The development of biological tools to aid in the genetic investigation of the black (Diceros bicornis) and white (Ceratotherium simum) rhinoceros mitochondrial genomes
- Authors: Parsons, Michelle
- Date: 2015
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/56059 , vital:26769
- Description: The black (Diceros bicornis) and white (Ceratotherium simum) rhinoceros are found in South Africa. A decline in the populations of these species has resulted due to human activities such as habitat fragmentation and poaching. This has contributed to the loss of genetic diversity amongst the black and white rhinoceros. Conservation and anti-poaching efforts are needed to help maintain genetic diversity. These efforts could be improved through the development of non-invasive techniques to examine DNA from threatened animals. The aim of this research was to develop a molecular technique which would allow for the identification of the black and white rhinoceros and to develop a molecular technique which would allow for intraspecies genetic variation to be examined. DNA extractions were performed on matched faecal and tissue samples that were collected from two regions in South Africa. Polymerase chain reaction (PCR) primer sets were designed to investigate several regions of the rhinoceros mitochondrial genome. PCR optimisation was completed for the target regions. Sequencing was conducted on all final PCR products. The cytochrome c oxidase subunit 1 (COIi) gene allowed for the rhinoceros family to be identified. This region was digested with the HindIII restriction enzyme, which allowed for the specific identification of either the black or white rhinoceros. A subsequent region of the cytochrome c oxidase subunit 1 (COIii) as well as the D-loop, hypervariable regions (HV1 and HV2), cytochrome b (cytb) and 16s rRNA regions were investigated. These regions displayed potential for establishing geographic origin for black rhinoceros samples, whereas the D-loop and HV2 show potential for the white rhinoceros. The white rhinoceros displayed sequence variation in the HV2 and cytb region, while variation was observed in the COIi and HV1 for the black rhinoceros. All investigated target regions allowed for the rhinoceros family to be identified. The COI (COIi and COIii), HV2 and cytb regions allowed for the subspecies of rhinoceros to be identified, however the D-loop was not able to identify the white rhinoceros species. The 16s rRNA and HV1 regions allowed for the correct subspecies of rhinoceros to be identified, however as the primers were only compatible for the black rhinoceros therefore a subsequent investigation is required for the white rhinoceros. The establishment of this novel PCR based technique to identify white and black rhinoceros will allow for efficient species identification in wildlife forensic cases. A biological method was established to study intraspecies variation for the white and black rhinoceros; however the investigated target regions did not yield sufficient genetic variation. The core techniques developed in this study will be valuable for future studies that wish to investigate genetic variation in mammal species.
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- Date Issued: 2015
Purification, characterisation and application of inulinase and transferase enzymes in the production of fructose and oligosaccharides
- Authors: Mutanda, Taurai
- Date: 2008
- Subjects: Fructose Transferases Oligosaccharides
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4448 , http://hdl.handle.net/10962/d1007734
- Description: Inulin hydrolysis can occur as a result of the action of exoinulinases and endoinulinases acting alone or synergistically. Exoinulinases cleave the non-reducing β-(2, I) end of inulin releasing fructose while endoinulinases act on the internal linkages randomly to release inulotrioses (F₃), inulotetraoses (F₄) and inulopentaoses (F₅) as major products. Fructosyltransferases act by cleaving a sucrose molecule and then transferring the liberated fructose molecule to an acceptor molecule such as sucrose or another oligosaccharide to elongate the short chain fructooligosaccharide. The production of high yields of oligosaccharides of specific chain length from simple raw materials such as inulin and sucrose is a challenge. Oligosaccharides of chain length up to degree of polymerisation (DP) 5 and fructose were produced using preparations of three commercial microbial enzymes. Production of these novel oligosaccharides was achieved by employing response surface methodology (RSM) with central composite experimental design (CCD) for optimising product yield. Using a crude Novozyme 960 endoinulinase preparation isolated from Aspergillus niger, the following conditions gave a high inulooligosaccharide (lOS) yield, temperature (60 ºC), 150 g/L inulin concentration, 48 h incubation; pH 6.0 and enzyme dosage of 60 U/ml. Under these conditions, inulotrioses (70.3 mM), inulotetraoses (38.8 mM), and inulopentaoses, (3.5 mM) were produced. Response surface regression predicted similar product levels under similar conditions. The crude endoinulinase was purified through a three step purification procedure with a yield of 1.11 % and 3.5 fold purification. The molecular weight of this endoinulinase was estimated to be 68 .1 kDa by SDS-PAGE and its endoinulinase nature was confirmed by native PAGE. The purified endoinulinase was more efficient in production of lOS than the crude endoinulinase preparation. The purified endoinulinase demonstrated a high affinity for the inulin substrate (Km[subscript] 3.53 mM, Vmax[subscript] 666.67 μmol/min/ml). Pectinex Ultra SP-L, a commercial crude enzyme preparation isolated from Aspergillus aculeatus is a cocktail of several enzymes including a fructosyltransferase. The crude enzyme showed both transfructosylation and hydrolytic activity in 200 to 600 g/L sucrose. The main fructooligosaccharides produced from sucrose were l-kestose (GF₂), nystose (GF₃) and fructofuranosyl nystose (GF₄). After the first RSM, with the coded independent variables of temperature, incubation time, pH and sucrose concentration, the highest levels of GF₂, was 68.61 mM, under sucrose concentration 600 g/L, temperature 60°C, enzyme dosage 20 U/ml , pH 5.6, after 4 h incubation. A sucrose concentration of 400 g/L favoured the synthesis of high levels of GF₃ and GF₄. In the second RSM the maximal yields of GF₂, GF₃ and GF₄ were 152.07 mM, 131.38 mM and 43.99 mM respectively. A purified fructosyltransferase did not synthesise GF₄. Ammonium ions were demonstrated to enhance the yield of FOS. A mixture of glucose and fructose was used as substrate for FOS synthesis and no FOS were formed. Glucose was shown to be an end product inhibitor of the fructosyltransferase and therefore hinders the formation of high FOS yield. Fructozyme, isolated from Aspergillus ficuum is a mixture of exo and endoinulinases with the former being predominant was used for fructose production from inulin hydrolysis. The exoinulinase was purified to electrophoretic homogeneity by a three step purification procedure. The molecular weight of the enzyme was estimated to be 53 kDa with a 2 I % yield and 4.2-fold. Response surface regression was used to predict the maximum fructose levels achievable under the combinations of temperature, enzyme dosage and incubation time. A reaction time (48 h), enzyme dosage (100 U/ml) and inulin concentration (150 g/l) at pH 5.0 at 50°C gave higher fructose levels (106.6 mg/ml) using crude exoinulinase as compared to 98.43 mg/ml using the purified exoinulinase. These findings indicate that higher levels of fructose require longer incubation periods and higher inulin substrate concentrations with higher enzyme dosage. The crude exoinulinase preparation gave fairly higher levels of fructose than the purified exoinulinase and this is due to the presence of other hydrolytic enzymes in the crude preparation. The conditions established by RSM and CCO were adequate in producing high yield of oligosaccharides and fructose and can therefore be applied for their industrial production since they are in high demand due to their health benefits as prebiotics.
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- Date Issued: 2008