Protein‐Polymer Bioconjugation Effects on Activity and Stability of Cellulase (FnCel5a) with Varying Molecular Weights of Polymer Chains

Abstract

An alternative method for energy production which decreases dependency on nonrenewable resources and utilizes a greater amount of the available plant biomass is cellulosic ethanol production. However, there is a need for a more cost effective and energy efficient method to produce a stable form of cellulase that can withstand the harsh chemical and physical industrial processes. One method to enhance protein stability has been demonstrated through protein‐polymer bioconjugation. Thermophilic cellulase, FnCel5a, has been shown to have increased activity when conjugated to polymers via amidation reactions onto lysine residues of the enzyme. However, due to numerous lysine residues there was a lack of site‐specific binding of the polymers. Therefore, a novel site‐specific approach was taken in this study to conjugate polymers onto cellulase via a maleimide method using thiolene click chemistry onto a cysteine residue. In addition, polymer chain length was manipulated to investigate its effects on enzymatic stability and activity using the amidation method. Polymerization methods of RAFT and ATRP were utilized to synthesize N, N‐dimethylacrylamide (DMAm) polymers and OEOA polymers of varying degrees of polymerization. These polymers were conjugated using a “graft to” method onto lysine residues. SDS‐PAGE electrophoresis confirmed conjugation of the polymers. And an activity assay using carboxymethylcellulose substrate indicated the presence of a maximum percent enzymatic activity with increased polymer chain length. For site‐specific conjugation, a mutagenesis at C296S and C102S was done for selective polymer binding. SDS‐PAGE electrophoresis confirmed DMam and OEOA polymer binding at the C296 position of FnCel5a. Overall this study confirmed the effective use of click chemistry method as a technique for site‐specific polymer binding of RAFT and ATRP at a cysteine residue. This method can also be utilized to better investigate how polymer chain length can affect the stability and activity of FnCel5a with controlled site‐specific reactions.Support or Funding InformationREU Site: Summer Research in Chemistry and Biochemistry at Miami University at Miami University, Award No. 1460862This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.