Developing a Novel Enzyme Immobilization Process by Activation of Epoxy Carriers with Glucosamine for Pharmaceutical and Food Applications

Abstract

In this paper, we describe the development of an efficient enzyme immobilization procedure based on the activation of epoxy carriers with glucosamine. This approach aims at both creating a hydrophilic microenvironment surrounding the biocatalyst and introducing a spacer bearing an aldehyde group for covalent attachment. First, the immobilization study was carried out using penicillin G acylase (PGA) from Escherichia coli as a model enzyme. PGA immobilized on glucosamine activated supports has been compared with enzyme derivatives obtained by direct immobilization on the same non-modified carriers, in the synthesis of different 3′-functionalized cephalosporins. The derivatives prepared by immobilization of PGA on the glucosamine-carriers performed better than those prepared using the unmodified carriers (i.e., 90% versus 79% cefazolin conversion). The same immobilization method has been then applied to the immobilization of two other hydrolases (neutral protease from Bacillus subtilis, PN, and bromelain from pineapple stem, BR) and one transferase (γ-glutamyl transpeptidase from Bacillus subtilis, GGT). Immobilized PN and BR have been exploited in the synthesis of modified nucleosides and in a bench-scale packed-bed reactor for the protein stabilization of a Sauvignon blanc wine, respectively. In addition, in these cases, the new enzyme derivatives provided improved results compared to those previously described.

Dual Valorization of Lignin as a Versatile and Renewable Matrix for Enzyme Immobilization and (Flow) Bioprocess Engineering

“…Nevertheless, no significant improvements over the resulting activity (U g −1 ) of the biocatalyst were detected (Figure S7). Additionally, lignin was functionalized with glucosamine to increase the hydrophilicity of the support following a modified protocol reported before for functionalization of acrylic resins [37] . Then, the Gs‐Lys6DH was immobilized on glucosamine‐lignin yielding a 2‐fold increase on the recovered activity compared to the enzyme immobilized on TALD‐lignin (Figure S8).…”

“…Additionally, lignin was functionalized with glucosamine to increase the hydrophilicity of the support following a modified protocol reported before for functionalization of acrylic resins. [37] Then, the Gs‐Lys6DH was immobilized on glucosamine‐lignin yielding a 2‐fold increase on the recovered activity compared to the enzyme immobilized on TALD‐lignin (Figure S8). Overall, however, this approach did not outperform the PEI‐lignin strategy.…”

“…Glucosamine‐lignin (Figures 1E and S2E): A previous protocol for activation of acrylic carriers was adapted [37] . TALD‐lignin (0.5 g) was incubated with 1.5 m glucosamine (5 mL) at pH 10 overnight.…”

“…Glucosamine‐lignin (Figures 1 E and S2E): A previous protocol for activation of acrylic carriers was adapted. [37] TALD‐lignin (0.5 g) was incubated with 1.5 m glucosamine (5 mL) at pH 10 overnight. After washing the resin, 1 mg mL −1 NaBH 4 in the same buffer (5 mL) was added and the suspension was incubated for 1 h. After a washing step, 15 m m NaIO 4 (10 mL) was added and the suspension was incubated for 1 h.…”

Enhanced Stability of Enzyme Immobilized in Rationally Designed Amphiphilic Aerogel and Its Application for Sensitive Glucose Detection

“…Natural enzymes that ubiquitously exist in the natural world, have wide applications in many aspects of life because of their impressive advantages like high specificities and high efficiency. − Their synergistically catalytic effect is extremely important due to the role of multienzyme complex (MEC) in interceding every biological process in all living bodies. − The existing form of most natural enzymes is protein, thus, the high catalytic efficiency, in essence, is owing to the unchanged conformation of the protein in mild conditions. − Nevertheless, several severe disadvantages greatly impede the practical applications of natural enzymes: (i) ease in denaturation under adverse environmental conditions; (ii) decomposition under existence of proteases; (iii) complicated and/or expensive preparation process. − An effective strategy for solving this problem is to spatially bring together the component enzymes in a well-controlled process to prevent the exposure of the enzymes to the environment. − For immobilization of multiple enzymes, attachment techniques always need to be carefully engineered because rigorous consideration is necessary in order to maintain enzyme activity in case the graceful functionality and feeble structures of enzymes would be destroyed under improper environmental conditions. ,− Adsorption is one of the promising ways to immobilize the enzymes because of the ease in manipulation, feasibility in loading amount modulation, etc. − According to the previous report, the support is preferably to have epoxy groups and hydrophilic nature for enzyme stabilization. ,,, Also, hydrogen bonding formed by oxygenated groups (e.g., carboxyl groups) and enzymes prevents the enzymes from leaking during catalysis processes while maintaining its high activity. , In other words, an amphiphilic support containing the hydrophobic (epoxy) and hydrophilic (−COOH, −OH, etc.) sites on surface not only facilitates the attachment of the enzyme to the interface, conserving the enzyme conformation, , but al...…”