Inhibition of enzymatic hydrolysis by residual lignins from softwood—study of enzyme binding and inactivation on lignin‐rich surface

Abstract

Lignin-derived inhibition is a major obstacle restricting the enzymatic hydrolysis of cell wall polysaccharides especially with softwood lignocellulosics. Enzyme adsorption on lignin is suggested to contribute to the inhibitory effect of lignin. The interaction of cellulases with softwood lignin was studied in the present work with commercial Trichoderma reesei cellulases (Celluclast) and lignin-rich residues isolated from steam pretreated softwood (SPS) by enzymatic and acid hydrolysis. Both lignin preparations inhibited the hydrolysis of microcrystalline cellulose (Avicel) and adsorbed the major cellulases present in the commercial cellulase mixture. The adsorption phenomenon was studied at low temperature (4°C) and at the typical hydrolysis temperature (45°C) by following activities of free and lignin-bound enzymes. Severe inactivation of the lignin-bound enzymes was observed at 45°C, however at 4°C the enzymes retained well their activity. Furthermore, SDS-PAGE analysis of the lignin-bound enzymes indicated that very strong interactions form between the residue and the enzymes at 45°C, because the enzymes were not released from the residue in the electrophoresis. These results suggest that heat-induced denaturation may take place on the surface of softwood lignin at the hydrolysis temperature.

Shotgun Approach to Increasing Enzymatic Saccharification Yields of Ammonia Fiber Expansion Pretreated Cellulosic Biomass

“…This result is consistent with studies indicating that residual lignin within pretreated biomass can bind cellulases nonproductively (Gao et al, 2011a(Gao et al, , 2014Rahikainen et al, 2011). Therefore, with an increased fraction of non-productively bound enzymes at lower protein loadings, the need for optimal enzyme mixtures becomes even more apparent.…”

Controlling the Size and Film Strength of Individualized Cellulose Nanofibrils Prepared by Combined Enzymatic Pretreatment and High Pressure Microfluidization

“…The huge morphological differences between Celluclast 1.5 L-and Fibercare-treated BSKP fiber indicated that the two cellulase enzymes worked differently. Fibercare, an extraglucanase deficient endoglucanase, is more selective, and enacts limited hydrolysis of the amorphous domain of cellulose (Wang et al 2012b), while Celluclast 1.5L, a complex cellulase mixture with endoglucanase and exoglucanase activity, provides extensive hydrolysis of amorphous and crystalline cellulose (Rahikainen et al 2011).…”

Efeito da lignina de bagaços de cana-de-açúcar pré-tratados na hidrólise enzimática da celulose

“…Como essas interações não são específicas, as enzimas podem ser ligar a outras superfícies, como a lignina, diminuindo a quantidade de enzimas disponíveis para ligar à celulose. Esse efeito, chamado de adsorção improdutiva, tem sido observado em diversos materiais lignocelulósicos, submetidos a diferentes pré-tratamentos, e tem sido considerado um importante efeito inibitório da lignina (BERLIN et al, 2005a;CHERNOGLAZOV;ERMOLOVA;KLYOSOVT, 1988;PALONEN et al, 2004;RAHIKAINEN et al, 2011).…”

“…Nessa temperatura, as enzimas que adsorvem na lignina podem ser recuperadas, com perdas mínimas na atividade catalítica. No entanto, na temperatura de hidrólise (acima de 45 ºC), as interações proteínas-lignina são intensificadas, e as enzimas perdem suas estruturas nativas, desnaturando e se ligando irreversivelmente à lignina (RAHIKAINEN et al, 2011).…”