Microbial, Biochemical and Food Biotechnology

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Open Access
Activation of the SARS-CoV-2 spike protein by 𝘤𝘳𝘺𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘯𝘦𝘰𝘧𝘰𝘳𝘮𝘢𝘯𝘴 secreted proteases
(University of the Free State, 2023) Mjokane, Nozethu; Sebolai, O. M.; Pohl, C. H.; Albertyn, J.; Gcilitshana, O. M. N.
The thesis is not structured in a classical way. As such, it is composed of a literature review section (Chapter 1) and two research chapters (Chapters 2 and 3). A general discussion section (Chapter 4) and addendums are also included. As some chapters are in a publication format, repetition of essential information could not be avoided. Chapter 1 reviews the emergence of SARS-CoV-2 and its impact. In particular, it considers the co-infection of this virus with respiratory fungal pathogens, which are major independent risk factors that complicate COVID-19 by causing a more severe infection resulting in higher mortality than that of either infection on its own. These fungal pathogens secreted furin-like proteases to further their virulence during host invasion. In this context, the thesis argues that it is foreseeable that the virus could also access these fungal furin-like proteases and pervert them in order to activate its latent spike protein. Therefore, this set up a number of questions, which are addressed in the thesis concerning the possible activation of the viral latent spike protein by fungal furin-like proteases. In Chapter 2, it was sought to characterise 𝘊𝘳𝘺𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 (𝘊.) neoformans proteases and assess if they could theoretically bind to the SARS-CoV-2 spike protein. To be specific, previous papers reporting on cryptococcal serine proteases were perused, and this made it possible to select a number of proteases, namely cryptococcal serine carboxypeptidase (CNBF4600), cryptococcal cerevisin (CNBJ2870) and cryptococcal peptidase (CNBA1340), cryptococcal peptidase (CNAG_00150) and cryptococcal cerevisin (CNAG_04625). By designing specific primers, it was possible to show that these serine proteases were expressed in 𝘊𝘳𝘺𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘯𝘦𝘰𝘧𝘰𝘳𝘮𝘢𝘯𝘴 H99, the prototypical cryptococcal strain used in this thesis. Therefore, the expressed gene products were expected to be secreted into the culture media. This was important for the work that follows in Chapter 3. Through using the computational programme, High Ambiguity Driven protein-protein DOCKing (HADDOCK), it was possible to show that some of the selected cryptococcal serine proteases could interact with the coronavirus spike protein and yield a binding affinity greater than and comparable to furin. However, as HADDOCK is a computational programme, the predicted binding affinities might not correlate with the experimental binding affinities in solution, more so since the used 𝘊𝘳𝘺𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘯𝘦𝘰𝘧𝘰𝘳𝘮𝘢𝘯𝘴 proteases structures were predicted and not solved. To account for this, Chapter 3 sought to provide enzymatic evidence using the collected culture media – in the form of supernatant. To do this, a mimetic fluorogenic peptide of the SARS-CoV-2 spike protein was designed and modified to have intra-molecular fluorescence quenching capability using 7-methoxycoumarin-4-yl acetyl (MCA) at the N-terminus and N-2,4-dinitrophenyl (DNP) at the C-terminus. The assay was performed using the cryptococcal supernatant. For reference, recombinant furin was included as this is the serine protease present in humans that catalyses the activation of the spike protein. Here, it was determined that cryptococcal serine proteases present in the supernatant could cleave the mimetic spike protein at S1/S2 site with biochemical efficiency comparable to furin. To test the veracity of these data, SARS-CoV-2 pseudovirion containing a full-length spike protein was used. It was possible to show that the pseudovirion could be transduced into HEK-293T cells in the presence of the cryptococcal supernatant. Chapter 4 takes into account the obtained results and provides a summary of the major observations. Of note, the thesis theorises that yeast kexin proteases are responsible for the observed activity. This is because there is a functional homology between yeast kexin proteases and furin (both are convertases); thus, it is reasonable that the supernatant (which contains yeast kexin proteases) could activate the latent SARS-CoV-2 spike protein. The thesis further proves that other respiratory fungal pathogens have yeast kexin proteases that activate the spike protein. This evidence is documented in Addendum no. 1. All things considered, the findings point to the regulation of protease activity as a viable approach to control the activation of the spike protein by either mammalian protease or fungal proteases. To this end, protease inhibitors could be used to control unwanted proteolysis. Addendum no. 2 attempted to show this. Here, it was possible to show that the South African-based medicinal plant Artemisia tea infusion extract and its active compound artemisinin could control the activation of the mimetic SARS-CoV-2 spike protein by furin but not the supernatant. The latter highlights the need to purify the supernatant and isolate yeast kexin proteases. The idea of exploring the control of unwanted proteolysis is also an interventional measure considered by Pfizer, the pharmaceutical company. This American multinational pharmaceutical and biotechnology corporation successfully piloted Paxlovid to control SARS-CoV-2. This drug contains an anti-protease (PF-07321332) that inhibits the protease (SARS-CoV-2 3CLp) responsible for viral replication.
Open Access
ADH2 regulation in the yeast Saccharomyces cerevisiae
(University of the Free State, 2004-05) Khoboko, Mojabatho Portia; Albertyn, J.; Du Preez, J. C.
English: The aim of this study was to investigate the ability of ethanol to repress the expression of ADH2 in the genome of Saccharomyces cerevisiae. To achieve this, an expression cassette (ADH2::LacZ) using LacZ as a reporter gene was constructed using the YIp356R shuttle vector. A 1000 bp up and downstream region, flanking the open reading frame of ADH2, was fused to the 5’-end and 3’-end of the LacZ gene in the YIp356R shuttle vector. Numerous attempts were made to transform the expression cassette (ADH2::LacZ) into S. cerevisiae (strain W303) containing a deleted ADH2 (adh2Δ::URA3), to displace the deletion cassette through homologous recombination, thereby placing ADH2::LacZ in the place of the ADH2 in genome of S. cerevisiae. This was unfortunately not successful and it was decided to use an alternative approach. In this case the expression cassette was cloned into the integrative vector YIplac211 and transformed into S. cerevisiae. For initial confirmation, the yeast transformants were grown on selective plates containing X-gal, which allows for the detection of β-galactosidase activity through the production of blue coloured colonies. The detection of the blue colour confirmed that the expression cassette was successfully constructed and integrated into the genome. Two randomly selected transformants were cultivated on 20 g glucose l-1 as sole carbon source, to study glucose repression and on three different ethanol concentrations to study the effect of ethanol on the expression of ADH2. Selection was maintained by growing the yeast in a URA– chemically defined media (pH 5.5) at 30ºC. Samples were taken at appropriate intervals to perform β-galactosidase assay, assess utilization of substrate (ethanol and glucose), ethanol formation and biomass determination. During growth on 20 g glucose l-1 the production of β-galactosidase was apparent only when glucose concentrations were very low (2.3 g l-1), indicating that glucose levels have to decrease to a critical level before ADH2 expression can resume. The highest final biomass was produced during growth on 20 g glucose l-1. During growth on the three different ethanol concentrations the highest β-galactosidase maximum specific activity was obtained during growth on 20 g ethanol l-1 (3 643 U mg-1) and the lowest during growth on 5 g ethanol l-1 (2 533 U mg-1). Although the maximum specific activity obtained during growth on 30 g ethanol l-1 were higher than that obtained during growth on 5 g l-1, the production rate was the lowest (93 U mg-1h-1) during growth on 30 g ethanol l-1, suggesting that 30 g ethanol l-1 concentration has negative effect on the expression of ADH2. However this slow production might have been due to the slow growth during this cultivation and not due to ethanol repression. The possible repression of ADH2 is further disputed by the high β-galactosidase production on 30 g ethanol l-1.
Open Access
Alcohol dehydrogenase mediated lactonization of 1,6-hexanediol
(University of the Free State, 2018) Dithugoe, Choaro David; Opperman, D. J.; Smit, M. S.
Alcohol dehydrogenases (ADHs) are oxidoreductases that catalyse the interconversion between alcohols and ketones. These are biotechnologically interesting enzymes due to their ability to produce optical active alcohols. Alternatively, lactonization of diols to corresponding lactones has been explored by researchers. The aim of this study was to screen ADHs for the lactonization of 1,6-hexanediol to Ɛ-caprolactone by evaluating a single ADHs enzyme system and a combinatorial ADHs enzyme system. Saccharomyces cerevisiae ADH 1 and ADH 6 together with Alcanivorax dieselolei B5 ADH 1 to ADH 3 genes were PCR amplified and subcloned into pET28b(+) expression vector. An additional 8 ADHs genes were synthesised and cloned into pET28b(+) by Genescript. The pET28b(+) ADHs were screened using cell-free extract (CFE), however, only four ADHs showed activity towards 1,6-hexanediol of which Aedes aegypti Farnesol dehydrogenase (AaSDR-1), a newly discovered ADH, showed activity towards 1,6-hexanediol. From the CFE reactions, an unknown side product was detected. Therefore, purified AaSDR-1 with Streptococcus mutant NADH oxidase 2 (SmNOX) for cofactor regeneration was used to study the reaction, however the same unknown side product also formed. Thereafter, it was speculated that tris buffer react with the products from 1,6-hexanediol. With NMR and GC-MS analysis, the product was identified to be a tricyclic-tris adduct. Different buffering systems, including sodium phosphate buffer, decreases the tricyclic-tris adduct. The AaSDR-1 and Equus caballus ADH (HLADH) were compared with low concentration of SmNOX. It was observed that at low SmNOX concentrations NADH regeneration is favoured, while, at high concentration NADPH regeneration is favoured. However, the single ADH enzyme system had limitation and a combinatorial ADH enzyme system was proposed to screen for the best primary and secondary ADH. From the combinatorial reaction, the combination of Thermus sp. ATN1 ADH (TADH)-AaSDR-1 gave the best activity. Unfortunately, during the single AaSDR-1 enzyme system and combinatorial TADH-AaSDR-1 system, a white-precipitate ‘plastic-like’ structure was formed over time. The precipitate could not be extracted and quantified. The precipitate could potentially be from 6-hydroxyhexanoic acid and therefore the Ɛ-caprolactone concentration might be underestimated. Chemical, photochemical regeneration and bi-convergent cascade systems were tested as alternatives to the SmNOX system. The FMN light driven reaction required high concentration of AaSDR-1 and also produced the white-precipitate ‘plastic-like’ structure. Although the bi-convergent system of NADPH-dependent Cyclohexanone monooxygenase (CHMO) and NADP+-dependent AaSDR-1 can produce Ɛ-caprolactone, the AaSDR-1 competes with the CHMO for substrate and cofactors resulting in low Ɛ-caprolactone yields.
Open Access
Alkane and fatty acid hydroxylating cytochrome P450 monooxygenases in yeast
(University of the Free State, 2008-11) Shuping, Daniel Sechaba Skake; Smit, M. S.
Yarrowia lypolytica, Candida tropicalis, Candida maltosa and Candida cloacae are extensively studied n-alkane degrading yeasts and are widely used in various industrial processes (Madzek et al., 2004, Mobley, 1999). Cytochrome P450 monooxygenases belonging to the CYP52 family are responsible for the terminal hydroxylation of n-alkanes and fatty acids. Candida species have been successfully used in the synthesis of long-chain α,ω-dicarboxylic acids (DCAs) which are difficult to produce using chemical processes (Wache et al., 2006 Mobley, 1999). These processes, which yield more than 100 g l-1 DCA at volumetric rates up to 1.9 g l-1h-1, are regarded as the most successful P450 dependent biotransformation processes developed thus far (Julsing et al., 2008). Because C. tropicalis and C. maltosa are related to Candida albicans (Eschenfeldt et al., 2003), they are regarded as potential pathogens. However, Y. lipolytica has GRAS status for a number of processes and the entire genome sequence is known (Fickers et al., 2005). Although Y. lipolytica has been shown to produce DCA, none of its mutant strains have produced DCA concentrations close to the DCA produced by Candida sp. (Kogure et al., 2007, Smit et al., 2005). In order to understand the differences in terminal hydroxylase activity that contribute to differences in DCA production, we compared the wild-type strains Y. lipolytica W29 and C. tropicalis ATTC20336 by using alkylbenzenes, 4-hexylbenzoic acid and 4-nonyloxybenzoic acid as substrates. We also cloned and expressed CYP52A13 and CYP52A17 from Candida tropicalis ATTC20336 into Y. lipolytica CTY021 and CTY022, two β-oxidation disrupted strains. Dodecane, tetradecane, hexadecane and 4-hexylbenzoic acid were used as substrates. One experiment was done in bioreactors using a control strain and a strain expressing CYP52A13. The experiments with the wild-type strains showed that Yarrowia lipolytica W29 and Candida tropicalis ATTC20336 responded differently to hydrocarbons. These differences are probably due to differences in the alkane and fatty acid hydroxylases of the two yeasts. In the biotransformation of alkylbenzenes by Y. lipolytica W29, nonylbenzene was the only substrate significantly converted to benzoic acid. The highest product formation (20.6 mM benzoic acid) was observed when the cultures were not induced with oleic acid or n-alkanes (C12, C14). In the case of C. tropicalis ATTC20336, both nonylbenzene and hexylbenzene were accepted as substrates and n-dodecane and oleic acid, reported inducers of CYP52 genes (Craft et al., 2003), significantly enhanced biotransformation of these substrates. Biotransformation of 4-hexylbenzoic acid by Y. lipolytica also occurred in the absence of an inducer, while in C. tropicalis it was only converted to the corresponding DCA after induction with oleic acid. This meant 4-hexylbenzoic acid could possibly be used as a marker substrate for monitoring the expression of C. tropicalis CYP52 genes in Y. lipolytica W29. Biotransformation of 4-nonyloxybenzoic acid by both these strains occurred when cultures were induced with oleic acid. The CYP52A17 and CYP52A13 were cloned under the pPOX2 promoter into Yarrowia lipolytica strains with β-oxidation disrupted. A transformant with at least three copies of the CYP52A17 gene cloned had relatively low activity towards alkanes when compared to the control strain. However, the CTY021:CYP52A13 transformant with at least two copies of the CYP52A13 gene showed improved activity towards n-alkanes. The biggest improvement (92 %) was observed with n-tetradecane. TLC analyses showed that after induction with oleic acid, palmitic acid and n-hexadecane slightly more product was formed from 4-hexylbenzoic acid by the strain with CYP52A13 cloned than by the control strain. However, this improvement was much less than anticipated and 4-hexylbenzoic acid was in the end not used to screen for strains expressing the cloned CYP52 genes. A bioreactor study was conducted in triplicate in a Sixfors multireactor to compare C16DCA production form hexadecane by the control strain and the strain with CYP52A13 cloned. The control strain (CTY026) and test strain CTY021:CYP52A13 grew differently. Although there was an indication that the strain with CYP52A13 cloned produced DCA faster, results were too varaible to reach a firm conclusion that cloning of the CYP52A13 gene significantly improved DCA production by Y. lipolytica.
Open Access
The Amylostereum symbiont of Sirex noctilio in South Africa
(University of the Free State, 1998-12) Slippers, Bernard; Wingfield, M. J.; Coutinho, T. A.; Wingfield, B. D.
English: In Chapter 1 of this thesis, the literature pertaining to the symbiosis between Sirex noctilio and Amy/ostereum areo/atum in the Southern Hemisphere, is reviewed. It is evident from this review that S. noctilio and A. areo/atum have become established throughout the pine growing regions of the Southern Hemisphere, despite measures to prevent its introduction. Unlike its relative unimportance as a pathogen in the Northern Hemisphere, this fungal-insect complex has resulted in great losses to softwood industries during a number of severe outbreaks in the Southern Hemisphere. The use of biological control agents in combination with preventative silvicultural practices, has been shown to be very effective in controlling Sirex in Australasia. It is, however, also evident from this review that despite the rather large collection of knowledge concerning the wasp and its control, information regarding the population structure and phylogenetic relationships of the fungal symbiont of Sirex, is scarce. The recent introduction of S. noctilio into South Africa and its confinement to a rather small area in this country provided the opportunity to study the population of its fungal symbiont in detail. Results from Chapter 2 suggest that the fungus has a very narrow genetic base in South Africa and that the introduction of Sirex into this country was limited. The genetic base of A. areolatum in Brazil and Uruguay is similarly uniform. Of even greater interest is the fact that South Africa and Brazil share a common vegetative compatibility group and, thus, a common origin of A. areo/atum and S. noctilio. Moreover, field isolates from the Southern Hemisphere appear to be closely related, which indicates that Sirex might have spread among countries of the Southern Hemisphere and were not necessarily new introductions from the Northern Hemisphere. Isolates of the fungus associated with the biocontrol nematode, De/adenus siricidicola, are, however, distinct from isolates from other Southern Hemisphere populations of the fungus. This could negatively influence the efficacy of the nematode as biocontrol agent in countries to which the nematode has been distributed. Boidin and Lanquetin (1984) report triangular mating incompatibility between isolates from the different Amy/ostereum spp. Results of Chapter 3 support their conclusions by clearly showing that A. areolatum is more distantly related to A. chailletii, A. laevigatum and A. ferreum, than these three species are to each other. The relationship between the latter three species is, however, more clearly defined in Chapter 3 where it is shown that A. ferreum and A. laevigatum are most closely related to each other. One isolate collected from Sirex areolatus, and, therefore, expected to be A. chailletii, was most closely related to A. laevigatum and A. ferreum. Neither of the latter species has, however, been implicated in associations with woodwasps. Furthermore, the data from this study show that Amylostereum spp. group with neither Stereum nor Peniophora, as has been previously hypothesised, but rather with Echinodontium tinctorium. This grouping was included in a larger clade that included species of Russula, Heterobasidion, Lentinellus and Auriscalpium. Analysis of DNA sequence data derived from the nuc-IGS-rDNA in Chapter 4 supported the phylogenetic relationships of the Amylostereum spp. inferred in Chapter 3. Similarly, the isolate obtained from S. areolatus, did not group with any of the four species of Amylostereum and might represent a new species or a distinct group in of one of the current species. Isolates of A. areolatum associated with both S. noctilio and S. juvencus contained four heterogenic sequences in the DNA region analysed. These heterogenic sequences were contained in each isolate of the fungus in one of five combinations. Neither the heterogenic sequences included in the fungal isolates, nor the different combinations of these sequences, separated the populations of A. areolatum associated with different wasp species. Despite the heterogenic nature of this DNA region in some isolates, RFLP analysis was used effectively to distinguish between the different species of Amylostereum. The work presented in this thesis represents the first molecular. view of the phylogeny of the genus Amylostereum, as well as that of some of the Amylostereum spp. associated with woodwasp species. It is clear from Chapter 5 that these findings now provide a powerful tool to give a clearer picture of the taxonomy and evolution of these fungi, as well the ecology of their symbiosis with woodwasps. The study of the genetic structure of the fungal populations associated with woodwasps also gives new insight into the geographical origin and history of both the insects and their associated fungi.
Open Access
Antibody fragments as a possible therapeutic treatment for infectious bronchitis in poultry
(University of the Free State, 2018-09) Coetzee, Janetta Magrieta; Bragg, R. R.; Boucher, C. E.; Van der Westhuizen, W. A.
Infectious bronchitis virus (IBV), a coronavirus, is the etiological agent for infectious bronchitis (IB), an acute respiratory disease of poultry. Infectious bronchitis is a notifiable disease, and taking in consideration that poultry is the second most consumed meat within South Africa, it highlights the importance of monitoring IBV outbreaks. Recombinant single chain variable fragments (scFv) have been used in therapeutic treatments for various human and veterinarian viruses, including other coronaviruses, such as SARS-CoV and MERS-CoV. The study aims to select scFv against the IBV antigen, with the use of phage display technology and commercial ELISA plates coated with the most prevalent IBV strains namely, H120 and M41. It has been proven that the S1 protein induces the binding of neutralising antibodies which provides protection against lethal CoV infections, thus, indicating a possible application for a therapeutic treatment. In this study, phage clones were selected from a human domain (dAb) library (Source BioScience, Australia). Panning was repeated three times using commercial ELISA plates coated with M41 and H120 IBV strains. Positive monoclonal phage clones were retrieved from the polyclonal mix by a sandwich ELISA and sequenced. The selected scFvs were expressed by Isopropyl β-D-1 thiogalactopyranoside induction and purified by immunoprecipitation with the Pierce Anti-c-Myc Agarose kit (Thermo Scientific, USA). After purification, binding ability of the scFvs were determined by means of a direct competitive ELISA. The neutralising ability of the scFvs was then determined by a virus neutralisation assay in ovo with the Avipro IBV H120 strain (Lohmann Animal Health Gmbh, Germany). This was performed in 9-day old SPF eggs over a time-period of six days. One set of eggs were injected with a dilution range of the IBV H120 strain and another set by a mixture of 2 μg/ml scFv with the IBV H120 strain. The end-point titres were determined and compared by the Spearman-Karber method (Spearman, 1908). Statistical analysis was performed using the student t-test with a p-value of 0.05. Round 1 of panning resulted in a total of 1.2 x 106 phages/ml and after round 3 a total of 3.0 x 1010 phages/ml were obtained. A total of 96 phage clones were manually selected from which only 12.5% showed a positive result during the sandwich ELISA. The 12 positive clones were sequenced and analysed based on nucleotide and amino acid composition. A total of five scFvs contained a complete variable heavy (VH) chain sequence, two of which was identical. This resulted in four unique and complete scFv sequences. These sequences showed a high variance in the nucleotide composition through- out the sequence. However, variation in the amino acid composition was only observed in the third complementary determining region. The scFvs were expressed and resulted in concentrations ranging from 204.38 μg/ml to 265.07 μg/ml. Detection of IBV antigen binding ability of the purified scFvs was conducted by a direct competitive ELISA. However, no statistical difference in absorbance values were observed, indicating insufficient binding of the scFvs. The in ovo virus neutralisation assay resulted in a one log reduction of the end-point titres. Statistical analysis proved one of the reductions to be statistically significant with a p- value less than 0.05, resulting in a partial neutralisation effect from the scFv. In conclusion, the selection process showed a progressive enrichment of antigen specific clones from the dAb library. The scFvs were successfully expressed, purified and characterised in terms of binding ability.
Open Access
Antigenic investigation of genetically different strains of Beak and feather disease virus
(University of the Free State, 2009-01) Hattingh, Albertha René; Bragg, R.R.
English: Psittacine beak and feather disease (PBFD) is a dermatological condition that affects both captive and wild psittacine birds worldwide. In Southern Africa, 10 – 20% of breeding stocks are lost due to the disease each year. PBFD threatens the survival of the indigenous endangered Cape parrot (Poicephalus robustus) as well as the black-cheeked lovebird (Agapornis nigrigenis). The disease is characterized by roughly symmetrical feather loss, feather abnormalities, anorexia and immunosuppression. In advanced cases of the disease beak and claw deformities are present. The causative agent of PBFD is Beak and feather disease virus (BFDV), a circovirus belonging to a diverse group of circoviruses within the family Circoviridae. BFDV has a circular single stranded DNA genome consisting of seven open reading frames (ORFs); three of these ORFs are conserved amongst all isolates of BFDV. ORF 1 encodes the Rep protein, ORF 2 the coat protein (CP) which is also the epitopic protein of the virus and ORF 5, whose function remains unclear. BFDV cannot be cultivated in tissue/cell culture or in embryonated eggs. The inability to cultivate the virus has hampered the development of diagnostic tests and a vaccine as preventative measure against the disease. BFDV is a genetically diverse virus. Researchers have demonstrated that there are at least eight different lineages of BFDV, where Southern African isolates group into three unique genotypes. Many studies have been performed which indicated the diversity of BFDV, but so far no studies have been done which link this genetic diversity to the possibility of the existence of more than one strain of BFDV. This led to the aims of the present study which were to investigate antigenicity of BFDV isolates belonging to different genotypes and then the subsequent bacterial expression of six isolates of BFDV that were genetically different. The entire CP genes of six isolates were amplified with polymerase chain reaction (PCR) and subsequently sequenced. Phylogenetic analysis of sequence data showed that the isolates from this study grouped into lineage one as was described by Heath and co-workers (2004). Amino acid sequences from the isolates from each lineage was applied in an in silico prediction algorithm in order to establish the possibility of more than one strain of BFDV. The predictions indicated that isolates from Australia and South Africa had the same antigenic profile. However, isolate BCL1-ZAM and LK-VIC each produced their own antigenic profile. This indicated the distinct possibility that there is more than one strain of BFDV and that at least one antigenic determinant was situated at the N-terminus of the CP. However, these results have to be confirmed by conducting in vitro studies. Attempts were made to express the full length CP genes of six isolates in BL21(DE3) Escherichia coli with the pET-28b(+) vector. Neither polyacrylamide gel electrophoresis (PAGE) nor Western blotting indicated the presence of recombinantly expressed protein in any of the studies conducted. The codon adaptation index (CAI) for BFDV was calculated to be 0.250, which indicated that the CP had a 25% possibility of being expressed in E. coli due to codon incompatibility. From this study it can be suggested that before attempting expression of any gene in E. coli the CAI should be calculated. It was also concluded from this work that no further attempts to express the CP gene in E. coli should be conducted.
Open Access
Application of amylases for the improvement of water drainage from recycled pulp fibre
(University of the Free State, 2003-05) Jansen van Vuuren, Hendrikus; Wolfaardt, J. F.
English: The use of recycled fibre to produce paper is increasing due to improved processes and consumer pressure. However, recycled fibre has slow drainage rates because of fines and residual starch. Slow drainage reduces machine speed, production rate, energy demands of the drying section and water consumption. Amylases have been used previously on mill scale to degrade residual starch in paper and improve drainage. This study evaluated commercial amylases to degrade starch and improve drainage. The enzymes BAN 480L, Duramyl 300L, Fungamyl 800L, Termamyl 120L and AMG 300L were used to treat recycled pulp and the influence of pH, temperature and shear forces was tested on the enzymes. BAN 480L and Duramyl 300L displayed activity over a wide temperature range, while Duramyl 300L had activity over a wide pH range. Termamyl 120L, BAN 480L, Duramyl 300L and Fungamyl 800L showed good tolerance to shear forces. The influence of BAN 480L, Duramyl 300L and Fungamyl 800L was tested on strength properties of pulp. Enzymatic treatment had no negative effect on Bursting Index, Tearing Index, Air Permeance and Handsheets Drainage Time. In some cases, strength properties improved. Laboratory-scale drainage evaluation (Canadian Standard Freeness, Drainage Time, Vacuum Drainage Time and the Water Retention Value) showed no significant improvements despite reduction of starch content. Pilot trials were conducted with different pulp grades and at different consistencies. Paper and backwater samples were evaluated for changes in moisture, starch content and Chemical Oxygen Demand (COD). The COD levels of backwater did not increase after enzymatic treatment. The reductions in starch and moisture content of paper indicated that amylases could improve papermaking, but that mill-scale trials were required to quantify possible benefits. Duramyl 300L was applied in a mill trial to treat K4 pulp for production of 195 Flute. Temperature, pH, starch, COD, Total Dissolved Solids (TDS), and moisture were measured at different sampling points. The machine speed, steam consumption, moisture and strength properties of the product were also recorded. Secondary starch degradation of 66 % was achieved and moisture of the trim and jumbo roll was reduced. Machine speed and steam consumption did not improve and no significant influence on strength properties was found. The backwater COD and TDS increased, but were still within control limits. Longer trials should be conducted in future to focus on optimisation of enzyme dosages and production of other fluting grades. The impact of the enzymatic treatment on effluent quality and microbial fouling should also be investigated.
Open Access
Application of fungi in biotechnological processes for the pulp and paper industry
(University of the Free State, 2001-11) Dunn, Carin; Wolfaardt, J. F.
English: Lignin, hemicellulose and cellulose occur together in wood and agricultural wastes that are used in industries such as the pulp and paper industry. Biodegradation could be applied by these industries to save cost and reduce environmental impact. Lignocellulose degradation is very complex and must be understood for the optimisation of biotechnological processes. White-rot fungi degrade lignin, cellulose and hemicellulose while brown-rot fungi modify lignin slightly, but also break down cellulose and hemicellulose. The most important enzymes in lignin degradation are manganese peroxidase, lignin peroxidase and laccase, which these fungi produce extracellularly. Decay fungi establish on organic material, which leads to the degradation of lignocellulose during the colonization process. A distinction must be made between primary and secondary colonisers during degradation, because of succession that takes place. Ophiostoma piliferum is an example of a primary coloniser, which is used to produce Cartapip 9if). This product was developed to treat wood chips during storage to reduce fibre degradation. The fungus was previously not available in South Africa because of a possible threat to local forest species. South African forestry companies wanted to test Cartapip 97® in industrial processes and it was, therefore, necessary to demonstrate that the fungus is not pathogenic. It also had to be confirmed that the fungus is a strain of 0. piliferum before certification and importation into South Africa would be allowed. Cultural and morphological characteristics of the anamorph of the Cartapip fungus were found to be similar to those of O. piliferum and it was released for field trials. The pathogenicity of the fungus was compared with Ophiostoma ips and Sphaeropsis sapinea, causes of sapstain on Pinus spp. in South Afiica, to demonstrate that O. piliferum does not pose a threat to forestry. Different pine species were inoculated and the results indicated that O. piliferum is not a pathogen and it is, therefore, safe to use Cartapip 9if) in South Afiica. Hardwood and softwood chips were subsequently treated with Cartapip 97® and pulped using different pulping methods, but the benefits were not obvious. It is, however, possible that the extractives content of the chips was reduced. A slight increase in strength of kraft pulp from softwood and hardwood and also of Soda-AQ pulp from A. mearnsii, was observed after pre-treatment with Cartapip 97®. Bagasse contains fibres that can be used for the production of paper. However, bagasse has to be stored for long periods during which time decay occurs. Stored bagasse could be pre-treated with fungi to preserve and possibly improve the quality of the bagasse. In this study, Lenzites betulina and Pycnoporus sanguineus were used to treat bagasse before pulping. Inoculum production, pulping processes and different incubation periods were evaluated to optimise biopulping. Ultrastructural studies of treated bagasse were used to determine the effect that fungal treatment has on bagasse. Pulping results obtained from bagasse treated with L. betulina were variable and P. sanguineus did not improve the pulping. These results indicated that colonisation and degradation strategies of biopulping fungi must be fully understood before an attempt is made to optimize pulping processes.
Open Access
Ascospore release and oxylipin production in the yeast Dipodascopsis
(University of the Free State, 2007-11) Goldblatt, Monique E.; Kock, J. L. F.; Van Wyk, P. W. J.; Pohl, C. H.
The genus Dipodascopsis was extensively studied with regards to reproductive cycles as well as the presence, distribution and function of 3-OH oxylipins. Most of this research was carried out on D. uninucleata var. uninucleata (Canadian strain) as well as D. tóthii. However, little is known concerning D. uninucleata var. uninucleata isolated from South African soil, as well as D. uninucleata var. wickerhamii. Consequently, using gas chromatography-mass spectrometry, electron microscopy and confocal laser scanning microscopy, the two varieties were compared regarding their morphologies, oxylipin production, mitochondrial activity as well as life cycles and ascospore release. According to literature, the two varieties differ only in their ability to assimilate certain carbon sources. During this study, differences in ascospore size as well as differences in ascospore clustering, after release, was observed. Furthermore, differences in the type of 3-OH oxylipin produced by the two varieties, also existed. 3-OH oxylipin production was found to be associated mainly with the sexual stage and concentrated in the ascus surrounding the ascospores, in both varieties. Furthermore, increased mitochondrial activity was also observed during the sexual stage and found to be concentrated in close vicinity of the ascospores. Since mitochondria produce 3-OH oxylipins, it is suggested that the increased activity during sexual development would be to aid in the production and release of the ascospores, as well as the accumulation of these 3-OH oxylipins. In addition, acetylsalicylic acid was found to inhibit the production of 3-OH oxylipins by probably decreasing mitochondrial activity resulting in the inhibition of ascospore release.
Open Access
Biocatalytic resolution of epoxides: epoxide hydrolases as chiral catalysts for the synthesis of enantiomerically pure epoxides and vic diols from α-olefins
(University of the Free State, 1999-06) Botes, Adriana Leonora; Smit, M. S.; Litthauer, D.
English: The synthesis of chiral pharmaceuticals in an enantiopure form had become increasingly important in the last few years. This same trend is now found in the synthesis of agrochemicals. Epoxides, due to their high reactivity with a large number of reagents, and vie diols, employed as their corresponding cyclic sulfates or sulfites as reactive intermediates, are versatile chiral synthons in the synthesis of many bioactive compounds. Extensive research efforts have thus been directed towards the synthesis of optically active epoxides and viel diols. Kinetic resolution of racemic epoxides by epoxide hydrolases has recently emerged as a very attractive strategy for the synthesis of enantiopure epoxides. Both chemical and biological catalysts that may be employed to obtain enantiopure epoxides from relatively inexpensive racemic substrates had been reviewed (Chapter 1). The potential use of microbial epoxide hydrolases, including those from yeasts as elucidated during this study, was emphasised in this review. At the onset of this study, epoxide hydrolase activity had been identified in only one yeast, Rhodotorula glutinis. The broad range of substrates that were hydrolyzed with excellent enantioselectivity by this yeast, indicated that yeast epoxide hydrolases might be very interesting catalysts. This had indeed been found to be true during the course of this study. Enantioselective hydrolysis of a homologous range of aliphatic 1,2- epoxyalkanes was accomplished in collaboration with the group of Jan de Bont (Division Industrial Microbiology, Wageningen AU, The Netherlands). No other microbial epoxide hydrolases have been found that display this unique enantioselectivity for epoxides lacking other substituents (Chapter 2). Extensive screening of yeasts from the renowned UOFS Yeast Culture Collection revealed that epoxide hydrolase activity was constitutively present in about 20% of the yeasts screened, and that other basidiomycetous yeasts from the genera Rhodotorula, Rhodosporidium and Trichosporon shared this unique enantioselectivity for 1,2- epoxyoctane with Rhodotorula glutinis (Chapter 3). he apparent association between carotinoid production and epoxide hydrolase activity in bacteria as well as the red yeasts Rhodotoru/a and Rhodosporidium, prompted us to investigate the epoxide hydrolase activity of the yellow pigmented bacterium Chryseomonas /uteo/a in our collection. Indeed, this bacterium displayed epoxide hydrolase activity, and moderate enantioselectivity for 1,2-epoxyalkanes (E =20) by a bacterial epoxide hydrolase was found for the first time (Chapter 4). A survey of the enantioselectivities of yeasts for a homologous range of 1,2- epoxyalkanes, 1,2-epoxyalkenes as well as the 2,2-disubstituted 2-methyl-1,2- epoxyheptane and benzyl glycidyl ether was conducted. Excellent biocatalysts for C-5 to C-8 epoxyalkanes and the C-8 epoxyalkene were found. The epoxide hydrolases from all the enantioselective yeasts were found to be membrane-associated (Chapter 5). The epoxide hydrolase from the yeast Rhodosporidium toru/oides was purified in an elegant one-step protocol from the microsomal fraction, using affinity chromatography (Chapter 6). However, initial attempts to obtain amino-acid sequences failed. In lieu of information about the primary structure of yeast epoxide hydrolases, inactivation of the enzyme by modification of specific amino acids was studied. Asp/Glu and His residues were found to be essential for catalytic activity. In addition, it was found that one or more Ser residues in the catalytic site are indispensible for catalytic activity. These results indicate that yeast epoxide hydrolases probably belong to the same subfamily of a,l3- hydrolase fold enzymes as the microsomal epoxide hydrolases from other eukaryotes. Unusual kinetic behaviour was observed during the hydrolysis of 1,2-epoxyalkanes by purified epoxide hydrolase. Hydrolysis was characterised by a strong dependence of enantioselectivity on the presence of the substrate as a second (Iypophilic) phase. The purified epoxide hydrolase was not very stable, with a half-life time at 35°C of 18 hours (Chapter 7).
Open Access
Bioflocculant produced by Bacillus velezensis and its potential application in brewery wastewater treatment
(Scientific Reports, 2022-06) Agunbiade, Mayowa; Oladipo, Babatunde; Ademakinwa, Adedeji Nelson; Awolusi, Oluyemi; Adesiyan, Ibukun Modupe; Oyekola, Oluwaseun; Ololade, Olusola; Ojo, Abidemi
This study was designed to evaluate the potential of bioflocculant producing strains isolated from wastewater sludge. According to the Plackett–Burman design, the response surface revealed glucose, magnesium sulfate, and ammonium sulfate as critical media components of the nutritional source, whereas the central composite design affirmed an optimum concentration of the critical nutritional source as 16.0 g/l (glucose), 3.5 g/l magnesium sulfate heptahydrate (MgSO4.7H2O), and 1.6 g/lammonium sulfate ( (NH4)2SO4), yielding an optimal flocculation activity of 96.8%. Fourier Transformer Infrared Spectroscopy (FTIR) analysis confirmed the presence of hydroxyl, carboxyl and methoxyl in the structure of the bioflocculant. Additionally, chemical analysis affirmed the presence of mainly a polysaccharide in the main backbone of the purified bioflocculant with no detection of protein. Energy Dispersive X-ray analysis affirmed the presence of chlorine, phosphorous, oxygen and chlorine as representatives of elemental composition. Thermogravimetric (TGA) analysis revealed over 60% weight was retained at a temperature range of 700 °C. The purified bioflocculant remarkably removed chemical oxygen demand, biological oxygen demand and turbidity in brewery wastewater. This study suggested that the bioflocculant might be an alternate candidate for wastewater treatment.
Open Access
Biogeochemical and kinetics characterization of sulfate reducing microbial communities enriched from mine drainages
(University of the Free State, 2015-02) Moloantoa, Karabelo Macmillan; Van Heerden, E.; Castillo, J. C.
English: Acid mine drainage (AMD) or acid rock drainage (ARD) is a global challenge contaminating a lot of the fresh surface and groundwater. The drainage is characterized by low pH, high metal and sulfate concentrations. It is a consequence of most mining activities as lead source of AMD. The metals and sulfate in the drainage result from oxidation of metal sulfide containing rocks also referred to as host rock. Oxidation of the host rock occurs during mining by water and oxygen or naturally where the oxidation process occur through weathering and both processes can be accelerated by iron oxidising bacteria such as Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans. The A. ferrooxidans catalyses the oxidation of iron containing sulfide minerals such as pyrite and arsenopyrite releasing iron, arsenic and sulfur which gets oxidized into sulfate generating sulfuric acid that lowers the pH of the water. The acidic environment induces dissolution of other metals. In most coal mines, AMD contains some neutralizing minerals such as calcium oxide (lime), calcium carbonate (calcite) and sodium carbonate which raise the pH of the water but do not precipitate all of the metals and sulfate. The pH of water can be raised to between 5 and 8 hence the drainage is termed Non-acid mine drainage (NMD). Toxic metals in AMD and NMD contaminate streams and rivers where they affect aquatic and terrestrial life. Treatments of these drainages have been developed and characterized into biotic and abiotic systems where chemical and biological methods are used. In this study, attention was given to extending knowledge that can contribute to developing and extending biotic remediation systems where sulfate-reducing bacteria (SRB) are employed to interact with AMD or NMD. SRB are a diverse group of microorganisms used in bioremediation and have been studied widely. SRB play a major role in the reversal process of AMD and NMD formation by reducing the sulfate concentrations. SRB use sulfate as their terminal electron acceptor releasing sulfide in a gas form that dissolves in the solution when the pH is above 4. This process occurs optimally in anaerobic environments and the dissolved sulfide, reacts with dissolved metals in the drainage forming metal sulfide precipitates. The sulfate reduction and metal removal processes from the drainages can be affected by numerous factors and a few discussed were explored and the knowledge base extended in this study. Environmental conditions always have an effect on most activities performed by biological entities. For sulfate reduction, pH, temperature, carbon source type and availability, metal concentrations and redox conditions have direct effects on SRB activities. In this study, three mine drainage study sites with generic names: Site-Ex, Site-Ka and Site-Po were selected. Water samples from the three sites as well as a sludge sample from Site-Po were collected and characterized chemically. Drainage from Site-Ex had characteristics of NMD while Site-Ka and Site-Po water samples had AMD characteristics. High concentrations of sulfate and transition metals were detected in the AMD samples. Microscopic analysis revealed high microbial cell counts in NMD and lower cell counts in AMD samples that could most probably be directly related to the diversity and toxicity of metals and low pH of the drainages. Molecular analysis revealed the presence of various SRB species in the drainages and sludge samples including the well-studied Desulfovibrio sp. Two media compositions: Postgate medium B (PSGM) and Anaerobic sulfate reducing medium (ASRM) were used to enrich anaerobic bacterial communities. Acclimation process with three passaging intervals of 20 days was conducted in anaerobic serum vials. After the third passaging stage, molecular identification of the enriched cells was performed and results revealed successful enrichment of SRB and other anaerobic (some novel) bacteria within the consortia. Scanning electron microscopy (SEM) was used to morphologically characterize the biogenic precipitates from the tertiary cultures. The SEM results showed bacterial biofilm associated with precipitates similar to those identified as framboid pyrite precursors. The enriched consortia from the three study sites were co-cultured in PSGM and ASRM respectively. The best bacterial growth was achieved in PSGM which was then used for subsequent experiments as the medium of choice. The sulfate reducing capabilities of the enriched SRB were tested in PSGM with sulfate concentrations ranging between 2 000 mg/L to 4 000 mg/L. An average of 72% sulfate reduction was achieved in all experiments with a positive response of SRB to higher sulfate concentrations. Effects of pH and temperature on sulfate reduction were evaluated at pH of 3.5 and 6.2 and temperatures of 10ºC and 25ºC. Low pH conditions showed negative effects on sulfate reduction activity and bacterial growth even when temperature was raised to 25ºC. Optimum SRB activity was observed in the experiment where pH was 6.2 at 25ºC. The results confirmed higher sulfate reducing conditions at higher pH (6.2) and temperature (25ºC). The carbon source utilisation by the enriched SRB between glycerol and sodium lactate was evaluated in batch operated bioreactors. The best sulfate reduction activity by SRB was observed when glycerol was used as a sole carbon source yielding greater amounts of dissolved sulfide concentrations. Glycerol was then used further as the main carbon source in PSGM. Metal-microbe interactions were evaluated where higher concentrations of zinc (Zn2+) and iron (Fe2+) were introduced in the bioreactors. Results showed 100%, 85% and 40% sulfate reduction in experiments where no metals, 200 mg/L of iron and 200 mg/L of zinc were added respectively. Effects of high Zn2+ concentrations were similar to those exerted by low pH conditions. However, 90% zinc and 97% iron were removed from the medium through biogenic precipitation. Precipitates were characterized by SEM, Transmission Electron Microscopy (TEM), X-ray Diffraction (XRD) and Energy Dispersive X-ray Spectroscopy (EDX) which confirmed the presence of biologically induced precipitates. Results in this study can be used to model the activity of SRB in evaluated conditions that affect sulfate reduction. The enriched bacterial communities also showed great potential to be used in the up-scaled “reactors” to reduce sulfate concentration while indirectly precipitating dissolved metals in AMD.
Open Access
A bioinformatic tool for analysing the structures of protein complexes by means of mass spectrometry of cross-linked proteins
(University of the Free State, 2013) Mayne, Shannon L. N.; Patterton, Hugh-G.
English: Multi-subunit protein complexes are involved in many essential biochemical processes including signal transduction, protein synthesis, RNA synthesis, DNA replication and protein degradation. An accurate description of the relative structural arrangement of the constituent sub-units in such complexes is crucial for an understanding of the molecular mechanism of the complex as a whole. Many complexes, however, lie in the mega-Dalton range, and are not amenable to X-ray crystallographic or Nuclear Magnetic Resonance analysis. Techniques that are suited to structural studies of such large complexes, such as cryo-electron microscopy, do not provide the resolution required for a mechanistic insight. Mass spectrometry (MS) has increasingly been applied to identify the residues that are involved in chemical cross-links in compound protein assemblies, and have provided valuable insight into the molecular arrangement, orientation and contact surfaces of sub-units within such large complexes. This approach is known as MS3D, and involves the MS analysis of cross-linked di-peptides following the enzymatic cleavage of a chemically cross-linked complex. A major challenge of this approach is the identification of the cross-linked di-peptides in a composite mixture of peptides, as well as the identification of the residues involved in the cross-link. These analyses require bioinformatics tools with capabilities beyond that of general, MS-based proteomic analysis software. Many MS3D software tools have appeared, often designed for very specific experimental methods. We review all major MS3D bioinformatics programs currently available, considering their applicability to different workflows, specific experimental requirements, and the computational approach taken by each. We also developed AnchorMS, a new bioinformatics tool for the identification of both the sequences and cross-linked residues of di-peptides within a post-digest peptide mixture based on MS1 and MS2 data. AnchorMS is intended as a component in the workflow of an MS3D experiment where the protein sequences, cross-linking reagent and protease are known. AnchorMS is freely available as a public web service at cbio.ufs.ac.za/AnchorMS via a simple, user-friendly web interface coded in PHP/XHTML. Experimental sample preparation information and MS data may be uploaded through the web form and analysed by AnchorMS. After analysis, the web interface displays the di-peptides detected, as well as the calculated maximum inter-residue distance between crosslinked residues. This distance information can be used in the optimization of sub-unit positioning within structural models using third party software. The computational core of AnchorMS was developed as an open-source Python project. We describe in detail the overall structure and workflow of the code as well as the functionality implemented in each section of the code. AnchorMS creates a digital library of possible di-peptides and generates expected precursor and fragment mass spectra for each. In order to identify di-peptides, the observed mass spectra are matched against the library of expected mass spectra. Features that are unique to AnchorMS are highlighted, including those for the analysis of di-peptides where the sequences are identical, but the cross-linked residues differ. AnchorMS considers their possible co-fragmentation and employs a specialised second score for distinguishing between such precursors. A unique mathematical model for estimating the level of false positive matching was derived based on an in silico simulation of false positive spectrum matching using randomly generated di-peptide sequences. Subsets of the simulation data were modelled using disparate functions, which were subsequently combined to yield a composite model that described expected false matching under various conditions. The refined calibration of this model against simulation data was performed using the R programming language. AnchorMS also implemented this model as a dynamic false positive threshold, where score values greater than the threshold were considered likely to be true spectrum matches.
Open Access
Biological reduction of soluble uranium by an indigenous bacterial community
(University of the Free State, 2013-07) Maleke, Mathews Maleke; Van Heerden, E.; Williams, P. J.; Botes, E.
English: Uranium (U) and chromium (Cr) in groundwater are a serious public health concern due to their chemical toxicity. Even so, microorganisms have developed mechanisms which permit them to thrive under previously perceived uninhabitable conditions. A number of bacteria have been isolated from areas impacted with the soluble heavy metals, and can be exploited as bioremediation agents since they are well adapted to these metals. To date, the use of microbial mechanisms for bioremediation processes is a growing industry since it provides green and sustainable technologies. In this study, the upflow bioreactors were used as a low cost, low maintenance effective bioremediation strategy in comparison to the available methods of remediation. Two metals known to be toxic in their soluble state were treated. The first was Cr(VI) from an impacted site in Limpopo and the second was U(VI) from the Wonderfonteinspruit catchment, North West Province. The system was efficient for the removal of soluble Cr(VI) and U(VI) from the impacted water through biostimulation of indigenous bacterial communities. This system can be up scaled and employed for the remediation of impacted sites, and it will be useful especially at low levels of U(VI). Indigenous bacterial community from impacted sites have the capability to reduce Cr(VI) and U(VI) effectively over a sustainable period. The shortage of electron donor and continuous oxygen exposure in the case of U(VI) act as a limiting factor. However, in this study successful Cr(VI) and U(VI) reduction rates were increased by the addition of an electron donor to stimulate the indigenous bacterial community. Furthermore, a third upflow bioreactor showed that it is even possible with gradual increases of U(VI) concentration that U(VI) bioreduction is possible at very high levels. The influent water was spiked step wise with uranyl acetate, allowed to reach maximal U(VI) reduction/removal and then the diversity was assessed. Despite the 10 mg/l U(VI) fed to the bioreactor, the established microbial community was able to tolerate, adapt and thereby remove the U(VI) from the spiked water. Even though biofilm could not sturdily adhere to the matrix from the bioreactor, high levels of U(VI) removal could be achieved and the planktonic community maintained. No biofilm could be observed from SEM analysis from the TEM it was observed that the planktonic microbial community have an interaction with uranium. Since no U(VI) could be detected from the effluent samples, it is thus postulated the uranium in contact with the microbial cells is in another form, probably U(IV) as previously shown in this laboratory. This study allows for the understanding of the metal microbe interactions in impacted environments, the use of this biome to remediate the water in an effective, low cost and maintenance bioreactor.
Open Access
Biological synthesis of gold nanoparticles by Thermus scotoductus SA-01
(University of the Free State, 2010-01) Van Marwijk, Jacqueline; Van Heerden, E.
English: The usual strategy to prepare gold nanoparticles involves the reduction of a gold salt in solution by various reducing agents in the presence of a stabilizer. These particles are mostly spherical with poor monodispersity. An alternative means is to use biological material to mediate particle synthesis. Microorganisms such as fungi have demonstrated the ability to produce nanoparticles of different shapes and sizes extending beyond the scope of chemical means, and the microbial interaction with metals also supply eco-friendly methods for nanoparticle production. It has been hypothesized that the proteins involved in nanoparticle synthesis require a co-factor such as NADH / NADPH, as previous studies have indicated that NADH- and NADPH-dependent enzymes are important factors in the biosynthesis of metal nanoparticles. Thermus scotoductus SA-01, a thermophilic bacterium, isolated from an AngloGold Ashanti mine near Carletonville, Republic of South Africa, was used for purification of a gold(III) reducing and nanoparticle synthesizing protein. This bacterium has the ability to produce gold nanoparticles, and more than one pathway can be followed to produce these particles. A protein was purified to homogeneity by using a combination of several liquid chromatography resins. The N-terminal sequence was obtained by using automated Edman degradation. The protein purified is not a classical oxido-reductase and was identified as an ABC transporter peptide-binding protein (~70kDa). This discovery shows that gold nanoparticles can be produced by proteins other than oxidoreductases. The interaction of the protein extracted and purified from Thermus scotoductus SA-01, as well as the recombinant proteins, with liquid gold under varying physico-chemical conditions have been studied using TEM, EDS, and by measuring the plasmon resonance band, to illustrate the effect on particle morphology and to elucidate the protein mechanism. The size and the shape of particles could, to an extent, be manipulated by controlling the environmental parameters. The purified protein as well as the recombinant proteins was only able to produce nanoparticles in the presence of sodium dithionite and it is thus hypothesized that the donation of electrons via the disulphide bridge in the protein is involved in the reduction of the gold ions. Even though the recombinant proteins had the ability toproduce nanoparticles they were not as efficient as the native protein, but when the optimum parameters for the recombinant proteins are established they could be used in the upscale production of gold nanoparticles or gold nanosheets.
Open Access
Biology of botryosphaeria dothidea and sphaeropsis sapinea as endophytes of eucalypts and pines in South Africa
(University of the Free State, 2001-12) Smith, Hendrik; Wingfield, M. J.; Coutinho, T. A.; Crous, P.W.
English: Botryosphaeria dothidea and Sphaeropsis sapinea both very important pathogens in the South African forestry context. These fungi are well established in the country and contribute substantially to annual losses incurred. Currently very little can be done to control the fungi and the damage they cause. The understanding of their respective disease etiologies is thus of great importance to develop relevant counter measures. The overall aim of this dissertation was to investigate various poorly understood aspects of these two fungi and to try and relate the results to practical contributions towards controlling the impact the two pathogens have. Studies have been conducted during the course of five years and each study represents an independent research investigation. The introductory chapter presents a review of the literature pertaining to all aspects of biology, history and taxonomy of B. dothidea and S. sapinea. The two fungi are clearly very similar in all these aspects and perhaps the only clear difference is that S. sapinea is restricted to pines in South Africa. Many other similarities and some differences between these two important pathogens are highlighted and many of these have provided the background for further investigations. In chapter two the presence of B. dothidea and S. sapinea lS demonstrated as symptomless endophytes in healthy, pine and eucalypt tissue. Botryosphaeria dothidea was found to be common in all the Eucalyptus spp. tested, occurring at high percentages in symptomless leaves of Eucalptus smithii, E. camaldulensis, E. grandis and E. nitens. Sphaeropsis sapinea was, in contrast, only present in young, green Pinus patuIa and P. radiata cones, but virtually absent from the cones of P. elliottii and P. taeda. Botryosphaeria dothidea is associated with die-back and canker diseases of eucalypts in South Africa. Despite this fact, little is known about the infection process. The fungus is known to occur endophytically in leaves of various Eucalyptus species in South Africa. In chapter three I consider the ability of B. dothidea to infect apparently healthy Eucalyptus leaves and the subsequent location and structure of these infections once inside leaf tissue. Scanning electron microscopy revealed that conidia of B. dothidea can infect healthy leaves through stomata. These infections ultimately reside amongst mesophyll cells and constitute a number of individual infections per leaf. Two morphologically similar fungi are associated with die-back and canker of eucalypts in South Africa. The one was identified as part of the Botryosphaeria dothidea-complex. In chapter four, the identity of the second fungus was determined by comparing morphology, pathogenicity and DNA sequence analysis of isolates of both taxa. Based on results obtained, Botryosphaeria eucalyptorum, and its anamorph Fusicoccum eucalyptorum, are described as a new species. I found that the teleomorph is morphologically similar to other taxa in the B. dothidea-complex, but conidial characteristics of the anamorph are distinct, as well as the sequences of the nrDNA internal transcribed spaeers ITS 1 and ITS2. As is the case with B. doth idea , the fungus is pathogenic to Eucalyptus, there do not, however, appear to be differences in pathogenicity between the two. Sphaeropsis sapinea is the most important pathogen of pines in South Africa. The fungus, which reproduces only asexually, occurs only on exotic pines. In chapter five, I investigated the diversity of the S. sapinea population in South Africa and compared it with a population from Northern Sumatra. Both populations were obtained from exotic P. patuIa plantations. The phenotypic diversity of these populations was assessed using vegetative compatibility tests. The percentage maximum genotypic diversity, based on Stoddard and Taylor's index, for the South African population was much higher than the Northern Sumatran population, thus indicating that the South African S. sapinea population was more diverse than the Northern Sumatran population. These results support the hypothesis that the population of S. sapinea in South Africa has been introduced from various parts of the world, during the last century. In chapter six, I investigated the role that latent S. sapinea infections in seed cones of P. patuIa, play in post-hail associated die-back. Pinus patuIa seed cones were found to be infected during the second year of development, with extensive colonization only occurring m the third year when cones mature, prior to seed discharge. Vegetative compatibility tests revealed that the presence of S. sapinea in individual third year seed cones is confined to a single genetic entity. Sphaeropsis sapinea colonisation of third year seed cones thus, apparently results from a single successful infection per cone. The probable role of latent infections by S. sapinea indicated that tree age and by implication, increased numbers of attached seed cones, contributes to more severe die-back after hail damage. The control of damage caused by S. sapinea is highly dependant on a dynamic hybridisation programme. Alternative species of pines is thus constantly evaluated for potential. In chapter seven, 65 families representing both the northern and southern populations of P. greggii were evaluated for their tolerance to infection and subsequent die-back caused by S. sapinea. Families were evaluated following natural infection after hail damage, as well as by artificial inoculation. Variation in tolerance occurred and was highly significant between the two provenances, with the northern provenance proving to be very tolerant. Pinus greggii trees of the southern provenances were comparable with P. patula. The potential of the families from northern origins has to be investigated further. Cultures of Cytospora isolated from Eucalyptus trees in South Africa, Congo, Thailand, Venezuela, Mexico, Uganda and Australia, as well as Cytospora-like isolates from Indonesia were compared in chapter eight. Comparisons were based on the homology of the internal transcribed spaeer regions and the 5.8S ribosomal DNA of the nuclear ribosomal DNA repeat unit. Isolates clustered into at least three unrelated groupings, with a fourth grouping that included isolates that morphologically resembled Cytospora. Results from this chapter indicated that the current description of Valsa ceratosperma encompasses several distinctly different species and needs to be further refined. Botryosphaeria dothidea and S. sapinea are two of the most important pathogens of eucalypts and pines in South Africa. The fact that they exist as symptom less endophytes in trees has added a fascinating aspect to our understanding of their role in tree diseases. In the past, they have generally been considered to be wound infecting opportunistic fungi. Results of these studies have shown that this is not so and that they are clearly able to infect healthy trees. They are unlikely to be able to infect dead or moribund tissue. The investigations presented in this dissertation have added considerable knowledge to our understanding of B. dothidea and S. sapinea and will also promote efforts to reduce disease caused by them. However, there are many questions that remain to be answered pertaining to them and it is my hope that this study will provide a foundation and stimulus for further work.
Open Access
Bioremediation of a bleach plant effluent from the pulp and paper industry
(University of the Free State, 2003-11) Van Driessel, Brian; Christopher, L.
English: Bleach plant effluent was characterised by physico-chemical methods. The chemistry of the bleach plant effluent was examined to devise effective treatment methods. Effluent contained trace amounts of nitrogen as well as carbohydrates and no ortho phosphate could be detected in the wastewater. The best decolourisation activities were obtained using adsorption as treatment method, with activated carbon removing > 99% colour from effluent. Chitosan (81%) and chitin (77%) could remove appreciable levels of colour from bleach plant effluent, followed by biomass from Rhizomucor pusillus, a mucoraelean fungus (71%). Chitosan and chitin from the cell wall of R. pusillus might be involved in the fungus decolourisation ability. Effluent pH was inversely related to effluent decolourisation when R. pusillus, chitosan or chitin was used as adsorbents. This might in part be due to acid catalysis during nucleophilic addition reactions, where amino groups of chitin/chitosan react with carbonyl groups in Eo-effluent. Also, chitin and chitosan amino groups can be protonated under acidic conditions and acquire positive charges that can interact with the chromophores found in Eo-effluent. However, pH exerted no significant effect on decolourisation when activated carbon was employed as adsorbent of effluent colour. Decolourisation employing commercial adsorbents seemed to be mainly due to chemisorption. Adsorption experiments conducted at various ionic strengths indicated that coulombic interactions are responsible for a fraction of the decolourisation activity of chitosan and chitin. Nevertheless, decolourisation obtained with RM7 and activated carbon was unaffected by the ionic strength. Flocculation of coloured compounds from Eo-effluent by chitosan containing solutions resulted in a maximum decolourisation of 75%. Anion-exchange treatment removed 96% colour from Eo-effluent. Ultraviolet irradiation could decolourise the Eo-effluent by about 42 to 43%. Decolourisation using organic solvent extraction proved ineffective with a highest colour removal efficiency of only 21% being achieved. Biological methods used for effluent remediation were: 1) Trickling filters, 2) Activated sludge reactors and 3) Rotating biological contactor reactors (RBC). Treatment using one biological system was followed by treatment in another system With trickling filters containing immobilised white-rot fungi, the highest decolourisation (61%) was obtained with Coriolus versicolor. This fungus required a co-substrate to efficiently decolourise the effluent. Effluent treatment in an activated sludge reactor reduced toxicity, COD and chlorophenol levels. However, colour and high molecular mass compounds were not affected significantly by this method of treatment. Decolourisation was studied in a RBC using immobilised C. versicolor and R. pusillus, respectively. The decolourisation rate by both fungi was proportional to initial colour intensities. Decolourisation was not adversely affected by colour intensity, except at the lowest level tested. Decolourisation of 53 to 74% could be attained using a hydraulic retention time of 23 h. Rhizomucor pusillus, removed 55% of AOX compared to a 40% AOX reduction by C. versicolor. Treatment employing R. pusillus and C. versicolor, respectively, rendered the effluent essentially non-toxic. Addition of nutrients to the decolourisation media stimulated colour removal by C. versicolor, but not significantly in the case of R. pusillus. Ligninolytic enzymes (manganese peroxidase and laccase) were only detected in effluent treated by C. versicolor. Decolourisation mechanisms were investigated using gel permeation chromatography. Rhizomucor pusillus decolourised the effluent by adsorption and C. versicolor removed effluent colour by adsorption as well as by biodegradation. Coriolus versicolor could decolourise the effluent for a period of 34 d whereas R. pusillus decolourised the effluent up to 54 d. Further improvements in effluent quality could be attained when treatment using one system was followed by treatment in another system, possibly because of toxicity reduction in the pre-treatment steps.
Open Access
Biotransformation of alkanes, alkylbenzenes and their derivatives by genetically engineered Yarrowia lipolytica strains
(University of the Free State, 2005-05) Van Rooyen, Newlandé; Smit, M. S.; Setati, E.
A variety of microorganisms, including yeasts, are capable of utilizing n- alkanes as carbon source (Schmitz et al., 2000; Watkinson & Morgan, 1990). The over expression of P450 genes such as the CYP52 family coding for the alkane hydroxylases may lead to an increase in activity and increased formation of possible useful products from hydrocarbon metabolism (Iida et al., 2000). Disruption of the -oxidation pathway by deleting the genes coding for acyl CoA-oxidases, also leads to the accumulation of products that would normally be broken down (Picataggio et al., 1991). The genetic engineering of these two points of control opens up many possibilities for the accumulation of different products from hydrocarbons. Although some work was done concerning these systems in Candida tropicalis very little work has been done in Yarrowia lipolytica. It was the aim of the project to investigate the biotransformation of alkanes, alkylbenzenes and their derivatives by different groups of genetically engineered Y. lipolytica strains in order to investigate a number of questions. The possible accumulation of monocarboxylic acids in Yarrowia lipolytica was inestigated by using substrates such as undecene and hexylbenzene. Y. lipolytica MTLY37 a -oxidation disrupted strain with POX2, POX3, POX4 and POX5 genes deleted could not accumulate any monocarboxylic acid from undecene. The undecene was however fully utilized indicating that this strain still had some -oxidation activity. Little phenylacetic acid was formed (0.4 mM) from hexylbenzene. Another product that could not be positively identified at the time, but which might have been phenylhexanoic acid accumulated (4mM). No monocarboxylic acids other than phenylacetic acid could also be accumulated from alkylbenzenes in strains with blocked - oxidation expressing CPR and CYP genes, leading to the conclusion that Y. lipolytica can not accumulate monocarboxylic acids. Y. lipolytica strains with disrupted -oxidation as well as a strain with functional -oxidation expressing additional YlCPR and CYP52F1 genes accumulated the full-length dioic acid from 5-methylundecane. All these strains also sequentially broke down the 5-methylundecanedioic acid to 5- methylnonanedioic acid, 3-methylheptanedioic acid and 3-methylpentanedioic acid. Y. lipolytica MTLY76 was the only strain that did not degrade the 5- methylundecanedioic acid completely. Using hexylbenzene as substrate it was possible to establish that ethanol delayed the induction of both the native ALK genes as well as the inserted CYP genes. However, the cloned genes were later induced quite strongly (probably by the phenylalkanoic acids formed from hexylbenzene) for an extended period, while the native genes were only weekly induced. The maximum activity of Y. lipolytica was slightly lower when ethanol was used as inducer (13µmol.min -1 l -1 ) than when oleic acid was used as inducer (19µmol.min -1 l -1 ). The alkane hydroxylase activity was however maintained for a longer time when ethanol was used as inducer. When dodecane was used as inducer native genes were strongly induced for a relatively long period, but not as long as the cloned genes after ethanol. Alkylbenzenes as substrate was also useful to distinguish between alkane hydroxylase activity of native and cloned monooxygenases. A significant difference in the activity of Y. lipolytica TVN356 expressing CPR together with CYP557A1 (putative fatty acid hydroxylase from Rhodotorula retinophila) and Y. lipolytica TVN91 expressing CPR together with CYP53 (benzoate para- hydroxylase from R. minuta) could be observed (14µmol.min -1 l -1 and 8µmol.min -1 l -1 respectively) when decylbenzene was used as substrate. To better study the hydroxylase activity of inserted P450s, it may be better to use the ICL1 promoter to drive the expression of the inserted CYP genes and use ethanol as inducer.
Open Access
Biotransformation of alkylbenzenes and alkylcylcophexanes by genetically engineered Yarrowia lipolytica strains
(University of the Free State, 2008) Ramorobi, Limpho Martha; Smit, M. S.
Y. lipolytica has the ability to utilise hydrophobic hydrocarbons as carbon sources. It is also an attractive host for heterologous expression of cytochrome P450 (CYP) genes. Y. lipolytica strains with CYP genes cloned under control of two different promoters, pPOX2 and pICL were used in this study. The purpose of this project was to detect the effect of cloned alkane hydroxylases in Y. lipolytica. Alkylbenzenes and alkylcyclohexanes were used to compare the hydroxylase activities of the genetically engineered strains with control strains. Butylbenzene and hexylbenzene were transformed to phenylacetic acid while pentylbenzene, heptylbenene and nonylbenzene yielded benzoic acid as product. Butylcyclohexane and limonene were transformed to cyclohexylacetic acid and perillic acid, respectively, as major products. The activity towards hexylbenzene was highest. Phenylhexanoic acid and phenylbutanoic acid were also for the first time observed as intermediates in the biotransformation of hexylbenzene. Y. lipolytica strains expressing alkane hydroxylases under pPOX2 were induced with oleic acid and harvested. A strain with multiple copies of a proven alkane hydroxylase, cloned, had in one experiment higher activity than the other strains, towards both hexylbenzene and nonylbenzene. However, these results could not be confirmed, because in subsequent experiments the resting cells had very low activities. Ethanol and sodium acetate were used as inducers in the experiments conducted with Y. lipolytica strains with CYP557A1, a putative alkane and/or fatty acid hydroxylase, cloned under pICL. The substrates were added directly to the cells. With single addition of ethanol, the strain with cloned CYP557A1 had in one whole cell experiment with butylbenzene as substrate higher activity than the control strains. With multiple additions of sodium acetate the strain with cloned CYP557A1 showed higher activity in two shake flask experiments when hexylbenzene was used as a substrate. However, when ethanol and sodium acetate were used as inducers the alkylbenzenes were often consumed without the equivalent formation of detectable products, complicating the interpretation of results. This also happened in bioreactor experiments. Biotransformation of alkylcyclohexanes also did not demonstrate the effect of the cloned gene, since the activities of the test strain with CYP557A1 cloned under pICL and the control strain were similar. Alkylcyclohexanes are not promising substrates to distinguish between hydroxylase activity of the cloned genes as they are very volatile and activity towards them is relatively low.