Binding modules alter the activity of chimeric cellulases: Effects of biomass pretreatment and enzyme source

Abstract

Improving the catalytic activity of cellulases requires screening variants against solid substrates. Expressing cellulases in microbial hosts is time-consuming, can be cellulase specific, and often leads to inactive forms and/or low yields. These limitations have been obstacles for improving cellulases in a high-throughput manner. We have developed a cell-free expression system and used it to express 54 chimeric bacterial and archaeal endoglucanases (EGs), with and without cellulose binding modules (CBMs) at either the N- or C-terminus, in active enzyme yields of 100-350 µg/mL. The platform was employed to systematically study the role of CBMs in cellulose hydrolysis toward a variety of natural and pretreated solid substrates, including ionic-liquid pretreated Miscanthus and AFEX-pretreated corn stover. Adding a CBM generally increased activity against crystalline Avicel, whereas for pretreated substrates the effect of CBM addition depended on the source of cellulase. The cell-free expression platform can thus provide insights into cellulase structure-function relationships for any substrate, and constitutes a powerful discovery tool for evaluating or engineering cellulolytic enzymes for biofuels production.

Identification and characterization of a multidomain hyperthermophilic cellulase from an archaeal enrichment

“…Assays on alternative substrates described in Table 1 were done as follows Pretreated substrates were treated as previously described 9 . All cellulolytic assays for insoluble substrates were carried out in quadruplicate in a final volume of 70 µl containing 1% (w/v) substrate (glucan loading), 0.2 µM of the EBI-244 and 100 mM sodium acetate buffer, pH 5.5 at 90 °C in a thermal cycler (Applied Biosystems).…”

“…Cellulase activities were measured for Avicel, Lichenan, AFEX pretreated corn stover, ionic-liquid pretreated avicel (IL-Avicel), Miscanthus (IL-Miscanthus), and corn stover (IL-corn stover). The mixtures were incubated at 90 °C for 15 h after which they were cooled to 4 °C before measuring the amount of soluble reducing sugar released using the glucose oxidase-peroxidase assay as previously described 9 .…”

“…These species have large and diverse inventories of glycosyl hydrolases (GHs), including multicopy hemicellulose and cellulose degrading-enzymes with various carbohydrate-binding domains. These cellulose-binding domains can be added to hyperthermophilic cellulases to increase degradation by domain addition, gene shuffling and other strategies 1,9 . For example, by adding a thermostable chitin-binding domain to the Pyrococcus horikoshii endoglucanase (EGph), higher activities on crystalline substrates were achieved 10 .…”

Computational Investigation of Glycosylation Effects on a Family 1 Carbohydrate-binding Module

“…Several routes exist for increasing cellulase performance, including rational and evolutionary methods to improve specific activity (8), screening for improved thermal tolerance (11,12), and the often overlooked strategy of increasing binding affinity to carbohydrates via CBM engineering (13)(14)(15)(16). In the lattermost case, several groups have shown that by increasing CBM binding affinity to cellulose via single point mutations or by swapping complete CBMs from other enzymes with higher binding affinity, cellulase enzyme activity can be improved (15,16). This effect is likely due to higher enzyme concentrations on the cellulose surface, which potentially leads to a higher fraction of catalytically engaged enzymes.…”

Lignocellulases: a review of emerging and developing enzymes, systems, and practices

“…Typically, cell-free systems are used for cofactor-independent reactions, and often exhibit reactionrate-limited kinetics, resulting from the direct access to substrate in solution (Smith et al 2015). Cell-free enzymes have been exploited both in single (Kengen et al 1993;Kim et al 2010;Böhmer et al 2012) and multiple domain systems (Kanafusa-Shinkai et al 2013). Several immobilization practices have been reported (Kazenwadel et al 2015).…”