Functional Characterization and Low-Resolution Structure of an Endoglucanase Cel45A from the Filamentous Fungus Neurospora crassa OR74A: Thermostable Enzyme with High Activity Toward Lichenan and β-Glucan

Abstract

Biomass is the most abundant and short-term renewable natural resource on Earth whose recalcitrance toward enzymatic degradation represents significant challenge for a number of biotechnological applications. The not so abundant but critically necessary class of GH45 endoglucanases constitutes an essential component of tailored industrial enzyme cocktails because they randomly and internally cleave cellulose molecules. Moreover, GH45 glucanases are core constituents of major-brand detergent formulations as well as enzymatic aid components in the cotton processing industry, clipping unwanted cellulosic fibers from cotton (cellulosic)-based tissues. Here we report on a recombinant high-yield Neurospora crassa OR74A NcCel45A production system, a single-band GH45 endoglucanase purification, and a complete enzyme functional characterization. NcCel45A is a bimodular endoglucanase showing maximum activity at pH 6.0 and 60 °C, while most active against lichenan and β-glucans and lesser active toward filter paper, carboxymethylcellulose, and phosphoric acid-swollen cellulose. Gluco-oligosaccharide degradation fingerprinting experiments suggest cellopentaose as the minimal length substrate and ThermalFluor studies indicate that NcCel45A displays excellent stability at elevated temperatures up to 70 °C and pHs ranging from 5 to 9. Remarkably, we show that NcCel45A is uniquely resistant to a wide-range of organic solvents and small-angle X-ray scattering show a monkey-wrench molecular shape structure in solution, which indicates, unlike to other known cellulases, a non-fully extended conformation, thus conferring solvent protection. These NcCel45A unique enzymatic properties maybe key for specific industrial applications such as cotton fiber processing and detergent formulations.

Engineering the conserved and noncatalytic residues of a thermostable β-1,4-endoglucanase to improve specific activity and thermostability

“…Enzymatic hydrolysis of cellulose into fermentable sugars for subsequent processes is of considerable practical interest because of the tremendous application potential in the bioconversion of lignocellulosic biomass 8 , 29 . Although many endoglucanases of different sources have been isolated and commercialized 30 , 31 , more effective enzymatic properties were optimized to satisfy the industrial applications 32 , 33 . To obtain enzymes with higher thermostability and specific activity, rationally engineering based on the homologously modelled structure provides an effective strategy in the improvement of enzyme performance 34 – 37 .…”

Production of cello-oligosaccharides from corncob residue by degradation-synthesis reactions

“…Six microbial cellulases classified into GH9, GH12, GH45, and GH131 families were selected for recombinant expression and tested for their ability to hydrolyze CCR. These include CpCel9 (Cphy_3367, GH9, GenBank accession number: NC_010001.1) from Clostridium phytofermentans (Warner et al 2011), CcCel9M (GH9, GenBank accession number: AF316823) from Clostridium cellulolyticum (Belaich et al 2002), NcCel45A (GH45, GenBank accession number: XM_952014.1) from Neurospora crassa OR74A (Kadowaki et al 2015), NfCel12A (GH12, GenBank accession number: KX507179) from Neosarorya fischeri P1 (Yang et al 2017), MtCel45 (GH45, GenBank accession number: XP_003659323) from Myceliophera thermophila (Berto et al 2019), and ChCel131A (GH131, GenBank accession number: CCF46974.1) from Colletotrichum higginsianum (Anasontzis et al 2019). CpCel9 and CcCel9M were synthesized by Genewiz (Suzhou, Jiangsu Province, China) and then cloned into the plasmid pET-28a( +) (Invitrogen, Carlsbad, CA, USA) between EcoRI and NotI.…”

“…Six endo-glucanases from the GH families of 5, 9, 12, and 45 were reported to generate COS with higher degrees of polymerization and selected as candidate enzymes in this study. Thus, these enzymes included CpCel9 from C. phytoplasmans (Warner et al 2011), CcCel9M from C. cellulolyticum (Belaich et al 2002), NfCel12A from N. fischeri P1 (Yang et al 2017), NcCel45A from N. crassa OR74A (Kadowaki et al 2015), MtCel45 from M. thermophila (Berto et al 2019), and ChCel131A from C. higginsianum (Anasontzis et al 2019). The endo-glucanases were recombinantly produced either in E. coli (CpCel9 and CcCel9M) or in P. pastoris (NfCel12A, NcCel45A, MtCel45, and ChCel131A) and purified to near homogeneity (Fig.…”

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

“…Finally, not all endoglucanases have extended molecular conformation in solution. Recent report on Neurospora crassa Cel45A endoglucanase revealed a monkeywrench molecular shape enzyme with the maximum dimension of 75 Å [50]. Clearly, more expensive low-resolution studies of the cellulases in solution are needed in order to understand in more detail their full-length molecular shape, conformation and flexibility.…”