Recyclable Thermoresponsive Polymer–Cellulase Bioconjugates for Biomass Depolymerization

Abstract

Here we report the construction and characterization of a recoverable, thermoresponsive polymer-endoglucanase bioconjugate that matches the activity of unmodified enzymes on insoluble cellulose substrates. Two copolymers exhibiting a thermoresponsive lower critical solution temperature (LCST) were created through the copolymerization of an aminooxy-bearing methacrylamide with N-isopropylacrylamide (NIPAm) or N-isopropylmethacrylamide (NIPMa). The aminooxy group provided a handle through which the LCST was adjusted through small-molecule quenching. This allowed materials with LCSTs ranging from 20.9 °C to 60.5 °C to be readily obtained after polymerization. The thermostable endoglucanase EGPh from the hypothermophilic Pyrococcus horikoshii was transaminated with pyridoxal-5’-phosphate to produce a ketone-bearing protein, which was then site-selectively modified through oxime linkage with benzylalkoxyamine or 5 kDa-poly(ethylene glycol)-alkoxyamine. These modified proteins showed activity comparable to the controls when assayed on an insoluble cellulosic substrate. Two polymer bioconjugates were then constructed using transaminated EGPh and the aminooxy-bearing copolymers. After twelve hours, both bioconjugates produced an equivalent amount of free reducing sugars as the unmodified control using insoluble cellulose as a substrate. The recycling ability of the NIPAm copolymer-EGPh conjugate was determined through three rounds of activity, maintaining over 60% activity after two cycles of reuse and affording significantly more soluble carbohydrates than unmodified enzyme alone. When assayed on acid-pretreated Miscanthus, this bioconjugate increased the amount of reducing sugars by 2.8-fold over three rounds of activity. The synthetic strategy of this bioconjugate allows the LCST of the material to be changed readily from a common stock of copolymer and the method of attachment is applicable to a variety of proteins, enabling the same approach to be amenable to thermophile-derived cellulases or to the separation of multiple species using polymers with different recovery temperatures.

Immobilization of cellulase onto a recyclable thermo-responsive polymer as bioconjugate

“…Some pH-responsive [10][11][12] and thermo-responsive polymers [13][14][15][16] reported have been used to immobilize enzymes. Specifically, polymers with lower critical solution temperature (LCST) behavior are interesting supports for enzymes [11,17,18].…”

Technical Barriers to Advanced Liquid Biofuels Production via Biochemical Route

“…This would maximize sugar yields and also produce valuable products from separate fermentation process. Another possible approach to process integration could be application of thermophilic bacteria that can ferment both glucose and xylose (Bai et al 2013;Ito et al 2013;MacKenzie and Francis 2013). A single system can be developed that can hydrolyze and ferment sugars at the same time.…”

Polymeric SpyCatcher Scaffold Enables Bioconjugation in a Ratio‐Controllable Manner

“…Inspired by nature, several kinds of materials have been used as scaffolds to co‐localize proteins for biosensing and biocatalysis applications. Immobilization of proteins onto protein scaffolds (i.e., streptavidin), nanoparticles, synthetic polymers, and DNA or RNA has been studied intensively and some of these systems have already been applied in industry . However, the loss of protein activity caused by commonly used chemical conjugation methods during the immobilization process and decreased diffusion efficiency imposed by the insoluble form of proteins have limited further applications of immobilized proteins.…”