Enzymes produced from live cells act as a natural biocatalyst and perform biochemical reactions under mild conditions with high substrate specificity (Girolamo et al., 2019). In comparison to chemical catalysts, the ease of production, catalytic efficiency, chemo-, regio-, and stereospecificity of the enzyme greatly promote its use in therapeutical and industrial applications (Zhang et al., 2013). However, poor stability of the enzyme, low shelf life, and high cost limits its use in industrial application. One of the best strategies to overcome this problem is to immobilize enzymes using a carrier such as a hydrogel, biopolymer, synthetic polymers, nanoparticles, etc (Bornscheuer, 2003, Homaei et al., 2013). According to a recent study, immobilization efficiency on nanostructured materials is greater due to their larger surface area, better enzyme loading, and higher activity (Ansari and Husain, 2012). The use of nanoparticles has brought significant medical applications in wound healing, cell imaging, tissue engineering, and drug delivery. AuNPs are thought to be an ideal model for studying the effects of immobilization on the activity and stability of various functional proteins (Ma et al., 2018, Saware et al., 2015). When AuNPs are introduced into biological organisms, their enormous surface area permits a huge number of proteins to be bound on their surface (Kong et al., 2017). PEGylated AuNPs exhibit promising advantages like enhanced biocompatibility, stability, etc. AuNPS serves as a promising SERS substrate for getting amplified Raman signals of the molecular fingerprint. SERS provides high sensitivity and molecular specificity along with its capability to resolve complex molecular level biological conformations. The integration of advanced technologies and minimal sample volume improved the utility of SERS in the quantitative and qualitative identification of various biomolecules as they provide characteristic signals. Herein, we made use of this technique to track the immobilization of eGFP and XylB on triglycine PEGylated AuNPs via Sortase E-mediated technique.

For industrial biocatalyst applications, enzyme immobilization offers a practical and recyclable design. Covalent immobilization appears to be the most appealing method among the numerous physical adsorption and chemical attachment options. Although chemical approaches for covalent immobilization are widely accessible, they often result in random and nonspecific immobilization, which may result in enzyme deactivation (Wong et al., 2009). As a result, developing site-specific and covalent immobilization techniques to preserve enzyme intrinsic activities is critical. Sortagging has emerged as a reliable and strong technique for site-specific peptide or protein attachment in recent years. The target proteins were altered based on the substrate specificity of the enzyme under mild conditions and without prior chemical modifications (Hata et al., 2015, Zou et al., 2019). Sortase A, a transpeptidase from Staphylococcus aureus, cleaves between the T and G residues in the sequence LPXTG and then forms a native peptide bond between the carboxyl group of the T residue and an amino group of N-terminal glycine oligomers. SaSrtA has been widely used in immobilizing proteins or enzymes on solid supports (Chan et al., 2007), nanoparticles (Hata et al., 2015), microgels (Zou et al., 2019), and hydrogels (Cambria et al., 2015) due to its high substrate selectivity. Sortagging has emerged as a reliable and strong technique for site-specific peptide or protein attachment in recent years. The target proteins were altered based on the substrate specificity of the enzyme under mild conditions and without prior chemical modifications (Hata et al., 2015, Zou et al., 2019). Although, there are several types of sortases found in Gram-positive bacteria, little is known about their expression and substrate specificity. However, C. glutamicum ATCC 13032, a non-pathogenic and well-known industrial microbe for amino acid production, contains a sortase E transpeptidase with high substrate selectivity with LAHTG (Susmitha et al., 2019). The Ca2+ independence, high substrate specificity, and ability to accept N-terminal (oligo) residues make C. glutamicum sortase E (CgSrtE) much more efficient than pathogenic SaSrtA in live cells and for industrial sortagging applications.

In this study, we employed CgSrtE to immobilize eGFP and XylB on triglycine functionalized AuNPs. The initial work was carried out on the model protein, eGFP to analyze the successful encapsulation of AuNPs. The second target protein XylB, catalyzes the oxidation of xylose to xylonic acid, which is used as a platform chemical in the industry (Lee et al., 2018). The resulting recombinant proteins were expressed in E. coli with LAHTG-tagged proteins at the C-terminal. Purified eGFP-LAHTG and XylB-LAHTG were immobilized separately on a functionalized triglycine AuNPs by a SrtE transpeptidation reaction and immobilization was confirmed by SERS and UV–vis spectral analysis. To the best of our knowledge, this is the first report on tag-mediated protein immobilization by Sortase E as well as detection of conjugated protein on AuNPs via Raman spectral analysis. The effect of XylB-oriented immobilization on activity and reusability was evaluated by comparing free enzymes by SrtE-mediated technique. In addition, the target protein was immobilized under mild conditions with no chemical modification.