Hen egg white lysozyme encapsulated in ZIF-8 for performing promiscuous enzymatic Mannich reaction

Abstract

Hen egg white lysozyme (HEWL) was exploited for the synthesis of β-amino carbonyl compounds through a direct and three-component Mannich reaction in aqueous, confirming high chemoselectivity toward imine. In order to further extend the applications of the enzyme, HEWL was encapsulated using a metal-organic framework (MOF). The reactivity, stereoselectivity, and reusability of the encapsulated enzyme were investigated. The reaction was significantly enhanced as compared to the non-encapsulated enzyme. A mutated version of the enzyme, containing Asp52Ala (D52A), lacking important catalytical residue, has lost the bacterial site activity against Micrococcus luteus (M. luteus) while the D52A variant displayed an increased rate of the Mannich reaction, indicating a different catalytical residue involved in the promiscuous reaction. Based on site-directed mutagenesis, molecular docking, and molecular dynamic studies, it was proposed that π-stacking, H-bond interactions, and the presence of water in the active site may play crucial roles in the mechanism of the reaction.

Keywords: Bioorganic chemistry; materials design.

Graphical abstract

Scheme 1

Using HEWL as biocatalyst in C-C coupling reaction Aldol, Michael, and Mannich reactions catalyzed by HEWL, reaction conditions: (A) A solution of 3-nitrobenzaldehyde (0.2 mmol), cyclohexanone (0.1 mmol), HEWL (10 mg), and water (1 mL) was stirred at 35°C for 20 h. (B) A solution of cyclohexanone (0.1 mmol), 2-cyclohexenone (0.1 mmol), HEWL (10 mg), and water (1 mL) was stirred at 35°C for 20 h. (C) A solution of 3-nitrobenzaldehyde (0.1 mmol), aniline (0.11 mmol), cyclohexanone (0.17 mmol) HEWL (10 mg), and water (1 mL) was stirred at 35°C for 20 h.

Figure 1

Biological activity of recovered HEWL (A) Catalytic performance of the reused HEWL after 4 runs on the yield of 4-(3-nitrophenyl)-4-(phenylamino)butan-2-one. Reaction conditions: a solution of 3-nitrobenzaldehyde (0.3 mmol), aniline (0.33 mmol), acetone (0.5 mmol), water (1 mL) HEWL 20 mg, reaction temperature of 35°C and reaction time 9 h. (B) Turbidimetric assay of recycled HEWL: 0.25 mg/mL of Micrococcus luteus (M. luteus) cell suspension was incubated with 1 mg/mL of each recycled lysozyme run. The decrease of turbidity at 450 nm confirmed that the muramidase activity of lysozyme would dramatically decrease after reusing the enzyme 4 times, error bars indicate standard deviation.

Figure 2

FT-IR spectra of different HEWL-biocomposites FT-IR spectra of pure HEWL, L-His, ZIF-8 crystal, HEWL/ZIF-8, HEWL-His/ZIF-8, PVP-modified HEWL-His/ZIF-8, and adsorbed HEWL on the surface of pre synthesized-ZIF-8 in the presence and absence of PVP.

Figure 3

SEM images of composites (A) ZIF-8. (B–D) HEWL/ZIF-8 in the presence of L-His (B) without, and (C) with PVP modification, and (D) reused catalyst after 7 cycles.

Figure 4

Turbidimetric assay of wild-type lysozyme, PHHZ, and mutant D52A against M. luteus The absorbance was measured as described in the legend of Figure 1B for the recycled HEWL; error bars indicate standard deviation.

Figure 5

Catalytic recyclability of PHHZ for the Mannich reaction The PHHZ biocomposite was used for a solution of benzaldehyde, aniline, and acetone as substrates in the model reaction.

Figure 6

Snapshots of randomly positioned of simulations for HEWL complex with substrates and the intermediate (A and B) Interactions of aniline and benzaldehyde with wild-type HEWL and mutated HEWL. (C and D) Schiff base and acetone interactions with wild-type HEWL and mutated HEWL respectively. Hydrogen bonds are depicted as dotted lines. The water molecules are displayed as white and red molecules. All images were generated using PyMOL.

Scheme 2

Proposed mechanism of HEWL-catalyzed Mannich reaction based on molecular docking and molecular dynamic studies.