Tyrosinase Magnetic Cross-Linked Enzyme Aggregates: Biocatalytic Study in Deep Eutectic Solvent Aqueous Solutions

Abstract

In the field of biocatalysis, the implementation of sustainable processes such as enzyme immobilization or employment of environmentally friendly solvents, like Deep Eutectic Solvents (DESs) are of paramount importance. In this work, tyrosinase was extracted from fresh mushrooms and used in a carrier-free immobilization towards the preparation of both non-magnetic and magnetic cross-linked enzyme aggregates (CLEAs). The prepared biocatalyst was characterized and the biocatalytic and structural traits of free tyrosinase and tyrosinase magnetic CLEAs (mCLEAs) were evaluated in numerous DES aqueous solutions. The results showed that the nature and the concentration of the DESs used as co-solvents significantly affected the catalytic activity and stability of tyrosinase, while the immobilization enhanced the activity of the enzyme in comparison with the non-immobilized enzyme up to 3.6-fold. The biocatalyst retained the 100% of its initial activity after storage at -20 °C for 1 year and the 90% of its activity after 5 repeated cycles. Tyrosinase mCLEAs were further applied in the homogeneous modification of chitosan with caffeic acid in the presence of DES. The biocatalyst demonstrated great ability in the functionalization of chitosan with caffeic acid in the presence of 10% v/v DES [Bet:Gly (1:3)], enhancing the antioxidant activity of the films.

Keywords: biocatalysis; caffeic acid; chitosan; deep eutectic solvents; enzyme; functionalization; immobilization; tyrosinase.

Figure 1

FTIR spectra of magnetic nanoparticles, tyrosinase CLEAs and tyrosinase mCLEAs.

Figure 2

XPS spectra of the Fe2p core level region of (a) iron oxide nanoparticles; (b) tyrosinase mCLEAs and XPS spectra of the C1s core level region of (c) iron oxide nanoparticles; (d) tyrosinase mCLEAs.

Figure 3

XRD patterns of iron oxide nanoparticles and tyrosinase mCLEAs.

Figure 4

(a) Reusability of tyrosinase mCLEAs in buffer: relative activity for 10 consecutive cycles; (b) storage stability of tyrosinase mCLEAs: relative activity after storing for different times at −20 °C. As 100% is defined the enzyme activity at the beginning of the test (initial activity). The reaction conditions were 1 mg mL⁻¹ of biocatalyst and 10 mM L-DOPA in 50 mM phosphate buffer pH 7, at 700 rpm and 30 °C.

Figure 5

Relative activity of tyrosinase mCLEAs and free tyrosinase in 10% v/v aqueous solutions of certain DESs. 100% is defined as the activity of tyrosinase mCLEAs or free tyrosinase in DES-free solution.

Figure 6

Relative activity of (a) tyrosinase mCLEAS and (b) free tyrosinase at increasing concentrations of certain DESs. 100% is defined as the activity of tyrosinase mCLEAs or free tyrosinase in DES-free solution.

Figure 7

Relative activity of free tyrosinase and tyrosinase mCLEAs in 10% v/v of various DESs after pre-incubation at 30 °C for 24 h. 100% is defined as the residual activity of free tyrosinase and tyrosinase mCLEAs after pre-incubation at 30 °C for 24 h in DES-free solution.

Figure 8

Maximum emission wavelength (λ_max) of free tyrosinase’s fluorescence emission spectra, for increasing concentrations of various DESs.

Figure 9

Photographs of a mCLEAs-modified chitosan film with caffeic acid (left), a blank chitosan film with caffeic acid (middle) and a neat chitosan film (right).

Figure 10

ATR spectra of a neat chitosan film (CS film) and a mCLEAs-modified chitosan film with caffeic acid derivatives (CA-CS film).

Figure 11

Antioxidant activity of a neat chitosan film (CS film), a chitosan film containing 10% v/v DES (DES-CS film), a mCLEAs-modified chitosan film with caffeic acid (CA-CS film), and a mCLEAs-modified chitosan film containing 10% v/v DES with caffeic acid (CA-DES-CS film).