Development of a Novel Bi-Enzymatic Nanobiocatalyst for the Efficient Bioconversion of Oleuropein to Hydroxytyrosol

Abstract

Lipase A from Candida antarctica (CalA) and β-glucosidase from Thermotoga maritima (bgl) were covalently co-immobilized onto the surface of chitosan-coated magnetic nanoparticles (CS-MNPs). Several parameters regarding the co-immobilization procedure (glutaraldehyde concentration, incubation time, CS-MNPs to enzyme mass ratio and bgl to CalA mass ratio) were evaluated and optimized. The developed nanobiocatalyst was characterized by various spectroscopic techniques. Biochemical parameters such as kinetic constants and thermal stability were also evaluated. The nanobiocatalytic system revealed an increase in the Km constant followed by a decrease in Vmax value compared with the native enzymes, while a significant increase (>5-fold higher) of the thermal stability of the immobilized CalA, both in individual and in co-immobilized form, was observed after 24 h incubation at 60 °C. Finally, the nanobiocatalyst was efficiently applied for the bioconversion of oleuropein to hydroxytyrosol, one of the most powerful naturally derived antioxidants, and it could be recycled for up to 10 reaction cycles (240 h of constant operation) at 60 °C, retaining more than 50% of its initial activity.

Characterization of Gentisate 1,2-Dioxygenase from Pseudarthrobacter phenanthrenivorans Sphe3 and Its Stabilization by Immobilization on Nickel-Functionalized Magnetic Nanoparticles

“…GDO's relative activity, though decreased, was also detectable for five more cycles (data not shown). This loss in activity could be probably attributed to any mechanical damage of the biocatalyst during the recycling process along with product inhibition, as has been previously reported [76]. Although no reusability data of gentisate 1,2-dioxygenase have been reported by any other study so far, similar data can be comparable to other enzymes immobilized on various supports [35,46,84,89,90].…”

“…The enzyme's activity recovery does not seem to be significantly affected at different mass ratios (Figure 3). However, the lowest activity recovery was observed at the highest mass ratio of 1:10, probably due to the insufficient available surface area of the nanocarrier to load the large amount of enzyme, resulting in decreased activity recovery [75,76]. The increase of the amount of MNPs led to an increase of the enzyme's activity recovery, and specifically, the maximum activity recovery was observed at the ratio of 4:2 (Figure 3).…”

Lignin Nanoparticles Support Lipase‐Tyrosinase Enzymatic Cascade in the Synthesis of Lipophilic Hydroxytyrosol Ester Derivatives

“…[32] Enzymatic cascade is an advanced tool for multiple synthetic transformations in one-pot condition, [33,34] promoting the fast exchange of substrate between adjacent enzymes. [35,36] Supported multienzyme cascades are reported, and their efficiency, selectivity, stability and reusability deeply discussed. [1,37] Here we describe the one-pot synthesis of lipophilic hydroxytyrosol esters from tyrosol and long side-chain carboxylic acids by lignin nanoparticles supported enzymatic cascade involving lipase M from Mucor javanicus (MJL) and tyrosinase (Ty) from Agaricus bisporus.…”

“…These compounds showed antioxidant and antiviral activity dependent from the number of carbon atoms in the side‐chain [32] . Enzymatic cascade is an advanced tool for multiple synthetic transformations in one‐pot condition, [33,34] promoting the fast exchange of substrate between adjacent enzymes [35,36] . Supported multienzyme cascades are reported, and their efficiency, selectivity, stability and reusability deeply discussed [1,37] .…”

A flexible kinetic assay efficiently sorts prospective biocatalysts for PET plastic subunit hydrolysis

“…The latter was shown to exhibit both MHETase and BHETase activities. 22,23 Both CalB and its paralog CalA (enzyme 13) 7,24–27 are popular in biotechnological applications due to their broad substrate promiscuity. We anticipated the commercial enzymes would react optimally at ∼37 °C, except enzyme 10, which comes from a Geobacillus stearothermophilus strain with a growth optimum of 55 °C.…”