Cellodextrin phosphorylase-catalyzed enzymatic polymerization from primers modified on poly(γ-glutamic acid) for the synthesis of moderate-molecular-weight celluloses

Abstract

Cellulose is an abundant natural polysaccharide composed of β(1→4)-linked repeating glucose units and is known to have a high molecular weight (MW). Although synthetic cellulose with a well-defined structure can be obtained through cellodextrin phosphorylase (CDP)-catalyzed enzymatic polymerization of α-D-glucose 1-phosphate (Glc-1-P) as a monomer, initiated from a cellobiose primer, the MWs of the products are low, due to their precipitation in an aqueous buffer medium, caused by crystallization. In this study, we successfully prepared cellulose graft chains with moderate MWs through the CDP-catalyzed enzymatic polymerization of Glc-1-P using cellobiose primer-modified poly(γ-glutamic acid)s (PGAs), where are difficult to prepare by the CDP catalysis using free cellobiose primer, as well as degradation of natural cellulose resources. After immobilizing the cellobiose primers via condensation between carboxylic acid groups of PGAs and cellobiosylamine, we carried out CDP-catalyzed graft polymerization from the resulting primer-modified PGAs at a Glc-1-P/primer feed ratio of 10/1 at 50 ^oC for 48 h in HEPES buffer. The ¹H NMR analysis of the precipitated products or a sample after acidic hydrolysis of the cellulose graft chains in the product, measured in NaOD/D₂O, confirmed the structures of the cellulose-grafted PGAs with moderate-MW cellulose graft chains. The powder X-ray diffraction profiles of the products indicated the formation of cellulose II crystalline structures originating from the graft chains among the PGA main chains. When the cellobiose primers were modified on PGAs, spatial separation was provided among the cellulose graft chains elongated from the primer ends. This favored chain propagation over crystallization, resulting in cellulose graft chains with higher MWs than those obtained by conventional CDP-catalyzed enzymatic polymerization using free cellobiose primers. The degree of primer substitution (DS) on PGAs also affected the MW values of the cellulose graft chains. We further investigated the isolation of the cellulose graft chains by alkaline hydrolysis of the PGA main chains in the product. The ¹H NMR analysis revealed that the MW of the isolated cellulose was comparable to that before isolation.