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.

Site-selective bioconjugation methods are valuable because of their ability to confer new properties to proteins by the chemical attachment of specific functional groups. Well-defined bioconjugates obtained through these methods have found utility for the study of protein function and the creation of protein-based materials. We have previously reported a protein modification strategy to modify the N-terminus of peptides and proteins using N-methylpyridinium-4-carboxaldehyde benzenesulfonate (Rapoport's salt, RS) as a transamination reagent, which oxidizes the N-terminal amino group to provide a uniquely reactive aldehyde or ketone. This functional handle can subsequently be modified with an alkoxyamine reagent of choice. Previous work had found glutamate terminal sequences to be highly reactive toward RS-mediated transamination. However, proteins of interest are often recombinantly expressed in E. coli, where the expression of a glutamate-terminal protein is rendered difficult because the N-terminal methionine derived from the start codon is not cleaved when Glu is in the second position. In this work, we describe a way to overcome this difficulty via the insertion of a Factor Xa proteolytic cleavage site to acquire the optimal glutamate residue at the N-terminus. Additionally, we present studies on alternative high-yielding sequences containing N-terminal residues that can be expressed directly. We have used site-directed mutagenesis to validate these findings on a model cellulase enzyme, an endoglucanase from the thermophilic Pyrococcus horikoshii. Activity assays performed with these mutants show that RS transamination and subsequent modification with alkoxyamines have no negative impact on cellulolytic ability.

There is currently much interest in the economic use of cellulosic biomass as a source of renewable fuels. This process typically involves the enzymatic hydrolysis of plant matter to afford soluble sugars for subsequent fermentation steps. The cost of cellulase enzymes presents a critical barrier to the commercialization of these processes. In this work, we demonstrate that a new family of polymer additives based on NIPAm can increase enzyme performance substantially. When applied to an industrially relevant combination of enzymes and lignin-containing biomass, polymer additives allow a 60% reduction in enzyme loading to achieve the same level of saccharification. Evidence presented herein suggests that these polymers function through multiple mechanisms, including (1) preventing enzyme denaturation through shear and interfacial interactions, (2) preventing non-productive adsorption to lignin, and (3) altering the cellulose structure. An advantage of these polymers over other additives is their thermoresponsive behavior, enabling their recovery and reuse.