83 research outputs found

    Fabrication, Characterization And Application Of Polyhydroxybutyrate-Titanium Dioxide Nanocomposite Materials

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    A serious need for a robust, low-cost and eco-friendly method to treat dye pollutants released by textile industries motivated this research. The dye adsorption effect of biodegradable poly-3-hydroxybutyrate [P(3HB)] and the photocatalytic degradation ability of inert titanium dioxide (TiO2) was exploited to fabricate a multifunctional nanocomposite film via solvent casting and electrospinning. The environmental degradation of the choice material, cast P(3HB) was first tested and confirmed to be comparable to its co-polymers; poly(3-hydroxybutyrate-co-5 mol% 3-hydroxyvalerate) [P(3HB-co-5 mol% 3HV)] and poly(3-hydroxybutyrate-co-5 mol% 3-hydroxyhexanoate) [P(3HB-co-5 mol% 3HHx)]. The weight loss was almost complete by 8 weeks in tropical mangrove sediment. The nanocomposite film was also degradable although to a lesser extent despite the microbial inactivation effect of TiO2. The optimum amount of P(3HB) and TiO2 loading in cast film was found to be 0.4 g and 40 wt% respectively. This film had an even distribution of TiO2 when mixed concurrently in chloroform followed by stirring for 24 h. It completely decolorized and detoxified real industrial Batik dye wastewater in 3 h and induced a chemical oxygen demand (COD) reduction of 80%. The 0.4 g P(3HB)-40 wt% TiO2 film exhibited good stability with decolorization percentage of ≥80% even after the sixth repeated usage

    Understanding Peroxidase immobilisation on Bioinspired Silicas and application of the biocatalyst for dye removal

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    Dyestuff industry is responsible for up to 20% of the industrial water pollution, due to dye loss in effluents. Compared to research on treatment of azo dyes (largest category), research of anthraquinone dyes (second largest category) is neglected. Environmental considerations about industrial chemical processes for water treatment have led to a shift towards green chemistry and biocatalysis. Although peroxidases are vastly applied in bioremediation, they cannot be industrially implemented due to low stability, lack of reusability and difficulty in scale-up. Immobilisation offers reusability and can improve the catalytic functions and operational stability of biocatalysts. Novel approaches, include bioinspired supports, synthesised fast and economically, avoiding the environmentally un-friendly methods used in “conventional” immobilisation. This project focused on understanding the immobilisation of Horseradish Peroxidase (HRP) on bespoke Bioinspired Silicas (BIS), by examining factors affecting the synthesis and performance of the biocatalysts. We immobilised HRP on BIS via in-situ encapsulation and adsorption, and compared the outcomes to that of HRP adsorbed on commercial silicas. We also examined the effect of the controlled presence of amine functionalisation on BIS, of the point of HRP addition during synthesis of the biocatalyst and of increasing HRP concentration, to the immobilisation efficiency and performance of biocatalysts. BIS showed high potential as immobilisation supports, offering high loading (about 20% HRP on BIS-HRP composite) of active enzyme and their ability to protect HRP under exposure to non-optimal conditions. Biocatalysts were characterised for their morphology and porosity before assessing their performance a standard peroxidase assay based on 2,2′-azino-bis(3-ethylthiazoline-6-sulfonate acid oxidation (ABTS assay) and an application assay based on enzymatic degradation of a model anthraquinone dye, Reactive Blue 19 (RB19 assay). Further examination of the best performing BIS-HRP samples, revealed a competitive action of BIS to enzymatic activity, where the support acts as an excellent adsorbent, hindering the diffusion of substrate and product(s) through the pore network. Although free HRP outperforms immobilised HRP (especially via encapsulation), immobilisation results in a highly reusable biocatalyst, for up to 20 times with 60% performance retention towards dye removal, with enhanced storage stability, retaining almost 100% activity over 50 days of storage, compared to 3 days of storage reached with free HRP. Through this work, we showed the importance of individual factors crucial for enzyme immobilisation, regarding both biocatalyst synthesis and expected performance, as well as the importance of the combination of enzyme, substrate and immobilisation support on biocatalyst performance. This work can be a great base for further optimisation of BIS as enzyme immobilisation support, and its exploration in other applications in the area of water treatment

    High-resolution spatial and genomic characterization of coral-associated microbial aggregates in the coral Stylophora pistillata

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    Bacteria commonly form aggregates in a range of coral species [termed coral-associated microbial aggregates (CAMAs)], although these structures remain poorly characterized despite extensive efforts studying the coral microbiome. Here, we comprehensively characterize CAMAs associated with Stylophora pistillata and quantify their cell abundance. Our analysis reveals that multiple Endozoicomonas phylotypes coexist inside a single CAMA. Nanoscale secondary ion mass spectrometry imaging revealed that the Endozoicomonas cells were enriched with phosphorus, with the elemental compositions of CAMAs different from coral tissues and endosymbiotic Symbiodiniaceae, highlighting a role in sequestering and cycling phosphate between coral holobiont partners. Consensus metagenome--assembled genomes of the two dominant Endozoicomonas phylotypes confirmed their metabolic potential for polyphosphate accumulation along with genomic signatures including type VI secretion systems allowing host association. Our findings provide unprecedented insights into Endozoicomonas-dominated CAMAs and the first direct physiological and genomic linked evidence of their biological role in the coral holobiont

    Novel fluorescent tools and techniques for 3D imaging of the cleared brain

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    Background: To better understand the complexity of the brain and how it becomes impaired under different pathological states, a considerably large number of brains would be needed for imaging to generate highly detailed maps in 3D. Chemical probes can offer a readily scalable labelling method that is robust, easy to use with the quick operation, and feasible for human tissue where genetic viral and toxin tracers are inappropriate. The drawbacks of immunostaining methods have spurred interest in developing alternative strategies to visualize the optically transparent brain, especially from fixed archived samples or human autopsies that are not optimally fixed. Purpose: We envision AIE-based probes and techniques as robust tools when paired with clearing methods for visualizing the human brain. This thesis aims to develop alternative strategies to tissue labelling using novel AIE-based fluorescent chemical probes and methods that offer easy operation, high brightness, photostability and contrast suitable for 3D visualization of neurons and nerve fibers in mouse brains. Paper I: The novel water-soluble silver-ion sensitive AIE probe TPE-4TA achieved by tetrazole-Ag+ coordination, allowed for the development of a new fluorescent silver (silver-AIE) method to visualize separated proteins following sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Compared with conventional silver nitrate stains, silver-AIE not only offers sensitive fluorogenic detection of proteins, but it is quantifiable, easy to use, has a broad linear dynamic range and a great contrast which rivals the popular commercial stain, SYPRO Ruby. Study II describes how to troubleshoot the fluorescent silver gel stain, alternative steps for rapid staining and techniques to carry out the procedure correctly to avoid suboptimal results. Paper II: We report a novel fluorescent silver stain for fixed mouse brain tissue compatible with multiplexed immunofluorescence imaging in paraffin sections. The Ag+-specific aggregation-induced emission (AIE) strategy outperforms the chromogenic detection employed by many conventional silver staining protocols to visualize neurites and fiber tracts in paraffin sections or passive Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging / Immunostaining / in situ-hybridization-compatible Tissue hYdrogel (CLARITY) -cleared tissue. This enables imaging using standard fluorescent widefield or optical sectioning microscopies. Not only does our method uses less hazardous reagents, but the highly sensitive TPE-4TA also uses silver nitrate concentrations up to two million-fold lower than the standard Yamamoto-Hirano’s modification of the Bielschowsky stain. Paper III: Development of the novel near-infrared AIE fluorescent probe PM-ML with D-π-A (donor-pi-acceptor) structure for the selective staining of myelinated fibers in the teased sciatic nerves, mouse brain cryosections and ClearT-cleared mouse brain tissue for 3D fluorescent imaging. We envision PM-ML as a potential tool for studying demyelination and evaluated its selectivity, photostability and signal-to-background (SBR) ratio which outperformed common commercial fluorescent myelin staining dyes

    Identification and characterisation of lignin degrading bacteria and enzymes fromlarval guts of the African Palm Weevil (Rhynchophorus phoenicis)

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    As developing economies continue to grow, the world’s demand for energy which currently stands at 84MB (million barrels oil) per day is projected to rise to 116 MB per day by the year 2030. The need to meet this continuous rise in demand for energy while lowering the emission of CO2 and other greenhouse gases has necessitated a shift in focus from the exploitation of fossil fuels which are limited, to more renewable and environmentally safe biological resources such as lignocellulosic biomass, the main structural components of plant cell walls. Lignocellulose, however, is resistant to degradation, thus there is a high cost and energy requirement associated with its pretreatment in order to access the lignin bound polysaccharides for subsequent hydrolysis, fermentation and conversion to biofuels and biomaterials.Xylophagous (wood-feeding) insects such as the African palm weevil (Rhynchophorus phoenicis) have developed the ability to effectively utilize lignocellulosic substrates as an energy source due to the synergistic association with their gut microbes. This makesthem viable resources to explore for novel lignocellulose degrading enzymes.Metagenomics allows access to the entire microbiome present in a particular environment and has been adopted in recent studies, rather than culture-based methods, thereby allowing for discovery of novel genes and enzymes from both culturable and non-culturable microbes.In this study, we carried out taxonomic profiling of the bacteria in the gut of the African palm weevil’s larvae using 16S rRNA gene amplicon sequencing with particular interest in identifying lignin degrading bacteria. We also performed functional metagenomicsanalysis from whole metagenome sequencing data derived from whole gut metagenomic DNA of APW larvae to identify genes and by extension, enzymes that can deconstruct lignocellulose and degrade its lignin component. The predominant bacterial genera found across all gut segments were Enterococcus, Lactococcus,Shimwellia, Lelliotia, Klebsiella and Enterobacter, with the foregut having the mostdiverse and abundant lignin degraders mostly from the Proteobacteria phylum. Onethousand, one hundred and forty-one (1,141) annotated genes identified from the R. phoenicis larval gut bacterial metagenome aligned with genes encoding CAZymes and 249 of these belonged to the “Auxiliary Activities” class which harbours the suite of genes implicated to play different roles in lignin deconstruction. Three genes of putative lignin degraders were successfully amplified by PCR, one of the three amplified genes B-38773 (encoding a putative deferrochelatase/ peroxidase of approximately 46kDa insize that has a conserved domain match to the dye decolourising peroxidase superfamily) was produced by heterologous expression and was found to exhibit activity on the peroxidase substrate ABTS, and the anthraquinone dye RB19 but no activity was observed with kraft lignin. Specific activity of 12.9Umg-1 at optimum temperature of 40°C and pH of 4 were recorded when B-38773 enzyme was assayed against ABTS as a substrate. Kinetic parameters: Vmax, Km, Kcat, and catalytic efficiency were determined to be 3.68 μMol/min, 1.089mM, 540.9S-1 and 4.96 X 105 M-1S-1respectively.This study elucidates the lignocellulose degrading potential of the gut community associated with the African palm weevil (APW) by robustly defining the bacterial community structure of the APW gut. Also, massive data from the metagenomic library generated will serve as a storehouse from where genes with various potential functions identified by the inhabitant gut bacteria can be harvested to contribute to areas of biotechnological relevance for industrial applications

    Multicomponent Approaches for the Generation of Structurally Diverse Microporous Coordination Polymers.

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    Microporous coordination polymers (MCPs), materials built from metal clusters bridged with organic linkers, constitute a rapidly growing class of porous solids. The conventional synthetic strategies for production of MCPs involve coordination polymerization of one type linker with a single metal. Despite the success of this method, there exist several challenges with regard to producing highly porous and commercially viable MCPs. These disadvantages include interpenetration of frameworks upon linker extension causing a reduction in porosity and the requirement for synthetically complex linkers to achieve high porosity leading to a drastic increase in cost. This thesis is focused on multicomponent approaches for synthesis of structurally diverse MCPs. In addition, using multiple building units enables suppression of interpenetration, higher predictability over network topology, and an overall reduction in cost. Chapter 2 describes a coordination copolymerization approach of three carboxylate linkers; this strategy was employed to generate a series of isoreticular (having the same network topology) pillared-layer MCPs wherein structures were tuned uniaxially via controllable replacement of pillar linker. Moreover, pillar linker extension occurred in absence of interpenetration resulting in highly porous MCPs. The coordination terpolymerization strategy described in chapter 2 is also exploited to alter the connectivity within the layer arrangement of a pillared-layer MCP in chapter 3. Using a mixture of commercially available linkers of differing lengths enabled formation of a pillared-layer MCP with a non-regular layer structure and no interpenetration. In chapter 4 the power of the multicomponent approach is leveraged through covalent bond formation in tandem with coordination chemistry. A linker was designed which allows either coordination processes or a combination of coordination processes and covalent bond formation to occur in presence of Zn(II). Upon addition of various reaction partners, materials with variable architectures and pore characteristics are obtained. This approach, in addition to being a compelling material discovery method, also offers a fundamental understanding of factors influencing the two distinct modes of assembly.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113422/1/anidutta_1.pd

    Metalloenzymes required for glycan processing and morphological development in Streptomyces lividans

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    Streptomyces are filamentous soil-dwelling bacteria with a complex life cycle. Elucidating the signals that regulate morphological change in microbes is of fundamental importance for biotechnology applications. In the case of Streptomyces a development switch occurs with the concomitant production of secondary metabolites, many of which have pharmaceutical properties. For the industrially used strain S. lividans this switch is dependent on the bioavailability of copper (Cu) in the environment. This thesis has explored the relationship between Cu-chaperones and a haem peroxidase, part of the sco operon, with the maturation of a Cu-containing oxidase, GlxA. In S. lividans the GlxA gene is part of the cslA/glxA operon that contains genes encoding putative enzymes involved in glycan processing. Both these gene clusters are highly conserved in streptomycetes. In Chapter 2 the characterisation of GlxA is reported. It was found to be membrane associated with a mononuclear Cu site and possess a Cys-Tyr redox cofactor capable of housing a protein radical, comparable to the fungal galactose oxidase (Gox). The tertiary structure of GlxA revealed a unique domain arrangement, atypical spectroscopic properties compared to Gox and a lack of enzymatic activity with classical Gox substrates. Generation of the ΔglxA null mutant was found to stall aerial hyphae development on solid media and dramatically change the morphology in liquid cultures. This was ascribed to the absence of the oxidation of a glycan by GlxA produced by CslA (a cellulose-synthase), required for morphogenesis on solid and liquid cultures. The molecular nature of this glycan is unknown. A number of GlxA variants were created in Chapter 3 to elucidate the proteins unique spectroscopic properties. It was found that the second coordination sphere residue, Trp288, plays a major role in tuning the electronic properties of the buried Cu site in GlxA. Its removal abolishes the Cys-Tyr radical and perturbs the spectroscopic properties such that they resemble Gox. Monoclonal antibodies were used to follow the maturation of GlxA through observing mobility differences on denaturing PAGE gels based on the presence or absence of the Cys-Tyr cross-link. X-ray crystallography provided structural insight into the maturation process. A surprising outcome of Chapter 3 was that upon removal of the crosslinking Cys121, a new protein radical is formed as opposed to the expected abolition. Chapter 4 addresses another surprising finding in that a putative haem peroxidase (DtpA), part of the sco operon, plays a role in GlxA maturation and in the Cu-dependent morphological development. DtpA is shown through enzymology and structural analysis to be a member of the dye-decolourising peroxidase (DyP) family. Crucially, it is shown that DtpA functions as a peroxidase in the presence of GlxA using the GlxA substrate, glycolaldehyde. Synthesis and modification of the CslA/GlxA glycan will inevitably require degradation during the life cycle. As part of the cslA-glxA gene cluster are two genes encoding for putative polysaccharide degrading enzymes. One of these is a putative Cu lytic polysaccharide monooxygenase, SliLPMO10E. Chapter 5 structurally characterises SliLPMO10E and also investigates the kinetics of Cu-binding. The latter brings to the attention that LPMOs are able to bind Cu in two forms at a single site before relaxing into a final substrate active form. Importantly, SliLPMO10E is found to be active only with chitin via a C1 sugar ring oxidation mechanism. This hints at the possibility that the glycan produced by CslA and modified by GlxA is chitin-like possessing N-acetyl glucosamine moieties. By combining the in vitro results from this thesis together with the in vivo results obtained through the duration of this work from collaborators at Leiden University an overall model of the Cu-dependent morphogenesis and glycan processing in the hyphal tips of S. lividans is presented. Chapters 2 through to 5 focus on events that occur under Cu limitations, i.e. homeostasis. Chapter 6 extends on previous work that characterised the CsoR regulon. The Cu sensitive operon repressor (CsoR) protein determines the set point of Cu(I) concentration in the cytosol. Under Cu stress, Cu(I) binds to CsoR and de-represses genes under its transcriptional control. Chapter 6 explores the possibility of whether CopZ-like Cuchaperones can traffic Cu(I) to the DNA-bound CsoR, resulting in the up-regulation of control systems to return the cell to homeostasis. Size-exclusion and EMSA studies showed that Cu(I) was transferred from CopZ to CsoR in a unidirectional manner. Re-analysis of previous RNA-seq data using the S. lividans genome as input, enabled for a more complete model for the CsoR regulon in S. lividans to be proposed

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    Department of Biological SciencesA variety of nano-sized materials are developed in the biotechnology fields. Developing nano-sized particles have become a critical issue in biomedical applications because they are closely related to quantum yield, large surface area and EPR effects. Despite these advantages of nanoparticles, it has become indispensable to use more advanced nanoparticles due to their chemical complexity, heterogeneity, difficulty in precisely controlling the size, and toxicity in vivo applications. In this regard, protein-based nanoparticles have biocompatibility, uniform size, shape, composition and stability, and they are quite suitable as multifunctional nanoplatforms. In addition, the structures of protein nanoparticles are based on the atomic resolution crystal structure allowing genetic and chemical modifications at the molecular level. The aim of this thesis was to describe of developing the multi-functional protein nanoparticles using protein cages and monomeric fusion proteins. Thus, Thermotoga maritima encapsulin protein cage whose outer diameter 24 nm was developed as in vitro theranostic nanoplatform. A novel protein cage, encapsulin have not been used for targeted delivery system before, and was prepared as a versatile template for targeted delivery through SP94 peptide insertion which known to bind with hepatocellular carcinoma cells. Functional plasticity and versatility of the engineered encapsulin allow us to apply for specifically detecting and effective treatment of diseases. Relatively small lumazine synthase which isolated from Aquifex aeolicus (AaLS) protein cage nanoparticles with outer diameter of 15.4 nm have been utilized to develop as uniform layer by layer assemblies. High ordered structures of two complementary AaLS protein cages were successfully constructed using simple recognition of histag and Ni-NTA. Furthermore, fluorescent imaging modular toolkits were established using monomeric fusion proteins and ligation proteins by giving cancer cell targeted capability of affibody and visualizing cancer cells of fluorescent proteins. These affibody-fluorescent protein conjugations are post-translationally generate, allowing simple and rapid binding between affibodies and fluorescent proteins. A variety of protein nanoparticles demonstrated that they have potential to be utilized as a multifunctional nanoplatforms in biomedical and biotechnology fields.ope
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