In this study, solid fermentation of Aspergillus niger with paracetamol in test culture media and no Paracetamol in the control media were carried out to generate Peroxidase. Effects of pH and temperature on peroxidase activity were determined by subjecting the enzyme to various pH and temperatures.
Paracetamol, usually called acetaminophen, is a drug used to relieve moderate pain such as headache, migraine, nerve pain, toothache, period pain, and muscular pain. Paracetamol can relieve pain by lowering the production of prostaglandins in the brain and in the spinal cord. Overdose of paracetamol leads to loss of appetite, nausea, vomiting, sweating, and whitening of the eye. Peroxidase forms a network in the body that has both beneficial and adverse effects.
Peroxidase has physiological functions that substantially contribute to immunity and several metabolic and signaling processes. However, under certain circumstances, they exert deleterious effects such as damage to macromolecules in the plasma and activate cell death.
TABLE OF CONTENT
LIST OF FIGURES
ABSTRACT
ACKNOWLEDGEMENT
DEDICATION
CHAPTER ONE: INTRODUCTION
CHAPTER TW0: LITERATURE REVIEW
CHAPTER THREE: MATERIALS AND METHOD
CHAPTER FOUR: RESULTS
CHAPTER FIVE: DISCUSSION
CHAPTER SIX: CONCLUSIONS AND RECOMMENDATIONS
CHAPTER SEVEN: REFERENCE
APPENDIX
LIST OF FIGURES
FIGURE 4.1. Peroxidase activity at different pH regions for both culture media
Figure 4.2 : Peroxidase activity for different temperature range for test media (media containing paracetamol) and the control media (media without paracetamol)
Figure 4.3 : Protein estimation for culture media with paracetamol and culture media without paracetamol
Fig 4.4.1 Effect of Peroxidase activity at different substrate concentrations in both culture media
Fig 4.4.2 Lineweaver-Burke plot for the effect of substrate concentration on enzyme preincubated with paracetamol and without paracetamol
ABSTRACT
Paracetamol, usually called acetaminophen, is a drug used to relieve moderate pain such as headache, migraine, nerve pain, toothache, period pain, and muscular pain. Paracetamol can relieve pain by lowering the production of prostaglandins in the brain and in the spinal cord. Overdose of paracetamol leads to loss of appetite, nausea, vomiting, sweating, and whitening of the eye. Peroxidase forms a network in the body that has both beneficial and adverse effects. Peroxidase has physiological functions that substantially contribute to immunity and several metabolic and signaling processes. However, under certain circumstances, they exert deleterious effects such as damage to macromolecules in the plasma and activate cell death. In this study, solid fermentation of Aspergillus niger with paracetamol in test culture media and no Paracetamol in the control media were carried out to generate Peroxidase. Effects of pH and temperature on peroxidase activity were determined by subjecting the enzyme to various pH and temperatures. The effect of substrate concentration on peroxidase activity was determined by subjecting the enzyme to 30% hydrogen peroxide, and o-dianisidine was used as the chromogen. The optimum pH and temperature for the enzyme peroxidase were 6.0 and 50℃, respectively, in the culture media containing paracetamol. It was also determined that the activity of Peroxidase was temperature-dependent as it increases with increasing temperature. Based on the findings, it can be concluded that paracetamol increases peroxidase activity.
ACKNOWLEDGEMENT
I am grateful to Rev. Bernard Gaglo for his support towards my stay at the university and Mrs. Monica Arthur. I really appreciate your love and kindness to me.
DEDICATION
To Maame Konadu
1.1 Background
CHAPTER ONE: INTRODUCTION
Paracetamol, usually called acetaminophen, is a drug used to relieve moderate pain such as headache, migraine, nerve pain, toothache, period pain, and muscular pain. Paracetamol relieves pain by lowering the production of prostaglandins in the brain and in the spinal cord (Davis & Lotter, 2009). Paracetamol also reduces fever by affecting the brain area that regulates our body temperature (the hypothalamic heat-regulating center). Since paracetamol is usually taken without a prescription, it is one of the most abused drugs. Overdose of paracetamol leads to loss of appetite, nausea, vomiting, sweating, and whitening of the eye (Narley, 1996). In research by Wolfe and Singh in 1999 to investigate the actual inhibitory activity of NSAIDS on germination, fungal growth, and production, most of the non-steroidal drugs were found to inhibit fungal growth; germination, and production with the most effective drugs were found to be ibuprofen and diclofenac. Similar results were obtained on Aspergillus niger and FusariumFusarium on different growth media; however, Aspergillus niger and FusariumFusarium exhibited a high level of resistance to Aspirin (Davis and
Lotter, 2009). Though paracetamol also possesses anti-inflammatory properties like NSAIDS, Wolfe and Singh did not determine its inhibitory effect. Aspergillus niger species are saprophytic, haploid filamentous fungi, which is an indispensable microorganism in microbiology. It is not only a xerophilic fungus but also temperature tolerant fungi, which grows in a hemophilic environment such as decaying vegetation, soil, organic debris, and an enclosed air environment (Bowen, 2007) . Aspergillus Niger usually yields masses of colonies made up of white felt wrapped with a dark asexual produces fungi spore(Bowen,2007). The major metabolic pathways in Aspergillus niger are carbohydrate and amino acids metabolism. In amino acid metabolism, Proline, alanine, and glutamine are mostly prospered in the environment they live (Jebbor, 2017). Aspergillus niger can break down copper, tin, aluminum, and nickel and leads to released energy. This energy obtained from the breakdown of the above compounds is used to produce oxalic acid, glucuronic acid, and citric acid (Jebbor, 2017). Production of glucuronic acid and citric acid makes their medium acidic and gives them a competitive advantage over most microbes living with them in the same habitat (Jebbor, 2017).
Many of the enzymes produced by Aspergillus Niger include citric acid enzymes , Amylases, Lipases, Cellulases, Xylanases, Proteases, and Peroxidases. Aspergillus niger has been used for waste treatment as well as treatment of chemicals; because of the numerous enzymes they can produce (Debets, 1990).
Among all the functions of protein (enzymes), the most critical significance is its ability to speed up a biochemical reaction. In the absence of this protein, most biological systems will defiantly undergo a prolonged reaction. Peroxidase has the notation (EC
1.11.1.1.7) this implies that the enzyme is an oxidoreductase. Peroxidase belongs to a family primarily found in fungi, plants, and vertebrates. Organisms that produce Peroxidase include Aspergillus awamori, Aspergillus niger, Hebeloma cylindrosporium, Phenerocheate Chrysosporium, Fusarium solani. (Debets,1990).
Since Peroxidase is biodegradable, it is safe for the ecosystem. Hence, it is a suitable replacement for harsh chemicals and eliminates harsh reaction conditions. Peroxidase can also replace heavy salt additions for bakery products bleaching process and act as an anti-microbial enzyme that can be used against the harsh side effects of drugs such as flucloxacillin, tetracycline and etc. (Bhati et al 2012).
The maximum generation of Peroxidase from Aspergillus niger and its peroxidase activity quantification mainly depends on the following factors: culture media, incubation period for peroxidase production, optimum pH for peroxidase production, optimum nitrogen source for peroxidase production, optimum carbon source to support the growth of the microbe (Stajic, 1996).
1.2 The problem of Statement and Justification
Due to the numerous functions peroxidase plays, it has been in an increasing demand by most industries. Most owners of the various pharmaceutical, oil, and textile industries spend much money in generating Peroxidase; this is because peroxidases are produced by few species of organisms and hence have limited sources. Because of that, this present study aims at analyzing the various interactions that occur when paracetamol is present in the culture media of Aspergillus niger, as well as its effects on
Peroxidase activity in other to determine the stability of fungal Peroxidase. This will add more to the existing literature on the toxicity and pharmacodynamics of paracetamol. it will also help industries that utilize Peroxidase to know how to regulate parameters to obtain high yields of peroxidase production through solid-state fermentation of fungus in other to cut down costs.
1.4 HYPOTHESIS
The presence of paracetamol in the culture media of Aspergillus niger decreases the activity of Peroxidase.
1.5 Aim
To investigate the effect of the presence of paracetamol in the cultural media of
Aspergillus niger on peroxidase activity.
1.6 Specific Objectives:
a. To stimulate the production of Peroxidase from Aspergillus niger.
b. To determine the effects of various concentrations of paracetamol on peroxidase activity obtained from Aspergillus niger.
c. To determine the effect of pH on peroxidase activity.
d. To determine the effect of temperature on peroxidase activity.
2.1 Paracetamol
CHAPTER TW0: LITERATURE REVIEW
Paracetamol is an international name in Europe, whereas acetaminophen is an international name in the USA. These are the official names derived from the chemical name N- acetyl- para aminophenol. The history of this drug dates back to the 19th century with a significant discovery by two doctors, namely: Arnold Chan and Paul Heppa, at the University of Strasburg. Intending to eradicate worms, by mistake, they dispensed acetanilide to a patient instead of naphthalene (www.chi.ic.uk/bristol.paracetamol/text.htm). They noticed that the drug had a negligible impact on intestinal parasites; however, it significantly decreased high temperatures. Due to the carcinogenic actions of damaging most patients' kidneys and liver, the drug was withdrawn from the American market in 1893, and Phenacetin in Poland also replaced it. The drug was accepted back into the market in 1948, where further studies by Bernard Brodie and Julius Axelrod finally demonstrated that; Paracetamol was the active metabolite of acetanilide, whereas Phenacetin was responsible for the analgesic and antipyretic actions of acetanilide.
2.11 Mode Of action of paracetamol
Paracetamol was formally grouped under non-steroidal anti-inflammatory drugs (NSAID). Most literature compared its mode of action to that of classical NSAIDs like Aspirin, which inhibits the cyclooxygenase pathway (Bertolini et al, 2008). However, paracetamol has been observed to have weak anti-inflammatory properties, and due to this, its site of action is found within the brain and the spinal cord. Paracetamol has also been reported to selectively inhibit the brain's COX-dependent pathway and not in any other peripheral tissue (Flower and vane 1972). This tissue selectivity of paracetamol upon inhibition of COX explains the contrasting adverse effects associated with NSAIDs other than Paracetamol. Hence paracetamol can be administered to pregnant and lactating women
The mechanism of action of COX inhibition is not directly binding to the enzyme's active site. However, paracetamol gains inhibition by decreasing the active form of the COX enzyme (Bertoloni, 2008).
2.2 Aspergillus niger
Aspergillus was named by Pier Antonio Michele, an Italian priest, and biologist, in 1729 when cataloging molds. The molds resembled an Aspergillum, a holy water sprinkler (from Latin Spargere-to sprinkle). In 1863 the species fumigatus was first described by physician George W. Fresenius. He observed that the spores had a green pigmentation
And had no septated fertile hyphae or conidiophores. Fumigatus is derived from Latin "fumigate," which means smoky, referring to the smoky blue-gray mycelium (Wikipedia.com / history of mould.uk.htm).
The discovery and the industrial use of Aspergillus niger became indispensable in biotechnology after an outstanding discovery by a food chemist by the name James Curric in 1917(Schuster et al., 2002). Curric was able to produce citric acid from citrus fruits. He also identified a technique to make it through the fermentation of sugar. Due to the number of biological compounds and enzymes Aspergillus niger can produce, it is considered to be one of the most versatile microorganisms used in the field of biotechnology. It also produces low titer of gluconic and Fumaric acid (Schuster et al., 2002). Most industries employ fermentation of Aspergillus niger to produce vital enzymes such as Glucoamylase, which is used in the production of high fructose syrup. Pectinase, also produced by A. niger is used in cider and wine production. In addition, glucose oxidase is used in the design of glucose biosensors. This shows that Aspergillus niger is indeed an indispensable microorganism in Biotechnology.
2.2.1 ECOLOGY.
Aspergillus niger is not only a species of plant-pathogen but also a group of Aspergillus that is made up of fifteen different varieties, all with black conidia. Group of fruits that harbor Aspergillus niger includes tomatoes, peanuts, grapes, onions, and mango. The fungus is mainly found in the soil, living as saprophytic in the decaying vegetation,
leaves, compost piles, and stored grains. It is also found in the indoor environment and in contaminated foods. Since the fungus is opportunistic, it can also be found in humans, birds, animals, etc., (jebbor, 2017). Aspergillus niger is a temperature tolerant fungus that grows in a hemophilic environment (Bowen, 2007). Filamentous fungus can grow under high freezing temperatures (Jebbor, 2017). Aspergillus niger usually yields masses or colonies made up of white felt wrapped with dark asexual produced fungi spores. It also contains mycelia, which are thread-like in nature and hyphae, divided by a very clear septum (Debets, 1990). Conidiophores which are asexual producing fungi spores of Aspergillus niger range from 900 to 1600 micrometers in length and contain globular vesicles ranging from 40 to 60 micrometers in diameter, and each vesicle is covered with phialides which is a projection from conidiophore (Debets, 1990).
Their metabolic system comprises the cytoplasm, mitochondrion, and peroxisomes in terms of metabolism. The central metabolism in Aspergillus niger is the carbohydrate and amino acids metabolism which takes place in both catabolic and anabolic reactions (Shuster, 2002). In amino acid metabolism, proline, alanine, and glutamine are mostly prospered in the environment they live. Aspergillus niger usually gains its energy through bioleaching. Bioleaching is the process of extracting metals from ores using bacteria (Schuster, 2002). A. niger usually breakdown various minerals into its fundamental element, and these breakdowns of these minerals into their respective essential elements lead to the release of energy to the
Aspergillus niger. The energy obtained by the Aspergillus niger is then used in the production of citric acid and glucuronic acid, as reported by Debits in 1990. Production of the citric acid and glucuronic acid by the Aspergillus niger makes their media acidic, giving them a competitive advantage over various organisms in the same media. Enzymes produced by Aspergillus niger include Glucoamylase, xylanases , Peroxidase, pe ptidase, etc.
2.2.2 Industrial use of Aspergillus niger
Aspergillus niger is very important in various industries due to the number of enzymes it can produce. Among the molds that play an essential role in the human economy and the natural ecosystem. Aspergillus niger serves as a cell factory for producing food ingredients in food preparation. Also, in pharmaceutical industries, it is used to produce anti-biotics. Aspergillus niger is used in wine and beer production to decompose plastic. The most crucial impact of Aspergillus niger is in the production of citric acid, which had led to the creation of employment, aiding the socio-economic development of most nations. From the environmental point of view, it is used as bioadsorpbent to detoxify and decolorize water waste samples (Shuster, 2002). Aspergillus niger leads to the production of Glucoside hydrolase, which is used in the conversion of biomass to biofuel. The enzyme functions by breaking down cellulose and hemicellulose from plant cell walls into a substance later used in the generation of ethanol. Glucose oxidase
Produced by Aspergillus niger is an extremely FAD-dependent enzyme that has found a wide range of applications in the electrochemical sensing of glucose in the blood (Shuster, 2002).
2.3.3 TOXICITY
Pathogenic Aspergillus spp. also produces mycotoxins. These toxins are known for their solid and varied biological activities. However, it also carries acute toxicity to various human cells such as hepatocytes, and renal cells. Therefore, a production under favorable conditions and new and unknown strains should be examined for ochratoxin before they are employed as production microorganisms. Nevertheless, they concluded that A. niger is generally a safe microorganism that can be used to produce lung epithelioid cells, etc., and various immunosuppressive activities (Bowen, 2007).
2.4 PEROXIDASE
Peroxidases are enzymes whose primary function is to oxidize hydrogen donors at the expense of peroxides. They are particular for hydrogen peroxide, but they accept a variety of hydrogen donors, including polyphenols (Al Ghouleh, 2011).
Peroxidases contain heme in their active center, which reacts with hydrogen peroxides to form a highly reactive intermediate known as peroxidase substrate (Reeder, 2017). The human peroxidase is grouped into two: the True Peroxidase and the Pseudo peroxidases (sies,2015). Heme peroxidases are enzymes with a heme prosthetic group in the catalytic site. The iron is bounded by four coordination to a protoporphyrin derivative. The rate constants of the interaction of heme in the active center of peroxidases with hydrogen peroxides are composed of several orders of magnitudes higher than that of the Fenton reactions. In the presence of hydrogen peroxides, heme peroxidase is activated to highly reactive intermediates. Peroxidases play a vital role in innate immunity and many physiologically important processes like apoptosis and cell signaling. On the other hand, unfavorable excessive peroxidase activity is implicated in oxidative damage of cells and tissues hence initiating a variety of diseases in humans (Reeder, 2017) .since Peroxidase differs in structure, properties, and location, mechanisms of controlling activity and the biological effects of peroxidase products are specific for each hemoprotein.
2.4 Diversity of Hemo proteins and peroxidase activity
A wide variety of Peroxidases exists, but most are plant, fungi, or bacteria peroxidases. Horseradish peroxidases, ascorbate peroxidases, yeast cytochrome c peroxidase, and lignin peroxidases. In the human body, fewer proteins display significant
Peroxidase activity. These enzymes are divided into true and pseudo Peroxidase (Du, 20015).
2.5 True human peroxidase
True human peroxidases are enzymes whose primary function is to generate oxidants and ROS. Myeloperoxidase, eosinophil peroxidase, lactoperoxidase, and thyroid Peroxidase are all members of the actual human peroxidases. Except for Thyroid peroxidases, all true human peroxidases are reduced or made catalytically inactive when halide ions are provided for them as substrate. The capacity of true human peroxidase to generate cytotoxic hypohalous and thiocynous acids accounts for their anti-microbial activity. The activity of human Peroxidase towards guaiacol, a hardly oxidizable compound can be used as a substrate for testing peroxidase compounds(Lazer,2009). True human peroxidase also uses phenolic compounds, aromatic amino acids, urate, ascorbate, and serotonin and oxidized them in the peroxide cycle into their respective radicals (Valsova,2012). The primary function of this enzyme is in the oxidation of specific molecules such as COX and plays an essential role in controlling the biosynthesis of various tyrosyl radicals formed in the peroxide cycle of Cox (Valsova, 2012).
Taking into consideration humans and animals, glutathione peroxidase (GPx) is the significant peroxidases involved in the regulation of cellular free radical levels. Due to the role GPx plays in curbing the deleterious effects of free radicals, it has been implicated in controlling and prevent diseases caused by these harmful reactive species, including diabetes, heart attack and arthritis (Lubos, Loscalzo & Handy, 2011). Because of the central role played by Peroxidase both in vivo and in vitro, studies that clarify the effect of paracetamol on the enzyme’s activity will greatly complement existing literature on the pharmacodynamics of these drugs. Studies like this one can also be exploited by industries that rely on Peroxidase to modify their production processes
2.6 Pseudo peroxidases
Pseudo peroxidases are hemoproteins that perform different important functions in the body and initially are not meant to interact with peroxide. However, due to changes in external conditions, the features of their active center can change, and they display Peroxidase-like activity (Matelitsa,2007). Pseudo peroxidases active sites are not specifically designed to catalyze the peroxide reduction to water. The rate of reaction of heme with peroxide is rather low; hence the first step of the pseudo peroxidases reaction is a rate-limiting step. By that, hydroxyl radicals can be formed in the active center of hemolytic splitting of peroxide (Cadenas, 2007).Pseudo Peroxidase are different in terms of their heme and activity features, but all of them have one common features that is the exposure to peroxide causes immediate oxidation of protein whose amino acids are very close to the active site (Du, 2015)
2.7 Industrial use of Peroxidase
Peroxidase is also significant in the textile and fabric industries in the sense that it is used in the removal of peroxides after bleaching of cotton fabrics. In the field of
optometry, Peroxidase is used in contact lens cleaning solution. Peroxidase is also an anti-oxidant hence helps in the management of oxidative stress by certain unstable compound.
2.8 Effect of different carbon, nitrogen sources as well as pH and incubation period activity of Peroxidase obtained from Aspergillus niger
Studies conducted by Mohammed A. Jebor, 2017 which deals with the isolation and purification of Peroxidase and the optimization of process parameters to obtain maximum yields of Peroxidase were reported. Solid fermentation of Aspergillus niger was carried out by the researcher to enhance the maximum production of Peroxidase. The peroxidase activity was also determined by the use of 30% hydrogen peroxide as the substrate for the Peroxidase obtained. The optimum conditions needed for the maximum yields of Peroxidase was determined as follows:
- Optimum carbon source for the media
- Nitrogen source for the media
- Optimum temperature at which the solid fermentation was carried out
- Optimum pH at which the Aspergillus niger can grow best
- Incubation period for peroxidase production.
2.8.1 Optimum incubation period for peroxidase production:
Peroxidase activity increased with increasing the incubation period of Aspergillus niger fermentation until it get to maximum activity after 12 days of Incubation, then it began
to decreased after 13,14,15 days of Incubation respectively Incubation. (Bhatti et al.,2007) 2.8.2 Optimum temperature for peroxidase activity
The influence of temperature on production of the enzyme showed that peroxidase activity reaching a maximum in 25°C, above this temperature there was a reduction in the peroxidase activity in 35, 40 °C respectively. Growth temperature is a very critical parameter which varies from organism to organism and slight changes in growth temperature may affect enzyme production, at higher temperature, due to the production of large amount of metabolic heat, the fermenting substrate temperature shoots up, thereby inhibiting microbial growth and enzyme formation
2.8.3 Optimum pH for peroxidase activity
Studies by Mohammed Jebor demonstrated that, the activity of Peroxidase increased with increasing the pH until reach to maximum activity in pH = 6.5, then it began to decreased in higher pH values in pH= 8 and 9.
2.8.4 Optimum nitrogen source for peroxidase activity:
The over production of enzyme by Aspergillus niger occurred using the peptone as nitrogen source, it gave high titer of peroxidase activity followed by ammonium nitrate
, while the other sources (beef extract, ammonium chloride, and ammonium tartrate) gave low titer of peroxidase activity . Similar results were also reported by Laura et al 2010 when they found the highest activity of Peroxidase from some white-rot fungi occurred using peptone as nitrogen source, while the other study like Stajic et al 2006 reported the maximum activity of Peroxidase from P. Pulmonarious using the KNO3 as nitrogen source
CHAPTER THREE: MATERIALS AND METHOD
3.1 Preparation of chemically defined medium
For the estimation of protein and activity of peroxidase enzyme, a culture media was prepared using 5.00g of glucose, 2.5g of KH2PO4, 0.5g of MgSO4.7H2O, 5g of NH4NO3, 0.01g of NaMoO4H2O, 0.05g of CaCl2 and 0.01g of FeSO4 in their respective order as stated with the aid of a chemical balance. The resulting mixture was then made up to 300ml using distilled water and was followed by determination of the initial pH using the pH meter and the probe. The initial pH (4.5) was adjusted using 0.0021M NaOH solution to pH of 5.5. The final volume was delivered into a well calibrated measuring cylinder and then made up to 500mL mark using distilled water. The resulting broth was then divided into 24 smaller conical flasks with each flask containing 20ml of the solution. Each conical flask was clogged using cotton wool and further covered with aluminum foil. The media were then autoclaved at 121°C for 20mins at 100Kpascal. The solutions were then cooled to room temperature after the sterilization.
3.2 Culturing of the Aspergillus nige
The A. niger used in this study was obtained from the University of Cape Coast’s Department of Molecular Biology and Biotechnology. With the aid of a sterilized inoculating loop, A. niger was introduced into the various sterile media whilst observing all standard microbiological practices to avoid any form of contamination. The inoculation loop was sterilized using the Bunsen burner. Aspergillus niger spores from Potassium Dextrose Agar (PDA) medium in a petri dish were introduced into the media using the sterilized inoculation loop. After this, the Cotton wool clog and aluminum foil were then sterilized with the conical flasks and reclogged and kept in a closed paper box in the shade and left to grow overnights.
3.3 Administration of drug into the media
On the fourth day of Incubation of Aspergillus niger, the drug acetaminophen was introduced into the media at a single concentration of 2mg/ml. Serial dilution of 1:2, 1:10 and 1:100 were prepared. Each 2mg/ml concentration was introduced into the culture media labelled with the above dilution factors. The introduction of the drug was done by tilting the flask to expose the medium for the introduction of the drugs. This was repeated for another set of cultured media
A total volume of 500ml of 2mg/mL acetaminophen drug was introduced into the media in bits for some set of media, thus 150mL in the morning of the first day, 150mL in the afternoon and the remaining 200mL in the next day and others at a time, thus some inoculum containing the Aspergillus niger were left for the drugs to be introduced after the period of Incubation. Here the medium was filtered and the drug was added to the spent medium. To one of the media, no drug was introduced and it served as general control. The set ups were then left again to stand for three days in the dark.
3.4 Filtration of media and determination of pH
After one week of Incubation, the various media were filtered into a well labelled container using funnel and filter papers. After this, the pH of each of the spent media was measured and recorded.
3.5 Protein estimation
A mass of 0.01g BSA was dissolved in 10mL distilled water. Using the serial dilution method, Series of BSA solutions were prepared in clean test tubes using 0.5, 1.0, 1.5, 2.0 mL volumes of the stock solution. The stock solutions in their various test tubes were made up to 2mL with the aid of a micropipette. A blank was prepared using 2mL distilled water. A 2.5mL volume of biuret reagent was added to each test tube. The solutions were mixed thoroughly and incubated at room temperature for 10 minutes. The absorbance w measured at 562nm using the UV-VIS spectrophotometer
3.5 Enzyme assay
Peroxidase was assayed by determining the rate of o-dianisidine oxidation in the presence of H2O2 according to the modified method of Malik and Singh (1980). To 2.5mL of each of the buffer solutions, 0.3mL H2O2 (24mM), 500μL of the crude peroxidase extract and 0.1mL of o-dianisidine (0.01M) were added. The reaction was allowed to proceed for 30 minutes
at room temperature. Using a micropipette 0.5mL of HCL was added to stop the reaction. The absorbance was measured at 430nm using a UV-VIS spectrophotometer.
3.5.1 Effect of pH
A 0.1M phosphate buffer solution of different pH ranging from 4 to 9 at an interval of 1 was prepared according to the Henderson hesselbelch’s equation. To 2.5mL of each of the buffer solutions, 0.3mL H2O2 (24mM), 500μL of the crude peroxidase extract and 0.1mL of o-dinisidine (0.01M) were added in an Eppendorf tube. The reaction was allowed to proceed for 30 minutes at room temperature. Using a micropipette 0.5mL of HCL was added to stop the reaction. The absorbance was measured at 430 using a UV-VIS spectrophotometer
CHAPTER FOUR: RESULTS
4.1 Peroxidase activity at different pH in test and control media
The activity of the enzyme preincubated with the paracetamol was labeled as test medium whereas media without paracetamol was labeled as control medium for which the activity of the enzyme was determined at different pH regions to obtain Peroxidase optimum pH
Abbildung in dieser Leseprobe nicht enthalten
Figure 4.1: Peroxidase activity at different pH for test and control culture media
4.2 peroxidase activity at different temperatures for culture media with Paracetamol and culture media without paracetamol
The temperature values for which Peroxidase works best was determined by assaying enzyme activity at different temperatures (30-70°C). This was done by initial Incubation of the culture media preparations containing the A. niger, the Peroxidase produced and the drug at temperatures ranging from 30-70°C, at intervals of 5. It was generally observed that for each culture medium, as temperature increases, the enzyme’s activity also increased, with the highest activity (2.9 mM/min) exhibited by the control (medium without any drug) at 70°C.
Abbildung in dieser Leseprobe nicht enthalten
Figure 4.2: Peroxidase activity for different temperature range for test media (media containing paracetamol) and the control media (media without paracetamol).
4.3 : Estimation of protein concentrations.
The amount of protein in each medium was determined using the biuret method of protein estimation. The test media gave the highest protein concentration in which the control culture media gave the lowest protein concentration.
Abbildung in dieser Leseprobe nicht enthalten
Figure 4.3: Protein estimation for culture media with paracetamol and culture media without paracetamol.
4.4 Effect of substrate concentration on peroxidase activity
Both the test and control media recorded their highest activity at a substrate concentration of 2.0mg/mL H2O2 whiles the least activity was observed at 0.2mg/mL H2O2 concentration. This information was then represented on Lineweaver Burk plot as well too.
Fig 4.4.1 Effect of Peroxidase activity at different substrate concentration in the test media and in the control media
Abbildung in dieser Leseprobe nicht enthalten
Fig 4.4.2 Lineweaver-Burke plot for the effect of substrate concentration on enzyme preincubated with paracetamol and without paracetamol
5.1 BACKGROUND
CHAPTER FIVE: DISCUSSION
Paracetamol, usually called acetaminophen is a drug which is used for relieving moderate pain such as headache, migraine, nerve pain, toothache, period pain and muscular pain .Paracetamol relief pain by lowering the production of prostaglandins in the brain and in the spinal cord (Davis and Lotter, 2009). Paracetamol also reduce fever by affecting the area of the brain that regulates our body temperature (the hypothalamic heat-regulating center).
Since paracetamol are usually taken without prescription, it is one of the most abused drugs. Peroxidase forms a network in the body that has both beneficial and adverse effects. Peroxidase has physiological functions that substantially contribute to immunity and a number of metabolic and signaling processes. However, under certain circumstances, they exert deleterious effects such as damage to macromolecules in the plasma and activate cell death. Apart from small like nitrogen dioxide and carbon dioxide that can interact with the heme iron, special proteins and lipid can bind to hemoproteins and regulate peroxidase activity. These mechanisms may be a basis for pharmacological strategies aiming to increase beneficial and to minimize adverse effects of peroxidase activity
The present study aim at investigating the effect of the presence of paracetamol in the culture media of Aspergillus niger on peroxidase activity as well as evaluation of various interactions that goes on in the growth media of Aspergillus niger so as to evaluate the stability of Peroxidase obtained from Aspergillus niger to tell why the pharmaceutical, oil and textile industries should choose Peroxidase as a source of Peroxidase for their industrial activity
5.2 Principal findings
5.2.1 Effect of pH on peroxidase activity.
From figure 4.1, it was observed that the culture media containing paracetamol gave the highest peroxidase activity at a pH value of 6, the activity was found to be 0.0644U/mL. Very low peroxidase activity was found at a pH range of 4 for both the test and control media. Also, it was observed from figure 4.1 that there was a decrease in peroxidase activity at pH 8 and 9, which varied widely with that of the test media. These result obtained from the study is similar to what( jebbor, 2017) reported. He was able to find maximum peroxidase activity at pH 6.5 and 7.0 respectively, and no activity at pH 2.5 and 9.5. Literature suggested that preincubation of the enzyme extract for 45 minutes at pH 2.5 or 9.5 completely inactivated the enzymes. Hence the low levels of enzyme activity recorded at pH 4 and 8 can also be attributed to the enzymes being inactivated in those pH regions. But the presence of paracetamol in the test media helped in resisting against change.(Campbell, 2009) also suggested that Low peroxidase activity observed in both culture media at pH of 4 and 8 was as a result of protonation and deprotonation of the amino acid residue present at the catalytic site of peroxidase enzyme, the protonation and deprotonation process occurred at pH of 4 and 8 respectively. Protonation and deprotonation of the amino acid residue which took place at the catalytic site of the Peroxidase caused a conformational change to the active site of Peroxidase such that the shape of the peroxide substrate wasn’t able to complementary fixed to the active site of the enzyme hence decreases the enzymes affinity for the substrate and by that decreases the enzyme activity is those pH regions.
5.2.2 Effect of temperature on peroxidase activity.
The results obtained from figure 4.2 gives an indication that peroxidase activity was temperature-dependent, since it keeps on increasing with increasing temperature in both the test media and the control media, but from temperatures of 30 to 40ºC, the activity of Peroxidase increased slowly in both media with the test medium containing paracetamol giving the highest activity at 50°C. But from 50 to 70ºC the peroxidase activity decrease was very sharp, with the control giving the least activity at a temperature value of 70°C. ). According to (Jebor, 2017), the optimum temperature for the activity of Peroxidase produced by A. niger is 35°C, and this conforms to the findings of the present study. The high value of optimum temperature observed in this study gives a clue that, strains of Aspergillus niger are highly thermophilic, and by that produces peroxidases that are highly heat resistant but on the other hand the culture media without paracetamol recorded the lowest peroxidase activity at a temperature region of 70 degrees , this shows that at temperature region of 70 degrees, the hydrogen and covalent bonds present at the active sites of the peroxidase enzyme were distorted and hence changes the conformation of the peroxidases active site which did not complementary fixed to its substrates to form more enzyme-substrate complexes(Campbell, 2009).This lead to the high decrease in enzyme activity in those regions. Growth temperature is a very critical parameter which varies from organism to organism and slight changes in growth temperature may affect enzyme production, at higher temperature, due to the production of large amount of metabolic heat, the fermenting substrate temperature shoots up, hence inhibits microbial growth and enzyme formation(Bertolin et al.,2003)
5.3 Protein determination
The amount of protein in each of the test and control media was determined by using the biuret method of protein estimation. From figure 4.3, it was observed that the culture media containing paracetamol gave the highest protein concentrations , this denotes that paracetamol was able to cause Aspergillus niger to produce more extracellular proteins, which eventually led to the high protein levels in the culture media. The culture media without paracetamol also gave an appreciable amount of protein concentrations in its media .This suggests that Aspergillus niger produces a lot of extracellular proteins in its habitat.
5.4 Effect of substrate concentration
In other to enhance microbial growth and harvest a large amount of enzyme production through solid-state fermentation, the most key factor is the choice of nutrient composition for the fermentation. The growth of an organism in culture medium is usually enhanced when the culture medium is made up of the optimum nutrients that can support the growth of the microbe under all conditions including stress conditions (Ellaiah et al., 2002). The source of enzyme production by Aspergillus niger in the present study was isolated from potato dextrose agar broth (PDA) medium and results obtained in figure 4.4.1 in this study indicates that peroxidase activity generally increased with increasing substrate concentration . Taking the plot in to consideration it was observed that, Velocity varies linearly with the substrate [S] for small [S]. As the substrate keeps on increasing [S] increases, the velocity V “plateaus” also shoots up indicating that V becomes independent of [S] at large values of [S]. A critical look at figure 4.4.2 which is the Lineweaver-burke plot indicate that the activity of the Peroxidase in the test media (KM =12.07M, Vmax =2.43 Min-1) was greatly enhanced by presence of paracetamol in that culture media with a KM of 18.12M and a Vmax of 4.53Mmin-1 which gave a higher harvest of peroxidase activity than the control media in all subjected substrate concentrations. Possession of a very small value of KM by an enzyme determines the affinity of the enzyme to its substrate, this implies that the enzyme achieves its maximum catalytic efficiency at low substrate concentrations. Hence, the smaller the value of KM, the more efficient is the catalyst. This may be attributed to the composition of the test media, since the presence of the paracetamol in the test media was able to intrinsically enhance the activity of Peroxidase obtained from Aspergillus niger, another important factor is the chemical composition of the the of the growth media, since it has a massive effect on the peroxidase activity this is because the affinity of an enzyme for a specific depends on the composition of the substrate. It also depends on the pH of the solution and the temperature at which the reaction is carried out. Literature also comments on the effect of carbon source to generate high peroxidase activity. The highest enzyme production by Aspergillus niger occurred using fructose as the carbon source , it gave high titer of peroxidase activity , followed by glucose , while the other sources (Glucose denoted ,sucrose denoted , mannitol denoted , lactose denoted ) gave low titer of peroxidase activity. When they show the highest activity of Peroxidase from Lentinus kauffmanii achieved using the fructose as the carbon source, while Prasher and (Chauhan 2013) reported the highest activity of Peroxidase from Grammothele fuligo achieved using the xylose as the carbon source
5.5 Significance
The optimum pH and temperature for the enzyme peroxidase was found to be 6.0 and 75℃ respectively. It was also observed that the activity of Peroxidase was temperature-dependent and therefore increased with increasing temperature. Results obtained on paracetamol indicated that the peroxidase enzyme works best at a pH of 6. Pharmaceutical, oil and textile industries that depend on peroxidase enzyme can use these findings in other to gain high yields of peroxidase production. Based on the results obtained it can be concluded that paracetamol indeed increases the activity of Peroxidase produced by Aspergillus niger.
CHAPTER SIX: CONCLUSIONS AND RECOMMENDATIONS
6.1 RECOMMENDATION
The aim of this study was to determine the effect of the presence of paracetamol in the culture of Aspergillus niger media on peroxidase activity. The aim of this study was met. In future approach where scientists want to investigate this same problem, they must take into consideration the incubation period of the Aspergillus niger fermentation, the source of carbon for the fermentation and the nitrogen source for the Aspergillus niger fermentation.
Again the study relied on Aspergillus niger as a source of enzyme, any researcher who will want to research into this same problem must choose a source of Peroxidase which will be much closer to human, pigs and etc. This is because there is a high difference in complexity and metabolism of these drugs between microbes and other complex organisms, and so to draw solid conclusions from such studies, one must consider a good source of peroxidase enzyme.
Again, subsequent studies of this nature should include isolation and purification of the enzyme before any further analysis. By doing this, it will help to reduce errors as a result of certain metabolites secreted by the enzyme or alongside the enzyme production.
6.2 CONCLUSION
The optimum pH and temperature for the enzyme peroxidase was found to be 6.0 and 50℃ respectively. It was also observed that the activity of Peroxidase was temperature-dependent and therefore increased with increasing temperature. Results obtained on paracetamol indicated that the peroxidase enzyme works best at a pH of 6. Pharmaceutical, oil and textile industries that depend on peroxidase enzyme can use these findings in other to gain high yields of peroxidase production. Based on the results obtained it can be concluded that paracetamol indeed increases the activity of Peroxidase produced by Aspergillus niger.
CHAPTER SEVEN: REFERENCE
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Bowen. N., Fordyce. A., Davidson. B,(2007.) induction of Contour sensing in Aspergillus niger by stress relevant fungal growth (4). pg:78-89
Bradford, M.M., ( 1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye-binding. Analytical Biochemistry (15 ) pg: 248-254
Bhatti, H; Mustafa G, Asgher, M (2007 ). Production of Glucoamylase by Fusarium moniliforme under solid-state fermentation. J. Chem. Soc. Pak ,( 29) pg: 161-165
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Campbell, M. (2009). Introduction to biochemistry. 1200 -1203.
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Jebbo, .A.M (2017).Production and purification of Peroxidase from Aspergillus niger.
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APPENDIX
Materials
Study materials for the present experimentation included, spores of Aspergillus niger obtain from the laboratory technology department, spectrophotometer, pH meter, micropipette, glasswares, thermometers, water bath all of which was obtain from the biochemistry laboratory
Drug used
Paracetamol
Reagents
O-dianisidine, Bovine serum albumen, H2O2, Biuret and Phosphate buffer
Buffer Preparation: Activity
pH= pKa + log [Salt] [Acid]
6=7.21 + log [Salt] [Acid]
-0.21= log[Salt] [Acid]
0.6166= [Salt]
1 [Acid]
[Na2HPO4] = 0.6166 x 0.1 = 0.02435M 1.6166
[NaH2PO4] = 0.1-0.02435 = 0.03814M [Na2HPO4] = C x M x V
= 0.03814 x 142 x 0.50
= 2.7079g
[NaH2PO4] = C x M x V
= 0.06186 x 121 x 0.50
= 3.7425g
Qualitative test for Peroxidase
Preparation of o-dianisidine solution was done according to modified Malik and Singh (1998). To 2.5mL of each of the buffer solutions, 0.3mL H2O2 (24mM), 500μL of the crude peroxidase extract and 0.1mL of o-dianisidine (0.01M) were added.
Biuret Preparation
0.2N NaOH Solution Preparation m= C x M x V
=0.2x 40g/mol x 50ml
= 0.4g
m(Na-K tartate) = 0.9
m(CuSO4) = 0.3g m(KI) = 0.5g
These masses dissolved in 50ml of 0.2NaOH
PROTEIN ESTIMATION
Absorbance(y) = 0.786
y = 0.278x + 0.034 ,0.786=0.278x +0.034 , 0.786– 0.034=0.278x ,X= 0.786–
0.034 34
X = 6.255mg/mL×2(dilution factor) = 12.511 Peroxidase Activity (U/mL)
= 𝑐ℎ𝑎𝑛𝑔𝑒 𝑖𝑛 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑥 𝑡𝑜𝑡𝑎𝑙 𝑎𝑠𝑠𝑎𝑦 𝑣(3.4𝑚𝐿) 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛
𝑓𝑎𝑐𝑡𝑜𝑟(1000)𝐸𝑥𝑡𝑖𝑛𝑐𝑡𝑖𝑜𝑛/ 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡(11.3)𝑥 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑒𝑛𝑧𝑦𝑚𝑒(0.3𝑚𝐿)𝑥 𝑡𝑖𝑚𝑒 𝑜𝑓
𝑖𝑛𝑐𝑢𝑏𝑎𝑡𝑖𝑜𝑛(10𝑚𝑖𝑛)
From the Lineweaver-burke plot analysis,
TEST MEDIA
KM/Vmax=4, 1/Vmax =0.2208 KM = 4 x (1/0.2208) =18.12M Vmax = 1/0.2208 =4.53 CONTROL MEDIA
KM/Vmax = 4.936, 1/Vmax =0.4111 KM = 4.936 x (1/0.4111) =12.07M Vmax = 1/0.4111 =2.43
Table 1: Varying Concentrations with corresponding Absorbances for BSA Calibration Curve
Standard BSA Calibration Curve
Table 3: Peroxidase activity at different substrate concentration in both culture media
Abbildung in dieser Leseprobe nicht enthalten
Table 4: Lineweaver-Burke Analysis
Table 6: Effect of pH on enzyme activity
Frequently Asked Questions about the Peroxidase Activity Study on Aspergillus niger
What is the purpose of this study?
This study aims to investigate the effect of paracetamol on peroxidase activity in Aspergillus niger cultures. It also evaluates various interactions within the Aspergillus niger growth media to assess the stability of fungal peroxidase.
What is paracetamol?
Paracetamol (also called acetaminophen) is a drug commonly used to relieve moderate pain, such as headaches, migraines, nerve pain, toothaches, period pain, and muscle pain. It also reduces fever.
What is Aspergillus niger?
Aspergillus niger is a saprophytic filamentous fungus that is found in various environments, including soil, decaying vegetation, and indoor environments. It is known for producing several enzymes, including citric acid enzymes, amylases, lipases, cellulases, xylanases, proteases, and peroxidases. It plays a critical role in industrial processes like citric acid production.
What is peroxidase?
Peroxidases are enzymes that oxidize hydrogen donors at the expense of peroxides. They are found in fungi, plants, and vertebrates, and contain heme in their active center. They are known for various roles, including immunity, apoptosis, and cell signaling, but may also have deleterious effects if unregulated.
What methods were used in this study?
The study involved preparing a chemically defined medium for culturing Aspergillus niger. Paracetamol was introduced into the media at varying concentrations. The cultures were incubated, filtered, and the pH was measured. Protein estimation was performed using the biuret method, and enzyme assays were conducted to determine peroxidase activity. The effect of pH, temperature and substrate concentration on enzyme activity was assessed.
What were the main findings regarding the effect of pH on peroxidase activity?
The culture media containing paracetamol showed the highest peroxidase activity at pH 6. Low peroxidase activity was found at pH 4 for both test and control media, and there was a decrease in peroxidase activity at pH 8 and 9. The presence of paracetamol in the test media appeared to mitigate the decrease in activity at higher pH levels.
What were the main findings regarding the effect of temperature on peroxidase activity?
Peroxidase activity was temperature-dependent. Enzyme activity generally increased with increasing temperature in both test and control media from 30-70°C. Test medium (paracetamol) saw peak activity at 50°C. Temperature increase beyond that resulted in a decrease in activity.
How was protein concentration estimated?
Protein concentration was estimated using the biuret method. The culture media containing paracetamol gave the highest protein concentrations, indicating that paracetamol may stimulate Aspergillus niger to produce more extracellular proteins.
How did substrate concentration affect peroxidase activity?
Peroxidase activity generally increased with increasing substrate concentration, reaching the highest activity at a substrate concentration of 2.0 mg/mL H2O2 for both test and control media. The Lineweaver-Burke plot revealed the difference in activity based on whether paracetamol was present. The presence of paracetamol increased Vmax from 2.43 to 4.53 (increased the amount of product created), while lowering the KM from 18.12M to 12.07M. (This improved affinity and enzyme efficiency)
What are the conclusions and recommendations of the study?
The study concluded that paracetamol increases the activity of peroxidase produced by Aspergillus niger. Future research should consider factors such as incubation period, carbon source, nitrogen source, isolation, and purification of the enzyme to further refine the understanding of the effects.
What industries can benefit from these findings?
Pharmaceutical, oil, and textile industries that depend on peroxidase enzymes can use these findings to optimize their production processes and achieve higher yields of peroxidase production. In this study, paracetamol increases peroxidase yields.
- Quote paper
- Renolph Gaglo (Author), 2022, Effect of the presence of paracetamol in the culture media of Aspergillus niger on activity of peroxidase, Munich, GRIN Verlag, https://www.grin.com/document/1255589
