Evaluation of bacteria laccase hybrid biosensor and application in the detection of phenolic contaminants in water
- Edoamodu, Chiedu Epiphany https://orcid.org/0000-0002-9254-3955
- Authors: Edoamodu, Chiedu Epiphany https://orcid.org/0000-0002-9254-3955
- Date: 2021-09
- Subjects: Laccase , Water -- Purification
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10353/22820 , vital:52802
- Description: The continuous outpour of aromatic pollutants in diverse groups, emanating from the industrial and domestic system due to population density, industrialisation and technological advancement is alarming. The increasing strength in wastewater discharge constitutes the main cause of the natural water pollution load, causing scarcity of potable water for consumption with an increasing health challenge. The physiochemical treatment approach has faced a series of limitations with little or no success. Hindrance to wastewater management can cause a point source contamination problem that might increase treatment cost and release a broad range of chemical contaminants in the environment. Hence, green, eco-friendly and cost-effective tools are imperative. The application of laccase has received much attention in bioremediation and bioprocessing matters owing to the oxidising capacity of a wide range of substrates. The process requires available molecular oxygen for its activation, releasing water as a by-product thus, establishing this research. This study was devised to examine the bioprocessing potentials of hybrid and amalgamated laccases extracted from the diverse environmental milieu of the Eastern Cape Province, South Africa. Bacteria producing laccase were isolated from marine sediment, cow dung, and wastewater samples via selective enrichment with some aromatic compounds. The axenic cultures were screened for laccase activity on various phenolic and non-phenolic substrates. The isolates were identified via molecular techniques and they belonged to the gammaproteobacteria and Bacilli classes under the following genera, Enterobacter and Bacillus. They were deposited in the NCBI database as Enterobacter asburiae ES1, Enterobacter sp. Kamsi, Enterobacter sp. AI1, and Bacillus sp. NU2 with the assigned accession number MN686602, MN686603, MN686605, MN686607, respectively. Optimisation of the laccase production via one factor at a time technique (OFAT) from the four bacteria species showed more enzyme yield in all lignocellulosic agro-waste media. However, wheat bran and mandarin peel maximally enhanced laccase production. In addition, xylose, galactose, fructose, and sorbitol were the best carbon sources utilised while (NH₄)₂SO8, KNO3 and NaNO3 were noted as the best nitrogen sources employed. Laccase yields were increased at pH 4 and 5, at temperatures 45 and 55 o C, and at 50 and 100 rpm, and precisely, at day eight of the incubation period. Further purification of the crude laccase yielded a purification fold of 4.18, 4.39, 2.78, 8.11, and the SDS-PAGE analysis showed a molecular size of 90, 55, 75 and 50 kDa for ES1, Kamsi, AI1, and NU2 laccases, respectively. The characterised purified laccase demonstrated polyextremotolerant potentials. The laccases were active through a wide temperature regime (30-90 o C) with maximum activity at 50 o C (ES1/AI1 and Kamsi/NU2) 60 o C (AI1), 70 o C (ES1, Kamsi, NU2); and were stable at 60 o C (ES1, AI1, NU2), 70 o C (ES1/AI1), 80 o C (Kamsi and Kamsi/NU2). Also, the laccases remained active through pH 3 - 8 and optimal at pH 4 (AI1, NU2), pH 5 (Kamsi, ES1/AI1), pH 7 (ES1), pH 8 (Kamsi/NU2), and the individual stability was measured at pH 4 (Kamsi, NU2), pH 5 (AI1), pH 6 (ES1), pH 7 (Kamsi/NU2), pH 10 (ES1/AI1). The purified laccases were either enhanced or left unchanged by a variable concentration of metallic salts, inhibitors, chelating agents and organic solvents. Clearly, the activities of the laccase were enhanced when pre-incubated with 1, 3, and 6 mm of CuCl2, FeCl3, MgCl2, ZnCl2 and AgCl, and 1, 2, 3 mm of Triton x-100, PMSF, EDTA, Tween 20, and NaCl. Additionally, 20, 30, and 10 percent v/v of acetone and DMSO were prominent organic solvents that also stimulated both the hybrid and amalgamated laccase activity. The gene of the purified laccases targeted showed a clear band size of 690 bp for the Enterobacter species laccases and 775 bp for the laccase from Bacillus sp. The protein sequence was deposited in NCBI database with the assigned accession numbers, MW251989, MW25990, MW251992, and MW251994 for ES1, Kamsi, AI1, and NU2 laccases, respectively. The optimised pH and temperature parameter examined on the decolourising potential of the bacteria laccases showed an effective dye removal on the five synthetic dyes (Congo Red (CR), Methyl Orange (MO), Malachite Green (MG), Reactive Blue 4 (RB4), Ramazol Brilliant Blue R (RBBR)) applied. The purified laccases were successfully immobilised in Na-alginate with cca. 88.49, 70.91, 76.04, 76.13, 90.07, and 91.99 laccase yield for the hybrid (ES1, ES1, Kamsi, AI1, NU2) and amalgamated (ES1/AI1 and Kamsi/NU2) laccases. The immobilised laccases were able to retain an average activity of 32 – 52 percent after eight dye decolourising cycles, exhibiting strong catalytic activity than the free laccases. Nonetheless, no significant difference was examined between the hybrid and amalgamated laccase activity. Also, the immobilised laccases were shown to be more efficient in biotechnological application than the free laccases. The result suggests that immobilising an enzyme in a carrier matrix served effectively as the remediation approach than the hybrid and the amalgamation of the free enzymes. Also, the application of lignocellulosic waste served as a cheaper substrate for commercial production of laccase and could help s in promoting es the biotechnology application and the bioeconomy. , Thesis (PhD) -- Faculty of Science and Agriculture, 2021
- Full Text:
- Date Issued: 2021-09
- Authors: Edoamodu, Chiedu Epiphany https://orcid.org/0000-0002-9254-3955
- Date: 2021-09
- Subjects: Laccase , Water -- Purification
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10353/22820 , vital:52802
- Description: The continuous outpour of aromatic pollutants in diverse groups, emanating from the industrial and domestic system due to population density, industrialisation and technological advancement is alarming. The increasing strength in wastewater discharge constitutes the main cause of the natural water pollution load, causing scarcity of potable water for consumption with an increasing health challenge. The physiochemical treatment approach has faced a series of limitations with little or no success. Hindrance to wastewater management can cause a point source contamination problem that might increase treatment cost and release a broad range of chemical contaminants in the environment. Hence, green, eco-friendly and cost-effective tools are imperative. The application of laccase has received much attention in bioremediation and bioprocessing matters owing to the oxidising capacity of a wide range of substrates. The process requires available molecular oxygen for its activation, releasing water as a by-product thus, establishing this research. This study was devised to examine the bioprocessing potentials of hybrid and amalgamated laccases extracted from the diverse environmental milieu of the Eastern Cape Province, South Africa. Bacteria producing laccase were isolated from marine sediment, cow dung, and wastewater samples via selective enrichment with some aromatic compounds. The axenic cultures were screened for laccase activity on various phenolic and non-phenolic substrates. The isolates were identified via molecular techniques and they belonged to the gammaproteobacteria and Bacilli classes under the following genera, Enterobacter and Bacillus. They were deposited in the NCBI database as Enterobacter asburiae ES1, Enterobacter sp. Kamsi, Enterobacter sp. AI1, and Bacillus sp. NU2 with the assigned accession number MN686602, MN686603, MN686605, MN686607, respectively. Optimisation of the laccase production via one factor at a time technique (OFAT) from the four bacteria species showed more enzyme yield in all lignocellulosic agro-waste media. However, wheat bran and mandarin peel maximally enhanced laccase production. In addition, xylose, galactose, fructose, and sorbitol were the best carbon sources utilised while (NH₄)₂SO8, KNO3 and NaNO3 were noted as the best nitrogen sources employed. Laccase yields were increased at pH 4 and 5, at temperatures 45 and 55 o C, and at 50 and 100 rpm, and precisely, at day eight of the incubation period. Further purification of the crude laccase yielded a purification fold of 4.18, 4.39, 2.78, 8.11, and the SDS-PAGE analysis showed a molecular size of 90, 55, 75 and 50 kDa for ES1, Kamsi, AI1, and NU2 laccases, respectively. The characterised purified laccase demonstrated polyextremotolerant potentials. The laccases were active through a wide temperature regime (30-90 o C) with maximum activity at 50 o C (ES1/AI1 and Kamsi/NU2) 60 o C (AI1), 70 o C (ES1, Kamsi, NU2); and were stable at 60 o C (ES1, AI1, NU2), 70 o C (ES1/AI1), 80 o C (Kamsi and Kamsi/NU2). Also, the laccases remained active through pH 3 - 8 and optimal at pH 4 (AI1, NU2), pH 5 (Kamsi, ES1/AI1), pH 7 (ES1), pH 8 (Kamsi/NU2), and the individual stability was measured at pH 4 (Kamsi, NU2), pH 5 (AI1), pH 6 (ES1), pH 7 (Kamsi/NU2), pH 10 (ES1/AI1). The purified laccases were either enhanced or left unchanged by a variable concentration of metallic salts, inhibitors, chelating agents and organic solvents. Clearly, the activities of the laccase were enhanced when pre-incubated with 1, 3, and 6 mm of CuCl2, FeCl3, MgCl2, ZnCl2 and AgCl, and 1, 2, 3 mm of Triton x-100, PMSF, EDTA, Tween 20, and NaCl. Additionally, 20, 30, and 10 percent v/v of acetone and DMSO were prominent organic solvents that also stimulated both the hybrid and amalgamated laccase activity. The gene of the purified laccases targeted showed a clear band size of 690 bp for the Enterobacter species laccases and 775 bp for the laccase from Bacillus sp. The protein sequence was deposited in NCBI database with the assigned accession numbers, MW251989, MW25990, MW251992, and MW251994 for ES1, Kamsi, AI1, and NU2 laccases, respectively. The optimised pH and temperature parameter examined on the decolourising potential of the bacteria laccases showed an effective dye removal on the five synthetic dyes (Congo Red (CR), Methyl Orange (MO), Malachite Green (MG), Reactive Blue 4 (RB4), Ramazol Brilliant Blue R (RBBR)) applied. The purified laccases were successfully immobilised in Na-alginate with cca. 88.49, 70.91, 76.04, 76.13, 90.07, and 91.99 laccase yield for the hybrid (ES1, ES1, Kamsi, AI1, NU2) and amalgamated (ES1/AI1 and Kamsi/NU2) laccases. The immobilised laccases were able to retain an average activity of 32 – 52 percent after eight dye decolourising cycles, exhibiting strong catalytic activity than the free laccases. Nonetheless, no significant difference was examined between the hybrid and amalgamated laccase activity. Also, the immobilised laccases were shown to be more efficient in biotechnological application than the free laccases. The result suggests that immobilising an enzyme in a carrier matrix served effectively as the remediation approach than the hybrid and the amalgamation of the free enzymes. Also, the application of lignocellulosic waste served as a cheaper substrate for commercial production of laccase and could help s in promoting es the biotechnology application and the bioeconomy. , Thesis (PhD) -- Faculty of Science and Agriculture, 2021
- Full Text:
- Date Issued: 2021-09
Assessment of some bacteria species isolated from woodlands of Raymond Mhlaba Local Municipality for high activity laccase production
- Authors: Gogotya, Asemahle
- Date: 2019
- Subjects: Laccase
- Language: English
- Type: Thesis , Masters , MSc (Microbiology)
- Identifier: http://hdl.handle.net/10353/19098 , vital:39879
- Description: The function of enzymes in keeping the earth clean is enormous; being executed in the biodegradation of different natural pollutants and biocatalysis of different responses by substituting the ecologically risky and harmful concoction impetuses offering a situation inviting option, laccases is an example of an enzyme described as it doesn’t produce harmful byproducts. Laccases are employed in several industrial processes that play a key responsibility in transformation of life and making the environment a better place. Bacteria have been described as best producers of laccases with a potential in the industrial scale processes. Laccase was produced from different bacterial isolates identified and named as Bacillus sp. strain GFN1 isolated from soil sample, Bacillus sp. strain GLN and Streptomyces sp. strain LAO both isolated from decaying wood samples in Raymond Mhlaba local municipality with accession numbers MK290988 to MK290990 respectively, as identified by partial sequencing, these were the best producers some of which were positive for Napthol and guaiacol; even upon quantitative screening they were better laccase producers. For quantitative laccase screening, 2,2'-azino-bis(3-ethylbenzothiazoline-6- sulphonic acid) was utilised as the substrate for laccase assays. These laccase producing bacteria were subjected to optimization of growth conditions using submerged fermentation which increased the activity of the produced laccase in great amounts. In optimizing basal medium growth conditions where laccase was harvested after every 72 hours where optimal activity was 16 obtained, studying several factors such as pH which turned out to be pH 5 for all isolates, effect of supplemented carbon and nitrogen sources with the be best being lactose and urea respectively with their effective concentrations using lignin as the main carbon and nitrogen source. Copper sulfate was used as the main inducer and the species preferred guaiacol and ferullic acid and the Fe2+ asthe best supplemented metal ion. The time course was done investigating parameters such as cell growth which was determined by observing the optical density, laccase activity, protein concentration and pH and the presented results suggested that when there was an increase in cell growth, enzyme activity decreased pH had no much effect on the enzyme production as it was almost stable all the time with protein concentration exhibiting no direct effect on enzyme activity also. Characterization of the crude enzyme was done to check the stability of the enzyme produced in various parameters. The enzymes produced by the different strains were thermophilic as they were able to withstand elevated temperatures between 90 and 100 C, with pH stability within an extensive variety of alkaline pH, typical of most bacterial laccases. Various metal ions affected the stability of the enzyme with CuSO4 increasing the stability of two of the bacterial enzyme and appeared to slightly decrease the stability of one enzyme. The studied inhibitors only decreased the stability on the enzyme and couldn’t completely inhibit the enzyme, and the enzymes showed specificity towards varying substrates. The studied bacterial laccases exhibit tremendous characteristics which are of great significance in the industries and will add to the novelty of bacterial laccases and their stability amongst the most studied fungal laccases.
- Full Text:
- Date Issued: 2019
- Authors: Gogotya, Asemahle
- Date: 2019
- Subjects: Laccase
- Language: English
- Type: Thesis , Masters , MSc (Microbiology)
- Identifier: http://hdl.handle.net/10353/19098 , vital:39879
- Description: The function of enzymes in keeping the earth clean is enormous; being executed in the biodegradation of different natural pollutants and biocatalysis of different responses by substituting the ecologically risky and harmful concoction impetuses offering a situation inviting option, laccases is an example of an enzyme described as it doesn’t produce harmful byproducts. Laccases are employed in several industrial processes that play a key responsibility in transformation of life and making the environment a better place. Bacteria have been described as best producers of laccases with a potential in the industrial scale processes. Laccase was produced from different bacterial isolates identified and named as Bacillus sp. strain GFN1 isolated from soil sample, Bacillus sp. strain GLN and Streptomyces sp. strain LAO both isolated from decaying wood samples in Raymond Mhlaba local municipality with accession numbers MK290988 to MK290990 respectively, as identified by partial sequencing, these were the best producers some of which were positive for Napthol and guaiacol; even upon quantitative screening they were better laccase producers. For quantitative laccase screening, 2,2'-azino-bis(3-ethylbenzothiazoline-6- sulphonic acid) was utilised as the substrate for laccase assays. These laccase producing bacteria were subjected to optimization of growth conditions using submerged fermentation which increased the activity of the produced laccase in great amounts. In optimizing basal medium growth conditions where laccase was harvested after every 72 hours where optimal activity was 16 obtained, studying several factors such as pH which turned out to be pH 5 for all isolates, effect of supplemented carbon and nitrogen sources with the be best being lactose and urea respectively with their effective concentrations using lignin as the main carbon and nitrogen source. Copper sulfate was used as the main inducer and the species preferred guaiacol and ferullic acid and the Fe2+ asthe best supplemented metal ion. The time course was done investigating parameters such as cell growth which was determined by observing the optical density, laccase activity, protein concentration and pH and the presented results suggested that when there was an increase in cell growth, enzyme activity decreased pH had no much effect on the enzyme production as it was almost stable all the time with protein concentration exhibiting no direct effect on enzyme activity also. Characterization of the crude enzyme was done to check the stability of the enzyme produced in various parameters. The enzymes produced by the different strains were thermophilic as they were able to withstand elevated temperatures between 90 and 100 C, with pH stability within an extensive variety of alkaline pH, typical of most bacterial laccases. Various metal ions affected the stability of the enzyme with CuSO4 increasing the stability of two of the bacterial enzyme and appeared to slightly decrease the stability of one enzyme. The studied inhibitors only decreased the stability on the enzyme and couldn’t completely inhibit the enzyme, and the enzymes showed specificity towards varying substrates. The studied bacterial laccases exhibit tremendous characteristics which are of great significance in the industries and will add to the novelty of bacterial laccases and their stability amongst the most studied fungal laccases.
- Full Text:
- Date Issued: 2019
Evaluation of some Eastern beach bacteria for high activity laccase production
- Authors: Ntlatywa, Nonkolisi
- Date: 2019
- Subjects: Laccase
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/${Handle} , vital:40025
- Description: Laccases are multi-copper oxidases endowed with huge applicability and versatility. They are found in nature, fungi, insects, archaea and bacteria; however, there is a dearth of information regarding bacterial laccases. Thus, the search for novel bacterial laccases becomes crucial. Therefore, the broad aim of the study was the assessment of some bacteria on marine water isolated from Eastern beach for the production of high activity laccases. In this study, marine water samples obtained from the Eastern beach in East London were investigated for bacterial laccase-producing isolates. The samples were analysed using different microbiological media and varied substrates via qualitative methods. The isolates were tested for laccase activity using ABTS (2, 2’- azinobis 3-ethyl-benzothiazoline-6-sulphonate), as a substrate. The present study also dealt with the optimization of culture conditions for laccase production and characterization of laccase. A total of forty-eight (48) bacterial isolates were obtained, which were identified based on their morphological and cultural characteristics. Many of the isolates (42) were categorized as Gram positive and a few (6) as Gram negative upon subjecting to microscopic examination. Result showed that on average, laccase activity ranged from 5.33 – 6.5 U/L as demonstrated by three bacterial isolates (Bacillus sp. NFN1, Bacillus sp. NLN and Escherichia coli LO). Laccase production was highly enhanced by the supplementation with vanillic acid by Bacillus sp. strain NLN with yield of 15.83 ± 0.7 U/L, Bacillus sp. strain NFN1, 12.72 ± 0.21 U/L. However, Escherichia coli LO demonstrated enhanced laccase production with guaiacol (12.84 ± 1.12 U/L) followed by ferrulic acid (12.8 ± 0.36 U/L). While optimization results revealed that the laccase yield was enhanced under the following conditions: 72 h of incubation, pH 5, 0.5mg/L lactose as a carbon source, 1. 5 mg/L yeast extract as a nitrogen source as well as in the presence of corn stover and mandarin peels as lignocellulosic substrates depending on the bacteria.The time course and stability of laccase against temperature, pH, metal ions and organic solvents were investigated using previously described methods. In the findings obtained for the time course, the laccase activity ranged from 22.2- 23.98 U/L. In general, the optimum parameters for the optimum laccase activities depended on the bacterial isolates and the time of incubation and included 80 °C and 70 °C, pH 3 and 9 while, the thermal stability was over 100%. Overall, NaCl, ZnSO4, NiCl and 20% acetone demonstrated positive effect on the residual activity of the laccases of all the bacterial isolates (Bacillus sp. NFN1, Bacillus v sp. NLN and Escherichia coli LO). In conclusion the findings of the study, suggest that marine water of the Eastern beach is a rich source for laccase producing bacteria with potential of high activity.
- Full Text:
- Date Issued: 2019
- Authors: Ntlatywa, Nonkolisi
- Date: 2019
- Subjects: Laccase
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/${Handle} , vital:40025
- Description: Laccases are multi-copper oxidases endowed with huge applicability and versatility. They are found in nature, fungi, insects, archaea and bacteria; however, there is a dearth of information regarding bacterial laccases. Thus, the search for novel bacterial laccases becomes crucial. Therefore, the broad aim of the study was the assessment of some bacteria on marine water isolated from Eastern beach for the production of high activity laccases. In this study, marine water samples obtained from the Eastern beach in East London were investigated for bacterial laccase-producing isolates. The samples were analysed using different microbiological media and varied substrates via qualitative methods. The isolates were tested for laccase activity using ABTS (2, 2’- azinobis 3-ethyl-benzothiazoline-6-sulphonate), as a substrate. The present study also dealt with the optimization of culture conditions for laccase production and characterization of laccase. A total of forty-eight (48) bacterial isolates were obtained, which were identified based on their morphological and cultural characteristics. Many of the isolates (42) were categorized as Gram positive and a few (6) as Gram negative upon subjecting to microscopic examination. Result showed that on average, laccase activity ranged from 5.33 – 6.5 U/L as demonstrated by three bacterial isolates (Bacillus sp. NFN1, Bacillus sp. NLN and Escherichia coli LO). Laccase production was highly enhanced by the supplementation with vanillic acid by Bacillus sp. strain NLN with yield of 15.83 ± 0.7 U/L, Bacillus sp. strain NFN1, 12.72 ± 0.21 U/L. However, Escherichia coli LO demonstrated enhanced laccase production with guaiacol (12.84 ± 1.12 U/L) followed by ferrulic acid (12.8 ± 0.36 U/L). While optimization results revealed that the laccase yield was enhanced under the following conditions: 72 h of incubation, pH 5, 0.5mg/L lactose as a carbon source, 1. 5 mg/L yeast extract as a nitrogen source as well as in the presence of corn stover and mandarin peels as lignocellulosic substrates depending on the bacteria.The time course and stability of laccase against temperature, pH, metal ions and organic solvents were investigated using previously described methods. In the findings obtained for the time course, the laccase activity ranged from 22.2- 23.98 U/L. In general, the optimum parameters for the optimum laccase activities depended on the bacterial isolates and the time of incubation and included 80 °C and 70 °C, pH 3 and 9 while, the thermal stability was over 100%. Overall, NaCl, ZnSO4, NiCl and 20% acetone demonstrated positive effect on the residual activity of the laccases of all the bacterial isolates (Bacillus sp. NFN1, Bacillus v sp. NLN and Escherichia coli LO). In conclusion the findings of the study, suggest that marine water of the Eastern beach is a rich source for laccase producing bacteria with potential of high activity.
- Full Text:
- Date Issued: 2019
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