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Lytic polysaccharide monooxygenases (EC1.14.99.53-56, LPMOs) are oxidative enzymes with the capability to enhance lignocellulose saccharification as well as nanofibrillation of cellulosic fibres. The parameters affecting the efficiency of oxidative modification of cotton linters and softwood kraft fibres by LPMO from Trichoderma reesei (Tr AA9A) were studied here. Circular dichroism experiments were carried out to analyze the thermal stability and to indicate the optimum temperature for the experiments. The enzyme was found to be most thermostable at pH 5 but can tolerate up to 40–50 °C at neutral pH. The enzymatic fibre oxidation efficiency was found to be dependent on reductant (gallic acid) and hydrogen peroxide supply in a pH dependent manner. A high resolution UHPLC-IM-MS method was used for identification of oxidized oligosaccharides released in the enzymatic treatments. A microscopy method was applied for analysis of single fibre dissolution kinetics of Tr AA9A pretreated softwood kraft fibres, demonstrating that the oxidative enzymatic treatment facilitated the fibre dissolution and degraded the fibre surface structures, which prevent swelling. Graphical Abstract

Hydrothermal methods are commonly applied in pretreatment of lignocellulose for conversion to biofuels and chemicals. This pretreatment partially disassembles and solubilizes hemicelluloses and lignin, releasing sugars and phenolic compounds, which are potential inhibitors for the subsequent enzymatic saccharification step. In this work, the inhibitory effect of soluble phenolics in hydrothermal pretreatment liquor on canonical Trichoderma reesei enzymes was investigated, using purified cellobiohydrolases, endoglucanases, a xylanase, and a lytic polysaccharide monooxygenase, as well as commercial enzyme cocktails Cellic Ctec2 and Celluclast. The pretreatment liquor was fractionated to enrich the phenolics, and thoroughly analyzed. The most sensitive to phenolics inhibition were cellobiohydrolases, the major enzymes in crystalline cellulose degradation and the primary component in cellulolytic mixtures. These were inhibited by the oligophenolics and phenol-carbohydrate complexes (lignin-carbohydrate complex type of compounds), the latter of which could be mitigated by other enzymes, i.e., xylanases and endoglucanases. Addition of lytic polysaccharide monooxygenase to the hydrolysis reactions was found to relieve the negative effect of phenolics. Similarly, hydrolytic activity of a commercial enzyme cocktail Cellic Ctec2 was enhanced in the presence of low concentrations of phenolics.

Fibre reactivity is essential for cellulose dissolution and derivatisation and a porous fibre structure is one key determinant for a highly reactive pulp. Mechanical and enzymatic treatments are known to improve fibre reactivity and more recently, the combination of mechano-enzymatic treatment has been shown to synergistically enhance the beneficial effect. The aim of this work was to do a systematic study on the effect of dry matter content during enzymatic modification of fibres and define the conditions that optimally improve fibre porosity. The combined mechano-enzymatic treatments at 15–25 w% consistency had the most pronounced effect on fibre porosity and morphology analysed by solute exclusion technique, nitrogen sorption and scanning electron microscopy. Light microscopy imaging confirmed that the combined mechano-enzymatic treatment at high consistency (> 10 w%) resulted in extensive fibrillation of the treated fibres which was not observed after sole mechanical or enzymatic treatments.

Enzymatic hydrolysis of lignocellulosic biomass into platform sugars can be enhanced by the addition of accessory enzymes, such as xylanases. Lignin from steam pretreated biomasses is known to inhibit enzymes by non-productively binding enzymes and limiting access to cellulose. The effect of enzymatically isolated lignin on the hydrolysis of xylan by four glycoside hydrolase (GH) family 11 xylanases was studied. Two xylanases from the mesophilic Trichoderma reesei, TrXyn1, TrXyn2, and two forms of a thermostable metagenomic xylanase Xyl40 were compared.Lignin isolated from steam pretreated spruce decreased the hydrolysis yields of xylan for all the xylanases at 40 and 50 °C. At elevated hydrolysis temperature of 50 °C, the least thermostable xylanase TrXyn1 was most inhibited by lignin and the most thermostable xylanase, the catalytic domain (CD) of Xyl40, was least inhibited by lignin. Enzyme activity and binding to lignin were studied after incubation of the xylanases with lignin for up to 24 h at 40 °C. All the studied xylanases bound to lignin, but the thermostable xylanases retained 22-39% of activity on the lignin surface for 24 h, whereas the mesophilic T. reesei xylanases become inactive. Removing of N-glycans from the catalytic domain of Xyl40 increased lignin inhibition in hydrolysis of xylan when compared to the glycosylated form. By comparing the 3D structures of these xylanases, features contributing to the increased thermal stability of Xyl40 were identified.High thermal stability of xylanases Xyl40 and Xyl40-CD enabled the enzymes to remain partially active on the lignin surface. N-glycosylation of the catalytic domain of Xyl40 increased the lignin tolerance of the enzyme. Thermostability of Xyl40 was most likely contributed by a disulphide bond and salt bridge in the N-terminal and α-helix regions.

Second-generation biorefineries converting lignocellulose to renewable fuels and chemicals require pretreatment to open the cell wall structure and enable enzymes to access polysaccharides. However, lignin from the commonly used hydrothermal pretreatments can restrict enzyme action by covering the cellulose surface and non-productively binding enzymes. The resulting residual lignin typically contains a relatively high amount of carbohydrates, restricting its solubility and utilisation in applications. The aim of this work was to elucidate how pretreatment severity affects lignin inhibition in enzymatic hydrolysis and how lignin from softwood and herbaceous plants changes during pretreatment. Understanding these factors can aid in developing pretreatments with simultaneously improved hydrolysis yields and improved applicability of the hydrolysis lignin. Spruce wood and wheat straw were hydrothermally pretreated at 180-220 °C with or without an acid catalyst. Increasing the pretreatment severity increased the inhibitory effect of lignin in Avicel hydrolysis. The inhibition arose from increased non-productive binding of enzymes to lignin. For both raw materials, the levels of aryl ether bonds decreased as pretreatment severity increased. Furthermore, for wheat straw lignin, a decrease in the amount of hydroxycinnamates and tricin was observed. Several publications have reported that introducing additives, such as 2-naphthol, to pretreatment can reduce lignin condensation resulting in improved hydrolysis yields. The flavonoid tricin, found in herbaceous plant, has an electron-rich aromatic ring structure similar to 2-naphthol. Softwood pine was hydrothermally pretreated at 200 °C with the additives tricin or 2-naphthol. Pretreatment with tricin resulted in higher sugar yields in the following enzymatic hydrolysis step when compared with the pretreatment with 2-naphthol. This may be due to modifications made to the lignin structure during pretreatment, which could be promising for both saccharification and lignin valorization pathways.

Background: Hydrothermal methods are commonly applied in pretreatment of lignocellulose for enhanced enzymatic hydrolysis and further conversion to biofuels and chemicals. The pretreatment partially disassemble and solubilize cell wall polymers. Besides mono- and oligosaccharides, the soluble degradation products include various phenolic compounds, which may affect the efficiency of enzymatic hydrolysis. Results: The phenolic compounds were isolated from pretreatment liquor and their effects on cellulolytic enzymes were investigated. The major enzymes in crystalline cellulose degradation, cellobiohydrolases, were inhibited by the oligophenolics and phenolic-carbohydrate conjugates the latter of which could be mitigated by other enzymes, i.e. xylanases and endoglucanases. Hydrolytic activity of a commercial enzyme cocktail containing lytic polysaccharide monooxygenase (LPMO) was enhanced in presence of low concentrations of phenolics.Conclusions: The effects of the pretreatment liquor phenolics is dependent on enzyme concentration and type of the phenolic compounds. For optimized performance, the pretreatment conditions and enzyme cocktail composition should be designed to promote oxidative activities for enhanced hydrolysis, degradation of the oligosaccharides linked to phenolics and resistance to oligophenolic compounds.

Enzymatic modification of bleached softwood kraft fibres for improved fibre reactivity was studied at high (20% w/w) and low (1% w/w) dry matter content. The role of enzyme family and structure in fibre modification was assessed using endoglucanases from three structurally different glycoside hydrolase (GH) families (5, 7 and 45) with and without a carbohydrate binding module (CBM). Based on the amount of dissolved sugars, enzyme action at high consistency was about sixfold higher compared to a fibre treatment at low consistency. The GH45 endoglucanase was found to be most specific in acting on pulp cellulose whereas the family 5 and 7 endoglucanases had activity also on pulp hemicelluloses. The GH45 endoglucanase was found to be most efficient in reducing molecular weight and viscosity of the pulp. In addition, treatment with the GH45 endoglucanase resulted in the highest micropore volume in fibres and thus an increase in cellulose accessibility. The increased accessibility could be seen as decreased dissolution time in cupriethylenediamine using recently developed analytical techniques: viscometric analysis and microscopic video analysis. At high consistency, CBM was not promoting enzyme action, although CBMs are known to be beneficial at low dry matter conditions. Graphical abstract: [Figure not available: see fulltext.].

Hydrothermal pretreatment is commonly used for enhancing enzymatic hydrolysis of lignocellulosics. Spruce and wheat straw were pretreated with increasing severity and lignin characteristics were analysed. The effect of enzymatically isolated lignin on the hydrolysis of Avicel and the adsorption of a cellobiohydrolase onto lignin was measured. Non-pretreated lignins had only a minor effect on Avicel hydrolysis. The structural changes in lignin accompanying hydrothermal pretreatment were associated with increased binding and inactivation of the cellulase on the lignin surface. The inhibitory effect was more pronounced in spruce than in wheat straw lignin. However, similar pretreatment severities caused similar levels of inhibition in Avicel hydrolysis for both biomass sources. The combined severity factor of the pretreatment correlated well with the inhibitory effect of lignin.

Adsorption of xylan on pulp is a potential method to improve its properties, especially refinability for high quality printing and writing (P&W) paper grades. In this study, elephant grass [ Pennisetum purpureum (Schumach.)] xylan was used for this purpose. The xylan was extracted using cold caustic extraction (CCE) from P. purpureum brown pulp, produced by the Soda-AQ process (kappa 20). Xylan resorption was accomplished during the oxygen delignification phase of eucalypt [ Eucalyptus globulus (Labill.)] pulp to avoid problems induced by the colour of the lignin-contaminated deposited xylan. Immunolabelling and enzymatic peeling methodologies were compared for the analysis of the spatial distribution of xylan in the fibre wall. The labelling appeared unevenly as faint and brighter patches on fibre surfaces. Increased labelling of xylan was detected on the samples with precipitated P. purpureum xylan. The enzymatic peeling method using a total hydrolysis enzyme mixture yielded a composition gradient as a function of time, showing clear xylose (Xyl) enrichment in the very beginning of the reaction, reflecting hydrolysis of fibre surfaces. Pure xylanase and endoglucanase hydrolyses yielded different product patterns and kinetics compared to total hydrolysis, but interpretation of those results in terms of xylan localisation was not straightforward. Deposition of xylan isolated from Pennisetum purpureum on fibres of Eucalyptus globulus and characterisation of the composition of the surface xylans by immunolabelling and enzymatic peeling | Request PDF. Available from: https://www.researchgate.net/publication/327595575_Deposition_of_xylan_isolated_from_Pennisetum_purpureum_on_fibres_of_Eucalyptus_globulus_and_characterisation_of_the_composition_of_the_surface_xylans_by_immunolabelling_and_enzymatic_peeling [accessed Sep 18 2018].

Adsorption of cellulases onto lignin is considered a major factor in retarding enzymatic cellulose degradation of lignocellulosic biomass. However, the adsorption mechanisms and kinetics are not well understood for individual types of cellulases. This study examines the binding affinity, kinetics of adsorption, and competition of four monocomponent cellulases of Trichoderma reesei during adsorption onto lignin. TrCel7A, TrCel6A, TrCel7B, and TrCel5A were radiolabeled for adsorption experiments on lignin-rich residues (LRRs) isolated from hydrothermally pretreated spruce (L-HPS) and wheat straw (L-HPWS), respectively. On the basis of adsorption isotherms fitted to the Langmuir model, the ranking of binding affinities was TrCel5A > TrCel6A > TrCel7B > TrCel7A on both types of LRRs. The enzymes had a higher affinity to the L-HPS than to the L-HPWS. Adsorption experiments with dilution after 1 and 24 hr and kinetic modeling were performed to quantify any irreversible binding over time. Models with reversible binding parameters fitted well and can explain the results obtained. The adsorption constants obtained from the reversible models agreed with the fitted Langmuir isotherms and suggested that reversible adsorption-desorption existed at equilibrium. Competitive binding experiments showed that individual types of cellulases competed for binding sites on the lignin and the adsorption data fitted the Langmuir adsorption model. Overall, the data strongly indicate that the adsorption of cellulases onto lignin is reversible and the findings have implications for the development of more efficient cellulose degrading enzymes.

The aim of this study was to investigate how fractionation of protein and lignin from brewer’s spent grain can be influenced by thermochemical and enzymatic pretreatments, including steam explosion, carbohydrase treatment and protease treatment. Following these pretreatments, protein and lignin were partially fractionated by alkaline extraction and acidic precipitation. Steam explosion and hydrolysis of cell wall polysaccharides substantially increased lignin solubilization and its recovery by acidic precipitation. Meanwhile, effective protein extraction required the use of protease, which increased protein solubilization from 15% to nearly 100% from otherwise untreated BSG. Steam explosion reduced the enzymatic protein solubilization but enhanced the extract recovery in the centrifugation step, presumably due to lower water-binding capacity of steam-exploded BSG compared to untreated BSG. Lignin and protein showed clear co-extraction and could only be partially separated by acidic precipitation, suggesting similar solubilities and strong interactions between these components. According to the results, protein and lignin are extractable from BSG after suitable pretreatments. This work provided increased understanding of the chemistry and interactions underlying the challenges in fractionation of BSG components.

Lignocelluloses are abundant raw materials for production of fuels, chemicals and materials. The purpose of this paper is to review the enzyme-types and enzyme-technologies studied and applied in the processing of the lignocelluloses into different products. The enzymes here are mostly glycoside hydrolases, esterases and different redox enzymes. Enzymatic hydrolysis of lignocellulosic polysaccharides to platform sugars has been widely studied leading to development of advanced commercial products for this purpose. Restricted hydrolysis or oxidation of cellulosic fibers have been applied in processing of pulps to paper products, nanocelluloses and textile fibers. Oxidation, transglycosylation and derivatization have been utilized in functionalization of fibers, cellulosic surfaces and polysaccharides. Enzymatic polymerization, depolymerization and grafting methods are being developed for lignin valorization.

BACKGROUNDThis study elaborates the possibility to apply combined ionic liquid (IL) and enzyme treatments for pulp fibre modification. The approach involves swelling of fibre surfaces with IL followed by enzymatic modification of the disrupted fibre surface using carbohydrate active enzymes. RESULTSThe capacity of seven cellulose-dissolving or cellulose-swelling ionic liquids to swell pulp fibres was compared. In addition, thirteen cellulases and five xylanases were screened for their IL tolerance, which determines their applicability in combined or sequential IL-enzyme treatments of fibres. Among the studied ionic liquids, 1-ethyl-3-methylimidazolium dimethylphosphate ([EMIM]DMP) and 1,3-dimethylimidazolium dimethylphosphate ([DMIM]DMP) had the strongest effect on fibre swelling. These solvents were also found to be the least inactivating for the studied enzymes. CONCLUSIONEnzyme compatibility and cellulose-dissolving capability are not two conflicting properties of an ionic liquid. (c) 2017 Society of Chemical Industry

Lignocellulosic plant biomass is a renewable and abundant source for the production of bio-based fuels, chemicals and chemical building blocks. Efficient fractionation and conversion of these feedstocks are an essential step in the valorization of the cellulose, hemicellulose and lignin fractions. The use of a new two-stage alkaline oxidation (AlkOx) process has been investigated for the pretreatment of softwood in presence of sodium carbonate. Within this study, the use of commercial polymeric and ceramic ultrafiltration membranes for the purification and concentration of lignin from the spent liquor of the AlkOx process has been evaluated enabling further valorization thereof. Higher permeation fluxes were observed, ranging from 30 to 139 L/m 2 h, depending on the hydrophilicity, pore size and structure/chemistry of the membrane. High lignin retentions have been obtained for all membranes. Diafiltration of the spent liquor using the ESP04 membrane enables the purification of the lignin fraction with an efficient removal of the impurities originating from both the lignocellulosic material and the pretreatment. The integration of both processes allows the co-valorization of the lignin fraction, besides the primary C5/C6 sugar fraction produced in the two-stage alkaline oxidation of softwood. Also some preliminary techno-economic calculations have been realized on the membrane separation process to asses the economic potential of this technology.

Oat and faba bean protein isolates were treated with transglutaminase from Streptomyces mobaraensis and tyrosinase from Trichoderma reesei to modify the colloidal properties of protein particles in order to improve their colloidal stability and foaming properties. Transglutaminase crosslinked faba bean protein extensively already with 10 nkat/g enzyme dosage. Oat protein was crosslinked to some extent with transglutaminase with higher dosages (100 and 1000 nkat/g). Transglutaminase increased the absolute zeta-potential values and reduced the particle size of oat protein particles. As a result, the colloidal stability and foaming properties were improved. Tyrosinase had limited crosslinking ability on both plant protein materials. Tyrosinase greatly reduced the solubility of oat protein despite limited crosslinking. Tyrosinase did not have effect on zeta-potential or colloidal stability of either protein, but it impaired foaming properties of both. Thus, the crosslinking enzymes studied caused significantly different end product functionality, presumably due to the different mechanism of action.

32 páginas.-- 6 figuras.-- 4 tablas.-- 53 referencias.-- SUPPORTING INFORMATION The following data are available online as complementary information: Assignments of lignin 13C/1H correlation signals in the 2D HSQC spectra of the MWL isolated from eucalyptus, wheat straw and spruce; size and zeta potential of the MWL measured in aqueous dispersions at different background electrolyte concentrations; water contact angles before and after CTec2 and CBH-I adsorption; AFM images of wheat straw films corresponding to SPR sensograms after rinsing with 50 mM background electrolyte; schematics of the different scenarios for enzymes adsorption on the surfaces for the calculation of surface coverage., Lignins were isolated from spruce, wheat straw and eucalyptus by using the milled wood lignin (MWL) method. Functional groups and compositional analyses were assessed via 2D NMR and 31P to realize their effect on enzyme binding. Films of the lignins were fabricated and ellipsometry, atomic force microscopy and water contact angle measurements were used for their characterization and to reveal the changes upon enzyme adsorption. Moreover, lignin thin films were deposited on quartz crystal microgravimetry (QCM) and surface plasmon (SPR) resonance sensors and used to gain further insights into the lignin-cellulase interactions. For this purpose, a commercial multicomponent enzyme system and a monocomponent Trichoderma reesei exoglucanase (CBH-I) were considered. Strong enzyme adsorption was observed on the various lignins but compared to the multicomponent cellulases, CBH-I displayed lower surface affinity and higher binding reversibility. This resolved prevalent questions related to the affinity of this enzyme with lignin. Remarkably, a strong correlation between enzyme binding and the S/G ratio was found for the lignins, which presented a similar hydroxyl group content (31P NMR): higher protein affinity was determined on isolated spruce lignin (99% G units) while the lowest adsorption occurred on isolated eucalyptus lignin (70% S units). The effect of electrostatic interactions in enzyme adsorption was investigated by SPR, which clearly indicated that the screening of charges allowed more extensive protein adsorption. Overall, this work furthers our understanding of lignin-cellulase interactions relevant to biomass that has been subjected to no or little pretreatment and highlights the widely contrasting effects of the nature of lignin, which gives guidance to improve lignocellulosic saccharification and related processes, This study was partially funded by the INDOX EU-project (KBBE-2013-7-613549); the LIGNOCELL, LIGNIN, NOESIS, and BIORENZYMERY Spanish MICINN (co-financed by FEDER funds) projects (AGL2011-25379, CTQ2014-60764-JIN, BIO2014-56388 R and AGL2014-53730-R) and the CSIC Project 201440E097. A.P. is grateful to the Spanish MINECO for a FPI fellowship. O.J.R. would like to thank the Academy of Finland for funding through its Centers of Excellence Program (2014–2019), under Project 132723612 HYBER.

Non-productive enzyme binding onto lignin is the major inhibitory mechanism, which reduces hydrolysis rates and yields and prevents efficient enzyme recycling in the hydrolysis of lignocellulosics. The detailed mechanisms of binding are still poorly understood. Enzyme-lignin interactions were investigated by comparing the structural properties and binding behaviour of fungal monocomponent enzymes, cellobiohydrolases TrCel7A and TrCel6A, endoglucanases TrCel7B and TrCel5A, a xylanase TrXyn11 and a ß-glucosidase AnCel3A, onto lignins isolated from steam pretreated spruce and wheat straw. The enzymes exhibited decreasing affinity onto lignin model films in the following order: TrCel7B > TrCel6A > TrCel5A > AnCel3A > TrCel7A > TrXyn11. As analysed in Avicel hydrolysis, TrCel6A and TrCel7B were most inhibited by lignin isolated from pretreated spruce. This could be partially explained by adsorption of the enzyme onto the lignin surface. Enzyme properties, such as enzyme surface charge, thermal stability or surface hydrophobicity could not alone explain the adsorption behaviour.

The effects of heat treatment (90 °C, 5 or 30 min) and enzymatic crosslinking with transglutaminase (TG, 10, 100 or 1000 nkat/g protein) on emulsifying and gelling properties of faba bean protein isolate (FPI) were studied. Heat treatment of FPI caused a clear shift in far-UV CD spectra towards random coil structure and an increase in surface hydrophobicity from 181 to 504 RFU. TG crosslinked heat-treated FPI more efficiently as compared to native FPI. TG-induced crosslinking caused a reduction of surface hydrophobicity from 504 to 435 RFU. Emulsifying activity and stability indexes of FPI stabilized emulsions ranged between 25 and 30 m2/g and 18–35 min, respectively. Rheological properties of the FPI gels induced by heating, followed by acidification or TG treatment (10 or 100 nkat/g protein) were analyzed. FPI showed gel-like characteristics at 10% concentration (G′ = 180 Pa). When the heat-treated FPI dispersion was further acidified or TG-treated (100 nkat/g), G′ values of >6500 and > 3500 Pa were achieved, respectively. Water holding capacity of the FPI gels were >93% and >98% for acid- and TG-induced gels, respectively. The gel microstructures showed mainly heterogeneously-sized protein aggregates, however, no differences were observed between acid- and TG-induced gels.

There is great interest in understanding changes that occur to cell wall constituents during saccharification of pretreated lignocellulose, particularly in relation to recalcitrance of the residues. This paper reports the effects of hydrothermal pretreatment followed by enzyme hydrolysis on the extractability and properties of recalcitrant wheat straw polymers. The results show that the undigested residue had lost much of its archestructure. Compositional analysis portrayed a considerable loss of cross-linking di-ferulic acid phenolics, hemicellulosic and cellulosic sugars. The remaining cellulosic and non-cellulosic polysaccharides were much more readily extractable in alkali and molecular profiling revealed the presence of low Mw oligomers in the fractions suggesting the partial enzyme hydrolysis of hemicelluloses and cellulose. Simultaneous saccharification and fermentation of the pretreated and enzyme-digested residues surprisingly resulted in ethanol yields of up to 99% of the theoretical. This is discussed in relation to the “recalcitrant” nature of the original pretreated and enzyme digested biomass.

Coupled binuclear copper (CBC) enzymes have a conserved type 3 copper site that binds molecular oxygen to oxidize various mono- and diphenolic compounds. In this study, we found a new crystal form of catechol oxidase from Aspergillus oryzae (AoCO4) and solved two new structures from two different crystals at 1.8-Å and at 2.5-Å resolutions. These structures showed different copper site forms (met/deoxy and deoxy) and also differed from the copper site observed in the previously solved structure of AoCO4. We also analysed the electron density maps of all of the 56 CBC enzyme structures available in the protein data bank (PDB) and found that many of the published structures have vague copper sites. Some of the copper sites were then re-refined to find a better fit to the observed electron density. General problems in the refinement of metalloproteins and metal centres are discussed.

Background Despite of the presence of sulfhydryl oxidases (SOXs) in the secretomes of industrially relevant organisms and their many potential applications, only few of these enzymes have been biochemically characterized. In addition, basic functions of most of the SOX enzymes reported so far are not fully understood. In particular, the physiological role of secreted fungal SOXs is unclear. Results The recently identified SOX from Aspergillus tubingensis (AtSOX) was produced, purified and characterized in the present work. AtSOX had a pH optimum of 6.5, and showed a good pH stability retaining more than 80% of the initial activity in a pH range 4-8.5 within 20 h. More than 70% of the initial activity was retained after incubation at 50 °C for 20 h. AtSOX contains a non-covalently bound flavin cofactor. The enzyme oxidised a sulfhydryl group of glutathione to form a disulfide bond, as verified by nuclear magnetic resonance spectroscopy. AtSOX preferred glutathione as a substrate over cysteine and dithiothreitol. The activity of the enzyme was totally inhibited by 10 mM zinc sulphate. Peptide- and protein-bound sulfhydryl groups in bikunin, gliotoxin, holomycin, insulin B chain, and ribonuclease A, were not oxidised by the enzyme. Based on the analysis of 33 fungal genomes, SOX enzyme encoding genes were found close to nonribosomal peptide synthetases (NRPS) but not with polyketide synthases (PKS). In the phylogenetic tree, constructed from 25 SOX and thioredoxin reductase sequences from IPR000103 InterPro family, AtSOX was evolutionary closely related to other Aspergillus SOXs. Oxidoreductases involved in the maturation of nonribosomal peptides of fungal and bacterial origin, namely GliT, HlmI and DepH, were also evolutionary closely related to AtSOX whereas fungal thioreductases were more distant. Conclusions AtSOX (55 kDa) is a fungal secreted flavin-dependent enzyme with good stability to both pH and temperature. A Michaelis-Menten behaviour was observed with reduced glutathione as a substrate. Based on the location of SOX enzyme encoding genes close to NRPSs, SOXs could be involved in the secondary metabolism and act as an accessory enzyme in the production of nonribosomal peptides. Electronic supplementary material The online version of this article (10.1186/s12858-017-0090-4) contains supplementary material, which is available to authorized users.

Milk fat was treated with the sn- 2 specific PAN-2 lipase from Pseudozyma antarctica with the aim of achieving selective removal of saturated fatty acids with chain lengths between C12 and C16, which are abundant at the sn- 2 position. Hydrolysis was also carried out using the non-specific CRU-NS from Candida rugosa and the 1,3-specific TLA-1,3 from Thermomyces lanuginosus . PAN-2 treatment decreased the proportion of the target fatty acids considerably (content of 28%, w/w) and increased the proportion of unsaturated ones (content of 52%, w/w) at the degree of hydrolysis of 48% (mol/mol) in relation to the unhydrolysed substrate (47%, w/w, C12:0-C14:0 and 32%, w/w, unsaturated). The fatty acid distributions of the TLA-1,3 and CRU-NS treated samples were similar to untreated milk fat. The proportion of crystalline fat was smaller and the firmness lower in the PAN-2 treated milk fat samples than in the untreated milk fat at 5 °C.

Fibres fractionated from solid recovered fuel (SRF), a standardised market combustion fuel produced from sorted waste, were considered as a source of lignocellulosic fermentable sugars. The fibre yield from four samples of SRF was 25-45%, and the separated material consisted of 52-54% carbohydrates, mainly glucan, with a high content of ash (12-17%). The enzymatic digestibility of recovered fibres was studied at low and high solids loading and compared with model substrates containing only chemical and mechanical pulps. Above 80% hydrolysis yield was reached at 20% solids loading in 48. h, but variation was observed between different samples of recovered fibres. Surfactants were found to improve the hydrolysis yield of recovered fibres especially in tumbling-type of mixing at low solids loading, where hydrolysis was found to stagnate without surfactants. The results suggest that SRF is a potential source of easily digestible lignocellulosic carbohydrates for use in biorefineries.

Chemical changes of lignin induced by the steam explosion (SE) process were elucidated. Wheat straw was studied as the raw material, and lignins were isolated by the enzymatic mild acidolysis lignin (EMAL) procedure before and after the SE treatment for analyses mainly by two-dimensional (2D) [heteronuclear single-quantum coherence (HSQC) and heteronuclear multiple-bond correlation (HMBC)] and 31P nuclear magnetic resonance (NMR). The β-O-4 structures were found to be homolytically cleaved, followed by recoupling to β-5 linkages. The homolytic cleavage/recoupling reactions were also studied by computational methods, which verified their thermodynamic feasibility. The presence of the tricin bound to wheat straw lignin was confirmed, and it was shown to participate in lignin reactions during the SE treatment. The preferred homolytic β-O-4 cleavage reaction was calculated to follow bond dissociation energies: G–O–G (guaiacyl) (69.7 kcal/mol) > G–O–S (syringyl) (68.4 kcal/mol) > G–O–T (tricin) (67.0 kcal/mol).

Ionic liquids (ILs) dissolve lignocellulosic biomass and have a high potential as pretreatment prior to total enzymatic hydrolysis. ILs are, however, known to inactivate cellulases. In this article, enzymatic hydrolysis of microcrystalline cellulose (MCC) and enzyme binding onto the cellulosic substrate were studied in the presence of cellulose-dissolving ILs. Two different ILs, 1,3-dimethylimidazolium dimethylphosphate ([DMIM]DMP) and 1-ethyl-3-methylimidazolium acetate ([EMIM]AcO), and two monocomponent cellulases, Trichoderma reesei cellobiohydrolase Cel7A and endoglucanase Cel5A, were used in the study. The role and IL sensitivity of the carbohydrate-binding module (CBM) were studied by performing hydrolysis and binding experiments with both the intact cellulases, and their respective core domains (CDs). Based on hydrolysis yields and substrate binding experiments for the intact enzymes and their CDs in the presence of ILs, the function of the CBM appeared to be very IL sensitive. Binding data suggested that the CBM was more important for the substrate binding of endoglucanase Cel5A than for the binding of cellobiohydrolase Cel7A. The CD of Cel7A was able to bind well to cellulose even without a CBM, whereas Cel5A CD had very low binding affinity. Hydrolysis also occurred with Cel5A CD even if this protein had very low binding affinity in all the studied matrices. Binding and hydrolysis were less affected by the studied ILs for Cel7A than for Cel5A. To our knowledge, this is the first systematic study of IL effects on cellulase substrate binding.

Over 250 microbial strains were screened for lipase activity specific towards saturated medium and long-chain fatty acids. Strains showing trimyristin (C14:0) hydrolysis on agar medium were further streaked on tricaprylin (C8:0) and on olive oil (mostly C18:1) agar to identify strains hydrolysing C12–C15 fatty acids with modest or no activity towards short-chain and unsaturated fatty acids. The twenty strains showing the desired specificity were grown as liquid cultures and analysed for p-nitrophenyl (p-NP) myristate hydrolysis in the presence of Triton X-100. Cells from the cultures showing the highest activity towards p-NP myristate were analysed for specificity towards p-NP esters of different fatty acid chain lengths. On the basis of the p-NP ester profiling, four lipase samples were selected for hydrolysis experiments on butter oil. Of these, the sample obtained from the cultivation of Didpodascus capitatus met the screening criteria: C12:0–C16:0 specificity and no preference towards C18:0 and C18:1 fatty acids.

The (hemi)cellulolytic systems of two novel lignocellulolytic Penicillium strains (Penicillium pulvillorum TUB F-2220 and P. cf. simplicissimum TUB F-2378) have been studied. The cultures of the Penicillium strains were characterized by high cellulase and β-glucosidase as well moderate xylanase activities compared to the Trichoderma reesei reference strains QM 6a and RUTC30 (volumetric or per secreted protein, respectively). Comparison of the novel Penicillium and T. reesei secreted enzyme mixtures in the hydrolysis of (ligno)cellulose substrates showed that the F-2220 enzyme mixture gave higher yields in the hydrolysis of crystalline cellulose (Avicel) and similar yields in hydrolysis of pre-treated spruce and wheat straw than enzyme mixture secreted by the T. reesei reference strain. The sensitivity of the Penicillium cellulase complexes to softwood (spruce) and grass (wheat straw) lignins was lignin and temperature dependent: inhibition of cellulose hydrolysis in the presence of wheat straw lignin was minor at 35 °C while at 45 °C by spruce lignin a clear inhibition was observed. The two main proteins in the F-2220 (hemi)cellulase complex were partially purified and identified by peptide sequence similarity as glycosyl hydrolases (cellobiohydrolases) of families 7 and 6. Adsorption of the GH7 enzyme PpCBH1 on cellulose and lignins was studied showing that the lignin adsorption of the enzyme is temperature and pH dependent. The ppcbh1 coding sequence was obtained using PCR cloning and the translated amino acid sequence of PpCBH1 showed up to 82% amino acid sequence identity to known Penicillium cellobiohydrolases.

Tyrosinases with different physico-chemical properties have been identified from various bacterial phyla such as Actinobacteria and Proteobacteria and their production is often inducible by environmental stresses. Tyrosinases are enzymes catalysing the oxidation of mono- and di-phenolic compounds to corresponding quinones with the concomitant reduction of molecular oxygen to water. Since the quinone produced can further react non-enzymatically with other nucleophiles, e.g. amino groups, many tyrosinases have a recorded cross-linking activity on proteins. Various bacterial tyrosinases oxidise tyrosine, catechol, l / d -DOPA, caffeic acid and polyphenolic substrates such as catechins. This substrate specificity has been exploited to engineer biosensors able to detect even minimal amounts of different phenolic compounds. The physiological role of tyrosinases in the biosynthesis of melanins has been used for the production of coloured and dyeing agents. Moreover, the cross-linking activity of tyrosinases has found application in food processing and in the functionalisation of materials. Numerous tyrosinases with varying substrate specificities and stability features have been isolated from bacteria and they can constitute valuable alternatives to the well-studied tyrosinase from common mushroom.

Steam explosion was studied as a means to improve the enzymatic digestibility of carbohydrates in Brewer’s spent grain, a protein and lipid-rich lignocellulosic by-product of the brewing industry. Having temperature, treatment time and the presence of acid catalyst as variables, a treatment at 200 °C for 10 min without an acid catalyst was found to be the most efficient, dissolving 12.1 % of the dry matter. Mainly oligomeric non-cellulosic glucan and arabinoxylan were dissolved, and the remaining insoluble carbohydrates could be efficiently hydrolysed by an enzyme cocktail (75 % hydrolysis yield). The process also caused partial protein degradation and dissolved over a third of the total nitrogen. Meanwhile, the insoluble protein appeared to become more strongly associated with acid-insoluble lignin. Compositional changes observed in the proteins and carbohydrates were supported by the results of epifluorescence microscopy. The process yielded three chemically different fractions which could serve as biorefinery products or intermediates.

Direct extraction and characterization of microbial community DNA through PCR amplicon surveys and metagenomics has revolutionized the study of environmental microbiology and microbial ecology. In particular, metagenomic analysis of nucleic acids provides direct access to the genomes of the “uncultivated majority.” Accelerated by advances in sequencing technology, microbiologists have discovered more novel phyla, classes, genera, and genes from microorganisms in the first decade and a half of the twenty-first century than since these “many very little living animalcules” were first discovered by van Leeuwenhoek. The unsurpassed diversity of soils promises continued exploration of a range of industrial, agricultural, and environmental functions. The ability to explore soil microbial communities with increasing capacity offers the highest promise for answering many outstanding who, what, where, when, why, and with whom questions such as: Which microorganisms are linked to which soil habitats? How do microbial abundances change with changing edaphic conditions? How do microbial assemblages interact and influence one another synergistically or antagonistically? What is the full extent of soil microbial diversity, both functionally and phylogenetically? What are the dynamics of microbial communities in space and time? How sensitive are microbial communities to a changing climate? What is the role of horizontal gene transfer in the stability of microbial communities? Do highly diverse microbial communities confer resistance and resilience in soils?, La lista completa de autores que integran el documento puede consultarse en el archivo., Facultad de Ciencias Exactas

Laccases are copper-containing enzymes used in various applications, such as textile bleaching. Several crystal structures of laccases from fungi and bacteria are available, but ascomycete types of fungal laccases (asco-laccases) have been rather unexplored, and to date only the crystal structure of Melanocarpus albomyces laccase (MaL) has been published. We have now solved the crystal structure of another asco-laccase, from Thielavia arenaria (TaLcc1), at 2.5 A resolution. The loops near the T1 copper, forming the substrate-binding pockets of the two asco-laccases, differ to some extent, and include the amino acid thought to be responsible for catalytic proton transfer, which is Asp in TaLcc1, and Glu in MaL. In addition, the crystal structure of TaLcc1 does not have a chloride attached to the T2 copper, as observed in the crystal structure of MaL. The unique feature of TaLcc1 and MaL as compared with other laccases structures is that, in both structures, the processed C-terminus blocks the T3 solvent channel leading towards the trinuclear centre, suggesting a common functional role for this conserved ‘C-terminal plug’. We propose that the asco-laccases utilize the C-terminal carboxylic group in proton transfer processes, as has been suggested for Glu498 in the CotA laccase from Bacillus subtilis. The crystal structure of TaLcc1 also shows the formation of a similar weak homodimer, as observed for MaL, that may determine the properties of these asco-laccases at high protein concentrations. Database Structural data are available in the Protein Data Bank database under the accession numbers 3PPS and 2VDZ Structured digital abstract • laccase binds to laccase by x-ray crystallography (View interaction)

Lignin‐derived inhibition is a major obstacle restricting the enzymatic hydrolysis of cell wall polysaccharides especially with softwood lignocellulosics. Enzyme adsorption on lignin is suggested to contribute to the inhibitory effect of lignin. The interaction of cellulases with softwood lignin was studied in the present work with commercial Trichoderma reesei cellulases (Celluclast) and lignin‐rich residues isolated from steam pretreated softwood (SPS) by enzymatic and acid hydrolysis. Both lignin preparations inhibited the hydrolysis of microcrystalline cellulose (Avicel) and adsorbed the major cellulases present in the commercial cellulase mixture. The adsorption phenomenon was studied at low temperature (4°C) and at the typical hydrolysis temperature (45°C) by following activities of free and lignin‐bound enzymes. Severe inactivation of the lignin‐bound enzymes was observed at 45°C, however at 4°C the enzymes retained well their activity. Furthermore, SDS–PAGE analysis of the lignin‐bound enzymes indicated that very strong interactions form between the residue and the enzymes at 45°C, because the enzymes were not released from the residue in the electrophoresis. These results suggest that heat‐induced denaturation may take place on the surface of softwood lignin at the hydrolysis temperature.

Sulfhydryl oxidases have found application in the improvement of both dairy and baking products due to their ability to oxidise thiol groups in small molecules and cysteine residues in proteins. A genome mining study of the available fungal genomes had previously been performed by our group in order to identify novel sulfhydryl oxidases suitable for industrial applications and a representative enzyme was produced, AoSOX1 from Aspergillus oryzae (Faccio et al. BMC Biochem 11:31, 2010). As a result of the study, a second gene coding for a potentially secreted sulfhydryl oxidase, AoSOX2, was identified in the genome of A. oryzae. The protein AoSOX2 was heterologously expressed in Trichoderma reesei and characterised with regard to both biochemical properties as well as preliminary structural analysis. AoSOX2 showed activity on dithiothreitol and glutathione, and to a lesser extent on D/L-cysteine and beta-mercaptoethanol. AoSOX2 was a homodimeric flavin-dependent protein of approximately 78 kDa (monomer 42412 Da) and its secondary structure presents alpha-helical elements. A. oryzae AoSOX2 showed a significant stability to pH and temperature.

The aim of this work was to study the potential of tyrosinase enzymes in structural engineering of acid-induced milk protein gels. Fat free raw milk, heated milk or a sodium caseinate solution were treated with tyrosinases from Trichoderma reesei (TrTyr) and Agaricus bisporus (AbTyr) and the reference enzyme transglutaminase (TG) prior to acid-induced gelation. TrTyr treatment increased the firmness of raw milk and sodium caseinate gels, but not that of heated milk gels, even though protein cross-linking was detected in heated milk. AbTyr did not cross-link proteins in any of the studied milk protein systems. TG was superior to TrTyr in gels prepared of heated milk. In acidified heated milk and sodium caseinate, TrTyr and TG treatment resulted in a decrease of the pore size. Scanning electron microscopy revealed more extensive particle interactions in the heated milk gels with TG than with TrTyr.

Growth of Irpex lacteus on polyurethane solid support led to secretion of various manganese-dependent peroxidase (MnP) isoenzymes with pI in the range of 3.8–6.7. The highest MnP activity was obtained at 8.9 mmol l−1 Mn2+ when predominantly a pI 4.8 isoenzyme was produced. Optimization of anion exchange chromatography (Mono Q) allowed the separation of MnP isoenzymes and collection of extensive sequence information by tandem LC–MS. The pI 4.8 isoenzyme was purified 109-fold (6% activity yield) by ion exchange chromatography, its specific activity of Mn2+ oxidation was 2800 nkat mg−1, molecular mass 37 kDa and pH optimum 5. The other properties were: respective half-lives (pH 4.5) of 575, 325 and 7 min at 40, 50 and 60 °C, Km values 46.7; 9.5; 21.4 μmol l−1 and kcat values 69.0; 64.2; 15.7 s−1 for Mn2+, H2O2 and DMP with Mn2+, respectively. The pI 4.8 isoenzyme was shown to be a true MnP affecting secondary substrates via the oxidation of Mn2+. Its biochemical properties and production by I. lacteus make the enzyme a candidate for biotechnological applications.

Different possibilities for protein crosslinking are examined in this review, with special emphasis on enzymatic crosslinking and its impact on food structure. Among potential enzymes for protein crosslinking are transglutaminase (TG) and various oxidative enzymes. Crosslinking enzymes can be applied in cereal, dairy, meat, and fish processing to improve the texture of the product. Most of the current commercial applications are based on TG. The reaction mechanisms of the crosslinking enzymes differ, which in turn results in different technological properties.

A homology search against public fungal genome sequences was performed to discover novel secreted tyrosinases. The analyzed proteins could be divided in two groups with different lengths (350–400 and 400–600 residues), suggesting the presence of a new class of secreted enzymes lacking the C-terminal domain. Among them, a sequence from Aspergillus oryzae (408 aa, AoCO4) was selected for production and characterization. AoCO4 was expressed in Trichoderma reesei under the strong cbh1 promoter. Expression of AoCO4 in T. reesei resulted in high yields of extracellular enzyme, corresponding to 1.5 g L−1 production of the enzyme. AoCO4 was purified with a two-step purification procedure, consisting of cation and anion exchange chromatography. The N-terminal analysis of the protein revealed N-terminal processing taking place in the Kex2/furin-type protease cleavage site and removing the first 51 amino acids from the putative N-terminus. AoCO4 activity was tested on various substrates, and the highest activity was found on 4-tert-butylcatechol. Because no activity was detected on L-tyrosine and on l-dopa, AoCO4 was classified as a catechol oxidase. AoCO4 showed the highest activity within an acidic and neutral pH range, having an optimum at pH 5.6. AoCO4 showed good pH stability within a neutral and alkaline pH range and good thermostability up to 60°C. The UV–visible and circular dichroism spectroscopic analysis suggested that the folding of the protein was correct.

Enzymatic modification of proteins, in order to produce functional materials such as hydrogels, adhesives and films via cross-linked networks or scaffolds of proteins, is a constantly evolving technology to create tailored micro- and nanostructured materials for food, cosmetic, and medical applications. For the successful utilization of oxidoreductases or transferases such as tyrosinases and transglutaminases, respectively, it is crucial to understand the action of these enzymes on protein substrates. In this study, cross-linking of the milk protein β-casein by Trichoderma reesei tyrosinase (TrTyr) was studied using size-exclusion chromatography (SEC) equipped with multi-angle light scattering (MALLS) and ultraviolet/visible (UV/Vis) detectors in order to determine the molecular mass (MM), radius of gyration (RG) and degree of polymerization (DP) of the reaction products. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect early polymerization states. The widely used Streptoverticillium mobaraense transglutaminase (Tgase) was used for comparison to tyrosinase from T. reesei. The results showed that cross-linking of β-casein by these two different types of enzymes resulted in the formation of polymerized reaction products with MM ranging from 500 to 1700 kg mol−1 depending on the enzyme dosage and incubation time. The DP varied from 21 to 71, respectively. In the case of TrTyr the polymerized reaction products were slightly colored, and formation of the covalent cross-linking of β-casein could be monitored by UV/Vis as a function of incubation time.

The C-terminus of the fungal laccase from Melanocarpus albomyces (MaL) is processed during secretion at a processing site conserved among the ascomycete laccases. The three-dimensional structure of MaL has been solved as one of the first complete laccase structures. According to the crystal structure of MaL, the four C-terminal amino acids of the mature protein penetrate into a tunnel leading towards the trinuclear site. The C-terminal carboxylate group forms a hydrogen bond with a side chain of His140, which also coordinates to the type 3 copper. In order to analyze the role of the processed C-terminus, site-directed mutagenesis of the MaL cDNA was performed, and the mutated proteins were expressed in Trichoderma reesei and Saccharomyces cerevisiae. Changes in the C-terminus of MaL caused major defects in protein production in both expression hosts. The deletion of the last four amino acids dramatically affected the activity of the enzyme, as the deletion mutant delDSGL559 was practically inactive. Detailed characterization of the purified L559A mutant expressed in S. cerevisiae showed the importance of the C-terminal plug for laccase activity, stability, and kinetics. Moreover, the crystal structure of the L559A mutant expressed in S. cerevisiae showed that the C-terminal mutation had clearly affected the trinuclear site geometry. The results in this study clearly confirm the critical role of the last amino acids in the C-terminus of MaL.

Trametes hirsuta laccase (EC 1.10.32) alone was not able to cross-link proteins effectively unless high dosages were used. Incorporation of ferulic acid enhanced the formation of intermolecular cross-links. Cross-linking was more effective when the reaction was carried out at 45 °C rather than at room temperature. Size exclusion chromatography showed that ferulic acid monomers disappeared from the solution in the presence of laccase. Force at rupture point of the caseinate gels treated with laccase without ferulic acid was not significantly different from the control, while stronger gels were achieved when ferulic acid was present. Rheological measurements showed that laccase treatment together with ferulic acid resulted in higher G′ values than the control. Gels were analyzed with confocal laser scanning microscope. A finer network was observed when the enzyme was used together with ferulic acid. Slight proteolytic activity resulted in a negative effect on the gel firmness and microstructure when laccase was used without ferulic acid.

We have solved a crystal structure from Melanocarpus albomyces laccase expressed in the filamentous fungus Trichoderma reesei (rMaL) at 1.3 Å resolution by using synchrotron radiation at 100 K. At the moment, this is the highest resolution that has been attained for any multicopper oxidase. The present structure confirmed our earlier proposal regarding the dynamic behaviour of the copper cluster. Thermal ellipsoids of copper atoms indicated movements of trinuclear site coppers. The direction of the type-3 copper motion was perpendicular to the type-2 copper. In addition, the structure at 1.3 Å resolution allowed us to describe important solvent cavities of the enzyme and the structure is also compared with other known multicopper oxidases. T2 and T3 solvent cavities, and a putative SDS-gate, formed by Ser142, Ser510 and the C-terminal Asp556 of rMaL, are described. We also observed a 2-oxohistidine, an oxidized histidine, possibly caused by a metal-catalysed oxidation by the trinuclear site coppers. To our knowledge, this is the first time that 2-oxohistidine has been observed in a protein crystal structure.

Cross-linking enzymes generate covalent bonds in and between food biopolymers. These enzymes are interesting tools for tailoring dough and bread structures, as the characteristics of the biopolymers significantly determine the viscoelastic and fracture properties of dough and bread. In this study, the influence of oxidative cross-linking enzymes, tyrosinase from the filamentous fungus Trichoderma reesei and laccase from the white rot fungus Trametes hirsuta, on dough and bread were examined. Oxidation of low molecular weight phenolic model compounds of flour, cross-linking of gluten proteins, dough rheology, and bread making were characterized during or after the enzymatic treatments. In the dough and bread experiments, laccase and tyrosinase were also studied in combination with xylanase. Of the model compounds tyrosine, p-coumaric acid, caffeic acid, ferulic acid, and Gly-Leu-Tyr tripeptide, tyrosinase oxidized all except ferulic acid. Laccase was able to oxidize each of the studied compounds. The phenolic acids were notably better substrates for laccase than L-tyrosine. When the ability of the enzymes to cross-link isolated gliadin and glutenin proteins was studied by the SDS-PAGE analysis, tyrosinase was found to cross-link the gliadin proteins effectively, whereas polymerization of the gliadins by laccase was observed only when a high enzyme dosage and prolonged incubation were used. Examination of large deformation rheology of dough showed that both laccase and tyrosinase made doughs harder and less extensible, and the effects increased as a function of the enzyme dosage. In bread making, interestingly, the pore size of the breads baked with tyrosinase turned out to be remarkably larger and more irregular when compared to that of the other breads. Nevertheless, both of the oxidative enzymes were found to soften the bread crumb and increase the volume of breads, and the best results were achieved in combination with xylanase.

The capability of Agaricus bisporus tyrosinase to catalyze the oxidation of tyrosine residues of silk sericin was studied under homogeneous reaction conditions, by using sericin peptides purified from industrial wastewater as the substrate. Tyrosinase was able to oxidize about 57% of sericin-bound tyrosine residues. The reaction rate was higher than with silk fibroin, but lower than with other silk-derived model peptides, i.e. tryptic and chymotryptic soluble peptide fractions of silk fibroin, suggesting that the size and the molecular conformation of the substrate influenced the kinetics of the reaction. The concentration of tyrosine in oxidized sericin samples decreased gradually with increasing the enzyme-to-substrate ratio. The average molecular weight of sericin peptides significantly increased by oxidation, indicating that cross-linking occurred via self-condensation of o-quinones and/or coupling with the free amine groups of lysine and, probably, with sulfhydryl groups of cysteine. The high temperature shift of the main thermal transitions observed in the differential scanning calorimetry curves confirmed the formation of peptide species with higher molecular weight and higher thermal stability. Fourier transform-infrared spectra of oxidized sericin samples showed slight changes related to the loss of tyrosine and formation of oxidation products. Oxidized sericin peptides were able to undergo non-enzymatic coupling with chitosan. Infrared spectra provided clear evidence of the formation of sericin–chitosan bioconjugates under homogeneous reaction conditions. Spectral changes in the NH stretching region seem to support the formation of bioconjugates via the Michael addition mechanism.

The effects of Trichoderma reesei tyrosinase-catalyzed cross-linking of isolated chicken breast myofibril proteins as a simplified model system were studied with special emphasis on the thermal stability and gel formation of myofibrillar proteins. In addition, tyrosinase-catalyzed cross-linking was utilized to modify the firmness, water-holding capacity (WHC), and microstructure of cooked chicken breast meat homogenate gels. According to SDS-PAGE, the myosin heavy chain (MHC) and troponin T were the most sensitive proteins to the action of tyrosinase, whereas actin was not affected to the same extent. Calorimetric enthalpy (DeltaH) of the major thermal transition associated with myosin denaturation was reduced and with actin denaturation increased in the presence of tyrosinase. Low-amplitude viscoelastic measurements at constant temperatures of 25 degrees C and 40 degrees C showed that tyrosinase substantially increased the storage modulus (G') of the 4% myofibrillar protein suspension in the 0.35 M NaCl concentration. The effect was the most pronounced with high-enzyme dosages and at 40 degrees C. Without tyrosinase, the G' increase was low. Tyrosinase increased the firmness of the cooked phosphate-free and low-meat chicken breast meat homogenate gels compared to the corresponding controls. Tyrosinase maintained gel firmness at the control level of the low-salt homogenate gel and weakened it when both salt and phosphate levels were low. Tyrosinase improved the WHC of the low-meat and low-salt homogenate gels and maintained it at the level of the corresponding controls of phosphate-free and low-salt/low-phosphate homogenate gels. Microstructural characterization showed that a collagen network was formed in the presence of tyrosinase.Chicken myofibrillar proteins; protein modification; cross-linking; tyrosinase; gelation; thermal stability; texture; water-holding capacity; microstructure.