Novel aromatic polymers for immobilizing Β-D-glucosidase and their possible application to cellulolysis

Abstract

We have synthesized, by enzymic and chemical means, a variety of novel polyaromatic-enzyme complexes that are extremely stable and show promise in the conversion of cellulose to glucose. Thus we have prepared a number of homo- and heteropolymeric supports (involvingl-tyrosine, pyrogallol, resorcinol, phloroglucinol, orcinol, catechol, protocatechuic acid, and various hydroxybenzoic acids) and discovered that, for example, a resorcinol-Β-d-glucosidase copolymer has high stability combined with lowK _m (10.5 mM vs commercial soluble (3-d-glucosidase 9.3 mM) and high V_max values (104 Μmol ρNP mg^-1H^-1 vs 85 Μmol ρNP mg^-1H^-1). These properties are enhanced when the copolymer is complexed with bentonite clay. The kinetic constants of the resorcinol-Β-d-glucosidase copolymer-bentonite complex wereK _m = 9.6 mM andV _max = 73.5 Μmol ρNP mg^-1H^-1. Stability has been assessed against proteolysis, organic solvents, elevated temperatures, storage, and incorporation into fresh soil.

A cellulase preparation fromTrichoderma viride has also been copolymerized with a variety of phenolic macromolecules and displays varying degrees of stability and activity against carboxymethyl cellulose.

The resorcinol Β-d-glucosidase-copolymer was immobilized on a PM10 ultrafiltration membrane (K _m = 16.8 mM; V_max = 42.4 (Μmol ρNP mg^-1H^-1) and showed enhanced thermostability, a broader pH range for maximal activity, and could be reused without loss of activity. An ultrafiltration cell, containing the membrane-immobilized resorcinol-Β-d-glucosida se copolymer, can be operated as a continuous reactor with substrate flow rates from 0.1 to 0.7 mL min^-1 without decrease in product formation.