Recombinant Trichoderma harzianum endoglucanase I (Cel7B) is a highly acidic and promiscuous carbohydrate-active enzyme

Abstract

Trichoderma filamentous fungi have been investigated due to their ability to secrete cellulases which find various biotechnological applications such as biomass hydrolysis and cellulosic ethanol production. Previous studies demonstrated that Trichoderma harzianum IOC-3844 has a high degree of cellulolytic activity and potential for biomass hydrolysis. However, enzymatic, biochemical, and structural studies of cellulases from T. harzianum are scarce. This work reports biochemical characterization of the recombinant endoglucanase I from T. harzianum, ThCel7B, and its catalytic core domain. The constructs display optimum activity at 55 °C and a surprisingly acidic pH optimum of 3.0. The full-length enzyme is able to hydrolyze a variety of substrates, with high specific activity: 75 U/mg for β-glucan, 46 U/mg toward xyloglucan, 39 U/mg for lichenan, 26 U/mg for carboxymethyl cellulose, 18 U/mg for 4-nitrophenyl β-D-cellobioside, 16 U/mg for rye arabinoxylan, and 12 U/mg toward xylan. The enzyme also hydrolyzed filter paper, phosphoric acid swollen cellulose, Sigmacell 20, Avicel PH-101, and cellulose, albeit with lower efficiency. The ThCel7B catalytic domain displays similar substrate diversity. Fluorescence-based thermal shift assays showed that thermal stability is highest at pH 5.0. We determined kinetic parameters and analyzed a pattern of oligosaccharide substrates hydrolysis, revealing cellobiose as a final product of C6 degradation. Finally, we visualized effects of ThCel7B on oat spelt using scanning electron microscopy, demonstrating the morphological changes of the substrate during the hydrolysis. The acidic behavior of ThCel7B and its considerable thermostability hold a promise of its industrial applications and other biotechnological uses under extremely acidic conditions.

A bifunctional cellulase–xylanase of a new Chryseobacterium strain isolated from the dung of a straw‐fed cattle

“…As the AA sequence of CbGH5 homologues is highly conserved in Chryseobacterium genus, they might potentially be interesting objects for mining cellulase or bifunctional cellulase–xylanase candidates. Bifunctional cellulase–xylanase activities were previously reported in GH7 (Nakazawa et al ., ; Karnaouri et al ., ; Pellegrini et al ., ), GH10 (Ding et al ., ; Hess et al ., ) and GH61 (Jagtap et al ., ) as well as in GH5_2 (Ghatge et al ., ), GH5_4 (Chang et al ., ; Hess et al ., ; Rashamuse et al ., ; Cheng et al ., ; Rattu et al ., ) and GH5_25 (Yuan et al ., ) (Table ), while CbGH5 is the first example in GH5_46. To date, CbGH5 and a metagenome‐derived protein 421339_68070/TW‐2 (GenBank accession number: ) (Hess et al ., ) are the only two glycoside hydrolases of GH5_46 that have been characterized.…”

Genome-wide characterization of cellulases from the hemi-biotrophic plant pathogen, Bipolaris sorokiniana, reveals the presence of a highly stable GH7 endoglucanase

“…Another EG from Trichoderma harzianum has also been reported to be very stable, with a little change in activity after 2 months of incubation (Additional file 1: Table S3). Here too, the enzyme has a very low turnover number at 0.45 s −1 on the substrate xyloglucan [63]. The alkaliphilic endoglucanase from the Bacillus sp.…”

Biophysical and biochemical studies of a major endoglucanase secreted by Xanthomonas campestris pv. campestris.

“…Several anaerobic cellulolytic bacteria produce multicomponent, multienzyme cellulosome complexes [16] whereas some other produce cellulases that are multi-domain enzymes comprised of endo-and exoglucanase catalytic domains linked to cellulose-binding modules, that "drill" into cellulose substrate instead of eroding it from the surface [17]. Finally, various endoglucanases are also enzymatically active against a number of hemicellulose polysaccharides, which could be related to their function in degradation of complex lignocellulosic biomass [18,19].…”