Primjena oksidoreduktaza iz gljiva roda Trametes spp. u biotehnologiji – obilje katalitičkog djelovanja

Those oxidoreductases that are part of the ligninolytic complex of basidiomycete and ascomycete fungi have played an increasingly important role in biotechnological applications during the last decade. The stability of these extracellular enzymes, their good solubility, and a multitude of catalyzed reactions contribute to this trend. This review focuses on a single genus of white-rot basidiomycetes, Trametes, to highlight the numerous possibilities for the application of this microorganism as well as three of its enzymes: laccase, cellobiose dehydrogenase, and pyranose 2-oxidase. Whereas laccase is without doubt a major player in biotechnology, the two other enzymes are less well known, but represent emerging biocatalysts with potential. Both cellobiose dehydrogenase and pyranose 2-oxidase are presumed to participate in lignin breakdown and will be used to exemplify the potential of less prominent oxidoreductases from this genus.Posljednjih deset godina oksidoreduktaze koje sudjeluju u procesu razgradnje lignina gljiva stapčara i mješinarki imaju sve važniju ulogu u biotehnološkoj primjeni. Tomu trendu je pridonijela stabilnost tih ekstracelularnih enzima, njihova dobra topljivost i mogućnost primjene u raznim katalitičkim procesima. U ovom je revijalnom prikazu osobito opisana jedna vrsta gljive stapčare koja uzrokuje bijelu trulež, Trametes, i istaknute razne mogućnosti primjene toga mikroorganizma i njegovih triju enzima: lakaze, celobioza-dehidrogenaze i piranoza-2-oksidaze. Dok lakaza bez sumnje ima glavnu ulogu u biotehnologiji, druga su dva enzima manje poznata, ali sve se više koriste kao biokatalizatori. Pretpostavlja se da celobioza-dehidrogenaza i piranoza-2-oksidaza sudjeluju u razgradnji lignina te se pomoću njih mogu objasniti mogućnosti manje poznatih oksidoreduktaza toga roda gljiva

Enzymatic colouration with laccase and peroxidases : recent progress

Enzymes have received significant attention as alternative catalysts to chemical auxiliaries in textile processing. For example, laccases and peroxidises are promising alternatives for bleaching and denim stone washing processes. Similarly, the ability to oxidise different phenolic substrates and dye precusors resulting in the formation of different coloured polymeric molecules is being exploited for developing green chemistry dyeing processes. The enzymatic process is simpler than conventional coloration processes, giving economic and environmental benefits. In this review, the applications of laccase and peroxidise enzymes in dyeing processes of various textile meterials is discussed.This work was supported by the Fundamental Research Funds for the Central Universities JUSRP211A02 and JUSRP21001; the Open Project Program of Key Laboratory of Eco-Textiles, Ministry of Education, Jiangnan University KLET1007 and the Open Project Program of State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, DongHua University LK1005

Semi-rational engineering of cellobiose dehydrogenase for improved hydrogen peroxide production

Abstract Background The ability of fungal cellobiose dehydrogenase (CDH) to generate H2O2 in-situ is highly interesting for biotechnological applications like cotton bleaching, laundry detergents or antimicrobial functionalization of medical devices. CDH’s ability to directly use polysaccharide derived mono- and oligosaccharides as substrates is a considerable advantage compared to other oxidases such as glucose oxidase which are limited to monosaccharides. However CDH’s low activity with oxygen as electron acceptor hampers its industrial use for H2O2 production. A CDH variant with increased oxygen reactivity is therefore of high importance for biotechnological application. Uniform expression levels and an easy to use screening assay is a necessity to facilitate screening for CDH variants with increased oxygen turnover. Results A uniform production and secretion of active Myriococcum thermophilum CDH was obtained by using Saccharomyces cerevisiae as expression host. It was found that the native secretory leader sequence of the cdh gene gives a 3 times higher expression than the prepro leader of the yeast α-mating factor. The homogeneity of the expression in 96-well deep-well plates was good (variation coefficient <15%). A high-throughput screening assay was developed to explore saturation mutagenesis libraries of cdh for improved H2O2 production. A 4.5-fold increase for variant N700S over the parent enzyme was found. For production, N700S was expressed in P. pastoris and purified to homogeneity. Characterization revealed that not only the kcat for oxygen turnover was increased in N700S (4.5-fold), but also substrate turnover. A 3-fold increase of the kcat for cellobiose with alternative electron acceptors indicates that mutation N700S influences the oxidative- and reductive FAD half-reaction. Conclusions Site-directed mutagenesis and directed evolution of CDH is simplified by the use of S. cerevisiae instead of the high-yield-host P. pastoris due to easier handling and higher transformation efficiencies with autonomous plasmids. Twelve clones which exhibited an increased H2O2 production in the subsequent screening were all found to carry the same amino acid exchange in the cdh gene (N700S). The sensitive location of the five targeted amino acid positions in the active site of CDH explains the high rate of variants with decreased or entirely abolished activity. The discovery of only one beneficial exchange indicates that a dehydrogenase’s oxygen turnover is a complex phenomenon and the increase therefore not an easy target for protein engineering.The authors thank the European Commission (FP7 243529-2-COTTONBLEACH) for financial support. CKP thanks the Austrian Science Fund (FWF) for financial support (grant P22094). IK is a member of the doctoral program BioToP (Biomolecular Technology of Proteins) of the Austrian Science Fund (FWF; W1224). MA thanks the Spanish Government for financial support (BIO2010-19697).Peer Reviewe

Mechanistic investigation of the effect of endoglucanases related to pulp refining

Endoglucanases are increasingly being touted as the ultimate solution for reducing energy consumption during the refining process in the pulp and paper industry. However, due to the high variety of endoglucanases in different enzyme formulations, these perform heterogeneously when applied to different pulps. In this study, the effect of four endoglucanases on softwood and hardwood pulp was studied using confocal laser scanning microscopy (CLSM) after addition of fluorescently labelled carbohydrate binding modules (CBMs). Nuclear magnetic resonance (NMR) analysis and high-performance liquid chromatography quantification of released oligo- and monosaccharides was performed for in-depth mechanistical investigation. Changes in the crystallinity levels caused by enzymatic degradation of amorphous regions were monitored by incubation with two different CBMs from Caldicellulosiruptor bescii and from Thermobifida fusca with high preference to either amorphous or crystalline regions of cellulose, respectively. When dosed at identical activity on the endoglucanase specific CellG5 substrate, CLSM analysis indicated the highest decrease of amorphous regions for those endoglucanases which were also most active in laboratory refining trials and which released highest amounts of cellooligomers from pulp. Using 13C-NMR analysis, an increase in para-crystalline cellulose caused by enzyme application was observed. Release of reducing sugars was determined at identical CellG5 dosage, indicating a high variance between the enzymes, especially when complex enzyme formulations were used. Scanning electron microscopy images were obtained for visualization of the endoglucanase activity. The results of mechanistical studies indicate that reduction of amorphous moieties of pulp by endoglucanases is especially beneficial for the refining proces

Bamboo fibre processing: insights into hemicellulase and cellulase substrate accessibility

A biological approach for degumming bamboo substrates has been assessed. The ability of various commercially available enzymes, including cellulase, xylanase, pectinase and mannanase, to hydrolyze bamboo powders was investigated. In addition, a commercial cellulase preparation was applied onto bamboo fibre bundles obtained by natural retting. It was found that almost all enzymes applied can use bamboo material as a substrate. Mild autoclave pre-treatment can enhance reducing sugar yield from different enzyme treatments. A most pronounced effect was observed with cellulase treatment in which the hydrolysis degree increased 1.7 fold as measured by reducing sugars for autoclave pre-treated bamboo powders versus non-treated powders after only a short period of incubation. The combined treatment of hemicellulase preparations showed no effect on the hydrolysis of bamboo substrates. The effect of autoclave pre-treatment on cellulase-treated samples was confirmed by the increase of sugar yield, protein absorption as well as by the enhancement of surface modification and enzyme penetration observed by CLSM (confocal laser scanning microscopy). This work establishes a base for future studies to develop enzymatic hydrolysis of bamboo materials, making them suitable for textile processing.This work was made possible by support from the earmarked fund for Modern Agro-industry Technology Research System (nycytx-19-E23), the European Union Biorenew Project [Sixth Framework Programme (FP6-2004-NMP-NI-4)] and the Fundamental Research Funds for the Central Universities (JUSRP211A02)

A fungal ascorbate oxidase with unexpected laccase activity

Ascorbate oxidases are an enzyme group that has not been explored to a large extent. So far, mainly ascorbate oxidases from plants and only a few from fungi have been described. Although ascorbate oxidases belong to the well-studied enzyme family of multi-copper oxidases, their function is still unclear. In this study, Af_AO1, an enzyme from the fungus Aspergillus flavus, was characterized. Sequence analyses and copper content determination demonstrated Af_AO1 to belong to the multi-copper oxidase family. Biochemical characterization and 3D-modeling revealed a similarity to ascorbate oxidases, but also to laccases. Af_AO1 had a 10-fold higher affinity to ascorbic acid (KM = 0.16 ± 0.03 mM) than to ABTS (KM = 1.89 ± 0.12 mM). Furthermore, the best fitting 3D-model was based on the ascorbate oxidase from Cucurbita pepo var. melopepo. The laccase-like activity of Af_AO1 on ABTS (Vmax = 11.56 ± 0.15 µM/min/mg) was, however, not negligible. On the other hand, other typical laccase substrates, such as syringaldezine and guaiacol, were not oxidized by Af_AO1. According to the biochemical and structural characterization, Af_AO1 was classified as ascorbate oxidase with unusual, laccase-like activityPeer ReviewedPostprint (published version

Antimicrobial and antioxidant linen via laccase-assisted grafting

A laccase from Ascomycetemyceliophthora thermophila was used to assist the binding of chitosan and catechin onto a previous enzymatically oxidized linen surface. The process consists of the pre-treatment of the linen with laccase followed by the application of chitosan in a first step and catechin plus laccase in a second step. The results presented here support the conclusion that laccase is able to oxidize phenols naturally existing in flax fibres, and that the o-quinones formed promote the attachment of chitosan or/and catechin. The pre-treatment of linen with laccase is therefore the key factor for the success of catechin and chitosan grafting. A multifunctional linen product with both antioxidant and antibacterial properties was obtained with an acceptable level of durability in terms of end user requirements.Carla Silva would like to acknowledge the Portuguese Fundacao para a Ciencia e a Tecnologia (FCT) for funding under the scholarship SFRH/BPD/46515/2008

Polymerization of lignosulfonates by the laccase-HBT (1-hydroxybenzotriazole) system improves dispersibility

The ability of laccases from Trametes villosa (TvL), Myceliophthora thermophila (MtL), Trametes hirsuta (ThL) and Bacillus subtilis (BsL) to improve the dispersion properties of calcium lignosulfonates 398 in the presence of HBT as a mediator was investigated. Size exclusion chromatography showed an extensive increase in molecular weight of the samples incubated with TvL and ThL by 107% and 572% from 28400 Da after 17 h of incubation, respectively. Interestingly, FTIR spectroscopy, 13C NMR and Py-GC/MS analysis of the treated samples suggested no substantial changes in the aromatic signal of the lignosulfonates, a good indication of the ability of TvL/ThL-HBT systems to limit their effect on functional groups without degrading the lignin backbone. Further, the enzymatic treatments led to a general increase in the dispersion properties, indeed a welcome development for its application in polymer blends.Financial support from the BIORENEW EU-project (NMP2-CT-2006-26456), Austrian Academic Exchange Programme (OEAD) and the Spanish projects BIO2007-28720-E, BIO2008-01533, and AGL2008-00709 is acknowledged

Polymerisation of lignosulfonates by the laccase-HBT (1-hydroxybenzotriazole) system improves dispersibility

Prasetyo, Endry Nugroho et al.--The ability of laccases from Trametes villosa (TvL), Myceliophthora thermophila (MtL), Trametes hirsuta (ThL) and Bacillus subtilis (BsL) to improve the dispersion properties of calcium lignosulfonates 398 in the presence of HBT as a mediator was investigated. Size exclusion chromatography showed an extensive increase in molecular weight of the samples incubated with TvL and ThL by 107% and 572% from 28400 Da after 17 h of incubation, respectively. Interestingly, FTIR spectroscopy, 13C NMR and Py-GC/MS analysis of the treated samples suggested no substantial changes in the aromatic signal of the lignosulfonates, a good indication of the ability of TvL/ThL-HBT systems to limit their effect on functional groups without degrading the lignin backbone. Further, the enzymatic treatments led to a general increase in the dispersion properties, indeed a welcome development for its application in polymer blends.Financial support from the BIORENEW EU-project (NMP2-CT-2006-26456), Austrian Academic Exchange Programme (ÖEAD) and the Spanish projects BIO2007-28720-E, BIO2008-01533, and AGL2008-00709 is acknowledged.Peer reviewe