Production, purification and characterization of a multifunctional, thermostable and acido/alkaline stable putative xylanase from the psychrotrophic bacterium, Sphingomonas aerolata
- Authors: Mathibe, Brian Nkanyiso
- Date: 2020
- Subjects: Sphingomonas , Sphingomonas aerolata , Psychrotrophic bacteria , Xylanases Purification , Thermodynamics , Characterization
- Language: English
- Type: thesis , text , Masters , MSc
- Identifier: http://hdl.handle.net/10962/164478 , vital:41122
- Description: Enzymes are biological catalysts produced by living organisms in order to increase the rate of chemical reactions that are essential for life. Enzymes are now applied as catalysts in a variety of fields in response to a worldwide shift towards sustainable industrial processes, with enzymatic processes having been found to be specific, fast and to result in savings in energy and raw materials, all while generating less hazardous wastes when compared to conventional chemical processes. Microorganisms, specifically fungi and bacteria, are the most common sources for the production of enzymes because they grow rapidly meaning the production of enzymes is also rapid and can be easily manipulated using genetic engineering to obtain enzymes with desired characteristics. Due to the harsh conditions required in most industrial processes, there have been growing interests in enzymes that can withstand such harsh conditions. It has been found that extremophiles, microorganisms that can survive and grow in extreme conditions that would otherwise be detrimental to other life forms, are a source of extremozymes, enzymes that can withstand harsh conditions and therefore be utilised industrially where production processes are often harsh. Psychrophiles are examples of extremophiles, having the ability to be prolific in extremely low-temperature environments, and have been found to produce cold-active enzymes that have high specific activity at low temperatures. Cold-active enzymes from psychrophiles are of industrial and biotechnological significance as they could potentially result in energy savings when applied in industries that do not require high temperatures such as the transformation of heat-sensitive substrates at low temperatures. In this study, a psychrophilic bacterium, Sphingomonas aerolata, was successfully sub-cultivated in both solid and liquid media and found to grow optimally at 28°C on media composed of (w/v): peptone, 1%; NaCl, 0.5%; yeast extract, 1%, and hydrolysed potato starch, 1%; with 1.5% agar incorporated to the solid medium formulation. This bacterium was found to produce an extracellular xylan degrading enzyme, which was purified to homogeneity using ultrafiltration with Amicon molecular weight cut-off filtration units. This enzyme was purified to a fold purification of 4.2 with percentage yield of 14.29%. A single band corresponding to approximately 36 kDa was observed using SDS-PAGE analysis of the purified putative xylanase sample. The xylanolytic activity of the purified sample was confirmed by a band of xylan hydrolysis using non-denaturing native activity-PAGE analysis, with 0.1% (w/v) beechwood xylan incorporated into the resolving gel. However, biochemical characterization of the enzyme revealed it to be also active on Avicel cellulose (22.64 U/mg), hydrolysed potato starch (19.11 U/mg), arabinogalactan (16.82 U/mg) as well as xylan substrates, beechwood xylan (23.69 U/mg) and wheat arabinoxylan (20.15 U/mg), making it a multifunctional glycoside hydrolase enzyme. Substrate competition assays between beechwood xylan and Avicel, and between beechwood xylan and hydrolysed potato starch, revealed that the enzyme uses the same active site to hydrolyse all three substrates, confirming the cross-specificity, or substrate promiscuity, of the enzyme. The enzyme, referred to as the putative xylanase in this study, was found to be cold-active, maintaining over 25% relative activity on beechwood xylan at 4˚C, consistent with other cold-active xylanases that have been reported in literature. The putative xylanase showed optimal xylanase activity at 40˚C and pH 6.0, and was found to be relatively thermostable, maintaining at least 78% relative activity on beechwood xylan after incubation for 24 hours at 50˚C. The enzyme was also found to be acido/alkaline stable, maintaining over 60% relative activity between pH 2.0 and 11.0, and also stable in the presence of various divalent cations, ethylenediaminetetraacetic (EDTA) and β-mercaptoethanol. The kinetic parameters of the putative xylanase on different substrates were determined using Hanes-Woolf plots. The Vmax values of the putative xylanase on beechwood xylan, wheat arabinoxylan, Avicel and hydrolysed potato starch were determined to be 24.63, 27.40, 12.20, and 19.34 U/mg, respectively. The KM values of the putative xylanase on beechwood xylan, wheat arabinoxylan, Avicel and hydrolysed potato starch were determined to be 3.03, 4.09, 4.49 and 2.21 mg/ml, respectively. In conclusion, the psychrophilic bacterium, Sphingomonas aerolata, was successfully sub-cultivated under laboratory conditions and found to produce an extracellular putative xylanase that was successfully purified and characterized, and determined to possess a number of novel features such as the ability to hydrolyse different substrates using one active site, activity at low temperatures coupled with high thermostability, and stability at acidic and alkaline pH conditions. This novel enzyme could be very useful as a biocatalyst in different fields due to its novel features, and further study into its structure especially around the active site that has been shown to be possibly fluid enough to maintain reaction rates at low temperatures while also stable enough to maintain high reaction rates after long incubation periods at high temperatures, can bind and hydrolyse different substrates and can maintain high reaction rates across acidic, moderate and alkaline conditions, could further expand on the existing knowledge on biocatalysts and their properties. , Thesis (MSc)--Rhodes University, Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text:
- Date Issued: 2020
- Authors: Mathibe, Brian Nkanyiso
- Date: 2020
- Subjects: Sphingomonas , Sphingomonas aerolata , Psychrotrophic bacteria , Xylanases Purification , Thermodynamics , Characterization
- Language: English
- Type: thesis , text , Masters , MSc
- Identifier: http://hdl.handle.net/10962/164478 , vital:41122
- Description: Enzymes are biological catalysts produced by living organisms in order to increase the rate of chemical reactions that are essential for life. Enzymes are now applied as catalysts in a variety of fields in response to a worldwide shift towards sustainable industrial processes, with enzymatic processes having been found to be specific, fast and to result in savings in energy and raw materials, all while generating less hazardous wastes when compared to conventional chemical processes. Microorganisms, specifically fungi and bacteria, are the most common sources for the production of enzymes because they grow rapidly meaning the production of enzymes is also rapid and can be easily manipulated using genetic engineering to obtain enzymes with desired characteristics. Due to the harsh conditions required in most industrial processes, there have been growing interests in enzymes that can withstand such harsh conditions. It has been found that extremophiles, microorganisms that can survive and grow in extreme conditions that would otherwise be detrimental to other life forms, are a source of extremozymes, enzymes that can withstand harsh conditions and therefore be utilised industrially where production processes are often harsh. Psychrophiles are examples of extremophiles, having the ability to be prolific in extremely low-temperature environments, and have been found to produce cold-active enzymes that have high specific activity at low temperatures. Cold-active enzymes from psychrophiles are of industrial and biotechnological significance as they could potentially result in energy savings when applied in industries that do not require high temperatures such as the transformation of heat-sensitive substrates at low temperatures. In this study, a psychrophilic bacterium, Sphingomonas aerolata, was successfully sub-cultivated in both solid and liquid media and found to grow optimally at 28°C on media composed of (w/v): peptone, 1%; NaCl, 0.5%; yeast extract, 1%, and hydrolysed potato starch, 1%; with 1.5% agar incorporated to the solid medium formulation. This bacterium was found to produce an extracellular xylan degrading enzyme, which was purified to homogeneity using ultrafiltration with Amicon molecular weight cut-off filtration units. This enzyme was purified to a fold purification of 4.2 with percentage yield of 14.29%. A single band corresponding to approximately 36 kDa was observed using SDS-PAGE analysis of the purified putative xylanase sample. The xylanolytic activity of the purified sample was confirmed by a band of xylan hydrolysis using non-denaturing native activity-PAGE analysis, with 0.1% (w/v) beechwood xylan incorporated into the resolving gel. However, biochemical characterization of the enzyme revealed it to be also active on Avicel cellulose (22.64 U/mg), hydrolysed potato starch (19.11 U/mg), arabinogalactan (16.82 U/mg) as well as xylan substrates, beechwood xylan (23.69 U/mg) and wheat arabinoxylan (20.15 U/mg), making it a multifunctional glycoside hydrolase enzyme. Substrate competition assays between beechwood xylan and Avicel, and between beechwood xylan and hydrolysed potato starch, revealed that the enzyme uses the same active site to hydrolyse all three substrates, confirming the cross-specificity, or substrate promiscuity, of the enzyme. The enzyme, referred to as the putative xylanase in this study, was found to be cold-active, maintaining over 25% relative activity on beechwood xylan at 4˚C, consistent with other cold-active xylanases that have been reported in literature. The putative xylanase showed optimal xylanase activity at 40˚C and pH 6.0, and was found to be relatively thermostable, maintaining at least 78% relative activity on beechwood xylan after incubation for 24 hours at 50˚C. The enzyme was also found to be acido/alkaline stable, maintaining over 60% relative activity between pH 2.0 and 11.0, and also stable in the presence of various divalent cations, ethylenediaminetetraacetic (EDTA) and β-mercaptoethanol. The kinetic parameters of the putative xylanase on different substrates were determined using Hanes-Woolf plots. The Vmax values of the putative xylanase on beechwood xylan, wheat arabinoxylan, Avicel and hydrolysed potato starch were determined to be 24.63, 27.40, 12.20, and 19.34 U/mg, respectively. The KM values of the putative xylanase on beechwood xylan, wheat arabinoxylan, Avicel and hydrolysed potato starch were determined to be 3.03, 4.09, 4.49 and 2.21 mg/ml, respectively. In conclusion, the psychrophilic bacterium, Sphingomonas aerolata, was successfully sub-cultivated under laboratory conditions and found to produce an extracellular putative xylanase that was successfully purified and characterized, and determined to possess a number of novel features such as the ability to hydrolyse different substrates using one active site, activity at low temperatures coupled with high thermostability, and stability at acidic and alkaline pH conditions. This novel enzyme could be very useful as a biocatalyst in different fields due to its novel features, and further study into its structure especially around the active site that has been shown to be possibly fluid enough to maintain reaction rates at low temperatures while also stable enough to maintain high reaction rates after long incubation periods at high temperatures, can bind and hydrolyse different substrates and can maintain high reaction rates across acidic, moderate and alkaline conditions, could further expand on the existing knowledge on biocatalysts and their properties. , Thesis (MSc)--Rhodes University, Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text:
- Date Issued: 2020
A study of petrol and diesel fuel blends with special reference to their thermodynamic propeties and phase equilibria
- Authors: Hayward, Caroline
- Date: 1986
- Subjects: Gasoline , Diesel fuels , Thermodynamics , Liquid-liquid equilibrium , Alcohol as fuel
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4065 , http://hdl.handle.net/10962/d1004902 , Gasoline , Diesel fuels , Thermodynamics , Liquid-liquid equilibrium , Alcohol as fuel
- Description: The ternary phase behaviour of the n-heptane-l-propanol-water system was studied and compared with the theoretical prediction based on the UNIQUAC model for non-electrolyte solutions. The results showed that this model adequately approximated experimental studies. The excess enthalpies and excess volumes for several binary mixtures were determined. The excess enthalpies were measured using a LKB flow microcalorimeter and the excess -volumes determined using a PAAR densitometer. The study showed that no significant enthalpy or volume changes occurred when petrol/n-heptane were mixed with alcohols . Ternary phase diagrams, including tie lines have been determined for a number of petrol-alcohol-water systems (including the Sasol blend of alcohols). The tie line results show that the concentration of water in the water-rich layer is strongly dependent on the type of alcohol used. The Sasol alcohol blended with petrol resulted in a high water concentration in the water-rich layer which forms on phase separation. This is believed to contribute significantly to the corrosion problems experienced by motorists using the Sasol blended fuel on the Witwatersrand. The effect of temperature on several of these blends was included in the study. Diesel-alcohol blends and the co-solvent properties of ethyl acetate investigated. Ethyl acetate ensures miscibility at low concentrations for diesel-ethanol blends. Octyl nitrate and two cetane improvers from AECI were assessed in terms of their ability to restore cetane rating of blended diesel fuel to that of pure diesel fuel. The results indicated that all three samples were successful in this application. , KMBT_363
- Full Text:
- Date Issued: 1986
- Authors: Hayward, Caroline
- Date: 1986
- Subjects: Gasoline , Diesel fuels , Thermodynamics , Liquid-liquid equilibrium , Alcohol as fuel
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4065 , http://hdl.handle.net/10962/d1004902 , Gasoline , Diesel fuels , Thermodynamics , Liquid-liquid equilibrium , Alcohol as fuel
- Description: The ternary phase behaviour of the n-heptane-l-propanol-water system was studied and compared with the theoretical prediction based on the UNIQUAC model for non-electrolyte solutions. The results showed that this model adequately approximated experimental studies. The excess enthalpies and excess volumes for several binary mixtures were determined. The excess enthalpies were measured using a LKB flow microcalorimeter and the excess -volumes determined using a PAAR densitometer. The study showed that no significant enthalpy or volume changes occurred when petrol/n-heptane were mixed with alcohols . Ternary phase diagrams, including tie lines have been determined for a number of petrol-alcohol-water systems (including the Sasol blend of alcohols). The tie line results show that the concentration of water in the water-rich layer is strongly dependent on the type of alcohol used. The Sasol alcohol blended with petrol resulted in a high water concentration in the water-rich layer which forms on phase separation. This is believed to contribute significantly to the corrosion problems experienced by motorists using the Sasol blended fuel on the Witwatersrand. The effect of temperature on several of these blends was included in the study. Diesel-alcohol blends and the co-solvent properties of ethyl acetate investigated. Ethyl acetate ensures miscibility at low concentrations for diesel-ethanol blends. Octyl nitrate and two cetane improvers from AECI were assessed in terms of their ability to restore cetane rating of blended diesel fuel to that of pure diesel fuel. The results indicated that all three samples were successful in this application. , KMBT_363
- Full Text:
- Date Issued: 1986
The thermodynamics of binary liquid mixtures
- Scoones, Brett Warren Hamilton
- Authors: Scoones, Brett Warren Hamilton
- Date: 1983 , 2013-03-15
- Subjects: Thermodynamics , Thermodynamics -- Tables , Solution (Chemistry) , Liquids
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4392 , http://hdl.handle.net/10962/d1005893 , Thermodynamics , Thermodynamics -- Tables , Solution (Chemistry) , Liquids
- Description: A systematic study of the excess thermodynamic properties of two-ring compounds and n-alkanes was conducted. The excess enthalpies were determined at 288,15 K and 298,15 K by using a flow microcalorimetric technique. The excess volumes of tetralin and cycloalkanes, and tetralin and --alkans at 288,15 K and 298,15 K were determined by using a vibrating tube densitometer. The results showed trends relating to the size and shape of the n-alkane and cycloalkan molecules. The theory developed by Flory was applied to the decalin + cycloalkane and + n-alkane systems and this gave qualitative predictions of the excess enthalpies from the excess volumes and vice versa. , KMBT_363 , Adobe Acrobat 9.53 Paper Capture Plug-in
- Full Text:
- Date Issued: 1983
- Authors: Scoones, Brett Warren Hamilton
- Date: 1983 , 2013-03-15
- Subjects: Thermodynamics , Thermodynamics -- Tables , Solution (Chemistry) , Liquids
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4392 , http://hdl.handle.net/10962/d1005893 , Thermodynamics , Thermodynamics -- Tables , Solution (Chemistry) , Liquids
- Description: A systematic study of the excess thermodynamic properties of two-ring compounds and n-alkanes was conducted. The excess enthalpies were determined at 288,15 K and 298,15 K by using a flow microcalorimetric technique. The excess volumes of tetralin and cycloalkanes, and tetralin and --alkans at 288,15 K and 298,15 K were determined by using a vibrating tube densitometer. The results showed trends relating to the size and shape of the n-alkane and cycloalkan molecules. The theory developed by Flory was applied to the decalin + cycloalkane and + n-alkane systems and this gave qualitative predictions of the excess enthalpies from the excess volumes and vice versa. , KMBT_363 , Adobe Acrobat 9.53 Paper Capture Plug-in
- Full Text:
- Date Issued: 1983
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