The Studies of Sepharose-Immobilized Lipases: Combining Techniques for the Enhancement of Activity and Thermal Stability
Thermal stability is one of the essential parameters characterizing biocatalysts with potential applications in the chemical and pharmaceutical industries. Therefore, it is extremely important to develop standardized procedures for enzyme stability studies. The paper attempts to assess the thermal s...
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2023
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ftmdpi:oai:mdpi.com:/2073-4344/13/5/887/ 2023-08-20T04:02:27+02:00 The Studies of Sepharose-Immobilized Lipases: Combining Techniques for the Enhancement of Activity and Thermal Stability Tomasz Siódmiak Jacek Dulęba Gudmundur G. Haraldsson Joanna Siódmiak Michał Piotr Marszałł 2023-05-15 application/pdf https://doi.org/10.3390/catal13050887 EN eng Multidisciplinary Digital Publishing Institute Biocatalysis https://dx.doi.org/10.3390/catal13050887 https://creativecommons.org/licenses/by/4.0/ Catalysts; Volume 13; Issue 5; Pages: 887 lipase from Candida rugosa lipase B from Candida antarctica Octyl-Sepharose CL-4B immobilization thermal stability incubation time climatic chamber aqueous buffers combining techniques Text 2023 ftmdpi https://doi.org/10.3390/catal13050887 2023-08-01T10:04:26Z Thermal stability is one of the essential parameters characterizing biocatalysts with potential applications in the chemical and pharmaceutical industries. Therefore, it is extremely important to develop standardized procedures for enzyme stability studies. The paper attempts to assess the thermal stability of immobilized lipases in aqueous buffers: lipase B from Candida antarctica (CALB) and lipase from Candida rugosa (CRL-OF) immobilized on the Octyl-Sepharose CL-4B carrier. As part of the optimization conditions of the immobilization, the influence of time on the catalytic activity and lipase loading, as well as the effect of temperature on lipase activity (optimal incubation—14 h at 4 °C), was determined. The thermal stability test procedure was carried out for 7 days using a climatic chamber (65 °C) and a refrigerator (4 °C). The studies of immobilized lipases included the assessment of the impact of various solvents (water, citrate buffer, 1,2-dichloropropane—DCP), temperature, light in the visible spectral range (400–800 nm), and additions of calcium ions. The highest value of residual activity (564.5 ± 21.6%) was received by storing the immobilized CALB in citrate buffer (pH 4.0, 500 mM) with the addition of calcium ions (Ca2+). On the other hand, residual activity values for immobilized CRL-OF after storage in the climatic chamber were lower than 5%. A combining of techniques: immobilization onto the support in high ionic strength and low pH, with a technique of extremally high-temperature applied in a climatic chamber, with the addition of Ca2+ allowed to achieve of excellent thermal stability of the immobilized CALB, with increasing of catalytic activity more than five-fold. Additionally, performing studies on the thermal stability of the tested lipases using a climatic chamber seems to be particularly promising in the context of unifying and standardizing storage guidelines, enabling the comparison of results between different laboratories, as well as enhancing catalytic activity. Text Antarc* Antarctica MDPI Open Access Publishing Rugosa ENVELOPE(-61.250,-61.250,-62.633,-62.633) Catalysts 13 5 887 |
institution |
Open Polar |
collection |
MDPI Open Access Publishing |
op_collection_id |
ftmdpi |
language |
English |
topic |
lipase from Candida rugosa lipase B from Candida antarctica Octyl-Sepharose CL-4B immobilization thermal stability incubation time climatic chamber aqueous buffers combining techniques |
spellingShingle |
lipase from Candida rugosa lipase B from Candida antarctica Octyl-Sepharose CL-4B immobilization thermal stability incubation time climatic chamber aqueous buffers combining techniques Tomasz Siódmiak Jacek Dulęba Gudmundur G. Haraldsson Joanna Siódmiak Michał Piotr Marszałł The Studies of Sepharose-Immobilized Lipases: Combining Techniques for the Enhancement of Activity and Thermal Stability |
topic_facet |
lipase from Candida rugosa lipase B from Candida antarctica Octyl-Sepharose CL-4B immobilization thermal stability incubation time climatic chamber aqueous buffers combining techniques |
description |
Thermal stability is one of the essential parameters characterizing biocatalysts with potential applications in the chemical and pharmaceutical industries. Therefore, it is extremely important to develop standardized procedures for enzyme stability studies. The paper attempts to assess the thermal stability of immobilized lipases in aqueous buffers: lipase B from Candida antarctica (CALB) and lipase from Candida rugosa (CRL-OF) immobilized on the Octyl-Sepharose CL-4B carrier. As part of the optimization conditions of the immobilization, the influence of time on the catalytic activity and lipase loading, as well as the effect of temperature on lipase activity (optimal incubation—14 h at 4 °C), was determined. The thermal stability test procedure was carried out for 7 days using a climatic chamber (65 °C) and a refrigerator (4 °C). The studies of immobilized lipases included the assessment of the impact of various solvents (water, citrate buffer, 1,2-dichloropropane—DCP), temperature, light in the visible spectral range (400–800 nm), and additions of calcium ions. The highest value of residual activity (564.5 ± 21.6%) was received by storing the immobilized CALB in citrate buffer (pH 4.0, 500 mM) with the addition of calcium ions (Ca2+). On the other hand, residual activity values for immobilized CRL-OF after storage in the climatic chamber were lower than 5%. A combining of techniques: immobilization onto the support in high ionic strength and low pH, with a technique of extremally high-temperature applied in a climatic chamber, with the addition of Ca2+ allowed to achieve of excellent thermal stability of the immobilized CALB, with increasing of catalytic activity more than five-fold. Additionally, performing studies on the thermal stability of the tested lipases using a climatic chamber seems to be particularly promising in the context of unifying and standardizing storage guidelines, enabling the comparison of results between different laboratories, as well as enhancing catalytic activity. |
format |
Text |
author |
Tomasz Siódmiak Jacek Dulęba Gudmundur G. Haraldsson Joanna Siódmiak Michał Piotr Marszałł |
author_facet |
Tomasz Siódmiak Jacek Dulęba Gudmundur G. Haraldsson Joanna Siódmiak Michał Piotr Marszałł |
author_sort |
Tomasz Siódmiak |
title |
The Studies of Sepharose-Immobilized Lipases: Combining Techniques for the Enhancement of Activity and Thermal Stability |
title_short |
The Studies of Sepharose-Immobilized Lipases: Combining Techniques for the Enhancement of Activity and Thermal Stability |
title_full |
The Studies of Sepharose-Immobilized Lipases: Combining Techniques for the Enhancement of Activity and Thermal Stability |
title_fullStr |
The Studies of Sepharose-Immobilized Lipases: Combining Techniques for the Enhancement of Activity and Thermal Stability |
title_full_unstemmed |
The Studies of Sepharose-Immobilized Lipases: Combining Techniques for the Enhancement of Activity and Thermal Stability |
title_sort |
studies of sepharose-immobilized lipases: combining techniques for the enhancement of activity and thermal stability |
publisher |
Multidisciplinary Digital Publishing Institute |
publishDate |
2023 |
url |
https://doi.org/10.3390/catal13050887 |
long_lat |
ENVELOPE(-61.250,-61.250,-62.633,-62.633) |
geographic |
Rugosa |
geographic_facet |
Rugosa |
genre |
Antarc* Antarctica |
genre_facet |
Antarc* Antarctica |
op_source |
Catalysts; Volume 13; Issue 5; Pages: 887 |
op_relation |
Biocatalysis https://dx.doi.org/10.3390/catal13050887 |
op_rights |
https://creativecommons.org/licenses/by/4.0/ |
op_doi |
https://doi.org/10.3390/catal13050887 |
container_title |
Catalysts |
container_volume |
13 |
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5 |
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887 |
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1774712895959990272 |