Gunnar Henriksson

A critical review of cellobiose dehydrogenases

Cellobiose dehydrogenase (CDH) is an extracellular enzyme produced by various wood-degrading fungi. It oxidizes soluble cellodextrins, mannodextrins and lactose efficiently to their corresponding lactones by a ping-pong mechanism using a wide spectrum of electron acceptors including quinones, phenoxyradicals, Fe(3+), Cu(2+) and triiodide ion. Monosaccharides, maltose and molecular oxygen are poor substrates. CDH that adsorbs strongly and specifically to cellulose carries two prosthetic groups; namely, an FAD and a heme in two different domains that can be separated after limited proteolysis. The FAD-containing fragment carries all known catalytic and cellulose binding properties. One-electron acceptors, like ferricyanide, cytochrome c and phenoxy radicals, are, however, reduced more slowly by the FAD-fragment than by the intact enzyme, suggesting that the function of the heme group is to facilitate one-electron transfer. Non-heme forms of CDH have been found in the culture filtrate of some fungi (probably due to the action of fungal proteases) and were for a long time believed to represent a separate enzyme (cellobiose:quinone oxidoreductase, CBQ). The amino acid sequence of CDH has been determined and no significant homology with other proteins was detected for the heme domain. The FAD-domain sequence belongs to the GMC oxidoreductase family that includes, among others, Aspergillus niger glucose oxidase. The homology is most distinct in regions that correspond to the FAD-binding domain in glucose oxidase. A cellulose-binding domain of the fungal type is present in CDH from Myceliophtore thermophila (Sporotrichum thermophile), but in others an internal sequence rich in aromatic amino acid residues has been suggested to be responsible for the cellulose binding. The biological function of CDH is not fully understood, but recent results support a hydroxyl radical-generating mechanism whereby the radical can degrade and modify cellulose, hemicellulose and lignin. CDH has found technical use in highly selective amperometric biosensors and several other applications have been suggested.

Wood-Derived Materials for Green Electronics, Biological Devices, and Energy Applications

With the arising of global climate change and resource shortage, in recent years, increased attention has been paid to environmentally friendly materials. Trees are sustainable and renewable materials, which give us shelter and oxygen and remove carbon dioxide from the atmosphere. Trees are a primary resource that human society depends upon every day, for example, homes, heating, furniture, and aircraft. Wood from trees gives us paper, cardboard, and medical supplies, thus impacting our homes, school, work, and play. All of the above-mentioned applications have been well developed over the past thousands of years. However, trees and wood have much more to offer us as advanced materials, impacting emerging high-tech fields, such as bioengineering, flexible electronics, and clean energy. Wood naturally has a hierarchical structure, composed of well-oriented microfibers and tracheids for water, ion, and oxygen transportation during metabolism. At higher magnification, the walls of fiber cells have an interesting morphology-a distinctly mesoporous structure. Moreover, the walls of fiber cells are composed of thousands of fibers (or macrofibrils) oriented in a similar angle. Nanofibrils and nanocrystals can be further liberated from macrofibrils by mechanical, chemical, and enzymatic methods. The obtained nanocellulose has unique optical, mechanical, and barrier properties and is an excellent candidate for chemical modification and reconfiguration. Wood is naturally a composite material, comprised of cellulose, hemicellulose, and lignin. Wood is sustainable, earth abundant, strong, biodegradable, biocompatible, and chemically accessible for modification; more importantly, multiscale natural fibers from wood have unique optical properties applicable to different kinds of optoelectronics and photonic devices. Today, the materials derived from wood are ready to be explored for applications in new technology areas, such as electronics, biomedical devices, and energy. The goal of this study is to review the fundamental structures and chemistries of wood and wood-derived materials, which are essential for a wide range of existing and new enabling technologies. The scope of the review covers multiscale materials and assemblies of cellulose, hemicellulose, and lignin as well as other biomaterials derived from wood, in regard to their major emerging applications. Structure-properties-application relationships will be investigated in detail. Understanding the fundamental properties of these structures is crucial for designing and manufacturing products for emerging applications. Today, a more holistic understanding of the interplay between the structure, chemistry, and performance of wood and wood-derived materials is advancing historical applications of these materials. This new level of understanding also enables a myriad of new and exciting applications, which motivate this review. There are excellent reviews already on the classical topic of woody materials, and some recent reviews also cover new understanding of these materials as well as potential applications. This review will focus on the uniqueness of woody materials for three critical applications: green electronics, biological devices, and energy storage and bioenergy.

A new scaffold for binding haem in the cytochrome domain of the extracellular flavocytochrome cellobiose dehydrogenase.

BACKGROUND The fungal oxidoreductase cellobiose dehydrogenase (CDH) degrades both lignin and cellulose, and is the only known extracellular flavocytochrome. This haemoflavoenzyme has a multidomain organisation with a b-type cytochrome domain linked to a large flavodehydrogenase domain. The two domains can be separated proteolytically to yield a functional cytochrome and a flavodehydrogenase. Here, we report the crystal structure of the cytochrome domain of CDH. RESULTS The crystal structure of the b-type cytochrome domain of CDH from the wood-degrading fungus Phanerochaete chrysosporium has been determined at 1.9 A resolution using multiple isomorphous replacement including anomalous scattering information. Three models of the cytochrome have been refined: the in vitro prepared cytochrome in its redox-inactive state (pH 7.5) and redox-active state (pH 4.6), as well as the naturally occurring cytochrome fragment. CONCLUSIONS The 190-residue long cytochrome domain of CDH folds as a beta sandwich with the topology of the antibody Fab V(H) domain. The haem iron is ligated by Met65 and His163, which confirms previous results from spectroscopic studies. This is only the second example of a b-type cytochrome with this ligation, the first being cytochrome b(562). The haem-propionate groups are surface exposed and, therefore, might play a role in the association between the cytochrome and flavoprotein domain, and in interdomain electron transfer. There are no large differences in overall structure of the cytochrome at redox-active pH as compared with the inactive form, which excludes the possibility that pH-dependent redox inactivation results from partial denaturation. From the electron-density map of the naturally occurring cytochrome, we conclude that it corresponds to the proteolytically prepared cytochrome domain.

Characterisation of lignin-carbohydrate complexes (LCCs) of spruce wood (Picea abies L.) isolated with two methods

Abstract A method for the quantitative isolation of lignin-carbohydrate complexes (LCCs) in a softwood is presented. The isolation steps involve partial enzymatic hydrolysis of cellulose, subsequent swelling in urea, and quantitative dissolution into four major fractions: (1) a galactoglucomannan LCC containing ∼8% of the wood lignin; (2) a glucane LCC containing ∼4% of the wood lignin; (3) a xylan-lignin-glucomannan network LCC (xylan>glucomannan) containing ∼40% of the wood lignin; and (4) a glucomannan-lignin-xylan network LCC (glucoman-nan>xylan) containing ∼48% of the wood lignin. Endoglucanase Novozyme 476, with only cellulase activity, and Ecopulp XM, with only xylanase and mannanase activities, were used as an enzymatic tool. From mildly ball-milled wood, all the lignin was isolated as LCCs. As a control, LCC was prepared from partially chlorite-delignified wood meal without ball milling, also in a mild procedure. The results were very similar to those obtained after ball milling. Thus, it can be safely concluded that the formation of new chemical linkages between lignin and carbohydrates during ball milling is improbable. Studies on isolated milled wood lignin (MWL) supported this conclusion and clearly showed that covalent linkages between lignin and carbohydrates are present. The study provide conclusive evidence of covalent linkages between lignin and carbohydrates in the native lignin in wood. It is concluded that carbohydrate-free lignin, i.e., lignin without covalent bonds to carbohydrates, probably cannot be present in spruce wood.

CELLOBIOSE DEHYDROGENASE (CELLOBIOSE OXIDASE) FROM PHANEROCHAETE-CHRYSOSPORIUM AS A WOOD DEGRADING ENZYME - STUDIES ON CELLULOSE, XYLAN AND SYNTHETIC LIGNIN

Degradation of carboxymethylcellulose (CMC), xylan and synthetic lignin was studied in a cellobiose dehydrogenase system, that reduced Fe(III) to Fe(II) with cellobiose as electron donor, which in the presence of hydrogen peroxide degraded all the above representatives of the main wood components, probably by forming Fentons reagent. The production of hydroxyl radicals was shown by benzoate decarboxylation. For the CMC and xylan studies viscometry was used, while lignin degradation was studied by measuring the passage of 14C-labelled synthetic lignin (DHP) through membranes of different molecular-mass cut-off. The possible participation of cellobiose dehydrogenase, Fe(III) and hydrogen peroxide in wood degradation by white-rot and brown-rot fungi is discussed.

Polymerization of Monolignols by Redox Shuttle-Mediated Enzymatic Oxidation: A New Model in Lignin Biosynthesis I

Lignin is one of the most abundant biopolymers, and it has a complex racemic structure. It may be formed by a radical polymerization initiated by redox enzymes, but much remains unknown about the process, such as how molecules as large as enzymes can generate the compact structure of the lignified plant cell wall. We have synthesized lignin oligomers according to a new concept, in which peroxidase is never in direct contact with the lignin monomers coniferaldehyde and coniferyl alcohol. Instead, manganese oxalate worked as a diffusible redox shuttle, first being oxidized from Mn(II) to Mn(III) by a peroxidase and then being reduced to Mn(II) by a simultaneous oxidation of the lignin monomers to radicals that formed covalent linkages of the lignin type. Furthermore, a high molecular mass polymer was generated by oxidation of coniferyl alcohol by Mn(III) acetate in a dioxane and water mixture. This polymer was very similar to natural spruce wood lignin, according to its NMR spectrum. The possible involvement of a redox shuttle/peroxidase system in lignin biosynthesis is discussed.

Lignins: Major Sources, Structure and Properties

Publisher Summary Lignin is one of the most predominant biopolymers present in plants. Together with cellulose and hemicelluloses, lignin builds up the cell wall in an arrangement which is regulated on the nano-scale and results in lignin–carbohydrate network structures. The molecular complexity of lignin renders all isolation and identification processes difficult and, consequently, many structural questions still remain. This chapter reviews the present knowledge about the formation of lignin in plants, its presence in different types of plants as well as several different approaches taken to reveal the chemical structure, is summarized. Furthermore, this chapter briefly discusses the chemical changes introduced in lignin as the result of different types of delignification processes, such as kraft and sulfite pulping and steam explosion, is included.

Carbohydrate reactions during high-temperature steam treatment of aspen wood.

Aspen wood was treated with steam at different time-temperature severity factors. Analysis of the amounts of acids released revealed a relationship between the acidity and the formation of furfural and hydroxymethyl furfural as degradation products from carbohydrates. It is suggested that two concurrent or consecutive mechanisms are responsible for the observed results: a homolytic cleavage and an acid hydrolysis of glucosidic linkages in the polysaccharides. By preimpregnating the wood with alkali, hydrolysis can be eliminated, resulting in a much cleaner depolymerization of the polysaccharides without any further acid-catalyzed degradation. The enzymatic digestibility of the steam-treated wood material for the formation of glucose was compared with that of steam-exploded wood. A more efficient route for glucose production from steam-exploded wood was found as long as the biomass-pretreated material was homogeneous and without shives.

The physical action of cellulases revealed by a quartz crystal microbalance study using ultrathin cellulose films and pure cellulases.

The effects of fungal cellulases on model cellulose films were studied using a high-resolution quartz crystal microbalance (QCM) sensitive to minute changes of the nanometer thick model cellulose films. It was found that endoglucanases not only produce new end groups but also cause a swelling of the cellulose film. The cellobiohydrolases degraded the films quickly, which was detected as a rapid decrease in the remaining amount of cellulose on the QCM crystal. However, changing viscoelastic properties of the films also indicated a softening of the film during the degradation. A defined mixture of selected cellulases caused a significantly higher rate of degradation than only cellobiohydrolases. Cellulase synergism is discussed with the endoglucanase swelling effects and film softening added.

Do the extracellular enzymes cellobiose dehydrogenase and manganese peroxidase form a pathway in lignin biodegradation

The extracellular enzyme manganese peroxidase is believed to degrade lignin by a hydrogen peroxide‐dependent oxidation of Mn(II) to the reactive species Mn(III) that attacks the lignin. However, Mn(III) is not able to directly oxidise the non‐phenolic lignin structures that predominate in native lignin. We show here that pretreatment of a non‐phenolic lignin model compound with another extracellular fungal enzyme, cellobiose dehydrogenase, allows the manganese peroxidase system to oxidise this molecule. The mechanism behind this effect is demethoxylation and/or hydroxylation, i.e. conversion of a non‐phenolic structure to a phenolic one, mediated by hydroxyl radicals generated by cellobiose dehydrogenase. This suggests that cellobiose dehydrogenase and manganese peroxidase may act in an extracellular pathway in fungal lignin biodegradation. Analytical techniques used in this paper are reverse‐phase high‐pressure liquid chromatography, gas chromatography connected to mass spectroscopy and UV‐visible spectroscopy.