Browsing by Subject "Biotekniikka (EYT)"

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  • Teikari, Jonna (Helsingfors universitet, 2013)
    Cyanobacteria are phototrophic organisms. They usually occur in water but many species also live in terrestrial habitats, e.g. in symbiotic relationships with fungus. Inorganic phosphorus (Pi) is usually considered to be a limiting factor for the growth of cyanobacteria living in water, since cyanobacteria can use only Pi as a direct source of phosphorus. It has been shown that cyanobacteria have pho-regulon similar to that of Escherichia coli. Pho-regulon can transport and assimilate inorganic phosphate. Cyanobacteria usually produce a wide range of bioactive compounds, which can e.g. be toxic or prevent growth of other bacteria, fungi or yeast. Many of these compounds are produced by non-ribosomal peptide synthetases (NRPS). The aim of this study was to investigate changes in Anabaena sp. 90 proteome, and differences in amounts of bioactive compounds produced by the strain, while growing it in media with high (5,5 mg/l) or low (0,05 mg/ml) Pi concentration. Before moving the culture into two different Pi media, phosphorus storages of the culture were emptied by growing the strain in the media without Pi. In this study, 2D differential gel electrophoresis (2D-DIGE) coupled with LC/MS was used to study proteomes of the organism. Bioactive compounds were also analyzed by LC/MS. Anabaena sp. 90 was chosen because of its fully sequenced and annotated genome. The strain has been found to produce microcystins, anabaenopeptins and anabaenopeptilides. Eleven protein spots with significantly increased or decreased protein quantities were identified in the low Pi media. Ten of them were identified as proteins, which participate in bacterial stringent response. Stringent response is activated when culture is achieving the stationary phase. These stringent response proteins participate in the amino-acid metabolism and translation. One of the proteins was shown to be a ribosomal protein. In addition, the identified proteins included ribulose-bisphosphate carboxylase oxygenase (RuBisCO), which had a significantly lower concentration in the cells in low-phosphorous media. There were no significant differences in amounts of bioactive compounds when growing the culture in low and high Pi media. More replicates could be used, when the study of bioactive compounds is repeated.
  • Huang, Liyang (Helsingfors universitet, 2016)
    Plant biomass consists largely of polymeric compounds of which diverse polysaccharides are the main components. Ferulic acid is a ubiquitous phenolic phytochemical in plant cell wall and forms the linkage between plant cell wall polymers. Therefore it is a major aspect in the recalcitrance of cell wall against microbial attack. Ferulic acid esterases (FAEs) are hydrolytic enzymes which participate in plant biomass degradation by removing ferulic acid from the polysaccharides in order to weaken the integrity of the cell wall. By using phylogenetic gene prediction strategy, three putative FAE gene models have been detected from the genome of the ascomycete fungus Aspergillus niger. The codon optimized putative FAE encoding genes have been synthetized for heterologous production in Pichia pastoris GS115. In this work, these three FAEs of A. niger, i.e. FAE796, FAE807 and FAE809, were produced in P. pastoris and their biochemical properties were characterized. The properties included substrate profiling, thermostability, pH optimum and solvent tolerance of the recombinant FAEs. The three A. niger FAEs were successfully produced in P. pastoris resulting as approximately 57 kDa molecular mass proteins. Substrate profiling was performed by using a set of 11 synthetic FAE model substrates and substrates for tannase and lipase activity. FAE796 and FAE809 preferred methoxy substrates, and thus were likely to belong to type A class of FAEs. FAE807 had activity towards a wider range of substrates including methyl sinapate, methyl cinnamate, chlorogenic acid and para-nitrophenyl ferulate, suggesting it to belong to type C class of FAEs. In addition, FAE807 had tannase activity which is a novel property described among the FAEs studied so far. The optimal temperature for FAE796, FAE807 and FAE809 were +37 °C, +55 °C and +55 °C, respectively. FAE809 was the most thermostable enzyme, and retained half of its activity up to +60 °C for 60 min. The studied FAEs were most active at pH 4.0-5.0. FAE809 was relatively stable towards the studied solvents retaining 70%-91% of its activity after solvent treatment.
  • Rouvinen, Vilma-Riitta (Helsingfors universitet, 2010)
    Psychrotrophs, which are mostly gram negative bacteria, can produce heat stable proteases and lipases. Even though these bacteria cannot survive pasteurization of milk, the enzymes can. This can cause problems for the dairy industry. Psychrotrophic milk isolates also have multidrug-resistant traits for antimicrobials and may act as reservoir for resistance genes. The aims of the study were to test denaturing gradient gel electrophoresis (DGGE) for PCR products obtained from organic and conventional raw milk bacteria, to optimize DNA-extraction protocols and PCR-conditions for the raw milk samples and study the bacterial population changes during the cold storage. The aim was also to study the antimicrobial susceptibility of the bacterial isolates. The DNA was extracted from the raw milk samples, when received and after 4 days cold storage, using commercial kits. Nested-PCR was performed and samples were analysed using DGGE. Susceptibility to antimicrobials was determined by growing bacteria on plates that contained two different concentrations of five antibiotics. From the five antibiotics two were used as a combination. The composition of the bacterial population changed during the cold (4ºC) storage: the difference in DGGE profiles was clear between 0 and 4 days cold storage. Fingerprint profile analysis showed that irrespective of the origin of the raw milk, the sample profiles were clustered according to the sampling date (day 0 or day 4). There was no clear difference between DGGE-profiles from conventional and organic milk. Proportion of psychrotrophic bacteria increased and antimicrobial resistance seemed to be more prevailing in conventional than in organic raw milk. Antimicrobial resistance decreased after four days storage at 4ºC, in most of the cases. We showed that the PCR-DGGE-method is an efficient tool to analyse the changes in bacterial populations in raw milk and that cold storage has an evident effect in population composition.
  • Alexanova, Anna (Helsingfors universitet, 2016)
    Long QT syndrome, LQTS, is a congenital or acquired cardiac disorder characterized by prolonged cardiac repolarization phase. It is observed as a prolonged QT period in electrocardiodiagraph and can cause life-threatening specific ventricular tachycardia, torsades de pointes. Hundreds of mutations in 15 genes (LQT1-15) are linked to congenital LQTS. Worldwide prevalence of congenital LQTS gene mutations is from 1:2000 to 1:5000. However, the prevalence in Finland is much higher due to four founder mutations that alone occur in one out of 250 individuals. Acquired LQTS is often drug-induced and the most common cause for the withdrawal of drugs on the market. Carriers of LQTS mutations are more susceptible to acquired LQTS than normal population. LQTS-specific cardiomyocytes can thus provide a thorough model for drug cardiotoxicity screening, better insight into disease mechanisms and assist in drug development. This thesis was a part of a bigger project concentrating on validation of LQT2-specific cell lines that could be used for the purposes mentioned above. Induced pluripotent stem (iPS) cell technology enables creation of disease-specific pluripotent stem cell lines, which can be differentiated into any cell type. In this thesis, two LQT2-specific iPS cell lines derived from a clinically symptomatic 44-year-old female were used. She is heterozygous for Finnish founder mutation L552 in KCNH2 gene, which encodes the α-subunit of the cardiac rapidly activating potassium rectifier channel. iPS cells were first verified to express pluripotency markers and to form embryoid bodies containing all germ layers. iPS cells were then differentiated into cardiomyocytes by culturing them with END-2 cells and mechanical beating of the cardiomyocytes was assessed from video recordings. Single LQT2-specific cardiomyocytes showed LQT2-related phenotype in vitro with 43% of single LQT2 cardiomyocytes showing abnormal beating patterns and prolonged contraction time. This phenotype was rescued in LQT2-specific cardiomyocyte clusters. Finally, the expression ratios of wild type and mutated KCNH2 alleles were compared between cardiomyocytes derived from the female and her son, a carrier of the same mutation but with asymptomatic phenotype. Cardiomyocytes from both individuals expressed KCNH2 alleles with the ratio between 1:2 and 1:1 (wt:mut), thus allelic imbalance does not explain differences in the clinical phenotypes. All in all, the results of this thesis suggest that after further validation, mainly electrophysiology studies, these cell lines are most likely suitable to be applied for disease modeling, cardiotoxicity screening and finding new therapies for LQT2.
  • Nuotio, Ulpukka (Helsingfors universitet, 2017)
    Neuropathic pain is pain caused by injury or damage to the nervous system. This adverse condition affects millions of people in all parts of the world, and no known cure has been developed. Existing treatments are mainly anti-depressants or opioids that alleviate symptoms instead of repairing damaged neurons. Glial cell line-derived neurotrophic factor (GDNF) and artemin, belonging to GDNF family ligands, have been shown to restore damaged neurons. However due to the poor pharmaceutical properties of these proteins, such as difficult administration and expensive production, their transition to clinics is complicated. That is why we have been developing small molecule GFL-mimetics as an alternative. One of these mimetics is a compound named BT44. Characterization of BT44 began with in vitro experiments, where we tested the compound’s ability to activate luciferase reporter gene in cells expressing GDNF (GFRalpha1 and RET) and artemin (GFRalpha3 and RET) receptors, as well as ability to induce RET phosphorylation and activate intracellular MAPK/ERK and Pi3-K/Akt pathways. Furthermore, we tested stimulation of neurite outgrowth by the compound from cultured dorsal root ganglion neurons. In a similar manner to GDNF and artemin, BT44 was shown to activate GFRalpha1/RET and GFRalpha3/RET receptors and induce RET phosphorylation and intracellular signaling, in addition to stimulating neurite outgrowth from cultured DRG neurons. Because of the promising in vitro results, we moved on to in vivo testing in rat spinal nerve ligation (SNL) model of neuropathic pain. Similarly to artemin, BT44 was able to alleviate mechanical nociception and cold allodynia in SNL rats. In addition, we found that BT44 normalized to a certain degree nociception-related markers influenced by SNL in the tissues of experimental animals, which emulates previously published results for artemin. To summarize, our results indicate that BT44 is effective in neuronal restoration and pain alleviation, suggesting it for further development as innovative neuropathic pain treatment.
  • Kiviniemi, Eero A. (Helsingin yliopisto, 2018)
    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.
  • Lohva, Henri (Helsingfors universitet, 2016)
    Saccharomyces cerevisiae is a popular organism in the production of biofuels, chemicals and pharmaceuticals. This is thanks to a good understanding of its metabolism, GRAS status and the ease of modification. Traditionally its genetic modification has been based on the use of selectable markers. Modifying multi gene pathways has required a sequential process consisting of multiple single gene disruptions together with marker recycling. Additionally, many industrial S. cerevisiae strains are polyploid and lack the same tools for their modification as laboratory strains. In this study we sought to develop CRISPR/Cas9 based genetic engineering method for the modification of industrial S. cerevisiae strains. The CRISPR/Cas9 system is based on the adaptive immunity system of bacteria. It makes use of the Cas9 endonuclease which produces double stranded DNA brake to any location determined by a gRNA molecule. This causes the activation of DNA repair mechanisms which can be utilized to for the genomic integration of a template DNA. This makes transformation events much more likely and thus enables producing multiple modifications at once and removes the need for the of use selectable markers. In our approach Cas9 and gRNA were transformed into the cell in a plasmid together with a separate template DNA molecule. We used this method to remove lyp1, ura3 and can1 genes from diploid and polyploid industrial S. cerevisiae strains multiple genes at a time. Simultaneously we evaluated the effect of the NHEJ repair mechanism on CRISPR/Cas9 by repeating the tests with a deletion strain missing the ku70 gene required by NHEJ. Finally the method was used for the metabolic engineering by integrating the five gene violacein metabolic pathway into two loci in a single transformation event. This study demonstrated the CRISPR/Cas9 method is well suited for the modification of industrial S. cerevisiae strains and is capable of modifying up to three loci at a time in a polyploid yeast strain.
  • Le, Thanh Ngoc Uyen (Helsingin yliopisto, 2017)
    The aims of this study were to use the heterologously expressed laccase in Pichia pastoris as oxidative biocatalysts for the degradation of BPA and to find out natural mediators that could assist this laccase to degrade BPA efficiently and environmental friendly. In addition, removal of the estrogenic activity of BPA by the white rot fungus Physisporinus rivulosus cultures and the role of its laccase 2 in the BPA disapperance was also focused. Firstly, the removal of BPA’s estrogenic activity by the white rot fungus Physisporinus rivulosus was confirmed in cultures both with and without the fungal mycelium. Next, the recombinant laccase 2 (rLac2) played a role in the BPA disapperance. At the similar laccase activity level, removal of BPA’s estrogenic activity was done more efficiently in the fungal cultures than in the cell-free enzymatic treatments. Metabolites present in the fungal cultures could possibly act as natural mediators that enhance the removal of BPA. In addition, combination of nine laccases present in the fungal cultures could possibibly enhance the degradation of BPA. Finally, none of the ten mediatiors used was found to act as an efficient rLac2 enhancer in degrading BPA. Degradation of BPA was followed using a bioreporter system. The yeast Saccharomyces cerevisiae has been genetically modified in order to express the estrogen receptor alpha and produce a bioluminescent signal upon contact with estrogenic substances such as BPA. This bioreporter system has been further developed to specifically detect the estrogenic activity of bisphenol A and to be used in a high-throughput manner.
  • Pasupulate, Avinash (Helsingin yliopisto, 2015)
    The aim of this study was to develop and apply a high throughput and cost effective method for screening food samples for antibiotic contamination. This method can be used to reduce the number of samples that have to be analysed using expensive chemical methods that are at currently being used for determining antibiotic concentrations. The primary objective of this study involved the construction of a macrolide sensitive bacterial bioreporter, which can be used to detect the levels of macrolide antibiotics in meat samples. The bioreporter was constructed by merging a macrolide promoter region to a bioluminescent signalling gene like the lux operon, this fused segment was then cloned into a suitable vector and transformed to a host E. coli along with the repressor (producing) plasmid. The bioreporter works in the presence of macrolides, when the repressor protein is released from the promoter region, resulting in the expression of the lux operon, which produces light. This light signal can be used for the detection and estimation of macrolide antibiotics using a luminometer. The secondary objective was to use the macrolide bioreporter along with a previously constructed tetracycline bioreporter to measure antibiotic concentrations in bovine meat samples acquired from EVIRA (Finnish Food Safety Authority). On analysis of the resulting luminescence data, calculated concentrations of oxytetracycline were found to be in correlation to the data acquired from EVIRA and also using solid meat samples in assays, instead of extracted muscle fluid produced more accurate results. This shows the usability of bioreporters in the detection of antibiotics in animal-based foods from different sources. The resulting data can also be used to monitor and control the spread of antibiotic resistance through animal farms.
  • Koirala, Prabin (Helsingin yliopisto, 2018)
    The current multidisciplinary interests on human intestinal microbiomes have stimulated large scale research initiatives, involving collection and processing of up to thousands of fecal samples within a single study. Hence, there is a need for high throughput protocols that are cost-efficient and validated for their performance to ensure that the relative abundance of different bacteria, the main outcome of microbiota studies, is not biased due to technical artefacts originating from sample processing. Infant’s microbiota colonization is one of the central research areas in human microbiome research because of the long-lasting and profound health implications of the pioneering microbes. This experimental study aimed to develop and validate a high throughput fecal sample collection and processing system for microbial DNA extraction operating in 96 well format. This newly developed method was used to extract DNA from 647 fecal samples collected from mother-infant pairs within a clinical study that will study the effect of antenatal antibiotic prophylaxis on infant’s gut microbiota development. A subset of 28 mother-infant pair samples (14 from each antibiotic and non-antibiotic groups) were selected to study the prevalence of a probiotic bacterium, Lactobacillus rhamnosus GG (LGG), among infants and their mothers longitudinally from birth to 3 months by using species-specific PCR amplification method targeting sortase C gene. In addition, the prevalence of L. rhamnosus GG in 3-month-old infants was compared between the above samples and those (n=30) collected in another clinical trial conducted ~10 years earlier. From extensive testing and validation, an efficient high throughput system for fecal sample collection and processing for extraction of microbial DNA in 96 well format was established. Tests were performed to validate the performance of a) fecal sample collection system b) commercial, readymade bead beating tubes for bacterial cell lysis and c) selfmade wash buffers as part of the automatic DNA purification system. Performance was evaluated based on the quality and quantity of the resultant DNA. We show that this new fecal processing system can yield high quality microbial DNA from 96 fecal samples within ~6 hrs. Based on the ratios of dominant gram-positive and gram-negative bacteria evaluated using PCRs and next generation sequencing, the new DNA extraction method resulted into similar microbiota composition than the previously validated manual DNA extraction method. However, the DNA yield per sample was markedly lower due to the lower input volume of the sample. Based on the sortase C gene PCR tests, the prevalence of LGG was similar (~60%) among 3-month-old children in both clinical studies conducted ~10 years apart, although false negatives among the recent samples due to the low amount of DNA cannot be excluded. Following the temporal pattern of colonization, we observed no evidence for the transfer of LGG at the time of birth from the mother to her child, instead the infants became positive for LGG typically between 1-3 weeks after birth. The carriage of LGG seemed to be dependent on their diet. During this project, we found out that the PCR method employed for detection of LGG was not fully specific for this strain, and hence a more specific qPCR assay was developed.
  • Tuomiranta, Petra (Helsingin yliopisto, 2018)
    Glucuronoyl esterase (GE) is an enzyme produced by plant biomass degrading basidiomycete and ascomycete fungi. GEs have been found to hydrolyse a specific ester bond between lignin alcohols and xylan hemicellulose, which is one of the crosslinks in plant biomass. GEs are the only known enzymes capable of hydrolysing both glycosidically bound and free methyl esters of 4-O-methyl-D-glucuronic acid. In biodegradation of plant biomass, fungal GEs are assumed to cleave the bonds between aromatic lignin alcohols and 4-O-methyl-D-glucuronic acid of hemicellulose. Due to these characteristics, GEs are promising candidates for future industrial applications, such as biofuel production. The genome of white-rot bacidiomycete Dichomitus squalens contains two GE encoding genes. The aim of this work was to express one wild-type GE from D. squalens, DsGE1, and its site-directed mutants as recombinant proteins in the yeast Pichia pastoris. The catalytic activity of the wild-type and mutated recombinant DsGE1 enzymes was examined in optimum conditions with synthetic benzyl glucuronate as a substrate. Recombinant GEs were then exposed to varying of temperature and pH values as well as organic solvents. The aim was to assess whether the mutated DsGE1 enzymes retained a higher GE activity than the wild type enzyme in the tested conditions. Hypothesis of this work was that mutated recombinant DsGE1 enzymes catalyse the hydrolysis of a synthetic benzyl glucuronase. The mutated enzymes were expected to have improved thermal resistance in temperatures higher to the optimum when compared to the wild-type GE. In addition, another hypothesis was that the DsGE1 mutants retain their specific activity better than the wild-type enzyme when pH changes from optimum as well as when the enzymes are exposed to organic solvents. The first research hypothesis was confirmed, while the results of the work disproved the following two hypotheses.
  • Lüüs, Helen (Helsingfors universitet, 2009)
    The present study was conducted to test the effect of an essential oil blend containing thymol and cinnamaldehyde on chicken pancreatic enzyme activities. Essential oils are oils from plant origin and have become an important alternative in animal nutrition after the European Union banned the use of antibiotics as growth promoters. Some previous studies have indicated that essential oils may stimulate the secretion of digestive enzymes, and thereby have a positive effect on the digestion and absorption of nutrients (Windisch et al. 2008). Pancreas samples originated from 20- and 28-day old Ross male chickens, that were fed with wheat soybean diets supplemented (or not) with the essential oil blend. Optimization of the colorimetric assays for detecting the enzyme activities in the supernatants of pancreatic homogenates was carried out. The ?-amylase, trypsin, chymotrypsin and lipase activities were expressed as U per mg of protein. The activity results were compared with previously collected digestibility and performance results. The addition of the essential oil blend at the applied concentrations to chicken diet did not have a significant effect on the activities of the chicken pancreatic enzymes. Overall, the enzyme activities did not show strong correlations to chicken performance or nutrient digestibilities. However, there were significant differences (P < 0.05) in the lipase and trypsin activities between the 20- and 28-day old chickens. It can be concluded that the studied essential oil blend did not stimulate pancreatic enzyme activities at the tested conditions. However, further studies are needed to explain the effect of the dietary supplement of essential oils on chicken digestion.
  • Peltonen, Kaisa (Helsingin yliopisto, 2018)
    New alternative feedstocks are needed for biofuel production to fulfil the growing demand in the coming years. The industry is moving away from second-generation biofuels, produced from food and feed crops, to using waste streams from industrial processes. An abundant, cheap and attractive waste stream for processing in Europe is the pectin-rich pulp from sugar beet processing and fruit juice industry. Sugar beet pulp is particularly rich in D-galacturonic acid and arabinose, but neither are naturally used by the yeast Saccharomyces cerevisiae, which would be an interesting candidate for the microbial fermentation of the biomass. S. cerevisiae is one of the most used organisms in the industrial biotechnology, and methods for the genetic engineering of the organism are highly developed. To overcome the natural limitations of the yeast for D-galacturonic acid fermentation, the metabolic pathways present in other organisms could be integrated in the yeast genome. Two bacterial and one fungal pathway are known to convert D-galacturonic acid into metabolites of the yeast glycolytic and ethanol fermentation pathways, and are thus considered promising for engineering in yeast. A major engineering challenge in integrating the fungal pathway in yeast is the redox imbalance caused by the two NADPH-specific reducing enzymes. The aim of this thesis was to review the potential of different D-galacturonic acid pathways for yeast fermentation. S. cerevisiae is a well-characterised organism for heterologous protein expression, but at times functional expression of foreign proteins is not achieved. One approach to study the pathways was to clone and express enzymes of the bacterial isomerase and dehydratase pathways in S. cerevisiae, and to test their activity in culture lysates. In addition, to overcome the redox imbalance in the eukaryotic pathway, two approaches were used to obtain an NADH-spesific D-galacturonic acid reductase. First, a mutant library of the Trichoderma reesei gar1 reductase was designed with the structure-guided cofactor specificity reversal tool CSR-SALAD. An automated high-throughput screening method for expression in Escherichia coli was developed, and the library was screened for enzymatic activity. The second approach was to try to identify the sequence for the characterised NADH-utilising reductase from the single-cell algae Euglena gracilis. A cDNA library of the algae was made and screened with PCR and in vivo methods. The reductase uxaB of the isomerase pathway and dehydrogenase kduD of the dehydratase pathway were functionally expressed in S. cerevisiae, with specific activities of 1.1 µmol min-1 mg-1 and 0.22 µmol min-1 mg-1 , respectively. The enzymes dehydratase uxaA and isomerase kduI did not exhibit activity in activity assays. The galurD of the dehydratase pathway was expressed in E. coli, and the purified enzyme was successfully used to convert D-galacturonate to 5-keto-4-deoxy galacturonate. The approaches to change the cofactor specificity of the NADPH-specific reductase of the eukaryotic pathway did not lead to a discovery of a NADH-specific enzyme. More research is needed for engineering active enzymes for S. cerevisiae expression and constructing a fully functional D-galacturonic acid pathway for feasible D-galacturonic acid fermentation.
  • Viinamäki, Miia (Helsingfors universitet, 2015)
    Ruisleipä on keskeinen elintarvike suomalaisten ravitsemuksessa. Sen valmistuksessa käytetään yhä perinteistä juuritaikinaa eli raskia, joka sisältää maitohappobakteereja ja mahdollisesti hiivoja. Ruis sisältää runsaasti fruktaaneja ja lisäksi raskin fermentoinnin aikana muodostuu mannitolia. Fruktaani ja mannitoli imeytyvät huonosti ohutsuolesta, mutta suoliston bakteerit fermentoivat ne nopeasti. Nopeasti fermentoituvat substraatit saattavat aiheuttaa vatsaoireita jopa yli 10 % väestöstä. Opinnäytetyössä tutkittiin erään teollisen raskin fruktaani- ja mannitolimetaboliaa, raskimikrobistoa sekä raskimikrobiston fruktaaninhajottamiskykyä. Tutkimuskohteeksi valittiin BF-raski. BF-raskin fruktaanin, mannitolin ja pelkistettyjen sokerien pitoisuutta mitattiin raskituksen edetessä. Mittausten jälkeen käyttövalmiista BF-raskista eristettiin maitohappobakteerit ja niiden fruktaanin hajottamiskykyä testattiin kromogeenisillä maljoilla ja puhdasviljelmistä tehdyillä fermentaatioilla. Lisäksi valmistettiin BF-raskin kaltainen spontaani raski pienessä mittakaavassa ja mitattiin sen fruktaanipitoisuutta. Tutkimuksessa havaittiin, että BF-raskin raskitusprosessin aikana fruktaani ja prosessissa syntynyt mannitoli hävisivät. Yhdellä raskin maitohappobakteereista havaittiin olevan kyky kuluttaa tehokkaasti fruktaania ja geenit, jotka koodaavat solunulkoista fruktaanihydrolaasi-entsyymiä sekä FOS/fruktoosi-kuljetussysteemiä. Lisäksi siltä löytyivät mannitolin käyttöön tarvittavat geenit. BF-raskin kaltainen fruktaania kuluttava raskisysteemi saatiin aikaan myös spontaanisti pienemmässä mittakaavassa. Fruktaanivapaan ruisraskin avulla voidaan valmistaa niukasti fruktaania ja mannitolia sisältävää ruisleipää. Tällainen ’vatsaystävällinen’ ruisleipä voisi sopia perinteisestä ruisleivästä vatsaoireita saaville kuluttajille.
  • Mäkkylä, Heidi (Helsingfors universitet, 2017)
    Bacteria can communicate with each other using phenomenon called quorum sensing (QS). In QS the bacteria produce and release small signaling molecules which they use to communicate. Bacteria use QS in situations where it is beneficial to act on population level. QS has an important role e.g. in the formation of virulence factors and biofilms. There are several different QS systems. Gram-negative bacteria use i.a AI-1, AI-2, AI-3, and CAI-1 systems to communicate. All QS systems are based on the accumulation of signaling molecules when the bacterial concentration increases. When the concentration of signal molecules reaches the threshold level, the system activates. The activation of the signaling system then activates the expression of the genes controlled by the QS system. AI-2 signaling is assumed to be universal. That means that bacteria can use AI-2 signaling system in interspecies communication. In AI-2 signaling bacteria produce and release 4,5-dihydroxy-2,3-pentanedione (DPD) which works as a signaling molecule in the AI-2 system. Escherichia coli and Salmonella typhimurium use an ATP binding cassette ABC-type transporter to transport DPD molecules into the cell where LsrK kinase phosphorylates the DPD molecules. The phosphorylated DPD molecules bind to the LsrR regulator protein which acts as a suppressor of the lsr operon. The binding of the phosphorylated DPD molecules releases the LsrR from the lsr promoter region enabling the expression of the lsr genes. In Vibrio harveyi the surface proteins LuxP and LuxQ form a protein complex that recognizes DPD molecules. When the DPD concentration increases, the LuxPQ complex transform from kinase to phosphatase and the reaction chain, where LuxU phosphate transfer protein transfers a phosphate group from LuxO regulator protein, activates. The dephosphorylation of of LuxO releases the LuxR transcription factor and activates the expression of QS controlled genes. The aim of this thesis was to optimize two assays which can be used to screen for compounds that disrupt AI-2 signaling. The first assay was a bioreporter based assay where V. harveyi BB120 bioreporter strain was used. The second assay was protein based LsrK assay where the LsrK activity was monitored using assay kit which measures the concentration of ATP or ADP. The concentrations of bacteria, LsrK, and DPD used in the assays were optimized. The dimethyl sulfoxide (DMSO) tolerance of both assays were tested, the stability of the kits used in the LsrK assays was tested and the reaction buffer for the LsrK assay was selected from the two tested buffer options. The selected bacterial concentration for the V. harveyi BB120 assay was 100000 CFU/ml and DPD concentration 1 µM. The selected enzyme concentration for the LsrK assay was 300 nM and DPD concentration 300 µM. The tested DMSO concentrations had no effect on the kit measuring ATP but the highest concentrations tested had a small effect on the kit measuring ADP. A buffer containing triethanolamine, magnesium chloride, and bovine serum albumin was selected as the reaction buffer for the LsrK assay. Using the optimized LsrK assay, a screening was performed for a synthesized compound library. None of the compounds showed any LsrK inhibiting activity. The optimized assay was also used to make dose-response experiment to one LsrK inhibiting compound, named FIMM000642, which was found in a separate screening. The FIMM000642 dose-response as-say was also done against glycerol kinase to see if the compound would inhibit another enzyme from the same protein family or if the compound was a specific inhibitor to LsrK. FIMM000642 inhibited also the activity of glycerol kinase.
  • Tikkanen, Jouni (Helsingin yliopisto, 2019)
    The Master's thesis was completed as part of the joint project (Haittaeläin) by the Finnish Food Authority (Ruokavirasto) and Natural Resources Institute Finland (Luke). In the project, rodents and shrews caught on farm premises were investigated for zoonotic bacterial pathogens. This thesis covers the thermophilic cam-pylobacter findings in the caught pests in autumn 2017.Rodents and shrews caught on farm premises were investigated for zoonotic bacterial pathogens. This thesis deals with the result of thermophilic campylobac-ters isolated from the pests in the autumn 2017. Thermophilic campylobacters (Campylobacter jejuni ja Cam-pylobacter coli) cause gastroenteritis called campylobacteriosis in humans, which is one of the most common cause for human gastroenteritis in the world. The literature review discusses the characteristics of thermo-philic campylobacters and their epidemiology, which focused on aspects of a public health and risk factors for campylobacteriosis and the major sources of campylobacters. Moreover, a current knowledge of campylo-bacters’ occurrence in rodents and shrews were summarized in the literature review. The presence of thermophilic campylobacters was investigated from 227 pooled samples, which comprised of a total of 442 intestinal samples collected from 12 different species. Three species (yellow-necked mouse, house mouse and bank vole) covered 81,3 % of all caught pests. The numbers of caught pests were not signif-icantly different between the farms when compared to a geographical position (south or north) or a type of farm (pig or cattle). The numbers of captured yellow-necked mice were significantly higher in farms in the south. Especially, yellow-necked mice and bank voles were detected to be campylobacter positive. Other campylobacter positive species were harvest mice, rats, field voles and southern voles. A total of 93 samples were detected as campylobacter positive and all of them were identified as C. jejuni. There was not signifi-cant difference on the numbers of campylobacter positive samples between the farms when compared the geographical position or the type of farm. 46 isolates of all 96 campylobacter positive samples were selected for a whole genome sequencing. After the raw read data was assembled, the contigs were analysed with MLST and cgMLST typing schemes by Ridom SeqSphere+. 41 isolates contained 14 new sequence types (STs). Instead, 5 isolates contained previously described STs: ST-1304, ST-2219 and ST-4791. According to MLST typing isolates obtained from voles differed from isolates from mice and rats. Moreover, cgMLST typing supported the conclusion. In the cgMLST typing scheme vole’s isolates were found to have more missing loci than isolates from mice or rats. Therefore, all isolates from voles, apart from two bank vole isolates belonging to ST-1304, contained less than 90 % loci compared to a reference genome. Furthermore, these same isolates were poorly identified in MALDI TOF analysis unlike other isolates. One interpretation to the difference between isolates could be that isolates from voles belong to a new C. jejuni subspecies.
  • Leamon, A K M Al Amin (Helsingin yliopisto, 2019)
    Cellulose is a major component of plant biomass that can be used to produce bio-derived materials, chemicals, and fuels. Whereas the conversion of cellulose to chemicals and fuels is limited by the surface functionality of cellulose fibers due to their highly compact structure. A group of non-catalytic proteins including expansins, loosenins and cerato-platanins are known to alter cellulose fiber structures, presumably by breaking hydrogen bonds between cellulose microfibrils. This makes them interesting targets for the chemical and biophysical modification of plant polysaccharides. In this work, seven non-catalytic target genes were selected from the transcriptome of Phanerochaete carnosa. Pichia pastoris was transformed with expression constructs harboring the respective genes under control of the AOX1 promoter. Protein expression was analyzed by colony blot assay and small-scale expression in liquid media. Two cerato-platanins (CP1, CP4) and 3 loosenin-like proteins (LOOL7, LOOL9 and LOOL12) were successfully produced in large-scale. The identity of CP1, LOOL7, LOOL9 and LOOL12 was confirmed by MALDI-TOF MS analysis. The proteins showed no hydrolytic activity when tested on carboxymethyl cellulose, xylan or glucomannan. Defibrillation assay results of CP1, CP4, LOOL7 and LOOL9 suggested that the non-catalytic proteins lack specific weakening effect on cellulose filter paper. Considerably high protein blank absorbance of LOOL7 indicates possibility for self-assembling abilities of the protein. Incubation of filter paper with CP1 seemed to improve the hydrolytic action of cellulases on Avicel and filter paper, as shown by complementation assays. The effect of LOOLs on enzymatic cellulose hydrolysis varied over the time course of different experiments. Most notably, LOOL7 and LOOL12 were able to increase the reducing sugar release from Avicel and filter paper by cellulase at different time points. The maximum increase of cellulose conversion achieved by LOOL12 was 30% after 10 min when non-catalytic proteins and Cellic CTec2 were added simultaneously. LOOL7 showed highest conversion improvement of 21% after 6 h pre-incubation assay with filter paper. Upon prolonged pre-incubation (72 h), significant improvement of filter paper hydrolysis was noticed for CP4 and CP1, while LOOLs unable to enhance the hydrolytic activity of the tested cellulase mixture. For the first time, a comparative study of cerato-platanins and loosenin-like proteins was carried out and the findings presented in this thesis suggest that especially CP1 can be a promising accessory protein for efficient bioconversion of cellulose. Nevertheless, a deeper understanding of the structure and function of these non-catalytic proteins will be helpful in determining their potential biotechnological applications.
  • Zhang, Angel (Helsingfors universitet, 2016)
    The enzymatic degradation of plant cell walls has being extensively explored during the past decade chiefly due to the current and potential uses of plant biomass found in several industries. In this process, the effective removal of the hemicellulosic portion is of paramount importance. For this purpose, hemicellulases have been intensively studied and produced from bacterial and fungal sources. Within this group of enzymes, mannanases, which are mostly used during the processing of softwood, have been found in several industrial applications. Currently, white rot basidiomycetes have gained increasing attention due to their ability to fully decompose wood. One of these fungi, Dichomitus squalens, has been regarded as an effective wood degrader; nonetheless, its enzymatic arsenal and catalytic potential have only recently been explored. In the present study, six mannan acting enzymes were selected from the genome of D. squalens and heterologously produced using the methylotrophic yeast Pichia pastoris. Four of these enzymes, consisting of two β-1,4-endomannanases, one β-1,4-mannosidase and one α-galactosidase, were successfully produced. The biochemical characterization of the concentrated enzymes using synthetic p-nitrophenyl substrates corresponding to their predicted activities showed optimal pH ranging from 3 to 5 and optimal temperatures ranging from 50 to 60 °C. In addition, three of the enzymes, namely MAN1, MND1 and GAL2, showed high thermostability retaining the majority of their enzyme activities after an incubation of 60 minutes at 60 °C. In contrast, MAN2 lost all of its activity after an incubation of 30 minutes at 60 °C. Furthermore, MAN1 and GAL2 showed optimal enzyme activities of 1922.2 (± 26.6) and 903.7 (± 7.4) U/mL respectively, with specific activities of 11.1 (± 1.0) and 27.9 (± 0.5) U/mg respectively. The four enzymes had a molecular mass ranging from 47 to 110 kDa, without showing evidence that any of them formed dimers or more complex structures. The results of the present study revealed the function of four putative mannan degrading enzymes from D. squalens and provided evidence of its mannolytic capacity. Future studies with other cell wall degrading enzymes or the synergistic effects of characterized enzymes of this species may further reveal its potential as a source of industrial enzymes.
  • Jian, Ching (Helsingfors universitet, 2016)
    Non-alcoholic fatty liver disease (NAFLD) is currently the most common liver disease in the western world. The human intestinal microbiota possesses enormous metabolic and immunomodulatory capabilities, and together with increased intestinal permeability, changes in the microbiota have been linked to the development of NAFLD. However, human studies so far have yielded contradictory findings regarding the compositional microbiota changes and provided little mechanistic understanding due to the predominance of cross-sectional studies. The aim of this study was to study human intestinal microbiota and gut permeability in NAFLD. Real-time PCR was employed to quantify the key intestinal bacterial groups in overweight or obese subjects with (n = 12) and without (n = 19) NAFLD, and in response to hypercaloric overfeeding, where participants were provided with three compositionally distinct diets to temporarily increase liver fat. In addition to the comparative analysis, the microbiota results were correlated to serum markers of intestinal permeability and metabolic endotoxemia, as well as clinical parameters related to NAFLD. The results show that host lipid metabolism and the gut microbiota, specifically Bacteroidetes and Clostridium cluster XIVa, are firmly intercorrelated. Bacteroidetes were found to be less abundant in subjects with NAFLD and correlate negatively with liver fat and serum triglycerides at baseline. Clostridium cluster XIVa, a dominant Firmicute group, was positively associated with serum triglycerides and pro-inflammatory markers but negatively with intestinal permeability. The relative abundance of Bacteroidetes as well as the markers of metabolic endotoxemia changed significantly in response to overfeeding, while no diet-induced systematic effects were found in Clostridium cluster XIVa, total bacteria, Escherichia coli group, Bifidobacterium or gut permeability. Our results based on a targeted microbiota analysis suggest that the role of the intestinal microbiota and gut permeability on triggering metabolic disarrangement and NAFLD in humans is inferior to other stimuli, such as diet.
  • Tran, Thu (Helsingin yliopisto, 2020)
    The impact of color on perception of food has been studied a lot with solid and liquid foods, but research with semi-solid foods, such as yogurt, has not been published as much. Semi-solid oat-based foods have great commercial potential, yet there are still challenges related to sensory properties. In order to meet consumer acceptance, more research on plant-based foods is needed. The aim of this thesis work was to study the impact of food color in two different oat-based yogurt-like products on consumers’ perception of flavor and texture, as well as acceptance and preference. For this purpose, descriptive analysis and consumer test were applied on six samples, Yosa and Oddlygood as such and with two levels of added yellow food colorant. Generic Descriptive Analysis (GDA) was done for 8 attributes (yellowness intensity, consistency, overall aroma intensity, overall flavor intensity, sourness intensity, oat flavor intensity, thickness and powderiness) with 30 trained panelists. Consumer test (106 panelists) included 9-point scales for pleasantness, flavor intensity, thickness, and how natural the color looks, a Just-About-Right (JAR) scale for thickness and a Check-All-That-Apply (CATA) question. Increased color intensity was found to increase flavor intensity, as well as sourness and aroma of Yosa product, but not of Oddlygood product. Color intensity did not affect consumers’ preference, but the closer the color was to that of commercial products, the more natural consumers considered its color to be. According to JAR result, it was advisable to improve thickness of all the samples. From CATA result, 5 words used the most to describe all the samples were creamy, viscous, runny, smooth, and watery. In conclusion, the effect of color on perception of flavor was found in Yosa product, but not Oddlygood product, and color had no significant effect on pleasantness of the products.