Analysis of the rate limiting steps in the enzymatic hydrolysis of lignocellulose: the effect of lignin and enzyme characteristics: Dissertation

Physicochemical lignocellulose pretreatment and subsequent enzymatic conversion of polysaccharides to platform sugars is an important technology in the valorisation of various lignocellulosic biomass streams. This technology is needed in the bio- and circular economy. Lignin, one of the main components in lignocellulose, is known to inhibit enzymatic hydrolysis by non-productively binding enzymes and sterically preventing enzymes to access cellulose. In this work, the aim was to elucidate how lignin in herbaceous plants and softwood is modified during pretreatment and what is the effect of pretreatment severity on lignin-derived inhibition in the enzymatic hydrolysis. Characteristics of cellulases and hemicellulases contributing to binding and inactivation on lignin were investigated.

Spruce and wheat straw were hydrothermally pretreated with or without an acid catalyst at increasing severities, followed by isolation of the lignin to explore the inhibitory effects. Lignin inhibition in the enzymatic hydrolysis of microcrystalline cellulose Avicel increased with increasing pretreatment severity. When spruce and wheat straw were pretreated at the same severity, as assessed by the combined severity factor, lignins from both biomasses were equally inhibitory. Furthermore, lignin from mild pretreatment severities did not have a significant effect on the hydrolysis of Avicel. This indicate that the changes in lignin structure during pretreatment are the main reasons for the inhibitory effect of lignin. A decrease in β-O-4 aryl ether bond as well as a change in molecular weight of lignin was observed after pretreatment. The molecular weight of spruce lignin decreased, whereas the molecular weight of wheat straw increased after pretreatment. Degradation and polymerisation reactions competed during pretreatment and the net effect depended on biomass type and pretreatment severity. Lignin-derived inhibition in Avicel hydrolysis strongly correlated with the binding and inactivation of the cellobiohydrolase TrCel7A to lignin. TrCel7A is the main component in the Trichoderma reesei cellulase cocktails.

The correlation between enzyme binding to lignin and inhibition in hydrolysis was studied using six purified enzymes common in cellulase cocktails, cellobiohydrolases TrCel7A, TrCel6A, endoglucanases TrCel7B and TrCel5A, a xylanase TrXyn2 from T. reesei and a β-glucosidase AnCel3A from Aspergillus niger. The cellobiohydrolases, an endoglucanase and a xylanase were all inhibited by isolated lignin. Interestingly, the most thermostable enzyme AnCel3A exhibited minor binding to lignin and was, in fact, activated by lignin. The activation of AnCel3A was the result of soluble lignin-derived compounds. The enzymes TrCel6A and TrCel7B, which exhibited strong binding to thin lignin films as analysed by quartz crystal microbalance (QCM), were also the enzymes that were most inhibited by lignin in hydrolysis assays.

The interactions contributing to enzyme binding to lignin were enzyme-specific, however, some common interactions were identified. Enzymes containing a carbohydrate binding module from family 1 (CBM1), TrCel7A, TrCel6A, TrCel7B and TrCel5A, exhibited greater adsorption to lignin than the enzymes without a CBM. Furthermore, enzymes having a net positive surface charge bound to lignin more than enzymes with a net negative surface charge in the experimental pH. Enzyme surface hydrophobicity was computationally determined. Enzymes containing large uniform hydrophobic patches on the enzyme surface had stronger binding to lignin as only a low amount of enzyme was released from the lignin surface during rinsing with buffer.

Thermal stability had a profound effect on lignin tolerance for family GH11 xylanases. Two T. reesei xylanases TrXyn1 and TrXyn2 as well as two forms of a metagenomic xylanase Xyl40 were all inhibited by lignin in hydrolysis assays and bound to lignin after incubation with enzymatically isolated lignin from steam pretreated spruce. Interestingly, the thermostable xylanases Xyl40 produced in Escherichia coli and the catalytic domain of Xyl40 produced in T. reesei remained partially active on the lignin surface, whereas the thermolabile TrXyn1 and TrXyn2 became inactive. N-glycosylation of the catalytic domain of Xyl40 did not affect the hydrolysis yield but had a significant effect on lignin tolerance. The glycosylated xylanase achieved higher hydrolysis yields in the presence of lignin than the deglycosylated xylanase. High thermal stability and structural glycans improved the lignin tolerance of the xylanases studied.