Lytic Polysaccharide Monooxygenase from Aspergillus fumigatus can Improve Enzymatic Cocktail Activity During Sugarcane Bagasse Hydrolysis

Abstract

Background: Lytic Polysaccharide Monooxygenases (LPMOs) are auxiliary accessory enzymes that act synergistically with cellulases and which are increasingly being used in secondgeneration bioethanol production from biomasses. Several LPMOs have been identified in various filamentous fungi, including Aspergillus fumigatus. However, many LPMOs have not been characterized yet. Objective: To report the role of uncharacterized A. fumigatus AfAA9_B LPMO. Methods: qRT-PCR analysis was employed to analyze the LPMO gene expression profile in different carbon sources. The gene encoding an AfAA9_B (Afu4g07850) was cloned into the vector pET- 28a(+), expressed in the E. coli strain RosettaTM (DE3) pLysS, and purified by a Ni2+-nitrilotriacetic (Ni-NTA) agarose resin. To evaluate the specific LPMO activity, the purified protein peroxidase activity was assessed. The auxiliary LPMO activity was investigated by the synergistic activity in Celluclast 1.5L enzymatic cocktail. Results: LPMO was highly induced in complex biomass like sugarcane bagasse (SEB), Avicel® PH-101, and CM-cellulose. The LPMO gene encoded a protein comprising 250 amino acids, without a CBM domain. After protein purification, the AfAA9_B molecular mass estimated by SDSPAGE was 35 kDa. The purified protein specific peroxidase activity was 8.33 ± 1.9 U g-1. Upon addition to Celluclast 1.5L, Avicel® PH-101 and SEB hydrolysis increased by 18% and 22%, respectively. Conclusion: A. fumigatus LPMO is a promising candidate to enhance the currently available enzymatic cocktail and can therefore be used in second-generation ethanol production.

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

“…To identify enzyme components that may be lacking in cellulase cocktails, commercial cellulase mixtures have also been supplemented with fungal culture broths or (semi)purified enzyme components. Celluclast has been studied extensively in spiking studies, revealing the positive impact of xylanase, mannanase and LPMO supplementation on the efficiency of cellulose saccharification [81,87,143,177,250,272,382], as also discussed above. In some cases, in-house fungal (e.g., T. reesei) culture broths have been used to showcase the positive effect of selected enzymes, such as three AA9s from Geotrichum candidum [205] or two AA14 LPMOs from Pycnoporus coccineus [68], on saccharification efficiency.…”

Current understanding of substrate specificity and regioselectivity of LPMOs

“…Studies have shown that adding LPMOs to cellulase cocktails can improve the degradation efficiency of cellulose biomass and reduce the required enzyme amount (de Gouvea et al 2019;Dimarogona et al 2013;Harris et al 2010;Hemsworth et al 2015;Zhang et al 2019). It is speculated that this synergy is due to the oxidative cleavage of polysaccharide crystalline regions by LPMOs, which provides more accessible sites for glycoside hydrolases ( Fig.…”

Characterization of two family AA9 LPMOs from Aspergillus tamarii with distinct activities on xyloglucan reveals structural differences linked to cleavage specificity

“…So far, the (putative) LPMOs of A. tamarii have not gained much attention. With regard to putative LPMOs from aspergilli, LPMO activity has been shown, to date, for five AA9s from A. fumigatus [27,28], seven AA9s from A. terreus [27], two AA9s [29,30] and one AA13 [31] from A. nidulans, an AA9 from A. niger [32], an AA11 [33] and AA13 [31] from A. oryzae, and an AA16 from A. aculeatus [34], whereas structural data are available for an AA9 (AfAA9B) from A. fumigatus [35] and an AA11 [33] and AA13 [31] from A. oryzae. So far, xyloglucan activity by these LPMOs has remained unexplored, except for one LPMO from A. nidulans (AN3046) for which xyloglucan activity has been proposed [29].…”

“…With regard to putative LPMOs from aspergilli, LPMO activity has been shown, to date, for five AA9s from A . fumigatus [ 27 , 28 ], seven AA9s from A . terreus [ 27 ], two AA9s [ 29 , 30 ] and one AA13 [ 31 ] from A .…”