Characterization of a Dual Cellulolytic/Xylanolytic AA9 Lytic Polysaccharide Monooxygenase from Thermothelomyces thermophilus and its Utilization Toward Nanocellulose Production in a Multi-Step Bioprocess

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are enzymes able to act on a great variety of polysaccharides by an oxidative cleavage mechanism, targeting not only crystalline substrates, such as cellulose and chitin, but also other amorphous structures including xylan, mannan, and pectin. The key role of LPMOs toward the isolation of nanocellulose from natural substrates has been demonstrated since these enzymes promote amorphogenesis of the substrate and facilitate the defibrillation process. In the present study, an AA9 LPMO from the thermophilic fungus Thermothelomyces thermophilus (TtLPMO9G) with C1-regioselectivity and a dual cellulolytic/xylanolytic activity was heterologously produced in Pichia pastoris and biochemically characterized. The enzyme was employed both as a pre- and a post-treatment step alongside with commercially available and in-house produced tailored cocktails of hemicellulases and cellulases in four-step multi-enzymatic processes for the isolation of nanoscale cellulose from OxiOrganosolv pretreated beechwood. Nanostructures obtained from each of these green bio-processes were examined for their morphological features and dimensions, crystallinity, colloidal stability, and the presence of carboxylate groups. The results demonstrate the formation of well-dispersed nanoscale cellulose in the complete absence of any chemical or mechanical treatment step and verify the importance of efficient hemicellulose removal for the isolation of nanocellulose.

Secretome Analysis of Thermothelomyces thermophilus LMBC 162 Cultivated with Tamarindus indica Seeds Reveals CAZymes for Degradation of Lignocellulosic Biomass

“…The fungus was grown in test tubes and suspended in sterile distilled water, and its spores were counted in a microscope through a Neubauer chamber. The suspension was inoculated into 125 mL Erlenmeyer flasks with 25 O (0.16%), distilled water q.s. (100 mL) (5.0 mL); yeast extract (0.1 g); carbon source (1.0 g); distilled water q.s.…”

“…Thermophilic fungi are a promising source of new enzymes for cost-effective industrial applications, including abundant thermostable enzymes for biomass degradation and generation of chemicals and biofuels [19][20][21]. Among them, a fungus that is described in the literature as a source of many CAZymes, especially glycoside hydrolases (GHs) and oxidative enzymes that aid in the breakdown of lignocellulosic materials, is the thermophilic ascomycete Thermothelomyces thermophilus (formerly Myceliophthora thermophila) [22][23][24][25]. This filamentous fungus has been shown to be safe for large-scale production processes and can utilize cost-effective sources of plant biomass [26], as waste from the fruit pulp industry, especially tamarind seeds [27].…”

Selective enhanced cytotoxicity of amino acid deprivation for cancer therapy using thermozyme functionalized nanocatalyst

“…Notably, thermozymes, which have research significance in bioengineering, offer a promising solution. These enzymes have good thermal stability and long half-life, overcoming the biological instability that often occurs in biomedical applications of mesophilic and cryophilic enzymes [ 13 – 15 ], while the low catalytic activity of thermozymes in vivo facilitates avoidance of biological damage to nontarget tissues. These advantages together point to thermozymes as important candidates for valuable drugs [ 16 , 17 ].…”

Evaluation of OxiOrganosolv pretreated hardwood and softwood lignocelluloses as substrates for the chemoenzymatic production of 5-hydroxymethylfurfural (HMF)

“…Among popular fields are the synthesis of chemicals, food additives, pharmaceuticals, and polymeric materials [2,3]. In particular, many biobased-polymers from lignocellulose have been developed, including nanocellulose, polylactic acid (PLA), and polyhydroxyalkanoates (PHAs), such as poly(3-hydroxypropionic acid) and polyhydroxybutyrate, by combining microbial fermentation and enzyme-mediated processes with chemical catalysis [4][5][6][7][8]. In this way, lignocellulosic biomass can serve as a substrate for the synthesis of green plastics, thus lowering the dependence of using petrochemicals and fossil fuels.…”