Browsing by discipline "Biotechnology (EYT)"

Vitamin D is either obtained through synthesis in the skin due to UVB-light (290-315 nm) or from the diet. The hydroxylased metabolite 25-hydroxyvitamin D (25(OH)D) is the metabolite to measure when vitamin D status wants to be determined. The active form of vitamin D is 1,25- dihydroxyvitamin D (1,25(OH)?D) which interacts with a large set of tissue cells (especially bone) through its nuclear receptor the vitamin D receptor (VDR). Vitamin D deficiency can lead to rickets in children and osteoporosis or osteomalacia in adults. Type 1 Diabetes (T1D) is an autoimmune disease which is caused by the destruction of the pancreatic ?-cells. The disease has genetic and environmental features but the whole mechanism of disease development is still unknown. The prevalence of T1D is constantly growing in the whole world. Therefore it is important to study possible environmental factors that can eventually serve as pathogenesis modifiers. Vitamin D and T1D have been associated among others because there is a seasonal and geographical variation in T1D incidence, more cases have been identified in the North and during winter. The aim of this study was to investigate if the serum 25(OH)D status during first trimester of pregnancy is associated with T1D development in the offspring. The subjects where mothers of T1D children (N=310) and the controls were mothers of healthy children (N=310). Serum samples were obtained from the Finnish Maternity Cohort (FMC) and analyzed for S-25(OH)D. S- 25(OH)D measurement was performed with an indirect enzyme immunoassay (EIA). No significant (p>0.05) difference was seen between S-25(OH)D mean concentrations in cases and controls. The mean concentration of cases was 43.3 ± 15.9 nmol/l and 43.0 ± 15.5 nmol/l (mean ± standard deviation (SD)) of controls. Insufficient and deficient S- 25(OH)D status was seen in 72% of the whole study population. As a result of this study it has been shown that the S-25(OH)D status during first trimester of pregnancy is not associated with T1D development in the offspring. Samples from later stages of pregnancy could be analyzed to determine if the overall status during pregnancy has an effect on T1D development in the offspring. Considering the possible health outcomes of vitamin D insufficiency, recommended vitamin D supplementation should be raised to improve maternal and fetal health.

As the sustainable energy is becoming increasingly important, utilization of lignocellulosic biomass for biofuel production is the central part of this area. Fungal enzymes play an important role in lignocellulose degradation. Glucuronoyl esterase (GE) is a less studied fungal enzyme which degrades the ester linkage between lignin alcohol and hemicellulose side chain 4-O-methyl D-glucuronic acids. Genes encoding GE have been identified from various fungal species and they have been expressed in different production systems to be able to study their biochemical properties in detail. The gene encoding GE from the basidiomycete litter-decomposing fungus Stropharia coronilla was cloned and heterologous expressed in Pichia pastoris yeast. The expression and secretion of GE was induced by growing S. coronilla on lignocellulose supplemented cultivations. ScGE activity can be detected after the fifth day cultivation and it peaked on the 14th day. The heterologous expression of ScGE in P. pastoris showed that ScGE was produced as an enzymatically active protein. The commercial K-URONIC kit supplemented with a GE specific substrate benzyl-D-glucuronate was used to determine GE activity.

In this research, lignocellulose decomposition and bioethanol production potentiality of the white rot fungus Phlebia radiata 79 was studied at different atmospheric conditions on several solid substrate mixtures containing spent brewery barley mash (SBBM), barley straw, spruce wood sawdust, and birch wood sawdust. The fungus was capable of growing on all substrate mixtures, subsequently converting them into fermentable sugars like glucose, producing various primary metabolites involving ethanol, acetate, and glycerol. Ethanol accumulation was always dominant under nitrogen flushed anaerobic conditions as well as semi-aerobic conditions. The highest concentration of ethanol accumulated on the second week of cultivation on all solid substrate mixtures. Under anaerobic conditions, the detected amount of ethanol was 88 mmol/l, 87 mmol/l, and 108 mmol/l, after two weeks of cultivation of the fungus on lignocellulose substrates containing SBBM mixed with barley straw, spruce wood sawdust, or birch wood sawdust, respectively. Under semi-aerobic conditions, corresponding concentrations of ethanol - 90 mmol/l, 61 mmol/l, and 105 mmol/l - accumulated in the cultures after two weeks of cultivation on the same substrate mixtures, respectively. Under aerobic conditions, only small amounts of ethanol were detected during the first two weeks of cultivation. Another part of the study was to establish an enzyme assay method for pectin degradation and conversion, in order to measure the activities of specific carbohydrate-active enzymes (CAZymes) involving pectinase, as well as cellulolytic β-glucosidase and cellobiohydrolase (CBH) activities. The highest β-glucosidase activity (5.2 nkat/ml) was observed under aerobic conditions in cultures on the substrate mixture of SBBM and barley straw. CBH activity was also prominent under aerobic conditions, and the maximal activity (0.7 nkat/ml) was detected on the substrate mixture of SBBM and spruce wood sawdust, while elevated pectinase activity (83 nkat/ml) was recorded under aerobic conditions on substrate mixtures containing SBBM and barley straw. Thus, the conclusions are that composition of the solid waste lignocellulose substrate mixture affected enzyme production by the fungus, whereas production of ethanol was mainly controlled by the cultivation atmosphere. Interestingly, both anaerobic and semi-aerobic atmospheric conditions supported similar bioconversion efficiency resulting with similar high levels of bioethanol production in the fungal cultures within two weeks.

Fungal ferulic acid esterases (FAEs) are important accessory enzymes that participate in degradation of plant cell wall hemicellulose in grasses, including cereals and many energy crops. They could be used to convert agricultural wastes into a variety of value-added products such as biofuel, feed and paper. In non-aqueous media, they have been shown to act as catalysts for enantioselective reactions. Putative faes are widespread in genomes of plant pathogenic and saprotrophic fungi. However, only few FAEs have been characterized in detail. Additionally, despite of their diverse biotechnological potential, fungal FAEs have not been produced recombinantly on a bioreactor scale, which is a necessary step for their commercial applications. The aim of the present study was recombinant production on a bioreactor scale and biochemical characterization of a putative FAE from Aspergillus terreus. The enzyme demonstrated a broad substrate profile and an excellent storage stability. Its catalytic activity was highest against methyl 3,4-dimethoxycinnamate, but the enzyme was also active against methyl ferulate. It preferred methoxy groups to hydroxyl groups on the substrate’s phenyl ring, while shortening of the aliphatic side chain diminished the activity. The enzyme was observed to be fully stable at 37 °C for 1 h, and it demonstrated thermal activation at the same temperature. At 45 ˚C, it retained 75 % of its initial activity for 1 h. McIlvaine’s buffer was observed to increase the activity by 85 % compared to the standardly used MOPS buffer. The results of this study have contributed to the biochemical knowledge of fungal FAEs and elucidated their substrate preferences.

The Baltic Sea is a geologically and biologically unique sea highly vulnerable to environmental hazards, and the most emphasized threat is the risk of oil spills. The microbiology of the Baltic Sea has not been extensively studied, and most studies have focused on bacteria, leaving archaea and fungi to less attention. In addition to the natural microbial communities of different parts of the Baltic Sea, the effects of diesel oil, and dispersants applied in case of an oil spill, on these microbial communities is yet to be elucidated. The focus of this Master’s thesis was to compare the bacterial, archaeal and fungal community compositions of the Baltic Sea surface water at three distinct locations; the open sea, a pristine archipelago, and putatively oil contaminated coastal water at an oil refinery. In addition, the short-term effects of diesel oil and dispersant on the three locations were studied during a 72-hour microcosm experiment. Next-generation Ion Torrent sequencing of bacterial V3–V4 and archaeal V4 regions of 16S rDNA and fungal ITS regions was used for a community composition analysis. Quantitative polymerase chain reaction (qPCR) was applied to determine the changes in the copy numbers of bacterial 16S rRNA genes and two genes associated with microbial hydrocarbon degradation, i.e. ring-hydroxylating dioxygenases (RHD) and alkane hydroxylases (AlkB). Based on the findings, the three sites under investigation harbored differing surface water microbiomes demonstrating differing responses to diesel and dispersant amendments, and furthermore, the results indicate that the putatively oil contaminated coastal site has higher natural petroleum hydrocarbon degradation potential compared to the pristine archipelago and especially the open sea. It is noteworthy, that over 90% of the fungal sequences from the open sea and the pristine archipelago, and over half of the fungal sequences from the putatively oil contaminated coastal site were unidentified even at phylum level. In addition, almost half of the archaeal sequences from the putatively oil contaminated site were unindentified. Assessing the petroleum hydrocarbon degradation potential of the indigenous microbiome in different parts of the Baltic Sea is of great importance, since the data can potentially be utilized when developing suitable biological oil spill response methods as well as predicting the rates of petroleum hydrocarbon degradation in different parts of the Baltic Sea area.

Ihmisen suolistomikrobisto on monimuotoinen ekosysteemi, jonka koostumuksen ja toiminnan on osoitettu olevan merkittävässä asemassa ihmisen terveyden kannalta. Mikrobien osallisuus erilaisissa tautitiloissa tekee niistä mahdollisia kohteita tai keinoja lääkehoidolle. Tutkimuksissa yhdeksi tehokkaista hoitokeinoista esimerkiksi toistuvissa Clostridium difficile -infektiossa on osoittau-tunut ulosteensiirto, jossa terveen henkilön ulostetta viedään sairastuneen henkilön suolistoon. Tämän pro gradu -tutkielman tavoitteena oli mitata onnistuneissa ulosteensiirroissa C. difficile -infektion hoidossa Helsingin yliopistollisen keskussairaalan (HYKS) gastroenterologian poliklinikalla käytetystä ulosteensiirteestä tutkimusryhmämme kehittämän menetelmän avulla eristettyjen Bacteroidales -lahkoon kuuluvien 16 eri bakteeri-isolaatin kykyä vähentää tulehdusta in vitro sekä niiden adheroitumista HT-29- ja Caco-2-soluihin sekä mukukseen. Lisäksi haluttiin selvittää, oliko tutkittavien bakteeri-isolaattien mahdollinen kyky vähentää tulehdusta riippuvaista adheesiosta. Menetelmä, jossa mitattiin bakteeri-isolaattien kykyä vähentää tulehdusta, perustui siihen että HT-29-solut inkuboitiin ensin tutkittavan isolaatin kanssa ja sen jälkeen soluihin indusoitiin tulehdus endotoksiini lipopolysakkaridin (LPS) avulla. Tämän jälkeen soluista tuottuneen proinflammatorisen interleukiini-8:n (IL-8) määrä mitattiin ELISA-kittiä käyttäen. Adheesion mittaus puolestaan perustui siihen, että bakteeri-isolaatit leimattiin ensin radioaktiivisella leimalla ja inkuboitiin epiteelisolujen tai mukuksen kanssa, jonka jälkeen adheroitumattomat bakteerit pestiin solujen päältä ja soluihin jäljelle jääneen radioaktiivisen säteilyn määrä mitattiin. Mitatun säteilyn määrästä voitiin laskea, kuinka paljon bakteeri-isolaateista oli prosentuaalisesti adheroitunut epiteelisoluihin. Menetelmä, jossa tulehdus-adheesio-riippuvuussuhdetta selvitettiin, perustui siihen, että HT-29-solut inkuboitiin ensin tutkittavan isolaatin kasvatusliuoksen kanssa, jonka jälkeen soluihin indusoitiin tulehdus LPS:n avulla. Tämän jälkeen soluista tuottuneen IL-8:n määrä mitattiin ELISA-kittiä käyttäen. Tulosten perusteella näytti siltä, että kymmenellä kuudestatoista tutkitusta isolaatista oli kantaspesifisesti kyky alentaa merkitsevästi proinflammatorisen IL-8:n tuottoa. Isolaattien adheroitumis-% vaihteli epiteelisolulinjojen sekä tutkittujen lajien ja niiden kantojen välillä. Isolaattien adheroituminen Caco-2-soluihin oli voimakkainta, kun taas mukukseen se oli erittäin heikkoa. Lisäksi adheesiomittauksen tulokset yhdessä tulosten, jossa tarkasteltiin isolaattien kasvatusliuoksen vaikutusta IL-8:n tuottoon, osoittivat että tutkittujen isolaattien kyky alentaa IL-8:n tuottoa ei välttämättä ole riippuvaista adheesiosta, mikä jatkossa lisää mielenkiintoa selvitää bakteerien tuottamien molekyylien merkitystä tulehduksen vähentämisen tai eston kannalta.

Natural products have enormous structural and chemical diversity and are either the source or direct inspiration for many drugs in use today. Cyanobacteria are prolific producers of complex natural products with serine protease inhibiting activity. Many of these natural products are the product of non-ribosomal peptide synthetase (NRPS) modular enzyme complexes. Suomilide is a complex tetrapeptide produced by strains of the benthic cyanobacterium Nodularia sphaerocarpa. It has a highly complicated structure and contains an unusual azabicyclononane moiety, a methylglyceric acid, a xylose unit with hexanoic acids and a terminal 1-amidino-3-(2-aminoethyl)-3-pyrroline moiety. Suomilide inhibits thrombin, plasmin and trypsin in low micro-molar concentrations. The biosynthetic of this unusual glycoside remain unclear. However, suomilide is long predicted to be part to the aeruginosin family of protease inhibitors. A 5.4 Mb draft genome of Nodularia sphaerocarpa HKVV was obtained in order to identify the suomilide biosynthetic. The 43.7 kb suomilide gene cluster was identified on a single contig by performing tBLASTn searches on the draft genome of Nodularia HKVV using aerDEF genes from aeruginosins gene cluster as query. This gene cluster encodes 27 genes including two complex NRPS enzymes and a set of tailoring enzymes for the assembly of suomilide. The suomilide gene cluster shares extensive homology to known aeruginosin gene clusters including two aerB and aerG genes encoding NRPS enzymes, 12 genes (aerC, aerD, aerE, aerF, aerI, aerK, two copies of aerN and four copies of aerH) encoding for the enzymes responsible for synthesis of precursor non-proteinogenic amino acids and 13 other tailoring enzymes. The suomilide gene cluster was much larger and encoded a greater number of biosynthetic enzymes reflecting the structural complexity of suomilide. We identified 10 aeruginosin gene clusters and 2 suomilide gene clusters from 12 strains of cyanobacteria by genome mining. Bioinformatics analyses suggested these gene clusters encoded an unanticipated chemical diversity of aeruginosins and suomilides. LC-MS and Q-TOF analysis detected aeruginosins or suomilide variants from 12 of the 15 strains. Surprisingly, inhibition assays with the crude extracts using all three isoforms of human trypsin suggest that these compounds may have potent and selective inhibition of human trypsin isoforms. Further work is required to prove that suomilide alone can carry out selective inhibition of trypsin isoforms or is it a result of synergism between the compounds produce by cyanobacteria. Phylogenetic analysis demonstrated that the aeruginosin evolved through the acquisition of multiple loading mechanisms and tailoring enzymes through horizontal gene transfers. Our results support the hypothesis that suomilides are a part of aeruginosin family as they are made through the same genetic pathway, however have gained a greater degree of structural diversity due to the acquisition of tailoring enzymes. These results together suggest that cyanobacteria produce an unexpected wealth of complex natural products belonging to the aeruginosin family and that some of these may be potent and selective inhibitors of isoforms of human trypsin.

In dairy industry starter cultues are used to acidify milk and, in addition, produce aroma compounds, such as diacetyl. Diacetyl have buttery aroma and it is a desired flavor compound in products like butter, buttermilk and cottage cheese. Diacetyl can be metabolised from pyruvate which is a common intermediate of carbohydrate and citrate catabolisms. The metabolism from pyruvate to lactic acid or citrate depends on the strains and their growth conditions. Lactococcus lactis ssp. lactis biov. diacetylactis is an exceptional lactic acid bacteria type, which can use citrate as a sole energy source. The citrate metabolism enzymes and the citrate uptake to the cell, have a key role, when diacetyl production is tried to increase in LABs with their growth conditions. New starter cultures of lactic acid bacteria are needed to solve for acidification problems caused by bacteriophages. Dairy industry has developed control methods for bacteriophages like starter rotation. For starter rotation there should be more than one starter culture, which has same properties in acidification and aroma compounds production but different bacteriophage insensitivity. The aim of this Master’s thesis was to find a strain, which can be used as an aroma producer starter culture in milk based products. Requirements for the strain were a diacetyl production of 120 ppm or more and different phage insensitivity than the strain in use. Diacetyl productions of 132 strains were determined with a colorimetric method, which is based on Voges-Proskauer reaction. The strains were grown in 1.1% citrate containing milk medium in aerobic conditions. Based on absorbance measurements 23 selected strains’ diacetyl production levels in same growth conditions were determinded with headspace gas chromatography–mass spectrometry. Two of the 23 strains produced more diacetyl than the strain in use, but they had the same phage insensitivity. Because of similarity to the features of the used strain, it was decided to find out growth conditions, which affect the diacetyl production by the strain L. diacetylactis H63, when growing in bioreactors. Factors for two experimental designs were chosen based on literature. Partial least square (PLS) regression method was used to analyse, which of the chosen parameters affects the diacetyl and acetoin production of the strain H63. Copper concentration, pH and the redox-potential of the growth medium, when samples were taken to determinations, explain the diacetyl and acetoin production of the strain H63 based on the model, which PLS method created. Also the initial pH of the growth correlates with diacetyl production. Result of this research is that by using same growth conditions it was not possible to find a strain from selected collection with the same diacetyl production capabilities but different phage insensitivity as the strain in use.