SphereZyme (TM) technology for enhanced enzyme immobilisation application in biosensors
- Authors: Molawa, Letshego Gloria
- Date: 2011
- Subjects: Immobilized enzymes , Hydrolases , Hydrolysis , SphereZyme , Biosensors , Proteolytic enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3989 , http://hdl.handle.net/10962/d1004048 , Immobilized enzymes , Hydrolases , Hydrolysis , SphereZyme , Biosensors , Proteolytic enzymes
- Description: Self-immobilisation enzyme technologies, such as SphereZyme™, suffer from the lack of applicability to hydrolyse large substrates. Solid support immobilisation is usually a method of choice, to produce a stable biocatalyst for large substrates hydrolysis in the industry. In order to investigate this limitation, a commercial protease called Alcalase® was chosen as a model enzyme due to its natural activity (hydrolysis of large substrates-proteins). Prior to immobilising through the SphereZyme™ technology, Alcalase® was partially purified through dialysis followed by CM Sepharose™ FF cation exchanger. Sample contaminants, such as salts and stabilisers can inhibit protein crosslinking by reacting with glutaraldehyde. Alcalase® was successfully separated into 3 proteases with the major peak correlating to a positive control run on native PAGE, indicating that it was likely subtilisin Carlsberg. A 16% alkaline protease activity for azo-casein hydrolysis was retained when 5% v/v PEI: 25% v/v glutaraldehyde solution was used as a crosslinking agent in Alcalase® SphereZyme™ production. An increase in activity was also observed for monomeric substrates (PNPA) where the highest was 55%. The highest % activities maintained when 0.33 M EDA: 25% v/v glutaraldehyde solution was initially used as crosslinking agent were 4.5% and 1.6% for monomeric and polymeric substrates, respectively. PEI is a hydrophilic branched polymer with an abundance of amine groups compared to EDA. A comparison study of immobilisation efficiencies of SphereZyme™, Eupergit® and Dendrispheres was also performed for large substrate biocatalysis. The two latter technologies are solid-support immobilisation methods. Dendrispheres reached its maximum loading capacity in the first 5 minute of the one hour binding time. Twenty minutes was chosen as a maximum binding time since there was constant protein maintained on the solid support and no enzyme loss was observed during the 1 hour binding time. PEI at pH 11.5, its native pH, gave the highest immobilisation yield and specific activity over the PEI pH range of 11.5 to 7. SphereZyme™ had the highest ratio for azocasein hydrolysis followed by Dendrispheres and Eupergit®. The SphereZyme™ was also shown to be applicable to biosensors for phenol detection. Different modifications of glassy carbon electrode (GCE) were evaluated as a benchmark for the fabrication of SphereZyme™ modified phenol biosensor. GCE modified with laccase SphereZyme™ entrapped in cellulose membrane was the best modification due to the broad catechol range (<0.950 mM), high correlation coefficient (R2, 0.995) and relative high sensitivity factor (0.305 μA.mM-1). This type of biosensor was also shown to be electroactive at pH 7.0 for which its control, free laccase, lacked electroactivity. From the catalytic constants calculated, GCE modified with laccase SphereZyme™ entrapped in cellulose membrane also gave the highest effectiveness factor (Imax/Km app) of 1.84 μA.mM-1. The modified GCE with Alcalase® SphereZyme™ was relatively more sensitive than GCE modified with free Alcalase®.
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The role of cellulases and glucohydrolases in the solubilisation of primary sewage sludge
- Authors: Ngesi, Nosisa
- Date: 2002 , 2013-05-09
- Subjects: Sewage sludge , Sewage sludge digestion , Cellulase , Glucosidase inhibitors , Hydrolases , Sulfates
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4080 , http://hdl.handle.net/10962/d1007454 , Sewage sludge , Sewage sludge digestion , Cellulase , Glucosidase inhibitors , Hydrolases , Sulfates
- Description: Biological sulph ate reduction has been identi fied as a potentially valuable process for removing sulphate and heavy metals from indllstrial effluents. The role of sulphate reducing bacteria (SRB) in this process has attracted the attention of biotechnologists and recently of enzymologists due to its fundamental properties and possible role in AMD bioremediation. These obligatory anaerobic sulphate-reducing bacteria are commonly known to dissimilate sulphate for energy. Under anaerobic conditions SRB oxidize simple organic compounds such as lactic acid with the sulphate and thereby generate hydrogen sulphide (a stTong reducing agent) and bicarbonate ions. The hydrogen sulphide in turn reacts with contaminant metals contained in AMD and precipitates them out of solution as metal sulphides. Bicarbonate ions neutralize AMD by reaction with protons to form carbon dioxide and water. Organic matter in the municipal sewage sludge has been identified as a potential source of electron donors for su lphate reduction. However, this organic matter is in the polymeric form that cannot be util ised by SRB. The latter depend on the activities of other hydrolytic bacteria for the degradation of complex polymers. Hence the availability of these monomeric substrates is a major factor, which may constrain further process development and is considered a rate-limiting step. Thi s study is therefore undertaken to investigate the bacterial glucohydrolase enzymes involved in the digestion of the polysaccharides present in the sewage sludge with specific interest in cellulases and/or p-glucosidase enzymes. The goals of the research are to: isolate, identify, purify and quantify these enzymes; study their distribution with respect to time, pH, and temperature; maximize and quantify the hydrol ys is products; study whether sulphide and sulphate have an enhancing or an inhibitory effect on the activity of enzymes; optimize the enzyme activity against substrate and/or product inhibition and soluble heavy metal salts. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
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