Recyclable Thermoresponsive Polymer−β-Glucosidase Conjugate with Intact Hydrolysis Activity

Abstract

β-Glucosidase (BG) catalyzes the hydrolysis of cellobiose to glucose and is a rate-limiting enzyme in the conversion of lignocellulosic biomass to sugars toward biofuels. Since the cost of enzyme is a major contributor to biofuel economics, we report the bioconjugation of a temperature-responsive polymer with the highly active thermophilic β-glucosidase (B8CYA8) from Halothermothrix orenii toward improving enzyme recyclability. The bioconjugate, with a lower critical solution temperature (LCST) of 33 °C withstands high temperatures up to 70 °C. Though the secondary structure of the enzyme in the conjugate is slightly distorted with a higher percentage of β-sheet like structure, the stability and specific activity of B8CYA8 in the conjugate remains unaltered up to 30 °C and retains more than 70% specific activity of the unmodified enzyme at 70 °C. The conjugate can be reused for β-glucosidic bond cleavage of cellobiose for at least four cycles without any significant loss in specific activity.

Elucidating the regulation of glucose tolerance through the interaction between the reaction product and active site pocket residues of a β-glucosidase from Halothermothrix orenii

“…Our initial studies on the model substrate Avicel support the potential gains of using a high specific activity and glucose tolerant β-glucosidase [14]. We had also previously reported the potential of recycling the wild-type enzyme towards industrial applications [49].…”

Hetero‐Diels–Alder Cycloaddition with RAFT Polymers as Bioconjugation Platform

“…P(eDEGA‐ co ‐mOEGA)s exhibit a lower critical solution temperature (LCST), which depends on the exact comonomer ratio, [29, 30] and will lead to thermoresponsive PPCs at possibly useful temperatures. Responsive PPCs [31, 32] are particularly interesting for control of biomolecular activity, [33] triggered reversible self‐assembly into biohybrid nanostructures [34–38] and fast removal/recovery of proteins from solution [39, 40] . Interestingly, similar polymers with short oligoethylene glycol side chains were shown not to exhibit the non‐desired antigenicity of PEG [41] .…”

“…Responsive PPCs [31,32] are particularly interesting for control of biomolecular activity, [33] triggered reversible self-assembly into biohybrid nanostructures [34][35][36][37][38] and fast removal/recovery of proteins from solution. [39,40] Interestingly, similar polymers with short oligoethylene glycol side chains were shown not to exhibit the nondesired antigenicity of PEG. [41] 2-cyanoprop-2-yl diethoxyphosphoryldithioformate (CPDPDT) was employed as CTA because it simultaneously enables the controlled polymerization of acrylates and the synthesis of polymers with a terminal C=S bond sufficiently electron-deficient for a rapid HDA cycloaddition to occur.…”

Hetero‐Diels‐Alder‐Cycloaddition mit RAFT‐Polymeren als Biokonjugationsplattform

“…P(eDEGA‐ co ‐mOEGA)s weisen eine untere kritische Lösungstemperatur (LCST) auf, die vom genauen Comonomer‐Verhältnis abhängt, [29, 30] und bei möglicherweise nützlichen Temperaturen zu thermoresponsiven PPK führt. Responsive PPK [31, 32] sind besonders interessant für die Kontrolle der biomolekularen Aktivität, [33] die gesteuerte reversible Selbstorganisation in biohybriden Nanostrukturen, [34–38] sowie die schnelle Entfernung/Wiedergewinnung von Proteinen aus der Lösung [39, 40] . Interessanterweise konnte gezeigt werden, dass ähnliche Polymere mit kurzen Oligoethylenglykol‐Seitenketten nicht die ungewünschte Antigenität von PEG aufweisen [41] .…”

“…Responsive PPK [31,32] sind besonders interessant für die Kontrolle der biomolekularen Aktivität, [33] die gesteuerte reversible Selbstorganisation in biohybriden Nanostrukturen, [34][35][36][37][38] sowie die schnelle Entfernung/Wiedergewinnung von Proteinen aus der Lçsung. [39,40] Interessanterweise konnte gezeigt werden, dass ähnliche Polymere mit kurzen Oligoethylenglykol-Seitenketten nicht die ungewünschte Antigenität von PEG aufweisen. [41] 2-Cyanoprop-2-yldiethoxyphosphoryldithioformiat (CPDPDT) wurde als CTA eingesetzt, weil es gleichzeitig die kontrollierte Polymerisation von Acrylaten und die Synthese von Polymeren mit einer terminalen C=S-Bindung ermçglicht, die ausreichend elektronenarm ist, um eine schnelle HDA-Cycloaddition zu bewirken.…”