Ewald Srebotnik

Degradation of nonphenolic lignin by the laccase:1-hydroxybenzotriazole system

Phenolic and nonphenolic (permethylated) synthetic [14C]lignins were depolymerized by Trametes villosa laccase in the presence of a radical mediator, 1-hydroxybenzotriazole (HOBT). Gel permeation chromatography of the treated lignins showed that approximately 10% of their substructures were cleaved. The system also cleaved a beta-O-4-linked model compound, 1-(4-ethoxy-3-methoxy-ring-[14C]phenyl)-2-(2-methoxyphenoxy)-propane- 1,3-diol, and a beta-1-linked model, 1, 2-bis-(3-methoxy-4-[14C]methoxyphenyl)-propane-1,3-diol, that represent nonphenolic substructures in lignin. High performance liquid chromatography of products from the oxidized models showed that they were produced in sufficient yields to account for the ability of laccase/HOBT to depolymerize nonphenolic lignin.

Oxidative degradation of high molecular weight chlorolignin by manganese peroxidase of Phanerochaete chrysosporium

Phanerochaete chrysosporium was able to degrade high molecular weight chlorolignins (Mr greater than 30,000) from bleach plant effluents, although a direct contact between ligninolytic enzymes and chlorolignin was prevented by a dialysis tubing. In the absence of the enzymes, Mn3+ depolymerized chlorolignin when complexed with lactate causing the color, chemical oxygen demand (COD) and dry weight to decrease by 80%, 60% and 40%, respectively. Manganese peroxidase effectively catalyzed the depolymerization of chlorolignin in the presence of Mn2+ and H2O2. It can be concluded from these results that manganese peroxidase plays the major role in the initial breakdown and decolorization of high molecular weight chlorolignin in bleach plant effluents by P. chrysosporium in vivo.

Chlorination of lignin by ubiquitous fungi has a likely role in global organochlorine production

Soils and decayed plant litter contain significant quantities of chlorinated aromatic polymers that have a natural but largely unknown origin. We used cupric oxide ligninolysis coupled with gas chromatography/mass spectrometry to show that Curvularia inaequalis, a widely distributed litter ascomycete, chlorinated the aromatic rings of lignin in wood that it was degrading. In aspen wood decayed for 24 weeks, two chlorolignin fragments, 5-chlorovanillin and 2-chlorosyringaldehyde, were each found at ≈10 μg/g of wood (dry weight). These levels resemble those of similar structures generally found in unpolluted environmental samples. Fractionation of the extractable proteins followed by tandem mass spectrometric analysis showed that the colonized wood contained a previously described C. inaequalis chloroperoxidase that very likely catalyzed lignin chlorination. Chlorolignin produced by this route and humus derived from it are probably significant components of the global chlorine cycle because chloroperoxidase-producing fungi are ubiquitous in decaying lignocellulose and lignin is the earths most abundant aromatic substance.

Chlorination and Cleavage of Lignin Structures by Fungal Chloroperoxidases

ABSTRACT Two fungal chloroperoxidases (CPOs), the heme enzyme from Caldariomyces fumago and the vanadium enzyme from Curvularia inaequalis, chlorinated 1-(4-ethoxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-1,3-dihydroxypropane, a dimeric model compound that represents the major nonphenolic structure in lignin. Both enzymes also cleaved this dimer to give 1-chloro-4-ethoxy-3-methoxybenzene and 1,2-dichloro-4-ethoxy-5-methoxybenzene, and they depolymerized a synthetic guaiacyl lignin. Since fungal CPOs occur in soils and the fungi that produce them are common inhabitants of plant debris, CPOs may have roles in the natural production of high-molecular-weight chloroaromatics and in lignin breakdown.

Ultrastructural localization of ligninase ofPhanerochaete chrysosporium by immunogold labeling

Ligninase was localized in the ultrastructure of hyphae ofPhanerochaete chrysosporium cultivated in liquid culture and wood, respectively, after immunolabeling. The application of different techniques of fixation is discussed. Ligninase was localized mainly in the hyphal cytoplasm close to the plasmalemma and was bound to the latter. No immunolabeling was observed in or on the hyphal wall or extracellular structures. Ligninase was detectable extracellularly only after fixation with picric acid. As ligninase was localized in the wood cell wall only in isolated cases, the question of penetration into the wood cell wall still remains unclear.

Fungal decay of spruce and beech wood assessed by near-infrared spectroscopy in combination with uni- and multivariate data analysis

Abstract Wood is colonised and degraded by a variety of micro-organisms, the most efficient ones are wood-rotting basidiomycetes. Microbial decay processes cause damage to wooden constructions, but also have great potential as biotechnological tools to change the properties of wood surfaces and of sound wood. Standard methods to evaluate changes in infected wood, e.g., EN350-1 1994, are time-consuming. Rapid FT-NIR spectroscopic methods are also suitable for this purpose. In this paper, degradation experiments on surfaces of spruce (Picea abies L. Karst) and beech (Fagus silvatica L.) were carried out with white rot basidiomycetes or the ascomycete Hypoxylon fragiforme. Experiments with brown rot or soft rot caused by Chaetomium globosum were also performed. FT-NIR spectra collected from the degraded wood were subjected to principal component analysis. The lignin content and mass loss of the specimens were estimated based on univariate or multivariate data analysis (partial least squares regression).

Laccase-catalyzed functionalization with 4-hydroxy-3-methoxybenzylurea significantly improves internal bond of particle boards

Abstract Enzymatic functionalization is an attractive tool to provide a reactive interface for further processing of lignocellulosic materials, such as wood particles and fibers. Here, spruce wood particles have been functionalized by fungal laccase combined with 4-hydroxy-3-methoxy-benzylamine (HMBA) or 4-hydroxy-3-methoxybenzylurea (HMBU). The expectation was crosslinking with resins in subsequent glueing processes, which should improve strength properties of particle boards. Essential process parameters, such as liquid to solid mass ratio and treatment time, were optimized on a laboratory scale resulting in HMBA and HMBU binding yields of 90% and above as determined by radiochemical mass balance analysis. We employed a multifactorial experimental design for board production from treated wood particles and urea/formaldehyde resin. Mechanical testing and multivariate data analysis revealed, for the first time, an increase of internal bond (IB) as a result of functionalization with HMBU. HMBA was not successful. Variance analysis of relevant parameters and their interactions demonstrated a highly significant difference (P>99.99%) between boards treated with laccase/HMBU versus untreated wood particles. Due to positive interactions, functionalization was most effective at high bulk density (750 kg m-3) and high resin content (10%) resulting in a calculated IB improvement of 0.12 N m-2 (21%).

Biocatalyzed approach for the surface functionalization of poly(L-lactic acid) films using hydrolytic enzymes.

Poly(lactic acid) as a biodegradable thermoplastic polyester has received increasing attention. This renewable polyester has found applications in a wide range of products such as food packaging, textiles and biomedical devices. Its major drawbacks are poor toughness, slow degradation rate and lack of reactive side‐chain groups. An enzymatic process for the grafting of carboxylic acids onto the surface of poly(L‐lactic acid) (PLLA) films was developed using Candida antarctica lipase B as a catalyst. Enzymatic hydrolysis of the PLLA film using Humicola insolens cutinase in order to increase the number of hydroxyl and carboxylic groups on the outer polymer chains for grafting was also assessed and showed a change of water contact angle from 74.6 to 33.1° while the roughness and waviness were an order of magnitude higher in comparison to the blank. Surface functionalization was demonstrated using two different techniques, 14C‐radiochemical analysis and X‐ray photoelectron spectroscopy (XPS) using 14C‐butyric acid sodium salt and 4,4,4‐trifluorobutyric acid as model molecules, respectively. XPS analysis showed that 4,4,4‐trifluorobutyric acid was enzymatically coupled based on an increase of the fluor content from 0.19 to 0.40%. The presented 14C‐radiochemical analyses are consistent with the XPS data indicating the potential of enzymatic functionalization in different reaction conditions.

Identification of the Main Regulator Responsible for Synthesis of the Typical Yellow Pigment Produced by Trichoderma reesei

ABSTRACT The industrially used ascomycete Trichoderma reesei secretes a typical yellow pigment during cultivation, while other Trichoderma species do not. A comparative genomic analysis suggested that a putative secondary metabolism cluster, containing two polyketide-synthase encoding genes, is responsible for the yellow pigment synthesis. This cluster is conserved in a set of rather distantly related fungi, including Acremonium chrysogenum and Penicillium chrysogenum. In an attempt to silence the cluster in T. reesei, two genes of the cluster encoding transcription factors were individually deleted. For a complete genetic proof-of-function, the genes were reinserted into the genomes of the respective deletion strains. The deletion of the first transcription factor (termed yellow pigment regulator 1 [Ypr1]) resulted in the full abolishment of the yellow pigment formation and the expression of most genes of this cluster. A comparative high-pressure liquid chromatography (HPLC) analysis of supernatants of the ypr1 deletion and its parent strain suggested the presence of several yellow compounds in T. reesei that are all derived from the same cluster. A subsequent gas chromatography/mass spectrometry analysis strongly indicated the presence of sorbicillin in the major HPLC peak. The presence of the second transcription factor, termed yellow pigment regulator 2 (Ypr2), reduces the yellow pigment formation and the expression of most cluster genes, including the gene encoding the activator Ypr1. IMPORTANCE Trichoderma reesei is used for industry-scale production of carbohydrate-active enzymes. During growth, it secretes a typical yellow pigment. This is not favorable for industrial enzyme production because it makes the downstream process more complicated and thus increases operating costs. In this study, we demonstrate which regulators influence the synthesis of the yellow pigment. Based on these data, we also provide indication as to which genes are under the control of these regulators and are finally responsible for the biosynthesis of the yellow pigment. These genes are organized in a cluster that is also found in other industrially relevant fungi, such as the two antibiotic producers Penicillium chrysogenum and Acremonium chrysogenum. The targeted manipulation of a secondary metabolism cluster is an important option for any biotechnologically applied microorganism.

Xpp1 regulates the expression of xylanases, but not of cellulases in Trichoderma reesei.

BackgroundThe ascomycete Trichoderma reesei is industrially used for the production of cellulases. During the production process xylanases are co-secreted, which uses energy and nutrients. Cellulases and xylanases share the same main regulators, which makes a knowledge-based strain design difficult. However, previously a cis-element in the promoter of the main xylanase-encoding gene was identified as binding site for a putative repressor. Subsequently, three candidate repressors were identified in a pull-down approach. The expression of the most promising candidate, Xpp1 (Xylanase promoter-binding protein 1), was reported to be up-regulated on the repressing carbon source d-glucose and to bind the cis-element in vitro.ResultsIn this study, Xpp1 was deleted and over-expressed in T. reesei. An in vivo DNA-footprint assay indicated that Xpp1 binds a palindromic sequence in the xyn2 promoter. Comparison of the deletion, the over-expression, and the parent strain demonstrated that Xpp1 regulates gene expression of xylanolytic enzymes at later cultivation stages. Xpp1 expression was found to be up-regulated, additionally to d-glucose, by high d-xylose availability. These findings together with the observed xyn2 transcript levels during growth on xylan suggest that Xpp1 is the mediator of a feedback mechanism. Notably, Xpp1 has neither influence on the d-xylose metabolism nor on the expression of cellulases.ConclusionsXpp1 as regulator acting on the expression of xylanases, but not cellulases, is a highly promising candidate for knowledge-based strain design to improve the cellulases-to-xylanases ratio during industrial cellulase production.