Abstract
In recent years, the development and application of site-specific immobilization technology for proteins have undergone significant advances, which avoids the unwanted and random covalent linkage between the support and active site of protein in the covalent immobilization. Formylglycine generating enzyme (FGE) can transform the cysteine from a conversed 6-amino-acid sequence CXPXR into formylglycine with an aldehyde group (also termed as “aldehyde tag”). Based on the frame of pET-28a, the His-tags were replaced with aldehyde tags. Afterward, a set of plasmids were constructed for site-specific covalent immobilization, their His-tags were knock out (DH), or were replaced at different positions: N-terminal (NQ), C-terminal (CQ), or both (DQ) respectively. Three different enzymes, thermophilic acyl aminopeptidase (EC 3.4.19.1) from Sulfolobus tokodaii (ST0779), thermophilic dehalogenase (EC 3.8.1.2) from Sulfolobus tokodaii (ST2570), and Lipase A (EC 3.1.1.3) from Bacillus subtilis (BsLA) were chosen as model enzymes to connect with these plasmid systems. The results showed that different aldehyde-tagged enzymes can be successfully covalently attached to different carriers modified with an amino group, proving the universality of the method. The new immobilized enzyme also presented better thermostability and reutilization than those of the free enzyme.
Highlights
Enzyme immobilization is an effective method for stabilizing and improving the efficiency of enzyme utilization
The results show that the enzyme with an aldehyde tag at the
The study showed that three aldehyde-tagged enzymes were successfully immobilized on different amino functionalized supports, and the highest immobilization efficiency of the model enzyme was over 90%, which indicated the universality of the site-specific immobilization
Summary
Enzyme immobilization is an effective method for stabilizing and improving the efficiency of enzyme utilization. Immobilized enzymes are widely used in biosensors, diagnostics, drug screening, and other fields due to its excellent green, economical, and sustainable properties [1,2]. Many researchers have concentrated on immobilization as an engineering implement to modulate and improve many of the catalytic properties of enzymes. A proper immobilization method is required to improve diverse enzymatic properties such as stability, reutilization, specificity, and selectivity [3,4,5]. Enzyme immobilization can be achieved by methods such as embedding, cross-linking, physical adsorption, and covalent bonding, according to the nature and application of the enzyme [6,7].
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