Isolation and modification of nano-scale cellulose from organosolv-treated birch through the synergistic activity of LPMO and endoglucanases.

Nanocellulose isolation from lignocellulose is a tedious and expensive process with high energy and harsh chemical requirements, primarily due to the recalcitrance of the substrate, which otherwise would have been cost-effective due to its abundance. Replacing the chemical steps with biocatalytic processes offers opportunities to solve this bottleneck to a certain extent due to the enzymes substrate specificity and mild reaction chemistry. In this work, we demonstrate the isolation of sulphate-free nanocellulose from organosolv pretreated birch biomass using different glycosyl-hydrolases, along with accessory oxidative enzymes including a lytic polysaccharide monooxygenase (LPMO). The suggested process produced colloidal nanocellulose suspensions (ζ-potential -19.4 mV) with particles of 7-20 nm diameter, high carboxylate content and improved thermostability (To = 301 °C, Tmax = 337 °C). Nanocelluloses were subjected to post-modification using LPMOs of different regioselectivity. The sample from chemical route was the least favorable for LPMO to enhance the carboxylate content, while that from the C1-specific LPMO treatment showed the highest increase in carboxylate content.

Effect of lignin fractions isolated from different biomass sources on cellulose oxidation by fungal lytic polysaccharide monooxygenases.

BACKGROUND: Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that oxidatively cleave recalcitrant lignocellulose in the presence of oxygen or hydrogen peroxide as co-substrate and a reducing agent as electron donor. One of the possible systems that provide electrons to the LPMOs active site and promote the polysaccharide degradation involves the mediation of phenolic agents, such as lignin, low-molecular-weight lignin-derived compounds and other plant phenols. In the present work, the interaction of the bulk insoluble lignin fraction extracted from pretreated biomass with LPMOs and the ability to provide electrons to the active site of the enzymes is studied. RESULTS: The catalytic efficiency of three LPMOs, namely MtLPMO9 with C1/C4 regioselectivity, PcLPMO9D which is a C1 active LPMO and NcLPMO9C which is a C4 LPMO, was evaluated in the presence of different lignins. It was correlated with the physicochemical and structural properties of lignins, such as the molecular weight and the composition of aromatic and aliphatic hydroxyl groups. Moreover, the redox potential of lignins was determined with the use of large amplitude Fourier Transform alternating current cyclic voltammetry method and compared to the formal potential of the Cu (II) center in the active site of the LPMOs, providing more information about the lignin-LPMO interaction. The results demonstrated the existence of low-molecular weight lignin-derived compounds that are diffused in the reaction medium, which are able to reduce the enzyme active site and subsequently utilize additional electrons from the insoluble lignin fraction to promote the LPMO oxidative activity. Regarding the bulk lignin fractions, those isolated from the organosolv pretreated materials served as the best candidates in supplying electrons to the soluble compounds and, finally, to the enzymes. This difference, based on biomass pretreatment, was also demonstrated by the activity of LPMOs on natural substrates in the presence and absence of ascorbic acid as additional reducing agent. CONCLUSIONS: Lignins can support the action of LPMOs and serve indirectly as electron donors through low-molecular-weight soluble compounds. This ability depends on their physicochemical and structural properties and is related to the biomass source and pretreatment method.

Side-by-side biochemical comparison of two lytic polysaccharide monooxygenases from the white-rot fungus Heterobasidion irregulare on their activity against crystalline cellulose and glucomannan.

Our comparative studies reveal that the two lytic polysaccharide monooxygenases HiLPMO9B and HiLPMO9I from the white-rot conifer pathogen Heterobasidion irregulare display clear difference with respect to their activity against crystalline cellulose and glucomannan. HiLPMO9I produced very little soluble sugar on bacterial microcrystalline cellulose (BMCC). In contrast, HiLPMO9B was much more active against BMCC and even released more soluble sugar than the H. irregulare cellobiohydrolase I, HiCel7A. Furthermore, HiLPMO9B was shown to cooperate with and stimulate the activity of HiCel7A, both when the BMCC was first pretreated with HiLPMO9B, as well as when HiLPMO9B and HiCel7A were added together. No such stimulation was shown by HiLPMO9I. On the other hand, HiLPMO9I was shown to degrade glucomannan, using a C4-oxidizing mechanism, whereas no oxidative cleavage activity of glucomannan was detected for HiLPMO9B. Structural modeling and comparison with other glucomannan-active LPMOs suggest that conserved sugar-interacting residues on the L2, L3 and LC loops may be essential for glucomannan binding, where 4 out of 7 residues are shared by HiLPMO9I, but only one is found in HiLPMO9B. The difference shown between these two H. irregulare LPMOs may reflect distinct biological roles of these enzymes within deconstruction of different plant cell wall polysaccharides during fungal colonization of softwood.

Recombinant expression of thermostable processive MtEG5 endoglucanase and its synergism with MtLPMO from Myceliophthora thermophila during the hydrolysis of lignocellulosic substrates.

BACKGROUND: Filamentous fungi are among the most powerful cellulolytic organisms in terrestrial ecosystems. To perform the degradation of lignocellulosic substrates, these microorganisms employ both hydrolytic and oxidative mechanisms that involve the secretion and synergism of a wide variety of enzymes. Interactions between these enzymes occur on the level of saccharification, i.e., the release of neutral and oxidized products, but sometimes also reflected in the substrate liquefaction. Although the synergism regarding the yield of neutral sugars has been extensively studied, further studies should focus on the oxidized sugars, as well as the effect of enzyme combinations on the viscosity properties of the substrates. RESULTS: In the present study, the heterologous expression of an endoglucanase (EG) and its combined activity together with a lytic polysaccharide monooxygenase (LPMO), both from the thermophilic fungus Myceliophthora thermophila, are described. The EG gene, belonging to the glycoside hydrolase family 5, was functionally expressed in the methylotrophic yeast Pichia pastoris. The produced MtEG5A (75 kDa) featured remarkable thermal stability and showed high specific activity on microcrystalline cellulose compared to CMC, which is indicative of its processivity properties. The enzyme was capable of releasing high amounts of cellobiose from wheat straw, birch, and spruce biomass. Addition of MtLPMO9 together with MtEG5A showed enhanced enzymatic hydrolysis yields against regenerated amorphous cellulose (PASC) by improving the release not only of the neutral but also of the oxidized sugars. Assessment of activity of MtEG5A on the reduction of viscosity of PASC and pretreated wheat straw using dynamic viscosity measurements revealed that the enzyme is able to perform liquefaction of the model substrate and the natural lignocellulosic material, while when added together with MtLPMO9, no further synergistic effect was observed. CONCLUSIONS: The endoglucanase MtEG5A from the thermophilic fungus M. thermophila exhibited excellent properties that render it a suitable candidate for use in biotechnological applications. Its strong synergism with LPMO was reflected in sugars release, but not in substrate viscosity reduction. Based on the level of oxidative sugar formation, this is the first indication of synergy between LPMO and EG reported.

Surface expression of ω-transaminase in Escherichia coli.

Chiral amines are important for the chemical and pharmaceutical industries, and there is rapidly growing interest to use transaminases for their synthesis. Since the cost of the enzyme is an important factor for process economy, the use of whole-cell biocatalysts is attractive, since expensive purification and immobilization steps can be avoided. Display of the protein on the cell surface provides a possible way to reduce the mass transfer limitations of such biocatalysts. However, transaminases need to dimerize in order to become active, and furthermore, they require the cofactor pyridoxal phosphate; consequently, successful transaminase surface expression has not been reported thus far. In this work, we produced an Arthrobacter citreus ω-transaminase in Escherichia coli using a surface display vector based on the autotransporter adhesin involved in diffuse adherence (AIDA-I), which has previously been used for display of dimeric proteins. The correct localization of the transaminase in the E. coli outer membrane and its orientation toward the cell exterior were verified. Furthermore, transaminase activity was detected exclusively in the outer membrane protein fraction, showing that successful dimerization had occurred. The transaminase was found to be present in both full-length and proteolytically degraded forms. The removal of this proteolysis is considered to be the main obstacle to achieving sufficient whole-cell transaminase activity.