Geranyl Functionalized Materials for Site-Specific Co-Immobilization of Proteins.

Jana Brabcova,Blanca De Las Rivas,Rosario Muñoz,Zaida Cabrera,Alicia Andreu,Jose M Palomo,David Aguilera

doi:10.3390/molecules26103028

Abstract

Different materials containing carboxylic groups have been functionalized with geranyl-amine molecules by using an EDC/NHS strategy. Chemical modification of the support was confirmed by XRD, UV-spectrophotometer, and FT-IR. This geranyl-functionalized material was successfully applied for four different strategies of site-selective immobilization of proteins at room temperature and aqueous media. A reversible hydrophobic immobilization of proteins (lipases, phosphoglucosidases, or tyrosinase) was performed in neutral pH in yields from 40 to >99%. An increase of the activity in the case of lipases was observed from a range of 2 to 4 times with respect to the initial activity in solution. When chemically or genetically functionalized cysteine enzymes were used, the covalent immobilization, via a selective thiol-alkene reaction, was observed in the presence of geranyl support at pH 8 in lipases in the presence of detergent (to avoid the previous hydrophobic interactions). Covalent attachment was confirmed with no release of protein after immobilization by incubation with hydrophobic molecules. In the case of a selenium-containing enzyme produced by the selenomethionine pathway, the selective immobilization was successfully yielded at acidic pH (pH 5) (89%) much better than at pH 8. In addition, when an azido-enzyme was produced by the azide–homoalanine pathway, the selective immobilization was successful at pH 6 and in the presence of CuI for the click chemistry reaction.

Highlights

The immobilization of biomolecules represents an important alternative for their technological applications in different areas such as pharmaceuticals, textile, environmental, etc. [1,2,3,4,5]
The incorporation of the geranyl groups to the support was confirmed by X-ray diffraction (XRD) (Figure 2b,c)
Scanning electron microscope (SEM) analysis of the CM support before and after the modification demonstrated that no changes in the material morphology was produced with the chemical introduction of the geranyl groups (Figure 2d,e, Figure S1 and Figure S2)

Summary

Introduction

The immobilization of biomolecules represents an important alternative for their technological applications in different areas such as pharmaceuticals, textile, environmental, etc. [1,2,3,4,5]. The first means the development of a strategy to achieve the synthesis of a particular product from a starting material through a cascade process, which involves the use of two or more enzymes [13,14,15,16,17] or the combination of enzymes, artificial metalloenzymes, or even hybrid systems with transition metal catalysts [18,19,20,21] This process will perform multiple catalytic steps in a single container, so it greatly diminishing disadvantages such as purification stages, synthetic steps, high amount of wastes, and the drastic reduction of the final performance of the process. Most of the reported methods focus on the manufacture of materials that present orthogonal functional reactive groups to immobilize proteins in different ways [23]

Methods

Results

Conclusion