Faculty of Applied Sciences
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Item Production of chitosan and lipids from a newly isolated Mucor circinelloides(2018) Zininga, Johnson Tungamirai; Permaul, Kugen; Singh, SurenFilamentous fungi are well-known sources of a wide variety of industrially-useful biomolecules. This study demonstrates the applicability of a newly isolated oleaginous fungi Mucor circinelloides ZSKP for lipid and chitosan production. Parameters affecting co-production were identified and were statistically optimized, which resulted in a 3–fold improvement in lipid production. The lipid profile showed a high content of unsaturated fatty acids including oleic, linolenic and linoleic acids, while palmitic acid was the major saturated fatty acid (21%). A comparative study to evaluate the efficacy of enzymatic and chemical treatments for biodiesel production from fungal lipids and sunflower oil revealed slightly enhanced production of biodiesel from fungal lipids, using a commercial lipase. The biodiesel synthesized using lipids from M. circinelloides ZSKP satisfied standard specifications and had a higher cetane number (56), lower kinematic viscosity (4.6 mm2/s) and lower acid number (0.03) compared to sunflower oil. Upon optimizing chitosan production and extraction processes the chitosan production was improved 2-fold. The fungal chitosan showed antimicrobial properties and was more effective against Aspergillus niger A chitosan spray was developed which was able to increase the shelf life of fresh fruit produce. These results indicate that Mucor circinelloides ZSKP is a promising candidate for concurrent production of lipids and the versatile bio-polymer chitosan.Item Utilization of shrimp waste for the recovery of valuable bioactive compounds(2018) Dlamini, Nosihle; Permaul, KugenShrimp waste is a major by-product of crustacean processing and represents an interesting source of bioactive molecules. In addition, its use increases the sustainability of processing fishery products. The present study reports a process developed for recovering bioactive molecules from shrimp waste through the use of chemical methods. The samples of shrimp were confirmed to be from the species Haliporoides triarthrus. The recovery of chitin was 30% of the processing waste and 30-60% chitosan (CH) from chitin. CH was characterized by FTIR analysis and exhibited a degree of deacetylation (DDA) of 72%. From the demineralization extract, CaCO3 was extracted and confirmed by FTIR. Based on a kinetic study of acid hydrolysis, it was demonstrated that chitin can be quantitatively hydrolysed into glucosamine (GlN), N-acetyl glucosamine (GlcNAc) and their respective oligomers with 32% hydrochloric acid at 60oC and qualitatively from CH with 32% hydrochloric acid at 80oC. The oligomer mixed fractions were desalted by activated charcoal extraction and the components of each fraction were analysed by TLC and HPLC. Chitooligosaccharides (COS) and N-acetly chitooligosaccharides (NAcCOS) with degrees of polymerization (DP) ranging from 2 to 6 were obtained from CH and chitin, respectively. The antimicrobial activities of chitosan, COS and NAcCOS were investigated against five gram-negative bacteria and five gram-positive bacteria. Chitosan exhibited stronger bacteriostatic effects against gram- positive bacteria than gram-negative bacteria in the presence of 1% chitosan. The oligomers showed no bacteriostatic or bactericidal effects on all tested bacteria. A total 30.74± 0.078 µg.g-1 astaxanthin was extracted with 90% acetone from the species; Haliporoides triarthrus and TLC analysis indicated that the species contained both astaxanthin and its esters. Chitosan films were obtained by solution casting of blends of chitosan with glycerol, polyethylene glycol 200 (PEG-200) and polyethylene glycol 600 (PEG-600) as plasticizers. Films were characterized by FTIR, XRD diffraction, TGA, and SEM analysis. The tensile strength and elongation at break properties of the films were also evaluated. CH films and CH/GLY blended films were translucent in appearance and the CH/PEG 200 and CH/PEG 600 films were opaque. The CH films yielded mechanically resistant films without the use of a plasticizer. These data point to the feasibility of an integrated process for isolating highly bioactive molecules, such as oligosaccharides, with a broad spectrum of applications from shrimp processing waste.Item Thermostable chitinase II from Thermomyces lanuginosus SSBP: Cloning, structure prediction and molecular dynamics simulations(Elsevier, 2015) Khan, Faez Iqbal; Govender, Algasan; Permaul, Kugen; Singh, Suren; Bisetty, KrishnaThermomyces lanuginosus is a thermophilic fungus that produces large number of industrially-significant enzymes owing to their inherent stability at high temperatures and wide range of pH optima, including thermostable chitinases that have not been fully characterized. Here, we report cloning, characterization and structure prediction of a gene encoding thermostable chitinase II. Sequence analysis revealed that chitinase II gene encodes a 343 amino acid protein of molecular weight 36.65 kDa. Our study reports thatchitinase II exhibits a well-defined TIM-barrel topology with an eight-stranded α/β domain. Structural analysis and molecular docking studies suggested that Glu176 is essential for enzyme activity. Folding studies of chitinase II using molecular dynamics simulations clearly demonstrated that the stability of the protein was evenly distributed at 350 K.Item The multi-chitinolytic enzyme system of the compost-dwelling thermophilic fungus Thermomyces lanuginosus(Elsevier, 2014-12-03) Zhang, Meng; Puri, Adarsh Kumar; Govender, Algasan; Wang, Zheng-Xiang; Singh, Suren; Perumal, KugenthirenThe recent sequencing of the Thermomyces lanuginosus SSBP genome by our group has revealed four putative family 18 chitinases. In this study, three novel chitinase genes (chit2, chit3 and chit4) and the previously-reported chit1 gene were cloned from T. lanuginosus SSBP. chit1, encoding a 44.1 kDa protein, and chit2, encoding a 36.6 kDa protein, were successfully expressed in Pichia pastoris. The recombinant Chit1 and Chit2 enzymes exhibited optimum activity at pH 5.0 and pH 4.0, respectively. Chit1 had optimal activity at 50 ◦C and retained 56% of its activity at 60 ◦C after 30 min, while Chit2 was optimally active at 40 ◦C and retained 71% of its activity at 50 ◦C after 60 min. Both enzymes produced chitobiose as the major product using different substrates. Chit2 displayed antifungal activity against Penicillium verrucosum and Aspergillus niger. These activities could be useful in the environmental degradation of chitinous wastes as well as for biotechnological applications.Item Thermostable chitinase II from Thermomyces lanuginosus SSBP : Cloning, structure prediction and molecular dynamics simulations(Elsevier, 2015-04-08) Khan, Faez Iqbal; Govender, Algasan; Permaul, Kugen; Singh, Suren; Bisetty, KrishnaThermomyces lanuginosus is a thermophilic fungus that produces large number of industrially-significant enzymes owing to their inherent stability at high temperatures and wide range of pH optima, including thermostable chitinases that have not been fully characterized. Here, we report cloning, characterization and structure prediction of a gene encoding thermostable chitinase II. Sequence analysis revealed that chitinase II gene encodes a 343 amino acid protein of molecular weight 36.65 kDa. Our study reports that chitinase II exhibits a well-defined TIM-barrel topology with an eight-stranded α/β domain. Structural analysis and molecular docking studies suggested that Glu176 is essential for enzyme activity. Folding studies of chitinase II using molecular dynamics simulations clearly demonstrated that the stability of the protein was evenly distributed at 350 K.