Browsing by Subject "biotekniikka"

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  • Ollikainen, Pia (Helsingin yliopisto, 2013)
    Bovine milk is a rich source of nutrients, such as protein, fat, carbohydrate, minerals and vitamins. In addition to the major protein components of casein and whey proteins, milk has low concentrations of compounds with bioactivity, growth factors and insulin. Insulin-like growth factor I (IGF-I) and transforming growth factor-β2 (TGF-β2) are the most studied because of their various growth-promoting activities and the possibility to use them as bioactive supplements in foodstuffs. In the present work heat-stability and separation of IGF-I, TGF-β2 and insulin were studied using ELISA immunoassay. Milk from a dairy farm was heat-treated at 65, 72, 90 or 135⁰C for 15 s using an indirect pilot heating processor. Both IGF-I and TGF-β2 showed good heat stability up to 90⁰C, but at 135⁰C both growth factors had lost most of their immunochemical activity. Heating also activated the latent forms of IGF-I and TGF-β2 to immunoreactive form, which was possible to analyse without any separate pre-treatment. Heat-activation of TGF-β2 was temperature-dependent and at 90⁰C all the TGF-β2 concentration was activated by heat. IGF-I was less sensitive, but at 90⁰C it showed a sharp increase in immunoreactivity. Heating as a pre-treatment method was introduced as a new method to analyse and compare IGF-I concentrations in samples with no or low whey protein concentrations. When heat-treated milks were separated to casein and whey fractions by acid, ultracentrifugation or membrane filtration, the heat-activated immunoreactive form of the growth factor followed the casein fraction and the latent form remained in whey. Bovine insulin was heat stable during pasteurization at 65 and 72⁰C, but lost some of its immunochemical activity at higher temperatures. During membrane filtration a higher share of insulin was concentrated in MF retentate than in UF retentate when compared to IGF-I. An in vitro digestion experiment was performed in order to compare the digestibility of the latent and active forms of TGF-β2. In the adult model TGF-β2 was hydrolysed in gastric phase, but in the infant model a small share of TGF-β2 survived the gastric and duodenal phases. There was no difference in survival or digestibility between the latent or active forms of the growth factor. The digestibility of IGF-I was tested in the infant model and this growth factor was hydrolysed during duodenal phase. The results obtained in this study have great importance when separating and concentrating the growth factors from milk or colostrum. If the separation and concentration is performed from whey, these results show that the heating history of milk has a great effect on the yields. The results also demonstrate that heating as a pre-treatment method and membrane filtration can be used to lead growth factors to different fractions, depending on the intended usage.
  • Mäntylahti, Sampo (Helsingin yliopisto, 2014)
    In the field of bioscience there is an ongoing explosive growth in discovery and information. Novel means in biotechnology as well as in medicines are introduced at an unseen rate. One of the aspects contributing to this development is the increased understanding of protein function and structure. Proteins have a role in almost every biological process. The function and structure of proteins are linked. Recent studies have discovered that the understanding of the protein structure has been biased. Namely, the studies have unearthed a previously dismissed protein structure state: intrinsically disordered proteins (IDPs). In this highly dynamic state a protein is without a globular fold, but does not meet the requirements of a random coil either. Rapid transition between folds renders most of the established research techniques to be poor methods to study the IDPs. Nuclear magnetic resonance (NMR) is a spectroscopy method, which enables the study of molecules at atomic resolution. The technique is based upon manipulation of the nuclear spins in specifically produced sample under strong magnetic field. In this method, spins of the system generate quantum coherence state(s), which is utilized to obtain information about the system. NMR is suitable for studying samples in solid and liquid mediums, but in case of biomolecules, water solution is preferable as it resembles in vivo environment. Highly mobile structure and chemical composition of IDPs cause many established NMR experiments to fail. Development of NMR pulse sequences is an obvious approach to solve the problem. This thesis presents a number of NMR pulse sequences, which are designed to improve acquisition of information from highly mobile sections of proteins. The key aspect is to utilize H atom instead of HN in coherence transfer. Additional improvements include limited residue specific identification and novel coherence transfer pathways. Articles I, II, and III present triple resonance experiments, which correlate protein backbone atoms. Combination of the spectra enables full sequential assignment. Article IV introduces an improved pulse sequence for measuring J couplings between nitrogen and amide proton. The experiments were subjected to experimental verification. Comparisons were drawn between established pulse sequences. In both globular proteins and IDPs the results show improvement over established pulse sequences. The proposed sequences yielded improved assignment coverage, resolution and sensitivity enhancement.
  • Nyyssönen, Mari (Helsingin yliopisto, 2009)
    Bioremediation, which is the exploitation of the intrinsic ability of environmental microbes to degrade and remove harmful compounds from nature, is considered to be an environmentally sustainable and cost-effective means for environmental clean-up. However, a comprehensive understanding of the biodegradation potential of microbial communities and their response to decontamination measures is required for the effective management of bioremediation processes. In this thesis, the potential to use hydrocarbon-degradative genes as indicators of aerobic hydrocarbon biodegradation was investigated. Small-scale functional gene macro- and microarrays targeting aliphatic, monoaromatic and low molecular weight polyaromatic hydrocarbon biodegradation were developed in order to simultaneously monitor the biodegradation of mixtures of hydrocarbons. The validity of the array analysis in monitoring hydrocarbon biodegradation was evaluated in microcosm studies and field-scale bioremediation processes by comparing the hybridization signal intensities to hydrocarbon mineralization, real-time polymerase chain reaction (PCR), dot blot hybridization and both chemical and microbiological monitoring data. The results obtained by real-time PCR, dot blot hybridization and gene array analysis were in good agreement with hydrocarbon biodegradation in laboratory-scale microcosms. Mineralization of several hydrocarbons could be monitored simultaneously using gene array analysis. In the field-scale bioremediation processes, the detection and enumeration of hydrocarbon-degradative genes provided important additional information for process optimization and design. In creosote-contaminated groundwater, gene array analysis demonstrated that the aerobic biodegradation potential that was present at the site, but restrained under the oxygen-limited conditions, could be successfully stimulated with aeration and nutrient infiltration. During ex situ bioremediation of diesel oil- and lubrication oil-contaminated soil, the functional gene array analysis revealed inefficient hydrocarbon biodegradation, caused by poor aeration during composting. The functional gene array specifically detected upper and lower biodegradation pathways required for complete mineralization of hydrocarbons. Bacteria representing 1 % of the microbial community could be detected without prior PCR amplification. Molecular biological monitoring methods based on functional genes provide powerful tools for the development of more efficient remediation processes. The parallel detection of several functional genes using functional gene array analysis is an especially promising tool for monitoring the biodegradation of mixtures of hydrocarbons.
  • Aranko, A. Sesilja (Helsingin yliopisto, 2014)
    Inteins are selfish but harmless autocatalytic proteins that perform a post-translational modification, termed protein splicing. In protein splicing an intein excises itself off from the precursor protein and simultaneously ligates the flanking proteins together with a peptide bond. Inteins are found sporadically distributed in unicellular organisms, but their biological functions remain obscure. Importanly, inteins that are split into two can remain active and perform protein ligation by protein trans-splicing (PTS). In principle, PTS allows ligation of any two protein-sequences, with the only requirement being Ser, Thr, or Cys as the first residue downstream of the intein. This has inspired development of numerous biotechnological applications including protein semisynthesis, segmental isotopic labeling, and cyclization. Protein ligation by split inteins is, however, limited by the lengths, substrate specificity, orthogonality, and the reaction yields of the split inteins. The objective of this thesis was to advance the development of protein splicing as a protein-ligation tool. First, the split site of a natively split DnaE intein was shifted in order to engineer a split intein with shorter C-intein that could be easily chemically synthetized. The newly engineered split intein could perform protein ligation in high yields and was demonstrated to be in certain cases even better than the natively split intein. Encouraged by this, 21 more split inteins were engineered starting from four different inteins, guided by the three dimensional structures of these inteins. Split inteins were systematically tested for activity and orthogonality to evaluate their potential for biotechnological applications. Next, the scope was widened to bacterial intein-like (BIL) domains. BIL domains belong to the same superfamily with inteins but are distinct by their distribution and functions and have a wider variety of residues at the downstream junction. The first structure of a BIL domain was solved. It highlighted their homology to inteins as well as allowed engineering of split BIL domains. The split BIL domains could perform protein ligation also with Ala at the downstream splicing junction, although in minute yields, which could be the first step towards nucleophile-free protein ligation. Finally, discovery of a previously not reported intermolecular protein-splicing reaction, termed intein-mediated protein alternative splicing (iPAS), was described. Structural studies revealed that three-dimensional domain swapping is the underlying mechanisms of iPAS. iPAS makes it possible to increase diversity at protein level, without altering the genetic code, and could be used to control protein functions in concentration and expression-order dependent manner. Discovery of this new phenomenon could allow protein interference and is opening new insights into the possible biological functions of inteins.