Magister Scientiae - MSc (Biotechnology)

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Novel dyp-type peroxidases from actinobacteria: a genome mining approach
(University of the Western Cape, 2024) Groep, Juandré
Twenty-one whole genome actinobacterial sequences were obtained from strains previously isolated from various environments. a genome-mining approach was applied to identify the presence of dye-decolourising (dyp-type or dyps) peroxidases. from genome sequence annotation twenty-six dyp-type sequences were identified and through bioinformatic analysis for the presence of twin-arginine translocation (tat) pathway signal peptides, was classified as belonging to class i (formerly class a) bacterial dyp-type peroxidases. phylogenetic analysis of the dyp sequences showed that the streptomyces derived dyps were predominantly grouping together and the nonstreptomyces derived dyps followed the same pattern. eight dyp-type peroxidases were selected for cloning, with the majority being derived from streptomyces spp., and primers were designed to amplify the dyp-type peroxidase genes. the pcr-amplicons were subjected to restriction digests and were ligated into pet20b(+). the constructs were transformed into escherichia coli jm109. only two of the constructs were successfully transformed: the dyp-type peroxidase genes from nocardia gamkensis czh20t and micromonospora sp. strain 30-1. further analysis using t7-primers confirmed sequence identity of the constructs.
Engineering robust yeast strains for the conversion of xylose derived from lignocellulosic biomass to xylitol
(University of the Western Cape, 2023) Maneveldt, Amber
To achieve a sustainable and economically viable 2G biofuels industry, biorefineries must coproduce high-value, low-volume bioproducts alongside high-volume, low-value cellulosic ethanol. This can be realised with the co-production of the low-calorie sugar substitute, xylitol which has a well-established market, as well as other chemicals. The construction of a xylitolproducing S. cerevisiae strain represents an economically feasible and environmentally friendly approach to xylitol production. Moreover, the exploitation of natural S. cerevisiae strain isolates as bioengineering hosts has the potential to be superior starting points due to their robustness towards process conditions. In this study, the xylitol-producing activities conferred to three natural isolate host strains via conventional and CRISPR-Cas9-mediated δintegration of three genes encoding a β-xylosidase, β-xylanase and xylose reductase (XR), was evaluated. The effect of over-expressed heterologous protein production on strain robustness and metabolism was also assayed. Our results revealed that the overexpressed XR failed to improve on the xylose reduction ability conferred to our strains, likely by their native GRE3 gene.
Green synthesis and characterization of silver nanoparticles from the cocktail of capparis sepiaria-tabernaemontana elegans extracts and assessment of their biological effects in vitro.
(University of the Western Cape, 2024) MASHILO, CATE MALOPE
Antimicrobial resistance (amr) is a global crisis that develops when bacteria, viruses, fungi, and parasites adapt and multiply in the presence of drugs that once negatively affected them. amr infections are commonly caused by the overuse and misuse of antimicrobial drugs, thus leading to severe illnesses, longer hospitalization, increased healthcare expenses, treatment ineffectiveness, and increased mortality. the increasing incidence of amr poses a serious threat to public health. as a result, alternative strategies that are both effective against amr pathogens and eco-friendly are urgently needed. green nanotechnology, in particular the use of silver nanoparticles (agnps), has been used as a solution in a wide range of applications, including antibacterial, antiviral, and antifungal therapies. over the last decades, several medicinal plant extracts have been used to synthesize agnps; however, combining extracts from two medicinal plants to synthesize agnps with enhanced properties has received less attention. therefore, this study reports on the green synthesis of agnps using a cocktail of capparis sepiaria–tabernaemontana elegans (cste) aqueous extracts as a reducing, stabilizing, and capping agent, and evaluation of their antimicrobial activities.
Green synthesis and characterization of gold nanoparticles from capparis sepiaria-tabernaemontana elegans cocktail and investigation of their biological effects in vitro.
(University of the Western Cape, 2024) MATSHAYA, ALUWANI
Wounds have a significant impact on the global healthcare system, adding heavy load on the economy and society. therefore, there is an urgent need to develop treatments that can facilitate rapid wound healing. to make matters worse wound infection by antimicrobial-resistant bacteria may result in prolonged debility of the patient and increased healthcare costs. avoidance of the development of antimicrobial resistance therefore needs increasing attention in the management of patients, with chronic wound infection. nanotechnology presents a groundbreaking method for synthesizing biocompatible nanomaterials (nms), revolutionizing wound healing with soft, flexible dressings that possess antimicrobial properties, gas barrier, and absorbency. these nms might fall into two categories: those with inherent wound-healing properties and those serving as delivery vehicles for therapeutic agents. gold nanoparticles (aunps) have more consideration due to their stability, oxidation resistance, electrical and optical properties. this study reports on the green synthesis and characterization of aunps synthesized using an aqueous extract of capparis sepiariatabernaemontana elegans (cste) and the investigation antibacterial and wound healing effects of the cste-aunps against amr pathogens, such as staphylococcus aureus, methicillin-resistant staphylococcus aureus, escherichia coli, and pseudomonas aeruginosa. wound healing properties of cste-aunps was also investigated using kmst-6 (fibroblast) cell line.
Identification of a transducin (beta)-like 3 protein as a potential biomarker of prediabetes from rat urine using proteomics
(University of the Western Cape, 2010) Mofokeng, Henrietta Refiloe
Obesity is a globally increasing disease particularly in developing countries and among children. It is mainly caused by intake of diets high in fat and the lack of physical activity. Obesity is a risk factor for diseases such as type 11 diabetes, high blood pressure, high cholesterol and certain cancers. Prediabetes is a condition where blood glucose levels are above normal but have not reached those of diabetes. It is difficult to diagnose, as there are no signs or symptoms. Some type II diabetes patients bear no symptoms at all and the disease is discovered late. Proteomics is a field that can provide opportunities for early diagnosis of diseases through biomarker discovery. The early diagnosis of diabetes can assist in the prevention and treatment of diabetes. Therefore there is a need for the early diagnosis of diabetes.
Green synthesis of silver nanoparticles from indian hawthorn (rhaphiolepis indica) and the investigation of their antimicrobial, antioxidant and cytotoxic effects
(University of the Western Cape, 2024) Nsumpi, Aurelie Ngalula
The global health concern pertaining the rapid spread of antimicrobial resistance (AMR) has led to a rise in mortality rate caused by infectious diseases that have become difficult to treat due to the misuse and lack of novel antibiotics. Nanotechnology is one innovative approach that has the potential to eradicate the burden of AMR with silver nanoparticles (AgNPs) well known for their antibacterial properties. Currently, AgNPs can be synthesised using a greener approach which makes use of the phytoconstituents in plants providing more advantages over the conventional chemical synthesis methods (i.e rapid, cost-effective, safer for the environment and sustainable) also enhancing the antioxidant properties of AgNPs for the treatment of degenerative diseases caused by oxidative stress. Indian Hawthorn (Rhaphiolepis indica) berries have been used to treat cardiovascular diseases, high blood pressure and heart failure and are known to have high content of polyphenols which are involved in bioreduction of metal-based NPs (MNPs) with enhanced bio-activities.
Engineering antimicrobial strains of saccharomyces cerevisiae as industrial platform for non-sterile bioprocesses
(University of the Western Cape, 2023) Van Lill, Gert Rutger
Microbial contamination is a major challenge in fuel ethanol facilities, especially during the fermentation stage. Bacterial contaminants compete with yeast for fermentable sugars and nutrients, which obstructs starch to ethanol conversion. This leads to a reduction in ethanol yield. At large production scales, prior sterilization of the feedstocks is economically unfeasible. Therefore, antibiotic supplementation is common practice to limit the growth of contaminating bacteria. In addition to the high cost of the antibiotics, concerns and evidence are mounting that antibiotic use in non-clinical settings is driving the emergence of drug resistant microbes. This study focused on heterologous expression of antimicrobial peptides (AMP) as an alternative strategy for combating microbial contamination. This work aimed to engineer an industrial Saccharomyces cerevisiae strain that produces AMPs active against lactic acid bacteria and Enterobacteriaceae which are some of the main bacterial contaminants found in industrial biofuel fermenters. Seven candidate AMPs were selected from curated online databases, and their amino acid sequence was analyzed using bioinformatic tools. AlphaFold, Clustal Omega, and SNAP2 were used to predict AMP tertiary structures, construct a phylogenetic tree, and analyse mutation effects, respectively.
Biogenic silver nanoparticles synthesized using eucomis autumnalis bulb aqueous extract, their characterization and in vitro antibacterial activity
(University of the Western Cape, 2023) WILLIAMS, LETOYA SHEILA
Antimicrobial resistance (AMR) is a growing global problem that poses a significant threat to public health. AMR arises when microorganisms become resistant to drugs that were initially designed to kill them. The application of antibiotics has proven effective in eliminating or inhibiting the growth of harmful microorganisms. Conversely, the primary cause of AMR is overprescribing and the misuse of antibiotics which is further increased by a reduced novel antibiotics discovery rate. AMR bacteria lead to hard-to-treat infections, causing longer hospital stays and higher healthcare costs, and is associated with a substantial risk of morbidity and mortality. Consequently, the development of infections caused by multi-drug resistant (MDR) microorganisms has resulted in a considerable reduction in the number of effective treatment options available. Thus, novel therapies are urgently required, considering the ineffectiveness of current therapy, moreover, further exacerbated by a reduced discovery rate of novel antibiotics. Alternative strategies are therefore urgently needed to treat AMR bacterial infections.
Screening for novel cold-adapted nitrile hydratases in Antarctic metagenornic libraries
(University of the Western Cape, 2024) Williams, Wesley Trevor; Cowan Don
Nitrile hydratases (NHase) catalyse the hydrolysis of nitriles to their corresponding amides, the first step in the nitrile degradation pathway. The second step is the conversion of the amide to a carboxylic acid, catalysed by an amidase. The NHases published to date have been isolated from mesophilic and thermophilic bacteria. The aim of this study was to identify a novel cold-adapted NHase from psychrophilic and psychrotrophic bacteria. The identification of a cold-adapted NHase is of interest for its potential use in chemical and pharmaceutical production. Enzymes isolated from psychrophilic bacteria typically have a high specific activ ity and low thermal stabi lity to cope with the inhibition of chemical reactions caused by low temperatures. However, culture based screening for these activities tend to reisolate already characterised isolates amiable to culturing. To access the majority of the bacterial biodiversity that the uncultured bacteria represent, the metagenomic approach was used to screen for nitrile degrading activity. Metage-nomic libraries constructed from soil taken from the McMurdo Dry Valleys in Antarctica were used for both functional and sequence based screens of NHases. Functional screens that were trialled were based on pH change and oligotrophic selective growth.
Metagenomic bioprospecting: discovery and characterization of a novel esterase for biomass conversion
(University of the Western Cape, 2024) Ohlhoff Colin W.; Tuffin Marla
Due to an increasing human population and ever-expanding industrial economies, fossil fuel reserves are rapidly being depleted. In order to reduce our dependence on non-renewable energy resources much global interest has been directed towards the development and production of biofuels. Plant biomass has been identified as a possible feedstock for alternative fuel production and typically consists of cellulose, hemicellulose and lignin. Although these components can serve as valuable substrates in the bioconversion process, they are generally recalcitrant to enzymatic degradation. Current hydrolytic methods limit the depolymerization of the lignocellulose component of plant biomass, which in turn decreases the yield of potential fermentable sugars. As microorganisms represent an intriguing, underexploited resource for the discovery of novel biocatalysts, this study employed a metagenomic approach towards the identification of enzymes for lignocellulose hydrolysis. A large-insert metagenomic fosmid library was constructed using total DNA extracted from thermophilic compost samples. The library comprised 150 000 clones, with an average insert size of 31 Kb, representing approximately 1300 prokaryotic genomes. Initial emphasis was placed on the assessment of the bacterial phylogenetic make-up of the metagenomic library and its validity for the mining of functional enzymes.
Production and characterization of therapeutic peptides with potential antimicrobial properties
(Univeristy of the Western Cape, 2024) Ratshilume, Raphaella; Keyster, Marshall
Bacterial drug resistance is a current and growing serious health problem worldwide because of the long-term and negligent use of antibiotics. Finding new antibiotics and shepherding them through the clinical trials and approval process is becoming increasingly difficult. In addition, the declining number of new innovative antibiotic candidates has worsened this burden, necessitating the development of new classes of antimicrobial agents. Antimicrobial peptides (AMPs) are protein molecules that often form part of the innate immune system and cellular defense mechanisms found in many organisms. AMPs are promising antibiotic alternatives for the treatment of drug-resistant infections. They are, however, constrained by their high manufacturing costs. Genetic engineering and heterologous recombinant peptide expression are promising approaches for producing such molecules at a low cost. In this study, peptide Gene 1 and peptide Gene 2 obtained from the bioinformatics analysis on marine metagenomes sampled from the TARA Oceans Project were produced using the heterologous recombinant peptide expression method. The peptide Gene 1 and peptide Gene 2 were cloned into the pPIC9 vector, transformed, and integrated into the P. pastoris genome. The expression was observed in BMMY medium, pH 6, 1% methanol, and 48 hr, 72 hr, 96 hr with a band at a molecular mass of 6.05 kDa for peptide Gene 1 and 11.37 kDa for peptide Gene 2 following SDS-PAGE analysis.
Effect of green synthesized metal nanoparticles on gene expression in an in-vitro model of non-alcoholic steatohepatitis
(University of the Western Cape, 2024) Nendouvhada, Livhuwani Portia
Metabolic dysfunction-associated liver disease (MASLD) occurs due to an excessive accumulation of fat in the liver (steatosis), independent of secondary causes such as excessive alcohol consumption, viral hepatitis, or certain medications. MASLD is a common chronic liver disease that can lead to end-stage liver disease eventually requiring a liver transplant. It is associated with metabolic syndrome (MetS), especially obesity, diabetes, hyperlipidemia, and hypertriglyceridemia. MASLD can advance to metabolic dysfunction-associated steatohepatitis (MASH), which causes inflammation and damage leading to scarring of the liver (cirrhosis). Only 43-44% of patients with steatosis progress to MASH, with 7-30% of MASH patients progressing to cirrhosis. To date, the mechanism of MASLD and its progression is not well understood, and there are no therapeutic strategies that are specifically tailored for MASLD/ MASH. The anti-obesity and anti-diabetic pharmacological approaches currently used were shown to reduce the MASLD progression, although they remain ineffective to completely treat or reverse its progression.
Development of gold nanoparticles based lateral flow assay for detection of food and water-borne pathogens
(Universty of the Western Cape, 2024) Mabhude, Yandiswa; Sibuyi, Nicole
Food and water are two of the necessities required for survival. With increasing global population, urbanization, and climate change, to name a few, the strain on these two precious resources puts pressure on health as well as the economic sector. Microbial contamination of food and water can lead to lethal infections that are detrimental to human health. More than 90% of reported food-borne illnesses were caused by bacterial species such as Staphylococcus, Salmonella, Campylobacter, Listeria, Bacillus etc. In the last decade, Salmonella was the leading cause of bacterial food-borne illnesses; followed by Campylobacter and Listeria. Listeriosis cases were reported in 2017 in Gauteng province, and the number of infected persons rapidly escalated nationwide prompting an investigation wherein a total of 937 cases were identified. Early detection of pathogens in food and water could prevent or slow down the spread of infections and deaths caused by consuming contaminated food and water. The laboratory and on-site approaches that are currently used for quality control technologies are precise and sensitive, but they have drawbacks such as being laborious and time-consuming. Therefore, there is an urgent need for approaches that may be utilized to detect microbial contaminations at a point-of-care (PoC), that are user-friendly, quick, robust, and sensitive. The use of gold nanoparticles (AuNPs)-based lateral flow assay (LFA) for the detection of these pathogens is gaining traction among researchers as an alternative to conventional methods for on-site testing in places where there is no proper infrastructure, particularly low-resourced areas. The current study aimed to develop an aptamer-AuNPs-based LFA for the detection of food and water pathogens at PoC.
Domestic greywater systems: a potential reservoir of antibiotic resistance genes transfer
(University of the Western Cape, 2024) Kariem, Min-ghah; McCullough, Bronwyn Kirby
During the Western Cape drought from 2015 to 2018, the use of greywater to alleviate pressure on the limited potable water available in the province was encouraged. Greywater, however, has the potential to harbour residual antibiotics and pathogenic bacteria, thereby potentially supporting the growth and proliferation of antibiotic resistant bacteria. This study combined traditional microbiology tests with molecular biology to detect resistant microorganisms within domestic greywater systems and their associated biofilms, while Quantitative PCR (qPCR) was used to determine the levels of clinically relevant antibiotic resistance genes (ARGs) (vanA, ampC and aadA), as well as the intI1 gene, which serves as a marker of horizontal gene transfer. Phenotypic resistance was confirmed using the Kirby-Bauer disc diffusion method. Notably, high levels of resistance to ampicillin and kanamycin were found in greywater and biofilm samples, as well as resistance to vancomycin. Bacterial viability was assessed using flow cytometry using the LIVE/DEAD BacLight Bacterial Viability kit, and it was revealed that a large portion of the cell population within greywater was dead or injured. qPCR analysis confirmed the presence of clinically relevant ARGs vanA and ampC, in domestic greywater samples, with abundance fluctuating in response to seasonal change. Additionally, the intI1 gene was detected in all greywater and biofilm samples, which suggests that genetic exchange occurs amongst bacteria in greywater and biofilm samples. Our findings support the hypothesis that greywater systems are colonised by resistant bacteria, which can form stable communities within the water environment which allows for high levels of genetic exchange. These findings support the use of qPCR and flow cytometry, in combination with traditional microbiology tests for monitoring antibiotic resistance in environmental samples. While the use of greywater is a viable method to reduce the demand for potable water, this study highlights the fact that the improper use of greywater poses a risk to the environment and public health. As such, the public should be better educated on how to safely use these systems.
Biochemical characterisation of an alpha-amylase with pullulan hydrolase type III characteristics derived from a hot spring metagenomics library
(University of the Western Cape, 2024) Boersma, Bianca; Trindade, Marla
The field of metagenomics has provided biotechnological researchers with new enzymes and insight into previously unknown enzyme families with potential for novel modes of action and being able to function under extreme conditions, such as extremes of temperature, acidity or alkalinity. The enzymes can also often act on multiple substrates and show increased affinity for certain substrates. In this study, the aim was to biochemically characterise a putative type III pullulan hydrolase (PHTIII). A sequence-based metagenomic screen from previous performed by Xiao Ping Hu (2010) was used to identify novel glycosyl hydrolase enzymes in shotgun metagenomic sequence data from the Mphizi hot spring in Malawi. An identified ORF, named Pull3.1, displayed low sequence identity to a characterised α-amylase (62.71%) and 61.66% to a characterised pullulan hydrolase type III, and was, therefore, hypothesized to be an α-amylase (EC 3.2.1.1) with pullulan hydrolase type III (EC 3.2.1.x) characteristics. Further sequence analysis indicated that Pull3.1 had a glycogen binding site and that it belongs to the GH13_20 family as well as having a family 48 carbohydrate-binding module. Pull3.1 displayed the highest substrate conversion rates for starch, pullulan and glycogen. The products released were of the following degrees of polymerisation 3, 2 or 1, in accordance with the products released from the PHTIII from Thermococcus aggregans. Pull3.1 is a thermozyme that shows maximum activity at 75°C and an optimum pH of 7. It was thermostable up to 80°C and had a half-life of 11.6 min at 50°C. Further kinetic characterisation showed that Pull3.1 had a high affinity for starch. The KM value of 0.048 mg/ml is within the range reported for other α-amylase enzymes, and Pull3.1 had a Vmaxof 0,729 (μmol mL−1). All of this data indicates that Pull3.1 is a good candidate for a starch conversion process that requires thermozymes.
South African medicinal plant extracts used in the treatment of fungal infections.
(University of the Western Cape, 2023) Ndlovu, Banele Michelle; Rahaman Fisher, F
The incidence of fungal skin infections is increasing at an alarming rate, especially in people with underlying immunosuppressive conditions. Currently, the most used methods of treatment are in the azole group of synthetic treatments. A major problem with these synthetic treatments’ is the increase in azole-resistant strains spreading worldwide. Additionally, an estimated 65–80% of all fungal infections are biofilm related, with biofilms found in Candida species such as Candida albicans, Candida dubliniensis, Candida glabrata and Candida tropicalis which infect the skin. There is an increased demand for alternative treatments that could be antifungals and medicinal plants could be the potential solution, as some plants possess antifungal properties. The overall aim of this study is to investigate the potential effect of South African medicinal plants used for the treatment of fungal infections against pathogens of dermatological relevance. The objectives were 1) to conduct a comprehensive literature search to identify indigenous medicinal plant extracts used to treat fungal skin infections, 2) to perform a cross-sectional study using an interview-based questionnaire to investigate the knowledge and practices of Cape bush doctors and validate their use of selected medicinal plants for treating dermatophyte infections in the Cape Metropole communities and 3) to investigate the in vitro bioactivity of selected plant extracts identified from the literature search and interviews. Fifteen medicinal plants were found using an in-depth literature search and all 15 plants were validated during the interviews as plants commonly used for fungal skin infections.
Valorisation of brewers’ spent grain: enzymatic hydrolysis in the production of xylooligosaccharides
(University of the Western Cape, 2023) Arries, Chelsey; den Haan, Riaan
The beverage industry constitutes approximately 26% of all food wastes, making it one of the largest contributors in this waste segment. By utilising waste or by-products from agriculture and food production in manufacturing value added compounds, the concepts of waste mitigation and green chemistry can contribute to establishing a circular bio-economy. In a biorefinery, bio-catalytic, thermal, chemical and physical techniques are used to extract valuable compounds from food and agricultural wastes. Brewers’ spent grain (BSG) is an ideal candidate for such a biorefinery approach. This high moisture, nutrient-rich by-product from beer production is either disposed of in landfills or used as an animal feed. However, high value products, such as xylooligosaccharides (XOS) can be extracted from BSG, thereby valorising this brewery waste. Xylooligosaccharides are sought after for their ability to function as a low caloric sweetener while exhibiting prebiotic effects in stimulating the growth of probiotic bacteria in the mammalian gut.
Antibacterial effects of biogenic silver nanoparticles synthesized using manihot esculenta (cassava) leaf aqueous extract
(University of the Western Cape, 2023) Oliver, Toni Charlize Alexia; Madiehe, Abram
Antimicrobial resistance (AMR) is a growing global health concern that poses a serious threat to the health of humans, animals, and plants. The major public health problem of AMR is primarily caused by the incorrect use of antibiotics, which is further compounded by the reduced novel antibiotics discovery rate. AMR bacteria cause infections that are difficult to treat, resulting in prolonged hospital stays and increased healthcare costs. Moreover, AMR is associated with a high risk of morbidity and mortality. Current therapeutic strategies for AMR infections are often inefficacious, associated with side effects, and may further exacerbate AMR. Therefore, there is an urgent need to develop alternative strategies to treat AMR bacterial infections.
Evaluation of the anticancer and molecular effects of green synthesized silver nanoparticles
(University of the Western Cape, 2023) Simon, Sohail; Madiehe, Abram
Globally, cancer is considered the second leading cause of death with a staggering tally of 10 million deaths. Breast, lung, colon, rectum and prostate cancers are amongst the most commonly diagnosed cancers. The current cancer treatment strategies such as chemotherapy, radiotherapy, and surgery; are accompanied with a long list of side effects. Nanotechnology has shown potential in improving biocompatibility of the chemotherapeutic drugs, and also provides a novel approach for the development of novel cancer treatment strategies through the application of the widely investigated nano-scaled materials known as nanoparticles (NPs). These NPs have been used as drug delivery systems and have, therefore, made a mark in the fight against cancer. Furthermore, their anti-cancer properties make them prospective anti-cancer agent.
Biosurfactant screening through metagenomic and culture-based approaches
(University of the Western Cape, 2022) Sithole, Nombuso Slindile; Trindade, Marla
The biosurfactant market is still predominantly represented by the three main products: surfactin, sophorolipids, and rhamnolipids, mainly as a result of the high cost and low yield associated with biosurfactant production. Moreover, only a few novel structures have been discovered in the last decade, due to a lack of interest, low hit rate, and high redundancy, in which the predominant microorganisms producing the same group of biosurfactants are isolated. Hence, there is the obvious need to open up the market with structurally and functionally diverse compounds that have the potential to tailor to specific needs or applications in different industries.

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