Philosophiae Doctor - PhD (Biotechnology)

Permanent URI for this collectionhttps://hdl.handle.net/10566/13468

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Biochemical characterisation of a novel Actinobacterial tyrosinase
(University of the Western Cape, 2012) Mavengere, William Nyasha
Tyrosinases are ubiquitously distributed melanogenic polyphenol oxidases that have applications in protein cross-linking, polyphenolic wastewater treatment and bioelectrode development. There are four groups of functional bacterial tyrosinases, three of which belong to the tyrosinase protein superfamily. The fourth group of functional bacterial tyrosinases exhibits structural similarities to multicopper oxidases. Tyrosinases are usually distinguished from other polyphenol oxidases (such as laccases) by their monophenolase activity. In this study, a Zambian hot spring isolate, denoted strain 8S1#2, was identified by a partial polyphasic approach as a possibly novel Dietzia species. Strain 8S1#2 forms orange-red colonies on agar plates. In addition, strain 8S1#2 is aerobic, Gram-positive, non-spore forming and non-motile. This thermotolerant haloalkalophile is the first confirmed tyrosinaseactive non-Streptomyces actinobacterium. Tyrosinase expression in strain 8S1#2 was shown to be induced. A multifaceted PCR approach for the amplification of the gene encoding the tyrosinase activity in 881#2 was used. Primers were designed based on a putative multicopper oxidoreductase, laccase gene sequence identified in the recently annotated genome of Dietzia cinnamea P4 (the closest phylogenetic relative of strain 881#2). The amplicon generated with the strain 8S1#2 genomic DNA as a template showed 100 % sequence similarity to the D. cinnamea P4 MPOL gene. The amplicon of the 8S1#2 MPOL gene was cloned and functionally expressed. The 8S1#2 MPOL protein was purified from the insoluble cell fraction and shown to be a 53 kDa homodimer with moderate stability after 3 hr incubation at 60 "C. The optimal tyrosinase activity was observed at pH 7, and the monophenolase-diphenolase ratio was determined as 0.06. The kcat / KM ratio of 8S1#2 MPOL, at 644.1 mM-1s-1, was the highest for tyrosinases using L-tyrosine as a substrate. The kcat / KM ratio for A8TS, a laccase substrate, was 0.9 mM-1s-1• A homology model of the BS1#2 MPOL protein was generated with Neisseria meningitides protein Nmb 0706 chain A (POB code 1 rv9) as the template. Topology analysis of 1 rv9 identified 24 cavities, with the largest cavity selected as a possible active site. 17 constituent residues were selected and correlated with those in BS1#2 MPOL. Of the four copper ligand binding sites (LBS) identified in MPOLs, three were conclusively identified in BS1#2 MPOL protein following a multiple sequence alignment based approach, with two LBSs located in the putative active site. This study showed that the BS1#2 MPOL protein, a member of the OUF152 protein superfamily, exhibited the functional properties of tyrosinases. However, the BS1#2 MPOL protein did not share any significant structural similarities with members of the tyrosinase protein superfamily. These observations provide evidence for tyrosinase functional convergence in strain BS1#2, a member of the suborder Corynebacterineae. Based on this study, MPOLs may be considered as the second structurally dissimilar group of functional bacterial tyrosinases. As such, the overall number of functional bacterial tyrosinase groups may be amended from four to five.
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. Phylogenetic analysis of the 16S rRNA gene clone library revealed the presence of bacteria belonging to the phyla Alpha- and Gamma-Proteobacteria, Bacteroidetes and Firmicutes, with many clones being most similar to uncultured microorganisms with high sequence novelty. High-throughput robotics was utilized to screen a portion of the library (110, 592 clones) for functionally active lignocellulosic enzymes. Over 100 clones, displaying cellulase, xylanase, lipase/esterase or laccase activities, were identified.
Evaluation of the antimicrobial and wound healing efficacy of extracts from Ehretia species and their silver nanoparticles.
(University of the Western Cape, 2025) Oselusi, Samson Olaitan
The rise of antimicrobial-resistant pathogens and the increasing prevalence of chronic wounds pose serious threats to public health and the global economy. Chronic wounds become difficult to treat, especially when they harbor polymicrobial infections, as these complex infections often encourage the development of drug resistance. Many existing conventional treatments are associated with constraints, such as limited efficacy, high costs, and adverse side effects on surrounding healthy tissues. These factors necessitate a paradigm shift. In recent years, extracts from medicinal plants and their chemical derivatives have attracted significant attention as promising alternative therapeutic agents. This is primarily attributed to their cost-effectiveness, widespread accessibility, and fewer side effects. Ehretia species, widely distributed medicinal plants across southern Africa and other parts of the world, have been traditionally used to treat various ailments, including respiratory disorders, gastrointestinal disorders, skin conditions, and inflammatory and pain-related ailments. In addition to their medicinal properties, plant extracts are increasingly valuable in green nanotechnology, offering a cost–effective, sustainable, and environmentally friendly method for fabricating nanoparticles (NPs).
Engineering xylan assimilation into industrial strains of Saccharomyces cerevisiae.
(University of the Western Cape, 2025) Kruger, Francois
Second-generation biofuels are attractive alternatives to environmentally damaging, non-renewable fossil fuels, as they are carbon neutral and produced from renewable lignocellulosic biomass (LCB). One of the main challenges facing LCB conversion to bioethanol is the incomplete use of all available sugars present in the biomass. To overcome this challenge, the hemicellulose fraction, consisting mostly of xylan, should be targeted for conversion in addition to the cellulose fraction. This study aimed to address this issue by developing strains of Saccharomyces cerevisiae capable of xylan degradation and efficient utilization of xylose. The laboratory strain S288C was the initial candidate used. The strain was previously engineered with a xylose isomerase (XI)pathway and subsequently further engineered using CRISPR-Cas9 technology. GH43xylosidase activity, either freely secreted or tethered to the cells, was introduced into the yeast, together with secreted xylanase activity. The strains were evaluated through enzymatic assays, growth on media containing xylo-oligosaccharides (XOS) and xylan, and fermentations on xylan media. It was found that the strain with tethered xylosidase and secreted xylanase activity showed the best growth on the polymeric substrates (XOS or xylan) and produced the highest ethanol titre of 0.47 g/L during fermentation on xylan as carbon source. Natural S. cerevisiae strain isolates YI13, YI59 and FIN1 were selected for potential industrial applications due to their robust fermentation performance and enhanced ethanol production compared to the reference strain S288C. Efficient xylose utilization was conferred to the natural strains through engineering with an XI gene cassette and a xylose transporter, combined with adaptive laboratory evolution (ALE) in minimal media with xylose as the sole carbon source. The xylose-utilizing strains were further engineered with cell-associated GH43 xylosidase activity and secreted xylanase activity. Enzymatic assays, growth trials on hemicellulosic substrates and fermentation on xylose and xylan showed that the strains were successfully engineered with xylan conversion capabilities and efficient xylose utilization. The final engineered version of YI13 showed the best xylose and xylan conversion, with ethanol titres of 4.51 g/L from xylose and ~ 3 g/L from xylan. This is the highest reported level of ethanol produced from polymeric xylan to date.
Cloning, expression and characterization of novel lipase and esterases from burkholderia multivorans UWCIO
(University of the Western Cape, 2005) Rashamuse, Konanani J
An esterase and lipase producing Burkholderia multivorans strain was isolated by culture Enrichment strategies. A shotgun library of Burkholderia multivorans genomic DNA (prepared in E. Coli/puc18) was screened for lipase and esterase activities. Three positive Recombinant clones, ptend5, phola6 and prashi4, conferring esterolytic and Lipolytic phenotypes respectively, were identified. Full-length sequencing of DNA inserts Was performed using subeloning and "primer-walking" strategies. Nucleotide sequence analysis revealed that the prash14 plasmid DNA consisted of two Open reading frames (ORPI and ORP2) encoding 356 and 350 amino acids, respectively. Database searches revealed that ORPI and ORP2 were homologous to lipases and Chaperones from subfamily I.2. In the ptend5 sequence, an open reading frame Consisting of 978 bp, encoding 326 amino acids, was identified. Database searches Revealed that this open reading frame was homologous to family Vesterases. Nucleotide Sequence analysis revealed that phola6, plasmid DNA consisted of 1194 bp encoding 398 amino acids and showed homology to family VIII esterases. The primary structures Of lipa, estefh5 and estbl from prashi4, ptend5 and phola6, respectively, Showed a classical gxsxg motif, which is conserved in many serine hydrolases. In Addition, estbl also showed a consensus sxxk motif, the serine of which acts as a Catalytic nucleophile in class C ~-lactames and some peptidases.
Metagenome sequencing and in silico gene discovery: From genetic potential to function
(University of the Western Cape, 2012) Anderson, Dominique Elizabeth
In a previous study, metagenomic DNA extracted from Antarctic Dry Valley soils was used to construct a large contig bacterial shotgun fosmid library (Anderson, 2008). In the current study, clones were selected based on a functional screen for putative lipolytic enzymes, which incorporated tributyrin in agar screening plates. Clones were subsequently subjected to next-generation sequencing and bioinformatic analysis, which allowed for further investigation of a portion of the Antarctic metagenome. Assembly and annotation of the genetic data encoded on three fosmid clones allowed for the identification of the genes responsible for tributyrin hydrolysis. Furthermore, hypotheses relating to survival and adaptation to abiotic conditions prevalent in the extreme Antarctic environment were developed (Chapter 3). A cold adapted esterase was subsequently characterised and showed substrate preference for para-nitrophenyl propionate. The optimum temperature and pH for the enzyme, DEaseI was 25 ° C and 8.5, respectively. In addition, results indicated that DEaseI was sensitive to thermal inactivation (Chapter 4). Furthermore, in fosmid clone LD13, one particular ORF annotated as a Water HYpersensitity response protein, became the focus of further study. When sub-cloned into a heterologous host, both ionic and osmotic stress tolerance was observed in vivo. The protein also exhibited a cryoprotective function in vitro, preventing cold denaturation of malate dehydrogenase during cycles of freeze-thaw (Chapter 5). This study demonstrates the value of combinatorial in silico and ‘-omic’ based techniques for the discovery and functional characterisation of potentially novel genes from bacteria which inhabit Antarctic Dry Valley soils.
Cloning, expression and characterization of novel lipase and esterases from Burkholderia multivorans UWC10
(University of the Western Cape, 2005) Rashamuse, Konanani J
An esterase and lipase producing Burkholderia multivorans strain was isolated by culture enrichment strategies. A shotgun library of Burkholderia multivorans genomic DNA (prepared in E. coli/pUC18) was screened for lipase and esterase activities. Three positive recombinant clones, pTEND5, pHOLA6 and pRASHI4, conferring esterolytic and lipolytic phenotypes respectively, were identified. Full-length sequencing of DNA inserts was performed using subeloning and "primer-walking" strategies. Nucleotide sequence analysis revealed that the pRASH14 plasmid DNA consisted of two open reading frames (ORPI and ORP2) encoding 356 and 350 amino acids, respectively. Database searches revealed that ORPI and ORP2 were homologous to lipases and chaperones from subfamily I.2. In the pTEND5 sequence, an open reading frame consisting of 978 bp, encoding 326 amino acids, was identified. Database searches revealed that this open reading frame was homologous to family Vesterases. Nucleotide sequence analysis revealed that pHOLA6, plasmid DNA consisted of 1194 bp encoding 398 amino acids and showed homology to family VIII esterases. The primary structures of LipA, EstEFH5 and EstBL from pRASHI4, pTEND5 and pHOLA6, respectively, showed a classical GxSxG motif, which is conserved in many serine hydrolases. In addition, EstBL also showed a consensus SxxK motif, the serine of which acts as a catalytic nucleophile in class C ~-lactames and some peptidases.
Actinobacteria associated with two diverse soil environments and their multicopper oxidase diversity
(University of the Western Cape, 2024) Prins, Alaric; McCullough, Bronwyn Kirby
The Cape Floristic Region (CFR) is a biodiverse region boasting unique plant diversity with a rich concentration of endemic plants. Aspalathus linearis (Rooibos) is an indigenous plant that grows in the Clanwilliam region of the Western Cape and is cultivated for its use as an herbal tea. Emerging peatlands in the CFR have gained increasing attention over recent years through research aiming to understand the microbial diversity associated with these environments. Little is known about the actinobacterial diversity of these regions, and as such, it is necessary to investigate the diversity of the actinobacteria associated with these environments, whilst simultaneously gaining knowledge on whether the associated actinobacteria may produce enzymes of biotechnological interest. Two CFR regions (the Rooibos environment – Clanwillian, and the Springfield emerging peatland environment – Agulhas) were explored through culture-based and genomic screening. Metabarcoding analyses using actinobacterial-specific 16S rRNA gene primers showed that the major taxa contributing to the Rooibos environment were members of the families Mycobacteriaceae, Pseudonocardiaceae, Frankiaceae and Geodermatophilaceae. Members of the families Mycobacteriaceaea, Pseudonocardiaceae, Acidimicrobiaceae and Nocardioiaceae was identified as the major taxa for the Springfield environment. Through selective isolation techniques, actinobacteria from rare (underrepresented) genera were isolated, including members of the genera Dactylosporangium, Actinokineospora, Curtobacterium, Modestobacter, Leifsonia and Actinomadura. The top strains, selected based on exhibiting extracellular multicopper oxidase (MCO) activity through culture-based screening, were subjected to whole genome sequence analysis. These rare genera are also vastly underrepresented among 3 400 bacterial MCO sequences found in the Laccase and Multicopper Oxidase Engineering Database (LccED).
The molecular characterization of trichoplusia ni single nucleocapsid nucleopolyhedrovirus: a study on early regulatory features
(University of the Western Cape, 2003) Wang, Weizhou; Davison, S
With the development of biological insecticides, many research efforts have been made in baculoviruses to investigate fundamental molecular aspects of these viruses, such as the function and regulation of genes, genome organization, mode of entry, DNA replication and virus factors that determine the host range and virulence. Previously, a South African Trichoplusia ni single capsid nuclear polyhedrosis virus (TnSNPV) isolate was partially characterized as a novel baculovirus. During the process of the characterization, a few late genes of the virus were identified. This thesis describes a molecular characterization of the TnSNPV early genes to gain insight into the functional roles of these genes, their unique features and further determination of the placement of TnSNPV in baculovirus phylogeny.
Genome-wide identification and comprehensive analysis of transcriptionsl desert regions
(University of the Western Cape, 2009) Schaefer, UIf; Bajic, Vladimir
The initiation of transcription in mammalian genomes predominatly occurs at 5' promoter regions, however increasingly initiation events have been observed within introns, coding exons and 3' UTRs. Nevertheless there are large segments of mammalian genomes that are not prone to transcription initiation. These locations can be understood to be 'transcription initiation deserts'. It is challenging and useful to demarcate these segments or locations of the genome. The availability of a huge number of transcript data has provided an opportunity to develop a methodology to predict and annotate these genomic segments. A comprehensive collection of data for Homo sapiens ard Mus musculus, consisting of CAGE tags and other evidence for the existence of ffanscription was used to develop a methodology that allows the annotation of locations of mammalian genomes as those that are highly likely to initiate tanscription and those that are unlikely to harbour transcription start sites (TSSs). The algorithm allows the recognition of TSSs with 100% sensitivity, which makes it the superior choice over other existing algorithms for promoter prediction for the task of annotating TSS deserts.
Mitigation of salinity stress using exogenously applied molybdenum in sorghum bicolor
(University of the Western Cape, 2022) Mabiya, Thembeka Confidence; Mulaudzi-Masuku, Takalani
The agricultural sector is the main producer of food throughout the world. However, the constant changes in environmental conditions, such as extreme weather, droughts, and salinity have impacted this sector negatively over the years. These stresses cause nutritional imbalance, delayed seed germination and decreased growth resulting in reductions in crop yield and hence affect food prices. The food and agricultural organization (FAO), reported that the average increase rate in crop production is below the amount required to cater for the growing population. Thus, to meet the food demands, discovery of several strategies to improve crop growth and yield under severe environmental conditions are imperative.
Inter-individual genetic variation and the development of hypertension in a Xhosa African population of Eastern Cape, South Africa
(University of the Western Cape, 2022) Mabhida, Sihle Ephraim; Benjeddou, Mongi
Cardiovascular diseases (CVD) are the leading cause of death globally, accounting for 18.6 million deaths. Hypertension (HTN) drives the global burden of CVD and is a leading cause of cardiovascular-related mortality with 1.4 billion affected adults and 10.4 million deaths globally. This public health condition has been escalating alarmingly in low and middle-income countries. In Sub-Saharan Africa, HTN is a major public health concern with South Africa having the highest prevalence between 27-58%. Accumulative evidence shows that HTN is driven by both modifiable and non-modifiable risk factors.
The interactive effects of salt stress and Fusarium proliferatum infection on maize seedlings
(University of the Western Cape, 2022) Badiwe, Mihlali; Klein, Ashwil
Field crops are often subjected to multiple co-occurring stress factors, and they evolved specific mechanisms to counteract the effects of these stress factors. Most studies explore the fundamental molecular mechanisms involved in plant abiotic and biotic stress interactions. These look at plant responses to individual stressors and not combinations. Studying the effects of individual stress responses is not an adequate approach as plants in nature are challenged by both abiotic and biotic stress factors occurring simultaneously. Modern studies have shown that plants possess the ability to tolerate co-occurring abiotic and biotic stresses through the utilization of tailored responses, which are impossible to understand by direct extrapolation from results of studies examining individual stress factors.
Identification of sub-clinical biomarkers that predict the risk of developing diabetic cardiomyopathy
(University of the Western Cape, 2022) Nxele, Xolisa; Benjeddou, Mongi
Cardiovascular disease (CVD) is the leading cause of death of people with obesity and type 2 diabetes (T2DM). According to a statistical report from the World Health Organization (WHO), approximately 17.9 million people die annually because of CVD and diabetic cardiomyopathy (DCM), a disease of the heart muscle occurring in the absence of coronary artery disease or hypertension. Although not fully elucidated, the pathophysiology of DCM includes myocardial left ventricular hypertrophy, impaired calcium handling, energy metabolism, inflammation, apoptosis and myocardial fibrosis.
Deciphering the determinants of molecular physiological responses to drought and heat stress in sorghum lines contrasting in stress tolerance via a proteomics approach
(University of the Western Cape, 2022) Ali, Ali Elnaeim Elbasheir; Ludidi, Ndiko
Sorghum is an important crop in Sub-Saharan Africa and South Asia. The predicted rise in global temperatures will increase the probability of exposing sorghum to heat waves in combination with drought. Thus, production and availability of sorghum and its products will be negatively affected. Although much progress has been made in identifying molecular processes involved in some crop responses to drought or heat stress, knowledge on such responses in sorghum is limited and in fact does not exist for combined drought and heat stress in sorghum lines that respond differently to these stresses.
Molecular detection and characterisation of RNA viruses of honeybees
(University of the Western Cape, 2009) Topley, Elize Lindsay; Davison, Sean
Honeybees have evolved through the centuries to inhabit most parts of the world except for the extreme Polar Regions. These insects have also been susceptible to pathogens and disease which has always been part of the honeybees’ ecology and has evolved and adapted accordingly. However disease has spread more rapidly into areas where no disease existed before with the transport and moving of hives. Disease has caused massive losses within the honeybee industry in recent history. Using new technology available to scientists, diseases and parasites can be identified and this information used to prevent damage to hives, the livelihood of many crop farmers and beekeepers around the world. Of these diseases honeybee viruses have become of some concern in recent times. Honeybee viruses’ black queen cell virus (BQCV) and acute bee paralysis virus (ABPV) were found to have genomes consisting of 8550 and 9490 nucleotides respectively. The viruses have two open reading frames (ORFs) which encode a non structural protein at the 5’ ORF and a structural protein at the 3’ ORF. Sacbrood virus (SBV) has a different organisation to BQCV and ABPV where it has a single ORF with the structural genes at the 5’ end and the non structural genes at the 3’ end. In an effort to rapidly identify honeybee viruses a multiplex reverse transcriptase polymerase chain reaction (RT-PCR) was developed Primers were designed within the 3’ open reading frame to amplify fragments of 434bp for SBV, 900bp for ABPV and 316bp for BQCV. RNA was extracted from laboratory infected and naturally infected samples. The PCR products were sequenced and found to be that of the appropriate virus. The primers were tested on naturally infected samples with SBV and BQCV being detected. Another well characterised honeybee virus Kashmir bee virus (KBV) was initially added to the multiplex RT-PCR. However inconsistencies with the multiplex PCR led to the sequencing of a 2 kilobase fragment of the KBV Indian (KBV-in) strain. Three overlapping cDNA fragments of KBV were sequenced and aligned with the full length sequence of KBV and a sequenced capsid region of KBV both from North America. Alignment to ABPV was also completed to observe the homology between KBV-in and ABPV. The KBV-in strain was not highly homologous to the North American strains over the region which was sequenced for KBV-in. ABPV was also not highly homologous over the entire 2 Kb region. However over the region where primers were designed for the RT PCR of KBV, ABPV was highly homologous at 80%. This could have led to the inconsistencies when PCR was done. Primer design and correct strain characterisation is needed before primers are designed to detect more than one virus per reaction. Further characterisation and sequencing of this strain is needed in order to make further comparisons. Propagation methods for honeybee viruses have not changed since these viruses were discovered. There are no suitable cell lines or cell culture techniques available for honeybee viruses. Honeybee viruses have to be manually injected with virus in order for the virus to multiply and be extracted. With the presence of inapparent viruses which could co-infect pupae, a method for pure virus propagations needs to be found. Recombinant baculovirus systems have been used extensively to produce foreign proteins from different viruses using vectors and recombinant technology. In this chapter we inserted the capsid gene from BQCV into a transfer vector under the control of the p10 promoter of Autographa californica. Fractions of the sucrose gradient containing the virus like particles (VLPs) were seen under the electron microscope. A Western blot showed the four capsid proteins at the expected sizes for BQCV capsid. This study therefore has shown that a heterologous system such as baculovirus can be used for virus like particle production. Infectious virus technology has helped gain insight into how viruses work. Using this technology altering honeybee viruses could be used to observe different functionalities of the viruses. An attempt was made to interchange the open reading frames of ABPV and BQCV to observe any changes in virus assembly and infectivity. A fusion PCR strategy was employed to interchange the 5’ and 3’ ORFs of APBV and BQCV. The strategy however was unsuccessful. Alternative strategies could improve the chances of obtaining a chimeric virus.
A proteomic analysis of drought and salt stress responsive proteins of different sorghum varieties
(University of the Western Cape, 2009) Ngara, Rudo; Ndimba, Bongani K.
Sorghum (Sorghum bicolorï, a drought tolerant cereal crop, is not only an important food source in the semi arid/arid regions but also a potential model for studying and gaining a better understanding of the molecular mechanisms of drought and salt stress tolerance in cereals. This study reports on a proteomic analysis of sorghum proteomes in response to salt and hyperosmotie stresses. Two-dimensional gel electrophoresis (2DE) in combination with mass spectrometry (MS) was used to separate, visualise and identify sorghum proteins using both sorghum cell suspension cultures and whole plants. The sorghum cell suspension culture system was used as a source of culture filtrate (CF) proteins. Of the 25 visualised CBB stained CF spots, 15 abundant and well-resolved spots were selected for identification using a combination of MALDI- TOF and MALDI- TOFTOF MS, and database searching. Of these spots, 14 were positively identified as peroxidases, germ in proteins, oxalate oxidases and alpha-galactosidases with known functions in signalling processes, defense mechanisms and cell wall metabolism. Following 200 mM NaCl and 400 mM sorbitol stress treatments, the expression/abundance of a protein spot similar to a rice wall-associated protein kinase was upregulated in the sorghum secretome in response to both stresses. Amino acid sequence alignment of the matching peptides between these two proteins showed that the sorghum CF spot possesses a protein kinase domain. Therefore, this protein could possibly participate in cell signalling functions, which link the external environment with the cell's cytoplasm. Using whole plant systems, a comparative study of leaf protein expression between two sorghum varieties, AS6 (salt sensitive) and MN1618 (salt tolerant) was conducted. Forty well resolved spots of varying abundances were picked for MS analysis. Of these, 28 were positively identified, representing proteins with functions in carbohydrate metabolism (60.7%), proton transport (17.9%), protein synthesis (7.1%), hydrolytic functions (7.1%), nucleotide metabolism (3.6%) and detoxification (3.6%). Using PDQuest™ Advanced 2D Analysis Software version 8.0.1 (BIO-RAD), a comparative analysis of leaf proteome expression patterns between the two sorghum varieties was conducted. The results indicated proteins with similar expression patterns as well as qualitative and quantitative differences between the two leaf proteomes. The effect of 100 mM NaCI on leaf proteome expression between the two sorghum varieties was also studied. Western blotting analysis of leaf, sheath and root tissues using Hsp70 antibodies showed that this treatment induced Hsp70 expression, a known stress protein, in both varieties. Thereafter, the partially annotated leaf proteome map was used to landmark other salt responsive proteins. Examples of differential expression patterns included glutathione S transferase and hydroxynitrile lyase proteins whose abundances were upregulated in both varieties, while the large subunit of RuBisCo was downregulated in AS6 but upregulated in MN1618. Qualitative spot expression differences in response to salt stress were also observed between the two sorghum varieties but these remained unidentified after both MALDI-TOF and MALDI-TOF-TOF MS, possibly indicating novel and previously uncharacterised sorghum proteins. The results of this study can be used as reference tools by proteomics researchers worldwide as well as a foundation for future studies.
Evaluation of the capacity of hydrogen sulfide to reduce infection of maize
(University of the Western Cape, 2020) Ntloko, A.; Ludidi, N
Maize (Zea mays L.) is grown globally as an important grain crop in South Africa, United States, China and Brazil and plays a major role in the worldwide economy. In South Africa, the grain is utilised for food consumption, livestock feed, for malting purposes and bioethanol production. Maize contains approximately 72% starch, 10% protein, 4% fat and supplying an energy density of 365 Kcal/100 g. The production of grain crops in South Africa is restricted by various factors such as abiotic and biotic stresses. The fungal genus Aspergillus is one of the most important biotic stresses affecting maize in the country. Aspergillus flavus can contaminate a wide range of agricultural commodities either in storage or field. Hydrogen sulfide appears to have a potential in the mechanism of resistance against pathogen attack by Aspergillus flavus.
Genomic and proteomic analysis of drought tolerance in Sorghum (Sorghum bicolor (L.) Moench)
(University of the Western Cape, 2014) Woldesemayat, Adunga,Abdi; Christoffels, Alan; Ndimba, Bongani.K
Drought is the most complex phenomenon that remained to be a potential and historic challenge to human welfare. It affects plant productivity by eliciting perturbations related to a pathway that controls a normal, functionally intact biological process of the plant. Sorghum (Sorghum bicolor (L.) Moench), a drought adapted model cereal grass is a potential target in the modem agricultural research towards understanding the molecular and cellular basis of drought tolerance. This study reports on the genomic and proteomic findings of drought tolerance in sorghum combining the results from in silica and experimental analysis. Pipeline that includes mapping expression data from 92 normalized cDNAs to genomic loci were used to identify drought tolerant genes. Integrative analysis was carried out using sequence similarity search, metabolic pathway, gene expression profiling and orthology relation to investigate genes of interest. Gene structure prediction was conducted using combination of ab initio and extrinsic evidence-driven information employing multi-criteria sources to improve accuracy. Gene ontology was used to cross-validate and to functionally assign and enrich genes. An integrated approach that subtly combines functional ontology based semantic data with expression profiling and biological networks was employed to analyse gene association with plant phenotypes and to identify and genetically dissect complex drought tolerance in sorghum. The gramene database was used to identify genes with direct or indirect association to drought related ontology terms in sorghum. Where direct association for sorghum genes were not available, genes were captured using Ensemble Biomart by transitive association based on the putative functions of sorghum orthologs in closely related species. Ontology mapping represented a direct or transitive association of genes to multiple drought related ontology terms based on sorghum specific genes or orthologs in related species. Correlation of genes to enriched gene ontology (GO)-terms (p-value < 0.05) related to the whole-plant structure was used to determine the extent of gene-phynotype association across-species and environmental stresses.
Cloning, expression and characterization of Novel Lipase and Esterases from Burkholderia multivorans UWC10
(2005) Rashamuse, Konanani J; Cowan, Don A
An esterase and lipase producing Burkholderia multivorans strain was isolated by culture enrichment strategies. A shotgun library of Burkholderia multivorans genomic DNA (prepared in E. coli/pUC18) was screened for lipase and esterase activities. Three positive recombinant clones, pTEND5, pHOLA6 and pRASHI4, conferring esterolytic and lipolytic phenotypes respectively, were identified. Full-length sequencing of DNA inserts was performed using subeloning and "primer-walking" strategies. Nucleotide sequence analysis revealed that the pRASH14 plasmid DNA consisted of two open reading frames (ORPI and ORP2) encoding 356 and 350 amino acids, respectively. Database searches revealed that ORPI and ORP2 were homologous to lipases and chaperones from subfamily I.2. In the pTEND5 sequence, an open reading frame consisting of 978 bp, encoding 326 amino acids, was identified. Database searches revealed that this open reading frame was homologous to family Vesterases. Nucleotide sequence analysis revealed that pHOLA6, plasmid DNA consisted of 1194 bp encoding 398 amino acids and showed homology to family VIII esterases. The primary structures of LipA, EstEFH5 and EstBL from pRASHI4, pTEND5 and pHOLA6, respectively, showed a classical GxSxG motif, which is conserved in many serine hydrolases. In addition, EstBL also showed a consensus SxxK motif, the serine of which acts as a catalytic nucleophile in class C B-lactames and some peptidases.

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