Maria Dimarogona

Lignin boosts the cellulase performance of a GH-61 enzyme from Sporotrichum thermophile

An enzyme belonging to the glycoside hydrolase family 61 from the thermophilic fungus Sporotrichum thermophile, was functionally expressed in the methylotrophic yeast Pichia pastoris under the transcriptional control of the alcohol oxidase (AOX1) promoter. The enzyme hydrolyzed barley β-glucan, carboxymethyl cellulose, lichenan, wheat arabinoxylan and birchwood xylan showing optimal activity at pH 8 and 65°C. A 2:1 mixture of Celluclast 1.5L and StCel61a was capable of increasing the degree of spruce conversion by 42%. The use of substrates with varying lignin content permitted the detection of a dependence of the enhancing capacity of StCel61a on the radical scavenging capacity of the different lignocellulosics. In the presence of a reductant, StCel61a boosted the efficiency of a mixture of purified cellulases (EGII, CBHI, β-GLUC) by 20%. The synergistic activity exhibited by StCel61a and its dependence on reducing substances provide guidelines for process design towards the production of economically viable bioethanol.

Structural and Functional Characterization of a Lytic Polysaccharide Monooxygenase with Broad Substrate Specificity

Background: The recently discovered lytic polysaccharide monooxygenases (LPMOs) are important in enzymatic conversion of lignocellulosic biomass. Results: We describe structural and functional studies of NcLPMO9C, which cleaves both cellulose and certain hemicelluloses. Conclusion: NcLPMO9C has structural and functional features that correlate with the enzymes catalytic capabilities. Significance: This study shows how LPMO active sites are tailored to varying functionalities and adds to a growing LPMO knowledge base. The recently discovered lytic polysaccharide monooxygenases (LPMOs) carry out oxidative cleavage of polysaccharides and are of major importance for efficient processing of biomass. NcLPMO9C from Neurospora crassa acts both on cellulose and on non-cellulose β-glucans, including cellodextrins and xyloglucan. The crystal structure of the catalytic domain of NcLPMO9C revealed an extended, highly polar substrate-binding surface well suited to interact with a variety of sugar substrates. The ability of NcLPMO9C to act on soluble substrates was exploited to study enzyme-substrate interactions. EPR studies demonstrated that the Cu2+ center environment is altered upon substrate binding, whereas isothermal titration calorimetry studies revealed binding affinities in the low micromolar range for polymeric substrates that are due in part to the presence of a carbohydrate-binding module (CBM1). Importantly, the novel structure of NcLPMO9C enabled a comparative study, revealing that the oxidative regioselectivity of LPMO9s (C1, C4, or both) correlates with distinct structural features of the copper coordination sphere. In strictly C1-oxidizing LPMO9s, access to the solvent-facing axial coordination position is restricted by a conserved tyrosine residue, whereas access to this same position seems unrestricted in C4-oxidizing LPMO9s. LPMO9s known to produce a mixture of C1- and C4-oxidized products show an intermediate situation.

Recalcitrant polysaccharide degradation by novel oxidative biocatalysts

The classical hydrolytic mechanism for the degradation of plant polysaccharides by saprophytic microorganisms has been reconsidered after the recent landmark discovery of a new class of oxidases termed lytic polysaccharide monooxygenases (LPMOs). LPMOs are of increased biotechnological interest due to their implication in lignocellulosic biomass decomposition for the production of biofuels and high-value chemicals. They act on recalcitrant polysaccharides by a combination of hydrolytic and oxidative function, generating oxidized and non-oxidized chain ends. They are copper-dependent and require molecular oxygen and an external electron donor for their proper function. In this review, we present the recent findings concerning the mechanism of action of these oxidative enzymes and identify issues and questions to be addressed in the future.

Cellulose degradation by oxidative enzymes

Enzymatic degradation of plant biomass has attracted intensive research interest for the production of economically viable biofuels. Here we present an overview of the recent findings on biocatalysts implicated in the oxidative cleavage of cellulose, including polysaccharide monooxygenases (PMOs or LPMOs which stands for lytic PMOs), cellobiose dehydrogenases (CDHs) and members of carbohydrate-binding module family 33 (CBM33). PMOs, a novel class of enzymes previously termed GH61s, boost the efficiency of common cellulases resulting in increased hydrolysis yields while lowering the protein loading needed. They act on the crystalline part of cellulose by generating oxidized and non-oxidized chain ends. An external electron donor is required for boosting the activity of PMOs. We discuss recent findings concerning their mechanism of action and identify issues and questions to be addressed in the future.

Interactions of a fungal lytic polysaccharide monooxygenase with β-glucan substrates and cellobiose dehydrogenase

Significance Copper-dependent lytic polysaccharide monooxygenases (LPMOs) are key players in the enzymatic conversion of biomass. LPMOs catalyze oxidative cleavage of glycosidic bonds in a process involving molecular oxygen and an electron donor, such as cellobiose dehydrogenase (CDH). Using protein NMR and isothermal titration calorimetry we have studied the interactions between a fungal LPMO and three soluble substrates and CDH. The results reveal which areas on the LPMO surface interact with the varying substrates and unambiguously show that both the substrate and CDH bind to a region that is centered around the copper site. The data presented here suggest that electron transfer occurs before substrate binding, providing important new leads for understanding the reaction mechanism of LPMOs. Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze oxidative cleavage of glycosidic bonds using molecular oxygen and an external electron donor. We have used NMR and isothermal titration calorimetry (ITC) to study the interactions of a broad-specificity fungal LPMO, NcLPMO9C, with various substrates and with cellobiose dehydrogenase (CDH), a known natural supplier of electrons. The NMR studies revealed interactions with cellohexaose that center around the copper site. NMR studies with xyloglucans, i.e., branched β-glucans, showed an extended binding surface compared with cellohexaose, whereas ITC experiments showed slightly higher affinity and a different thermodynamic signature of binding. The ITC data also showed that although the copper ion alone hardly contributes to affinity, substrate binding is enhanced for metal-loaded enzymes that are supplied with cyanide, a mimic of O2−. Studies with CDH and its isolated heme b cytochrome domain unambiguously showed that the cytochrome domain of CDH interacts with the copper site of the LPMO and that substrate binding precludes interaction with CDH. Apart from providing insights into enzyme–substrate interactions in LPMOs, the present observations shed new light on possible mechanisms for electron supply during LPMO action.

The structure of a novel glucuronoyl esterase from Myceliophthora thermophila gives new insights into its role as a potential biocatalyst

The increasing demand for the development of efficient biocatalysts is a consequence of their broad industrial applications. Typical difficulties that are encountered during their exploitation in a variety of processes are interconnected with factors such as temperature, pH, product inhibitors etc. To eliminate these, research has been directed towards the identification of new enzymes that would comply with the required standards. To this end, the recently discovered glucuronoyl esterases (GEs) are an enigmatic family within the carbohydrate esterase (CE) family. Structures of the thermophilic StGE2 esterase from Myceliophthora thermophila (synonym Sporotrichum thermophile), a member of the CE15 family, and its S213A mutant were determined at 1.55 and 1.9 Å resolution, respectively. The first crystal structure of the S213A mutant in complex with a substrate analogue, methyl 4-O-methyl-β-D-glucopyranuronate, was determined at 2.35 Å resolution. All of the three-dimensional protein structures have an α/β-hydrolase fold with a three-layer αβα-sandwich architecture and a Rossmann topology and comprise one molecule per asymmetric unit. These are the first crystal structures of a thermophilic GE both in an unliganded form and bound to a substrate analogue, thus unravelling the organization of the catalytic triad residues and their neighbours lining the active site. The knowledge derived offers novel insights into the key structural elements that drive the hydrolysis of glucuronic acid esters.

Structural and functional studies of a Fusarium oxysporum cutinase with polyethylene terephthalate modification potential.

BACKGROUND Cutinases are serine hydrolases that degrade cutin, a polyester of fatty acids that is the main component of plant cuticle. These biocatalysts have recently attracted increased biotechnological interest due to their potential to modify and degrade polyethylene terephthalate (PET), as well as other synthetic polymers. METHODS A cutinase from the mesophilic fungus Fusarium oxysporum, named FoCut5a, was expressed either in the cytoplasm or periplasm of Escherichia coli BL21. Its X-ray structure was determined to 1.9Å resolution using molecular replacement. The activity of the recombinant enzyme was tested on a variety of synthetic esters and polyester analogues. RESULTS The highest production of recombinant FoCut5a was achieved using periplasmic expression at 16°C. Its crystal structure is highly similar to previously determined Fusarium solani cutinase structure. However, a more detailed comparison of the surface properties and amino acid interactions revealed differences with potential impact on the biochemical properties of the two enzymes. FoCut5a showed maximum activity at 40°C and pH 8.0, while it was active on three p-nitrophenyl synthetic esters of aliphatic acids (C(2), C(4), C(12)), with the highest catalytic efficiency for the hydrolysis of the butyl ester. The recombinant cutinase was also found capable of hydrolyzing PET model substrates and synthetic polymers. CONCLUSIONS The present work is the first reported expression and crystal structure determination of a functional cutinase from the mesophilic fungus F. oxysporum with potential application in surface modification of PET synthetic polymers. GENERAL SIGNIFICANCE FoCut5a could be used as a biocatalyst in industrial applications for the environmentally-friendly treatment of synthetic polymers.

Marine-Derived Biocatalysts: Importance, Accessing, and Application in Aromatic Pollutant Bioremediation

The aim of the present review is to highlight the potential use of marine biocatalysts (whole cells or enzymes) as an alternative bioprocess for the degradation of aromatic pollutants. Firstly, information about the characteristics of the still underexplored marine environment and the available scientific tools used to access novel marine-derived biocatalysts is provided. Marine-derived enzymes, such as dioxygenases and dehalogenases, and the involved catalytic mechanisms for the degradation of aromatic and halogenated compounds, are presented, with the purpose of underpinning their potential use in bioremediation. Emphasis is given on persistent organic pollutants (POPs) that are organic compounds with significant impact on health and environment due to their resistance in degradation. POPs bioaccumulate mainly in the fatty tissue of living organisms, therefore current efforts are mostly focused on the restriction of their use and production, since their removal is still unclear. A brief description of the guidelines and criteria that render a pollutant POP is given, as well as their potential biodegradation by marine microorganisms by surveying recent developments in this rather unexplored field.