Theses and dissertations (Applied Sciences)
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Item The chitinolytic enzyme system of the compost-dwelling thermophilic fungus Thermomyces lanuginosus(2014) Zhang, Meng; Singh, Suren; Permaul, Kugen; Wang, Zheng-XiangChitin, a highly insoluble 1,4- -linked polymer of N-acetyl- -D-glucosamine, is the second-most abundant bio-polysaccharide in nature after cellulose. Most chitinolytic fungi are known to produce more than one kind of chitinase. The 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 (chitl, chit2 and chit3) and the previously reported chit4 gene were cloned from Thermomyces lanuginosus SSBP and their gene structures were analysed. chit3, encoding a 36.6 kDa protein, and chit4, encoding a 44.1 kDa protein, were successfully expressed in Pichia pastoris. The recombinant Chit3 and Chit4 enzymes exhibited optimum activity at pH 4.0 and 5.0 and at 40oC and 50°C, respectively. Chit3 was stable at 40oC and retained 71% of its activity at 50°C after 60 min, while Chit4 was stable at 50°C and retained 56% of its activity at 60°C after 30 min. Both enzymes produced chitobiose as the major product using colloidal chitin, chitooligosaccharides and shrimp shell powder as substrates. Of the fungal strains tested, Chit3 displayed antifungal activity against Penicillium sp. and Aspergillus sp. This is the first report on the multi-chitinolytic system of T. lanuginosus and enzyme characterization has shown the potential of the enzymes to be used in degradation of the under-utilized bio-resource chitin.Item Xylanase hyper-producer : the genome of the thermophilic fungus Thermomyces lanuginosus(2014-08-08) Mchunu, Nokuthula Peace; Permaul, Kugen; Singh, SurenThe global demand for green technology has created a need to search for microbes that can play an active role in advancing a greener and cleaner future. Microbial enzymes are nature’s keys to life and their efficiency, specificity and environmental-friendliness has lead to their increased use in industrial processes. Thermomyces lanuginosus is a thermophilic fungus that can degrade plant biomass and produces a variety of enzymes that have industrial application. The fungus T. lanuginosus SSBP has been reported in literature to produce the highest level of xylanase among other Thermomyces strains and some of its enzyme s viz., amylase and lipase are already being used. Because of this ability, it has been identified as one of the organisms that can have various industrial applications. Although a few proteins from this fungus have been cloned and used commercially, the vast majority are still unknown. In order to identify new protein candidates and understand their biochemical interactions, the T. lanuginosus genome (DNA) and the transcriptome (mRNA) were sequenced using 454 Roche and Solexa sequencing platforms. Genome and transcriptome data was assembled using Newbler software forming a genome size of 23.3 Mb contained 30 scaffolds. Protein prediction identified 5105 candidates as protein-coding genes and these gene models were supported by expressed sequence tag and transcriptomic data. The annotated data was assembled into metabolic pathways in order to identify functional pathways and validate the accuracy of the annotation process. T. lanuginosus is usually found in composting plant material thus protein related to plant hydrolysis were analysed. The total number of plant biomass-degrading and related proteins that fall into the carbohydrate-active enzyme (CAZy) family was 224. Most of these proteins were similar to proteins found in other filamentous fungi. Surprisingly, T. lanuginosus contained a single gene coding for xylanase which hydrolyses xylan although this organism is well known for being among the highest producers of this enzyme. An important subset of the above group of proteins is the cellulose degrading-proteins as this can be used in biofuel production. Eight candidates belonging to this group were identified, making this fungus significant in the biofuels. Among the eight cellulase candidates, phylogenetic analysis revealed that three of them were closely related to Trichoderma reesei, a well known industrial cellulase-producer. Utilization of cellulase-related compounds was validated by phenotypic microarray experiments, with cellobiose having inducing biomass in T. lanuginosus. Proteins that are involved in high temperature survival are vital for the survival. of this thermophilic fungus. Interestingly, T. lanuginosus contains 19 heat shocking proteins which are responsible for thermostability. Another adaptation identified in this fungus is the accumulation of trehalose to combat heat stress. Furthermore, T. lanuginosus contains the highest reported number methyltransferases, which have been linked to producing thermostable proteins and higher energy production. Also because of this organism’s ability to grow on composting environments, the assimilation and ability to produce biomass on different carbon sources were analysed using phenotypic microarray technique. The results showed that xylose was the best compound to induce biomass followed by trehalose, maltose and maltotriose. The genomic sequencing of this fungus has provided valuable information that can be used for various biotechnological applications, as well as providing greater insights into its thermostability. Understanding the metabolic pathways involved may allow for manipulation to increase production of these enzymes or cloning into other hosts. This can have an impact in the field of biofuel production and other plant biomass-related processes.