Boosting of enzymatic softwood saccharification by fungal GH5 and GH26 endomannanases

Abstract

BackgroundSoftwood is a promising feedstock for lignocellulosic biorefineries, but as it contains galactoglucomannan efficient mannan-degrading enzymes are required to unlock its full potential.ResultsBoosting of the saccharification of pretreated softwood (Canadian lodgepole pine) was investigated for 10 fungal endo-β(1→4)-mannanases (endomannanases) from GH5 and GH26, including 6 novel GH26 enzymes. The endomannanases from Trichoderma reesei (TresMan5A) and Podospora anserina (PansMan26) were investigated with and without their carbohydrate-binding module (CBM). The pH optimum and initial rates of enzyme catalysed hydrolysis were determined on pure β-mannans, including acetylated and deacetylated spruce galactoglucomannan. Melting temperature (Tm) and stability of the endomannanases during prolonged incubations were also assessed. The highest initial rates on the pure mannans were attained by GH26 endomannanases. Acetylation tended to decrease the enzymatic rates to different extents depending on the enzyme. Despite exhibiting low rates on the pure mannan substrates, TresMan5A with CBM1 catalysed highest release among the endomannanases of both mannose and glucose during softwood saccharification. The presence of the CBM1 as well as the catalytic capability of the TresMan5A core module itself seemed to allow fast and more profound degradation of portions of the mannan that led to better cellulose degradation. In contrast, the presence of the CBM35 did not change the performance of PansMan26 in softwood saccharification.ConclusionsThis study identified TresMan5A as the best endomannanase for increasing cellulase catalysed glucose release from softwood. Except for the superior performance of TresMan5A, the fungal GH5 and GH26 endomannanases generally performed on par on the lignocellulosic matrix. The work also illustrated the importance of using genuine lignocellulosic substrates rather than simple model substrates when selecting enzymes for industrial biomass applications.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1184-y) contains supplementary material, which is available to authorized users.

Crystal structure and substrate interactions of an unusual fungal non-CBM carrying GH26 endo-β-mannanase from Yunnania penicillata

“…Pans Man26A and the GH26 endomannanase from Aspergillus nidulans , Anid Man26A, were shown to have a significant −4 subsite, and to accommodate galactopyranosyl units not only in the −1 subsite, but also in −2 and +1, in contrast to the GH5 counterparts from A. nidulans Anid Man5A and Anid Man5C 24–26 . Several fungal GH26 endomannanases were found to have higher initial rates on soluble galactomannans than the tested GH5 endomannanases, with the GH26 endomannanase from Yunnania penicillata, Ypen Man26A, having the highest initial hydrolysis rate, closely followed by Anid Man26A and the GH26 endomannanase from Westerdykella sp , Wsp .Man26A 1 . However, the tested fungal GH26 endomannanases discriminated differently between the soluble mannans 1 , exemplified by the Ypen Man26A and the Wsp .Man26A which both had high initial hydrolysis rates on locust bean gum, but different rates on more heavily substituted galactomannan.…”

“…Several fungal GH26 endomannanases were found to have higher initial rates on soluble galactomannans than the tested GH5 endomannanases, with the GH26 endomannanase from Yunnania penicillata, Ypen Man26A, having the highest initial hydrolysis rate, closely followed by Anid Man26A and the GH26 endomannanase from Westerdykella sp , Wsp .Man26A 1 . However, the tested fungal GH26 endomannanases discriminated differently between the soluble mannans 1 , exemplified by the Ypen Man26A and the Wsp .Man26A which both had high initial hydrolysis rates on locust bean gum, but different rates on more heavily substituted galactomannan. While Ypen Man26A also showed high hydrolysis rate on guar gum, Wsp .Man26A appeared more restricted by the extra galactose substitutions.…”

“…Endo- β (1 → 4)-mannanases (endomannanases, EC 3.2.1.78) are important enzymes, catalysing the degradation of abundant plant β -mannans (hereafter mannan) in nature. Endomannanases are currently used in various applications including plant biomass conversion 1 , food and feed 2,3 , detergent formulations 4 and oil drilling 5 . An understanding of the intimate interactions between endomannanases and their substrates is key to optimising their utilisation and industrial performance.…”

“…the exo-acting β -mannosidases and α -galactosidases. Soluble substrates are often accessible to all these enzymes, but attack on mannan by endomannanases may also occur on water-insoluble substrate matrices 1,2,13 . Endomannanases are classified into four glycoside hydrolase (GH) families: 5, 26, 113 and 134 based on sequence similarity 14 .…”

Enzymatic processing of lignocellulosic biomass: principles, recent advances and perspectives

“…As an example, Agrawal et al have shown that the performance of Cellic CTec2 on acid or alkali pretreated bagasse and rice straw can be boosted by addition of two AA9 LPMOs from the thermophilic fungi Scytalidium thermophilum and Malbranchea cinnamomea [8]. Very recently, von Freiesleben et al have reported that supplementation with GH5 and GH26 mannanases leads to improved saccharification of pretreated lodgepole pine by Cellic CTec3 [367], confirming previous indications concerning suboptimal levels of mannanase activities in Cellic CTec3 for softwood saccharification [61]. As another example, d'Errico et al showed that a Cellic CTec preparation and the β-glucanase preparation UltraFlo possess only low amounts of glucuronoyl esterase activity and that supplementing these products with CE15 glucuronoyl esterases boosts their saccharification efficiency on pretreated corn fiber [77].…”