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Este trabalho teve como objetivo a utilização de glicerol, principal subproduto da produção de biodiesel, como fonte de carbono para a produção de biomassa e ribonucleotídeos de leveduras. Com este propósito, foi realizado um screening de leveduras para identificar aquelas cepas com maior capacidade de crescimento em glicerol. As leveduras Hansenula anomala CCT 2648 e Kluyveromyces marxianus var. lactis CCT 4086 apresentaram os maiores valores de fator de conversão (YX/S) e produtividade volumétrica em células (QX) sob condições fixas de glicerol (30 g/L), temperatura (30 oC) e pH (5,5). Foi também detectada a produção de etanol e ácidos orgânicos, por algumas das leveduras estudadas em meios contendo glicerol. Com base no potencial de produção de biomassa das cepas selecionadas na etapa anterior, foram realizados estudos para avaliar a influência da concentração de glicerol (10 - 50 g/L), extrato de levedura (1 - 3 g/L), pH(4,5 - 6,5) e temperatura (28 - 40oC) no crescimento destas linhagens, utilizando planejamento experimental 24. De acordo com os resultados, a levedura Hansenula anomala CCT 2648 foi selecionada para a produção de biomassa e ribonucleotídeos em experimentos conduzidos em bioreator por apresentar maior concentração de biomassa (4,59 g/L) quando comparada com a levedura Kluyveromyces marxianus var lactis CCT 4086 (3,37 g/L). Nesta fase, foi avaliada a influência da agitação (300 - 700 rpm), aeração (0,5 - 2 vvm) e concentração de glicerol (10 - 50 g/L) com o objetivo de estabelecer as melhores condições de processo, por meio de planejamento 23. Os maiores valores de YX/S (0,57- 0,60 g/g) foram obtidos nos ensaios conduzidos sob a menor concentração de glicerol (10 g/L), entretanto, o maiores valores de QX (0,44 - 0,62) foram obtidos em cultivos quando se utilizou valores de agitação superiores a 500 rpm. A análise estatística confirmou a significância das variáveis, concentração de glicerol e agitação, e permitiu estabelecer os modelos matemáticos representativos da influência destas variáveis na produção de biomassa e ribonucleotídeos por Hansenula anomala CCT 2648. Os resultados obtidos neste trabalho demonstraram o potencial de utilização do glicerol derivado da fabricação de biodiesel como fonte de carbono de baixo custo para a produção de biomassa e biomoléculas, como por exemplo os ribonucleotídeos. / Glycerol is considered to be the principle sub-product derived from biodiesel production process. In this work, glycerol was used as the only carbon source for biomass accumulation and ribonucleotides production. For this purpose screening of different strains was achieved, where the yeasts defined as Hansenula anomala CCT 2648 and Kluyveromyces marxianus var. lactis CCT 4086 showed high biomass yield (Y X/S) and productivity (QX) as well as better ribonucleotides production, using an initial concentration of glycerol of 30 g/L, temperature of 30oC and pH of 5,5. In addition, ethanol and organic acids were detected during glycerol assimilation by these two studied yeasts. Since these two selected yeast showed a promising potential in biomass and ribonucleotides production, a 24 complete factorial design was employed in order to study the influence of different parameters: glycerol initial concentration (10 - 50 g/L), yeast extract initial concentration (1-3 g/L), pH (4,5 - 6,5) and temperature (28 - 40oC) on the fermentation yield and productivity. According to the obtained results, the yeast Hansenula anomala CCT 2648 achieved higher biomass production (4,59 g/L) when compared to Kluyveromyces marxianus var lactis CCT 4086 (3,37 g/L). Such fact lead to the selection of Hansenula anomala CCT 2648 for a scale-up production of ribonucleotides using a stirred stank bioreactor, where the influence of the following fermentation parameters were studied: agitation (300 - 500 rpm), air input (0,5 - 2 vvm) and glycerol initial concentration (10 - 50 g/L) using a 23 full factorial design. High values of YX/S (0,57 - 0,6 g/g) were observed when the experiments were carried out at lower glycerol concentration (10 g/L), while high values of QX (0,44 - 0,62 g/L.h) were observed at high agitation rates of 500 rpm and above. Mathematical models were also created to confirm this influence of glycerol initial concentration and bioreactor stirring on biomass and ribonucleotides production by Hansenula anomala CCT 2648. Obtained results in this work demonstrated the feasibility of using glycerol derived from biodiesel production as a promising low-value carbon source for biomass and ribonucleotides accumulation.

Este trabalho teve como objetivo a utilização de glicerol, principal subproduto da produção de biodiesel, como fonte de carbono para a produção de biomassa e ribonucleotídeos de leveduras. Com este propósito, foi realizado um screening de leveduras para identificar aquelas cepas com maior capacidade de crescimento em glicerol. As leveduras Hansenula anomala CCT 2648 e Kluyveromyces marxianus var. lactis CCT 4086 apresentaram os maiores valores de fator de conversão (YX/S) e produtividade volumétrica em células (QX) sob condições fixas de glicerol (30 g/L), temperatura (30 oC) e pH (5,5). Foi também detectada a produção de etanol e ácidos orgânicos, por algumas das leveduras estudadas em meios contendo glicerol. Com base no potencial de produção de biomassa das cepas selecionadas na etapa anterior, foram realizados estudos para avaliar a influência da concentração de glicerol (10 - 50 g/L), extrato de levedura (1 - 3 g/L), pH(4,5 - 6,5) e temperatura (28 - 40oC) no crescimento destas linhagens, utilizando planejamento experimental 24. De acordo com os resultados, a levedura Hansenula anomala CCT 2648 foi selecionada para a produção de biomassa e ribonucleotídeos em experimentos conduzidos em bioreator por apresentar maior concentração de biomassa (4,59 g/L) quando comparada com a levedura Kluyveromyces marxianus var lactis CCT 4086 (3,37 g/L). Nesta fase, foi avaliada a influência da agitação (300 - 700 rpm), aeração (0,5 - 2 vvm) e concentração de glicerol (10 - 50 g/L) com o objetivo de estabelecer as melhores condições de processo, por meio de planejamento 23. Os maiores valores de YX/S (0,57- 0,60 g/g) foram obtidos nos ensaios conduzidos sob a menor concentração de glicerol (10 g/L), entretanto, o maiores valores de QX (0,44 - 0,62) foram obtidos em cultivos quando se utilizou valores de agitação superiores a 500 rpm. A análise estatística confirmou a significância das variáveis, concentração de glicerol e agitação, e permitiu estabelecer os modelos matemáticos representativos da influência destas variáveis na produção de biomassa e ribonucleotídeos por Hansenula anomala CCT 2648. Os resultados obtidos neste trabalho demonstraram o potencial de utilização do glicerol derivado da fabricação de biodiesel como fonte de carbono de baixo custo para a produção de biomassa e biomoléculas, como por exemplo os ribonucleotídeos. / Glycerol is considered to be the principle sub-product derived from biodiesel production process. In this work, glycerol was used as the only carbon source for biomass accumulation and ribonucleotides production. For this purpose screening of different strains was achieved, where the yeasts defined as Hansenula anomala CCT 2648 and Kluyveromyces marxianus var. lactis CCT 4086 showed high biomass yield (Y X/S) and productivity (QX) as well as better ribonucleotides production, using an initial concentration of glycerol of 30 g/L, temperature of 30oC and pH of 5,5. In addition, ethanol and organic acids were detected during glycerol assimilation by these two studied yeasts. Since these two selected yeast showed a promising potential in biomass and ribonucleotides production, a 24 complete factorial design was employed in order to study the influence of different parameters: glycerol initial concentration (10 - 50 g/L), yeast extract initial concentration (1-3 g/L), pH (4,5 - 6,5) and temperature (28 - 40oC) on the fermentation yield and productivity. According to the obtained results, the yeast Hansenula anomala CCT 2648 achieved higher biomass production (4,59 g/L) when compared to Kluyveromyces marxianus var lactis CCT 4086 (3,37 g/L). Such fact lead to the selection of Hansenula anomala CCT 2648 for a scale-up production of ribonucleotides using a stirred stank bioreactor, where the influence of the following fermentation parameters were studied: agitation (300 - 500 rpm), air input (0,5 - 2 vvm) and glycerol initial concentration (10 - 50 g/L) using a 23 full factorial design. High values of YX/S (0,57 - 0,6 g/g) were observed when the experiments were carried out at lower glycerol concentration (10 g/L), while high values of QX (0,44 - 0,62 g/L.h) were observed at high agitation rates of 500 rpm and above. Mathematical models were also created to confirm this influence of glycerol initial concentration and bioreactor stirring on biomass and ribonucleotides production by Hansenula anomala CCT 2648. Obtained results in this work demonstrated the feasibility of using glycerol derived from biodiesel production as a promising low-value carbon source for biomass and ribonucleotides accumulation.

Thesis (MSc (Microbiology))--Stellenbosch University, 2011. / ENGLISH ABSTRACT: The link between infection with Human Papillomavirus (HPV) and the development of cervical cancer has been established by several epidemiology studies. Cervical cancer is the second most common cancer among women and it occurs at a rate of 22.8 cases per 100 000 women in South Africa. Approximately 86% of newly reported cases of cervical cancer occur in developing countries where limited access to medical facilities hampers efforts to prevent and screen for HPV infection. Two commercial virus-like particle (VLP) vaccines consisting of HPV major structural protein L1, which protect against the most common high-risk HPV-types, are currently available. The high cost and type specificity of these commercially available vaccines have necessitated the development of a low cost, broad-spectrum HPV vaccine. Inclusion of the minor structural protein L2 has been shown to induce broadly cross-neutralizing antibodies and therefore a chimera was constructed that contains an epitope of L2 inserted within the L1 sequence. This construct, renamed SAF, was shown to be highly immunogenic and thus has the potential to be used as a prophylactic cervical cancer vaccine. Methylotrophic yeasts are known to be excellent producers of recombinant proteins due to their strongly inducible promoters that allow culturing of these yeasts to very high cell densities. Pichia pastoris and Hansenula polymorpha have been employed in several studies for heterologous protein production and levels of protein higher than 1 g/L have been reported. These yeasts also have GRAS status and can therefore be used to manufacture products for use in humans. In this study, the potential of H. polymorpha and P. pastoris to produce SAF intracellularly was evaluated. The effect of increased gene dosage and peroxisomal targeting on SAF production was examined as possible strategies to increase the yield of SAF. Peroxisomal targeting was achieved by fusing the SAF gene at the C-terminal end with the Peroxisomal Targeting Sequence 1 (PTS1) which consists of a short tri-peptide: –SKL. The functionality of PTS1 was confirmed using green fluorescent protein (GFP), fluorescence microscopy and peroxisome isolation. Peroxisomal targeting was shown to have a negative effect on SAF production levels in both H. polymorpha and P. pastoris. An increase in gene dosage had no discernable effect on SAF yield in H. polymorpha which is in contrast to previous research. The highest production levels were achieved by P. pastoris KM71 (24.86 mg/L) which compares well to levels of L1 achieved by other research groups. The most significant insight emerging from this work was that all the strains that produced SAF at detectable levels were equally efficient at the production of SAF. Increased biomass was therefore the biggest contributor to high SAF levels (mg/L) in the P. pastoris strains as significantly higher cell densities were achieved during culturing of these strains. With the necessary optimisation, the methylotrophic yeasts have the potential to be used as hosts for the production of a broad-spectrum HPV vaccine. / AFRIKAANSE OPSOMMING: Die skakel tussen infeksie met Mens Papilloomvirus (HPV) en die ontwikkeling van servikale kanker is deur verskeie epidemiologiese studies bevestig. Servikale kanker is die tweede mees algemene kanker onder vroue en dit kom voor teen ‘n tempo van 22.8 gevalle per 100 000 vroue in Suid Afrika. Ongeveer 86% van alle nuwe gevalle kom voor in ontwikkelende lande waar beperkte toegang tot mediese fasiliteite pogings om HPV infeksie te voorkom en te behandel, belemmer. Twee pseudovirale-partikel (VLP) entstowwe teen HPV is tans op die mark beskikbaar en hierdie entstowwe verleen immuniteit teen die mees algemene hoë-risiko HPV tipes. Die hoë koste en nou spektrum van hierdie entstowwe het dit nodig gemaak om ‘n goedkoop, wye-spektrum HPV entstof te ontwikkel. Navorsing het bewys dat die insluiting van die strukturele L2 proteïen in die VLP entstof, lei tot die indusering van neutraliserende teenliggame, wat wye spektrum antigenisiteit tot gevolg het. ‘n Chimeriese proteïen wat ‘n epitoop van L2 binne die L1 volgorde bevat is gekonstrueer, en hierdie proteïen is benoem SAF. SAF het hoë immunogenisiteit en kan dus potensieel as ‘n voorkomende servikale kanker entstof gebruik word. Metielotrofiese giste is bekend vir hulle vermoë om hoë vlakke rekombinante proteïene te produseer as gevolg van hulle induseerbare promotors wat groei tot baie hoë sel digthede toelaat. Pichia pastoris en Hansenula polymorpha is in menigte studies gebruik om heteroloë proteïene te produseer tot vlakke bo 1 g/L. Hierdie giste en die proteïen produkte wat hulle vorm word algemeen aanvaar as veilig vir menslike gebruik. In hierdie studie het ons die potensiaal van H. polymorpha en P. pastoris om SAF intrasellulêr te produseer, geevalueer. Die effek op SAF produksie van verhoogde geen dosering asook die teiken van SAF na die peroksisoom was ondersoek as moontlike strategieë om die opbrengs van SAF te verhoog. Die teiken van SAF na die peroksisoom is behaal deur die Peroksisomale Teiken Volgorde 1 (PTS1) aan die C-terminaal van SAF te heg. Die funksionaliteit van PTS1 was bevestig deur gebruik te maak van groen fluoroserende proteïen (GFP), fluoressensie mikroskopie en isolering van peroksisome. Teiken van SAF na die peroksisoom het ‘n negatiewe uitwerking gehad op SAF uitdrukking in beide H. polymorpha en P. pastoris. ‘n Verhoging in geen dosering het geen onderskeibare effek gehad op SAF opbrengs in H. polymorpha nie wat in teenstelling is met vorige navorsing. Die hoogste produksie vlakke is opgelewer deur P. pastoris KM71 (24.86 mg/L) wat goed vergelyk met vlakke van L1 wat deur ander navorsings groepe behaal is. Die belangrikste gevolgtrekking wat gemaak kan word uit hierdie studie is dat al die rasse wat SAF geproduseer het in meetbare hoeveelhede ewe effektief was. Verhoogde biomassa was dus die grootste bydraende faktor tot hoë SAF vlakke (mg/L) in die P. pastoris rasse as gevolg van die hoë sel digthede wat hierdie rasse kan bereik. Dit is duidelik dat metielotrofiese giste, met die nodige optimisering, oor die potensiaal beskik om as gasheer sisteme te dien vir die produksie van ‘n wye spektrum HPV entstof. / The NRF and the Department of Microbiology for financial support

2-Nitropropane dioxygenase from Hansenula mrakii is a flavin-dependent enzyme that catalyzes the oxidation of anionic nitroalkanes into the corresponding carbonyl compounds and nitrite, with oxygen as the electron acceptor. Although nitroalkanes are anticipated to be toxic and carcinogenic, they are used widely in chemical industry for a quick and effective way of synthesizing common reagents. Consequently, the biochemical and biophysical analysis of 2-nitropropane dioxyganase has a potential for bioremediation purposes. In this study, recombinant enzyme is purified to high levels, allowing for detailed characterization. The biochemical analysis of 2-nitropropane dioxygenase presented in this study has established that enzyme utilizes alkyl nitronates as substrates by forming an anionic flavosemiquinone in catalysis. The enzyme is inhibited by halide ions, does not contain iron and has a positive charge located close to the N(1)-C(2)=O locus of the isoalloxazine moiety of the FMN cofactor.

Killer yeasts secrete polypeptide toxins which kill sensitive cells of their own species and frequently those of other species and genera of yeasts. These protein compounds are designated as killer toxins. Also killer toxins of certain yeast strains have potential growth inhibitory activity on gram-positive pathogenic bacteria and plant pathogenic fungi. The yeasts are immune to their own killer protein. The killer phenomenon can be utilized for the protection of fermentation process against contaminating yeasts and for biological control of undesirable yeasts in the preservation of foods. The killer trait can also be used to produce large amount of foreign proteins in yeast. In the medical field , it is thought that their anti-microbial and anti-mycotic activity could be exploited in a therapeutic strategy. Yeast killer toxins are classified into 11 types according to their killing spectra and immunity-specificities such as K1, K2, etc. Altough there is considerable amount of published information concerning the applications of yeast killer toxins , among the 11 types , only K1 , K2 and K6 have been characterized. In this study , it was aimed to purify and characterize the K4 type yeast killer toxin secreted by the Hansenula anomala NCYC 432. Gel permeation chromatography was performed to isolate the killer toxin by using a HPLC system. The toxin was shown to be a glycoprotein having a molecular mass of between 49.08 kDa and 47.25 kDa and isoelectric point of between 3.77 and 3.41.

This diploma thesis focuses on the photocatalytic effect of titanium dioxide and ultraviolet light. Theoretical part shows the effect of titanium dioxide and aplication in various branches. Antibacterial effect of titanium dioxide iluminated by ultraviolet light was observed in the flow tube reactor on the yeast Hansenula anomala. After adjusting the optimal flow rate experimented with different inicial concentration of yeast cells. To increase desired effect also silver was used in the photocatalytic system. As observed in many previous studies, silver decreases the time needed for killing the microorganisms. The optained date were analysed.

This master thesis is oriented on study antimicrobial effects extracts and macerates from cure Stevia rebaudiana Bertoni. Teoretical part describes basic information about plant Stevia, summary of health significant matters contained in Stevia and posobilities preparing extracts from Stevia. Antimicrobial effects extracts and macerates from cure Stevia were testing on food-borne bacteria (Bacillus cereus and Micrococcus luteus) and yeasts (Geotrichum candidum and Hansenula anomala). Microorganism, extracts and macerates were chosen on basis previous study (Study of antimicrobial effects Stevia Rebaudiana extracts, Eva Rakovská). For screening antimicrobial activity were determined the growth curves by using turbidimetrie for bacteria and direct treetment metod of cells number for yeasts. Antimicrobial effects were confirmed aplication with diffusion pit method on the agar ranges. From the results flow the testing extracts and macerates from stevia analysed antimicrobial effects. The best effect was demostrated on macerates and the most sensitive was bacteria Micrococcus luteus with the best inhibitoring effects.

Today, recombinant expression of proteins is used for a variety of purposes. One of these is the production of allergens, which are vital components in allergy diagnostics. However, traditional expression systems such as ​Escherichia coli​ and ​Pichia pastoris​ might not have the capacity to express all proteins of interest. Thermo Fisher, which is a leading producer of allergy tests, has requested an evaluation of different microorganisms and their capacity for heterologous protein expression in order to expand their existing toolbox of expression systems. This summary was made through a literature study, where twelve organisms were evaluated. Six eukaryotic and six prokaryotic expression systems are compared based on their ability to properly glycosylate protein, need for specific culture conditions, safety, protease activity, duration, protein yield and protein solubility. The prokaryotic systems – Corynebacterium glutamicum​ , ​Lactococcus lactis​ , ​Pseudomonas fluorescens​ , Pseudoalteromonas haloplanktis​ , ​Ralstonia eutropha​ and ​Streptomyces lividans​ – are characterized by being easy to cultivate, operating in different temperature ranges and providing relatively high yields of recombinant protein. The eukaryotic systems – ​Aspergillus fungi, the green algae ​Chlamydomonas reinhardtii​ , the yeast ​Hansenula polymorpha​ , the parasite ​Leishmania tarentolae​ , the moss ​Physcomitrella patens​ and suspension-based plant cells – all have very different morphology and properties. In comparison with the prokaryotic systems, it can be concluded that they are generally better at folding and providing the correct glycosylation patterns for mammalian and plant proteins. However, they require more time and effort to establish a competent cell line. Furthermore, the resulting protein yield is usually less than for the prokaryotic systems. The conclusion can be drawn that no expression system is perfect. The solution is a toolbox, containing various expression systems and vector systems, providing the basis for successful expression of all kinds of complex proteins. Based on the evaluation of expression systems in this review, such toolbox can be obtained.