Browsing by Author "Kruus, Kristiina"

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Beyond the Surface: A Methodological Exploration of Enzyme Impact along the Cellulose Fiber Cross-Section
(2024-05-13) Sulaeva, Irina; Sto̷pamo, Fredrik Gjerstad; Melikhov, Ivan; Budischowsky, David; Rahikainen, Jenni L.; Borisova, Anna; Marjamaa, Kaisa; Kruus, Kristiina; Eijsink, Vincent G.H.; Várnai, Anikó; Potthast, Antje
A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
Despite the wide range of analytical tools available for the characterization of cellulose, the in-depth characterization of inhomogeneous, layered cellulose fiber structures remains a challenge. When treating fibers or spinning man-made fibers, the question always arises as to whether the changes in the fiber structure affect only the surface or the entire fiber. Here, we developed an analysis tool based on the sequential limited dissolution of cellulose fiber layers. The method can reveal potential differences in fiber properties along the cross-sectional profile of natural or man-made cellulose fibers. In this analytical approach, carbonyl groups are labeled with a carbonyl selective fluorescence label (CCOA), after which thin fiber layers are sequentially dissolved with the solvent system DMAc/LiCl (9% w/v) and analyzed with size exclusion chromatography coupled with light scattering and fluorescence detection. The analysis of these fractions allowed for the recording of the changes in the chemical structure across the layers, resulting in a detailed cross-sectional profile of the different functionalities and molecular weight distributions. The method was optimized and tested in practice with LPMO (lytic polysaccharide monooxygenase)-treated cotton fibers, where it revealed the depth of fiber modification by the enzyme.
Characterization of sulfhydryl oxidase from Aspergillus tubingensis
(2017-12-08) Nivala, Outi; Faccio, Greta; Arvas, Mikko; Permi, Perttu; Buchert, Johanna; Kruus, Kristiina; Mattinen, Maija Liisa
A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
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. 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.
Effect of cellulase family and structure on modification of wood fibres at high consistency
(2019-05) Rahikainen, Jenni; Ceccherini, Sara; Molinier, Matthieu; Holopainen-Mantila, Ulla; Reza, Mehedi; Väisänen, Saija; Puranen, Terhi; Kruus, Kristiina; Vuorinen, Tapani; Maloney, Thaddeus; Suurnäkki, Anna; Grönqvist, Stina
A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
Abstract: 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.].
The effect of heat and transglutaminase treatment on emulsifying and gelling properties of faba bean protein isolate
(2021-03) Nivala, Outi; Nordlund, Emilia; Kruus, Kristiina; Ercili-Cura, Dilek
A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
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 – 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.
Effect of laccase and xylanase on the structure formation of wheat flour and gluten doughs
(2004) Selinheimo, Emilia
Helsinki University of Technology | Master's thesis
In the literature survey, properties of wheat flour and dough as well as the structure formation of wheat bread were discussed, with special emphasis on rheology. Also the theory and a few measurement methods of the rheology were shortly introduced. Considering the influence of enzymes on bread making, a structure formation by an oxidative mechanism was discussed. The enzymes that were viewed more precisely on their effect on the structure formation of wheat bread included lipoxygenase, glucose oxidase, peroxidase, laccase and transglutaminase. In the experimental part of the study, the effect of Aspergillus oryzae and Bacillus subtilis xylanases and Trametes hirsuta and Melanocarpus albomyces laccases, separately and together, on the structure of wheat flour dough and gluten dough were examined. The experiments of added free ferulic acid (FA) were done to clarify the role of FA in dough structure formation. The rheological experiments were performed with Kieffer dough and gluten extensibility rig, when the dough extensibility Ex and the resistance to stretching Rmax, were determined. In a rheology point of view, laccases increased the maximum resistance Rmax of dough and decreased the dough extensibility Ex at Rmax, whereas xylanases decreased the Rmax of dough and increased the Ex at Rmax in flour and gluten doughs. Considering the protein-AX fraction, hardening by laccases and softening by xylanases were weaker in gluten doughs, which was presumably due to the lower AX content in gluten. Like xylanases, the added free FA softened the flour dough structure, as well. As a function of dough resting time, the structure of laccase treated doughs was observed to soften. The softening effect was strengthened as a function of laccase activity. The reason for the softening phenomenon was probably the laccase-mediated depolymerisation of cross-linked arabinoxylan network, resulting from mobile FA radicals. Combined laccase and xylanase experiments led to doughs of higher Rmax, with no remarkable change in Ex. The effect of laccase seemed to be predominant, especially at low xylanase dosages, but when xylanase was added to flour dough at high concentration, the hardening effect of laccase on dough was decreased. Presumably, when the AX fraction was hydrolyzed effectively by xylanase, laccase was not able to create a strong AX network. Similar decrease in laccase mediated hardening in doughs was not seen, when performing the combined laccase and xylanase test with gluten, which was presumably due to low AX content of gluten. The results indicated the critical role of feruloylated arabinoxylan fraction in laccase-catalysed structure formation both in flour and gluten, although in gluten doughs, protein fraction might have also been affected.
The effect of soluble phenolic compounds from hydrothermally pretreated wheat straw on Trichoderma reesei cellulases and commercial enzyme cocktails
(2024-01) Borisova, Anna S.; Pihlajaniemi, Ville; Kont, Riin; Niemelä, Klaus; Koitto, Taru; Mikkelson, Atte; Väljamäe, Priit; Kruus, Kristiina; Marjamaa, Kaisa
A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
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.
High consistency mechano-enzymatic pretreatment for kraft fibres: effect of treatment consistency on fibre properties
(2020-06) Rahikainen, Jenni; Mattila, Outi; Maloney, Thaddeus; Lovikka, Ville; Kruus, Kristiina; Suurnäkki, Anna; Grönqvist, Stina
A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
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.
The impact of the carbohydrate-binding module on how a lytic polysaccharide monooxygenase modifies cellulose fibers
(2024-08-24) Støpamo, Fredrik G.; Sulaeva, Irina; Budischowsky, David; Rahikainen, Jenni; Marjamaa, Kaisa; Kruus, Kristiina; Potthast, Antje; Eijsink, Vincent G.H.; Várnai, Anikó
A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
Background: In recent years, lytic polysaccharide monooxygenases (LPMOs) that oxidatively cleave cellulose have gained increasing attention in cellulose fiber modification. LPMOs are relatively small copper-dependent redox enzymes that occur as single domain proteins but may also contain an appended carbohydrate-binding module (CBM). Previous studies have indicated that the CBM “immobilizes” the LPMO on the substrate and thus leads to more localized oxidation of the fiber surface. Still, our understanding of how LPMOs and their CBMs modify cellulose fibers remains limited. Results: Here, we studied the impact of the CBM on the fiber-modifying properties of NcAA9C, a two-domain family AA9 LPMO from Neurospora crassa, using both biochemical methods as well as newly developed multistep fiber dissolution methods that allow mapping LPMO action across the fiber, from the fiber surface to the fiber core. The presence of the CBM in NcAA9C improved binding towards amorphous (PASC), natural (Cell I), and alkali-treated (Cell II) cellulose, and the CBM was essential for significant binding of the non-reduced LPMO to Cell I and Cell II. Substrate binding of the catalytic domain was promoted by reduction, allowing the truncated CBM-free NcAA9C to degrade Cell I and Cell II, albeit less efficiently and with more autocatalytic enzyme degradation compared to the full-length enzyme. The sequential dissolution analyses showed that cuts by the CBM-free enzyme are more evenly spread through the fiber compared to the CBM-containing full-length enzyme and showed that the truncated enzyme can penetrate deeper into the fiber, thus giving relatively more oxidation and cleavage in the fiber core. Conclusions: These results demonstrate the capability of LPMOs to modify cellulose fibers from surface to core and reveal how variation in enzyme modularity can be used to generate varying cellulose-based materials. While the implications of these findings for LPMO-based cellulose fiber engineering remain to be explored, it is clear that the presence of a CBM is an important determinant of the three-dimensional distribution of oxidation sites in the fiber.
Inhibitory effect of lignin on the hydrolysis of xylan by thermophilic and thermolabile GH11 xylanases
(2022-05-14) Kellock, Miriam; Rahikainen, Jenni; Borisova, Anna S.; Voutilainen, Sanni; Koivula, Anu; Kruus, Kristiina; Marjamaa, Kaisa
A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
Background: 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. Results: 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. Conclusions: 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. Graphical Abstract: [Figure not available: see fulltext.]
The mechanism of cellulase adsorption on isolated lignin fractions
(2010) Mikander, Saara
School of Chemical Engineering | Master's thesis
Unproductive binding of cellulolytic enzymes to lignin is one of the restricting factors in the cost-effective production of lignocelluloses bio ethanol. The aim of the present work was to study the mechanism of cellulase adsorption on lignin. Lignin was isolated from steam pre-treated spruce and wheat straw both enzymatically (EnzP-lignin) and by alkali extraction (alkali-lignin). The adsorption of a thermostable Melanocarpus albomyces endoglucanase Ce145A and its fusion proteins containing a native or mutated carbohydrate binding module (CBM) and an associated linker peptide from Trichoderma reesei Ce17A was studied by quantifying the amount of unadsorbed protein using SDS-PAGE combined with a Criterion Stain Free Gel Imaging system. The most interesting variants were purified to homogeneity and used for the construction of Langmuir adsorption isotherms. The adsorption followed the Langmuir isotherm relatively well. EnzP-lignin from spruce showed higher adsorption capacity (GAMMA_max=1.66 - 3.31 µmol/g lignin) compared to wheat straw EnzP-lignin (GAMMA_max=0.38 - 74 µmol/g lignin). Both alkali-lignins adsorbed the enzymes very efficiently and the differences between the enzymes were so small that reliable conclusions could not be made. The CBM with the linker peptide increased significantly the adsorption of Ce145A on EnzP-lignin's. In contrast, the replacement of tyrosine amino acids (Y31, Y32) in the CBM by neutral alanines decreased the level of adsorption. Amino acids Y31 and Y32 have been shown to be crucial in the binding of the CBM to crystalline cellulose. The replacement of the tyrosine residue by a more hydrophobic tryptophan at position 31 increased the adsorption of the enzyme on EnzP-lignins. Furthermore, a deletion removing practically all of the linker peptide significantly decreased the binding of the enzyme to lignin. The results strongly indicate that the CBM has a significant effect on the adsorption of the enzymes on lignin and that the CBM binds to lignin mainly with a similar mechanism which binds it to crystalline cellulose.
New fungal enzymes for total hydrolysis of biomass
(2011) Luode, Roosa
School of Chemical Engineering | Master's thesis
Enzymatic hydrolysis of lignocellulosic biomass is one of the bottlenecks in feasible production of lignocellulosic ethanol. The aim of this thesis was to discover novel lignocellulose-degrading enzymes that could enhance the hydrolysis performance of existing enzyme mixtures in the total hydrolysis of biomass. The objective was to screen novel fungal strains secreting enzymes which improve the hydrolytic efficiency of known enzyme mixtures. First, the strains (numbered from 1 to 31) were screened on the basis of their secreted cellulolytic and hemicellulolytic enzyme activities and their performance in the hydrolysis of microcrystalline cellulose and hydrothermally pre-treated wheat straw. Second, synergistic effects were studied between the most interesting fungal culture supernatants and two known enzyme mixtures (a commercial enzyme product Celluclast produced by the fungus Trichoderma reesei and a thermostable enzyme mixture developed in the HYPE-project) in standardised hydrolysis of cellulose and pre-treated wheat straw. Detailed enzyme activity profiles were determined in the most interesting culture supernatants selected on the basis of the synergy study. Several fungal culture supernatants enhanced the hydrolytic performance of the known enzyme mixtures. Clearest synergistic effects with T. reesei Celluclast and the thermo enzyme mixture were observed with the culture supernatants of strains 2, 6, 19, 22 and 27. The cultures of strains 22 and 27 showed the best overall synergy with both enzyme mixtures in the hydrolysis of both substrates. The cultures of strains 6 and 19 showed remarkable synergy with Celluclast in the hydrolysis of wheat straw, whereas the culture of strain 2 acted synergistically with the thermo enzyme mixture, especially in the hydrolysis of wheat straw. High xylanase activity was detected in the cultures of strains 2, 22 and 27, which could at least partly explain their good synergistic effects in the hydrolysis of wheat straw. The culture of strain 27 showed a versatile enzyme profile including high xylanase, endoglucanase and ß-xylosidase activities. Altogether, high xylanase activity appeared to be at least partly responsible for the high level of synergy between the novel fungal broths and the known enzyme mixtures. Several interesting strains secreting enzymes having clear synergistic effects with existing enzyme preparations were discovered in this work.
A novel approach to analyze the impact of lytic polysaccharide monooxygenases (LPMOs) on cellulosic fibres
(2024-03-15) Sulaeva, Irina; Budischowsky, David; Rahikainen, Jenni; Marjamaa, Kaisa; Støpamo, Fredrik Gjerstad; Khaliliyan, Hajar; Melikhov, Ivan; Rosenau, Thomas; Kruus, Kristiina; Várnai, Anikó; Eijsink, Vincent G.H.; Potthast, Antje
A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
Enzymatic treatment of cellulosic fibres is a green alternative to classical chemical modification. For many applications, mild procedures for cellulose alteration are sufficient, in which the fibre structure and, therefore, the mechanical performance of cellulosic fibres are preserved. Lytic polysaccharide monooxygenases (LPMOs) bear a great potential to become a green reagent for such targeted cellulose modifications. An obstacle for wide implementation of LPMOs in tailored cellulose chemistry is the lack of suitable techniques to precisely monitor the LPMO impact on the polymer. Soluble oxidized cello-oligomers can be quantified using chromatographic and mass-spectrometric techniques. A considerable portion of the oxidized sites, however, remain on the insoluble cellulose fibres, and their quantification is difficult. Here, we describe a method for the simultaneous quantification of oxidized sites on cellulose fibres and changes in their molar mass distribution after treatment with LPMOs. The method is based on quantitative, heterogeneous, carbonyl-selective labelling with a fluorescent label (CCOA) followed by cellulose dissolution and size-exclusion chromatography (SEC). Application of the method to reactions of seven different LPMOs with pure cellulose fibres revealed pronounced functional differences between the enzymes, showing that this CCOA/SEC/MALS method is a promising tool to better understand the catalytic action of LPMOs.
A novel Colletotrichum graminicola raffinose oxidase in the AA5 family
(2017) Andberg, Martina; Mollerup, Filip; Parikka, Kirsti; Koutaniemi, Sanna; Boer, Harry; Juvonen, Minna; Master, Emma; Tenkanen, Maija; Kruus, Kristiina
A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
We describe here the identification and characterization of a copper radical oxidase from auxiliary activities family 5 (AA5_2) that was distinguished by showing preferential activity toward raffinose. Despite the biotechnological potential of carbohydrate oxidases from family AA5, very few members have been characterized. The gene encoding raffinose oxidase from Colletotrichum graminicola (CgRaOx; EC 1.1.3.-) was identified utilizing a bioinformatics approach based on the known modular structure of a characterized AA5_2 galactose oxidase. CgRaOx was expressed in Pichia pastoris, and the purified enzyme displayed the highest activity on the trisaccharide raffinose, whereas the activity on the disaccharide melibiose was three times lower and more than ten times lower activity was detected on D-galactose at a 300 mM substrate concentration. Thus, the substrate preference of CgRaOx was distinguished clearly from the substrate preferences of the known galactose oxidases. The site of oxidation for raffinose was studied by 1H nuclear magnetic resonance and mass spectrometry, and we confirmed that the hydroxyl group at the C-6 position was oxidized to an aldehyde and that in addition uronic acid was produced as a side product. A new electrospray ionization mass spectrometry method for the identification of C-6 oxidized products was developed, and the formation mechanism of the uronic acid was studied. CgRaOx presented a novel activity pattern in the AA5 family.
Optimization of the ray fungus fermentation
(1985) Kruus, Kristiina
Helsinki University of Technology | Master's thesis
Oxidation of cellulose fibers using LPMOs with varying allomorphic substrate preferences, oxidative regioselectivities, and domain structures
(2024-04-15) Støpamo, Fredrik G.; Sulaeva, Irina; Budischowsky, David; Rahikainen, Jenni; Marjamaa, Kaisa; Potthast, Antje; Kruus, Kristiina; Eijsink, Vincent G.H.; Várnai, Anikó
A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
Lytic polysaccharide monooxygenases (LPMOs) are excellent candidates for enzymatic functionalization of natural polysaccharides, such as cellulose or chitin, and are gaining relevance in the search for renewable biomaterials. Here, we assessed the cellulose fiber modification potential and catalytic performance of eleven cellulose-active fungal AA9-type LPMOs, including C1-, C4-, and C1/C4-oxidizing LPMOs with and without CBM1 carbohydrate-binding modules, on cellulosic substrates with different degrees of crystallinity and polymer chain arrangement, namely, Cellulose I, Cellulose II, and amorphous cellulose. The potential of LPMOs for cellulose fiber modification varied among the LPMOs and depended primarily on operational stability and substrate binding, and, to some extent, also on regioselectivity and domain structure. While all tested LPMOs were active on natural Cellulose I-type fibers, activity on the Cellulose II allomorph was almost exclusively detected for LPMOs containing a CBM1 and LPMOs with activity on soluble hemicelluloses and cello-oligosaccharides, for example NcAA9C from Neurospora crassa. The single-domain variant of NcAA9C oxidized the cellulose fibers to a higher extent than its CBM-containing natural variant and released less soluble products, indicating a more dispersed oxidation pattern without a CBM. Our findings reveal great functional variation among cellulose-active LPMOs, laying the groundwork for further LPMO-based cellulose engineering.
Screening for microbes producing phenoloxidases
(2000) Paasivirta, Laura-Leena
Helsinki University of Technology | Master's thesis
Screening for microbes producing tyrosinase
(2003) Aurio, Jani
Helsinki University of Technology | Master's thesis
Tämän työn tarkoituksena oli seuloa tyrosinaaseja tuottavia mikrobeja VTT:n kantakokoelmasta. Työssä kehitettiin maljatesteihin perustuva tyrosinaasiaktiivisuuden seulontamenetelmä. Positiivisen maljaindikaation antaneilla mikrobeilla suoritettiin nestemäisiä ravistelukasvatuksia. Työn kirjallisuusosassa käsitellään tyrosinaasientsyymiä, sen rakennetta, katalysoimia reaktioita ja tehtäviä. Tyrosinaasilla on esim. elintarvike-, lääke- ja tekstiiliteollisuudessa monia mielenkiintoisia sovelluksia. Tutkimusosassa kehitettiin maljatesteihin perustuva seulontamenetelmä tyrosinaasipositiivisten mikrobien seulontaan. Tyrosinaasin tuotto havaittiin indikaattorien värireaktiona. Indikaattoreina käytettiin L-tyrosinaasia, p-kresolia, tyramiinia, p-kumariinihappoa, 2-amino-3-hydroksibentsoehappoa ja (+)-katekiinia. Tällä menetelmällä seulottiin VTT:n kantakokoelmasta 14 eri homekantaa ja 18 eri bakteerikantaa. Seulotuista homekannoista 11 ja bakteerikannoista kolme oli tyrosinaasipositiivisia. Uusia tuottajia löydettiin kuusi, viisi hometta ja yksi bakteeri. Positiiviseksi todettuja kantoja kasvatettiin nestemäisillä alustoilla. Tyrosinaasin tuottoa seurattiin aktiivisuusmittauksilla sekä solun sisä- että ulkopuolelta. Tyrosinaasin tuotto seulonnassa löytyneillä uusilla kannoilla osoittautui jatkotutkimusten kannalta vaikeaksi, sillä entsyymien tuottotasot jäivät alhaisiksi.