title: 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 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 12 h, 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.
authors.affiliation: Department of Chemistry, University of California, Berkeley, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720-1460, United States.
authors.forename: Katherine J
authors.initials: KJ
authors.lastname: Mackenzie
authors.forename: Matthew B
authors.initials: MB
authors.lastname: Francis
mesh_terms: D002480:Cellulase / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D008970:Molecular Weight; D058105:Polymerization; D011108:Polymers / Q000138:chemical synthesis / Q000737:chemistry*; D013696:Temperature
chemical_list: D011108:Polymers; D002480:Cellulase