015: Synthesis of Stabilized Dual Enzyme Cross-Linked Enzyme Aggregates to Valorize Lactose From Whey Permeate

Introduction

Immobilized enzyme systems have been widely used for transforming food coproduct streams due to their enhanced stability and facile recyclability relative to free enzyme systems. Notably, the conversion of D-lactose to lactose-fructose syrup (containing D-lactose, D-galactose, D-glucose, and D-fructose) using co-immobilized β-Gal and GI has been of high interest due to the abundance of D-lactose in dairy coproduct streams. However, the wide differences in thermal stability and optima between β-Gal and GI limit the use co-immobilized β-Gal and GI. To overcome these limitations, nanostructures containing the enzyme-stabilizing disaccharide, trehalose, were incorporated into co-immobilized β-Gal and GI in this study and its effect on enzyme performance was determined.

Methods

Trehalose nanostructures were synthesized at 0%, 10%, and 50% PAMAM surface coverage by carbodiimide coupling of succinylated trehalose to poly(amidoamine) [PAMAM] dendrimer nanostructures. Combined cross-linked enzyme aggregates of β-Gal and GI were prepared with and without trehalose nanostructures by precipitation of 100 mg/mL β-Gal and GI mixtures with 70% t-butanol, followed by crosslinked with 100 mM glutaraldehyde. The effect of varying concentrations of β-Gal and GI within Combi-CLEAs during preparation without trehalose nanostructures on the Combi-CLEAs conversion of D-lactose to D-fructose was evaluated using high-performance liquid chromatography. The stabilizing effect of adding trehalose nanostructures during β-Gal and GI Combi-CLEAs preparation was determined by the resulting stabilizer-added Combi-CLEAs’ thermal and pH optima, thermostability, and reusability.

Results

Combi-CLEAs of β-Gal and GI exhibited optimum activity (360 Uβ-Gal/gCombi-CLEAs and 83 UGI/gCombi-CLEAs) when prepared with 100 mg/mL enzyme mixtures with a β-Gal to GI activity ratio of 100 Uβ-Gal/UGI. Trehalose nanostructures’ structure was confirmed by Fourier-transform infrared spectroscopy and nuclear magnetic resonance, and upon addition to β-Gal and GI Combi-CLEAs, β-Gal activity increased by up to 51% and enhanced thermostability 2.26-fold while maintaining GI activity relative to Combi-CLEAs without trehalose nanostructures. Combi-CLEAs containing trehalose nanostructures were able to be reused five times while maintaining 40% and 7% β-Gal and GI activity.

Significance

This work demonstrates the valorization of D-lactose to lactose-fructose syrup with enhanced performance at elevated temperatures through use of β-Gal and GI Combi-CLEAs containing trehalose nanostructures. Trehalose nanostructures thus offer a promising solution for stabilizing biocatalytic systems.

Authors: Joshua G. Scott, Julie M. Goddard