Browsing by Subject "entsyymi"

Microbial cellulases, e.g. cellobiohydrolases, are able to degrade cellulose and lignocellulosic biomass to smaller glucose-containing monomers and oligomers. Cellulases are often multi-domain enzymes comprised of different protein domains (i.e. modules), which have different functions. The main two components, which often appear in cellulases, are the cellulose-binding module (CBM) and the catalytic domain. The CBMs bind to cellulose, bringing the catalytic domains close to their substrate and increasing the amount of enzymes on the substrate surface. The catalytic domain performs the cleavage of the substrate, e.g. in the case of cellobiohydrolases hydrolyses or “cuts” the crystalline cellulose chain into smaller soluble saccharides, mainly cellobiose. Unlike aerobic fungi, which utilize free extracellular enzymes to break down cellulose, anaerobic microbes often use a different kind of strategy. Their cellulases are organized and bound to the cell surface in a macromolecular protein complex, the cellulosome. The core of the cellulosome is formed of a scaffolding protein (the scaffoldin) consisting mainly of multiple consecutive cohesin domains, into which the catalytic subunits of enzymes attach via a dockerin domain. This creates a protein complex with multiple different catalytic domains and activities arranged in close proximity to each other. Dockerins and cohesins are known to bind each other with one of the strongest receptor-ligand -pair forces known to nature. Dockerin containing fusion proteins have also been successfully combined in vitro with proteins containing their natural counterparts, cohesins, to create functional multiprotein complexes. In this Master’s thesis work the goal was to 1) produce fusion proteins in which different CBMs were connected to dockerin domains, 2) combine these fusions with cohesin-catalytic domain fusion proteins to create stable CBM and catalytic domain containing enzyme complexes, 3) to characterize these enzyme complexes in respect of their thermostability and cellulose hydrolysis capacity and 4) to ultimately create a robust and fast domain shuffling method for multi-domain cellobiohydrolases (CBH) to facilitate their faster screening. The hypothesis of the experiments was that different CBMs fused with a dockerin domain and the cellobiohydrolase catalytic domain fused with a cohesin domain could be produced separately and then be combined to produce a functional two-domain enzyme with a dockerin-cohesin “linker” in between. In this way time and work could be saved because not every different CBM- catalytic domain -pair would have to be cloned and produced separately. Several CBM-dockerin fusion proteins (in which the CBM were of fungal or bacterial origin) were tested for expression in heterologous hosts, either in Saccharomyces cerevisiae or Escherichia coli. The purified proteins were combined with a fungal glycoside hydrolase family 7 (GH7) cellobiohydrolase-cohesin fusion protein produced in S. cerevisiae. The characterization of the catalytic domain-CBM -complexes formed through cohesin-dockerin interaction included thermostability measurements using circular dichroism and activity assays using soluble and insoluble cellulosic substrate. The results were compared to enzyme controls comprising of the same CBM and catalytic domain connected by a simple peptide linker. The results showed that the cohesin-dockerin –linked cellobiohydrolase complex performed in the cellulose hydrolysis studies in a similar manner as the directly linked enzyme controls at temperature of 50˚C and 60 ˚C. At temperatures of 70 ˚C the complex did not perform as well as the control enzymes, apparently due to the instability of the dockerin-cohesin interaction. The thermostability measurements of the enzymes, together with the previously published data supported the hydrolysis results and this hypothesis. The future work should be aimed at enhancing the thermostability of the cohesin-dockerin interaction as well as on verifying the results on different cellulase fusion complexes.

Japanese quince (Chaenomeles japonica) is a fruit crop that can be used for example in the production of jams, marmalades, juices, soft drinks, and alcohol beverages. Pressing juice from Japanese quince fruits produces large amounts of pomace as a side stream. The aim of the study was to examine the chemical composition of Japanese quince cultivars that are being cultivated in Finland. Both fruits and fruit pomaces were analysed. The effect of enzyme-assisted water extraction on dry matter yield from Japanese quince pomace was also investigated. Fruits and pomaces of cultivars Sirius, Venus, and seed offspring of cultivar Cido were used as research material. Determination of chemical composition was conducted with the analyses of dry matter, lipids, dietary fibre, ash, protein, and carbohydrate content. Fatty acids were extracted with accelerated solvent extraction and were analysed with gas chromatography (GC-FID). With pomaces four different extractions were made: one with plain water extraction, one assisted with pectinase enzyme, one assisted with cellulase enzyme, and one assisted with both pectinase and cellulase enzymes. Dry matter yield of extracts was measured, and extracts were also filtered with ultrafiltration (UF). UF permeates were analysed with ELSD-UPLC to detect sugars and organic acids from the extracts. According to this study cultivar had a significant impact (p < 0.05) on dry matter, protein and lipid content and fatty acid composition of fruits. With pomaces there were also significant differences in dietary fibre contents. Most of the dietary fibre was insoluble dietary fibre. The main detected fatty acids were C16:0, C18:1 (n-9) and C18:2 (n-6). Water extraction assisted with both pectinase and cellulase enzymes had significantly higher extraction yield (29.5%) compared to water extraction assisted with cellulase enzyme (24.2%) and plain water extraction (26.4%) which did not differ statistically (p < 0.05). Extraction yield from pectinase assisted extraction (28.4%) differed only with the yield from cellulase assisted extraction (p < 0.05). All the permeates contained mostly malic acid. Fructose, glucose, and little amount of sucrose were also detected. There were only little differences between composition of permeates. Chemical composition of fruits may vary between growing seasons and thus, repeating the analyzes during several harvest seasons would provide more information on what kind of effect growing season has on fruit composition. This study also raised further questions on what kind of impact of pretreatments prior enzyme assisted extraction could have on the extraction yield.

There is a vast diversity of fungi of Finnish forests that include species of, for example, the wood decaying polypore Bacidiomycota, wood-inhabiting Ascomycota, tree-associated mycorrhizal fungi and litter-decaying fungi living on the top surface of the soil. These fungi have an influence to tree growth and carbon circulation in the forest ecosystems of the Northern Hemisphere. Brackets, aphyllophoroid fungi as well as some gilled mushrooms are used as indicator species in the conservation of forests, forest environments and meadows. Fungal community in wood is in a constant change. Some fungal species or isolates of the same species affect positively on others, and may increase the growth of mycelia. In contrast, some species may have negative effect. Depending on the nature of the vegetative interaction, the fungal species may be either strong or weak combetitors. A change in the combative situation can affect the outcome of the interaction greatly. Some species are strong combatant upon the interaction of two species. Upon interaction of several species, or between different isolates, the outcome may dramatically change, and a previously weaker combatant may prove to be strong. This study examined the influence of Fomitopsis pinicola, which is a common wood-decaying brown rot polypore species in Finnish forests, on the growth and enzyme production of five wood-decaying white rot fungal species. Activities of several wood-degrading enzymes (laccase, manganese peroxidase, xylanase, endoglucanase, β-glucosidase) were studied for individual species, and in co-cultures of various combinations of the species for eight weeks on liquid media including coniferous wood shavings. All co-cultures included F. pinicola, and either one or two additional species.The hyphal extension growth rate on malt agar medium was quantified for each species, and the fungal biomass increment (as a dry weight) and acidification of the growth liquid were measured from malt extract broth media cultures. F. pinicola proved to be by far supreme colonizer on malt agar and its hyphae were advancing over the mycelia of the white rot species studied. Phlebia radiata formed mycelial blocks against the other white rot fungi but not against F. pinicola. In most cases, acidification of the liquid medium proved to be beneficial for fungal growth (dry weight). Except for F. pinicola, acidification of the culture fluid was moderate in the single species cultivations, and in the co-cultivations including P. radiata. Number of fungal species had no clear effect on the enzyme activity values in the co-cultivations, yet P. radiata increased the activities of laccase and manganeseperoxidase. Also Trichaptum abietinum had an influence on laccase activity. In conclusion, in polyporous fungal co-cultivaltions, a few species had an impact to fungal growth and and production of wood-decaying enzymes when cultivated on coniferous wood.

The aim of the study was to compare the effect of xylanase and β-glucanase enzymes on feed conversion ratio and growth compared to the control group. The Master's thesis also aimed to assess whether the amount of enzyme supplementation had an effect on feed conversion ratio or growth. The study was performed as a meta-analysis with 12 studies for xylanase and 13 studies for β-glucanase. The data collected included the amount of feed eaten, weight gain, the amount of enzyme supplementation and a negative control group. The data from the data set were compiled in a spreadsheet (Microsoft 365 Excel), after which the feed conversion ratio was calculated for each experimental group in the data set. The experimental groups were compared to the negative control group of the data by dividing the feed efficiency ratios of the experimental groups by the negative control group of the data and the results were tabulated in a separate table. The control group for the meta-analysis was set at 100 and the results from the data were compared to this value. The results were consistent with previous studies. Xylanase and β-glucanase enzyme supplementation had positive effects on feed conversion ratio and broiler growth. Meta-analysis showed that the optimal supplementation level for xylanase enzyme is slightly higher than for β-glucanase. On the other hand, there was some variation in the results between feed efficiency ratios, which may be explained by the main feed ingredients, environmental factors, or the breed of birds. Both xylanase and β-glucanase supplementation in feed improved feed utilization compared to the control group in the meta-analysis, which also supported the hypothesis of the study. So far, relatively little is known about the interactions between the different enzyme supplements and therefore more studies on the optimal level of enzyme supplementation should be conducted. However, the meta-analysis provided guidelines that the use of enzymes has positive effects on both animal welfare and feed efficiency. In conclusion, the use of enzymes in broiler feeding should be continued to improve bird performance, intestinal health, and feed efficiency.