Multienzyme Coimmobilization on Triheterofunctional Supports

Abstract

Immobilized multienzyme systems are gaining momentum in applied biocatalysis; however, the coimmobilization of several enzymes on one carrier is still challenging. In this work, we exploited a heterofunctional support activated with three different chemical functionalities to immobilize a wide variety of different enzymes. This support is based on agarose microbeads activated with aldehyde, amino, and cobalt chelate moieties that allow a fast and irreversible immobilization of enzymes, enhancing the thermostability of most of the heterogeneous biocatalysts (up to 21-fold higher than the soluble one). Furthermore, this trifunctional support serves to efficiently coimmobilize a multienzyme system composed of an alcohol dehydrogenase, a reduced nicotinamide adenine dinucleotide (NADH) oxidase, and a catalase. The confined multienzymatic system demonstrates higher performance than its free counterpart, achieving a total turnover number (TTN) of 1 × 105 during five batch consecutive cycles. We envision this solid material as a platform for coimmobilizing multienzyme systems with enhanced properties to catalyze stepwise biotransformations.

Self-Sufficient Heterogeneous Biocatalysis through Boronic Acid-Diol Complexation of Adenylated Cofactors

“…Finally, samples were diluted with 75 μL of acetonitrile and properly filtered. HPLC analysis was conducted as described elsewhere . The samples were monitored at a wavelength of 340 nm and eluted at a flow rate of 1 mL min –1 by using a dual mobile-phase system.…”

“…Benzaldehyde and pyruvate synthesized in batch mode were quantified by HPLC . Samples containing benzaldehyde were diluted by a 10 to 100 factor in a mix of water/acetonitrile 30/70 (v:v), while pyruvate samples were diluted in 50 mM sodium phosphate buffer at pH 2.9.…”

“…HPLC analysis was conducted as described elsewhere. 50 The samples were monitored at a wavelength of 340 nm and eluted at a flow rate of 1 mL min −1 by using a dual mobile-phase system. Mobile phase A consisted of 0.1% trifluoroacetic acid in water, while mobile phase B comprised acetonitrile.…”

“…mode were quantified by HPLC. 50 Samples containing benzaldehyde were diluted by a 10 to 100 factor in a mix of water/acetonitrile 30/70 (v:v), while pyruvate samples were diluted in 50 mM sodium phosphate buffer at pH 2.9. The samples were monitored at 254 nm (benzaldehyde) and 220 nm (pyruvate) and were eluted at 1 and 0.7 mL min −1 flow rates, respectively, using two mobile phases for benzaldehyde detection as described for L-alanine.…”

Heterogeneous biocatalytic reduction of 5-(hydroxy)methyl furfural using two co-immobilised alcohol dehydrogenases

“…The IDA and epoxy densities were determined by titrating the remaining epoxy groups after each functionalisation step following a stepwise hydrolysis/oxidation route. 50 After incubation the carriers were washed with abundant water and stored at 4 °C.…”