoid adverse conditions
All of but the enzyme with double-sided aldehyde tags lost almost all activity. These results indicated that the aldehyde-tagged ST2570 reduced the level of expression, which can affect the enzyme the fused polypeptide tag affects protein folding and the tertiary structure
The stability of immobilized ST2570CQ on SBA-15 by physical adsorption was examined in the batch wise reaction (Figure 5B); which turned out the physically immobilized enzyme lost 40% of its original activity when used for the second time; and only retained less than 20% of its original activity in the fifth use; which may be ascribed to significant deprival of the enzyme from the support during the reaction agitation and recovery process
Summary
Enzymes are biocatalysts that catalyze reactions under mild conditions with high efficiency, and stereospecificity or enantioselectivity, and they reduce or avoid adverse conditions. Enzymes exhibit limited mechanical and storage stability for some reactions and cannot be reused. With the emergence of protein modification and immobilization techniques, enzymes have become conducive to automatisation and continuous use for industrial production. Physical methods including physical adsorption and entrapment etc., which are simple, the immobilized enzymes with those methods maybe leaked out from the matrix during operation [2]. The immobilization of enzymes via multi-point chemical covalent binding sometimes may result in a certain loss of observed activity, the chemical stability will be significantly enhanced and the enzyme leakage could be minimized [3]
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