Enhanced Thermostability and Enzymatic Activity of cel6A Variants from <i>Thermobifida fusca</i> by Empirical Domain Engineering
Cellulases are a set of lignocellulolytic enzymes, capable of producing eco-friendly low-cost renewable bioethanol. However, low stability and hydrolytic activity limit their wide-scale applicability at the industrial scale. In this work, we report the domain engineering of endoglucanase (cel6A) of...
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MDPI AG
2020-08-01
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| author | Imran Ali Hafiz Muzzammel Rehman Muhammad Usman Mirza Muhammad Waheed Akhtar Rehana Asghar Muhammad Tariq Rashid Ahmed Fatima Tanveer Hina Khalid Huda Ahmed Alghamdi Matheus Froeyen |
| author_facet | Imran Ali Hafiz Muzzammel Rehman Muhammad Usman Mirza Muhammad Waheed Akhtar Rehana Asghar Muhammad Tariq Rashid Ahmed Fatima Tanveer Hina Khalid Huda Ahmed Alghamdi Matheus Froeyen |
| author_sort | Imran Ali |
| collection | DOAJ |
| description | Cellulases are a set of lignocellulolytic enzymes, capable of producing eco-friendly low-cost renewable bioethanol. However, low stability and hydrolytic activity limit their wide-scale applicability at the industrial scale. In this work, we report the domain engineering of endoglucanase (cel6A) of <i>Thermobifida fusca</i> to improve their catalytic activity and thermal stability. Later, enzymatic activity and thermostability of the most efficient variant named as cel6A.CBC was analyzed by molecular dynamics simulations. This variant demonstrated profound activity against soluble and insoluble cellulosic substrates like filter paper, alkali-treated bagasse, regenerated amorphous cellulose (RAC), and bacterial microcrystalline cellulose. The variant cel6A.CBC showed the highest catalysis of carboxymethyl cellulose (CMC) and other related insoluble substrates at a pH of 6.0 and a temperature of 60 °C. Furthermore, a sound rationale was observed between experimental findings and molecular modeling of cel6A.CBC which revealed thermostability of cel6A.CBC at 26.85, 60.85, and 74.85 °C as well as structural flexibility at 126.85 °C. Therefore, a thermostable derivative of cel6A engineered in the present work has enhanced biological performance and can be a useful construct for the mass production of bioethanol from plant biomass. |
| first_indexed | 2024-03-10T17:47:17Z |
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| institution | Directory Open Access Journal |
| issn | 2079-7737 |
| language | English |
| last_indexed | 2024-03-10T17:47:17Z |
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| spelling | doaj.art-dfafc87133f7415baddfd39623151a9d2023-11-20T09:29:11ZengMDPI AGBiology2079-77372020-08-019821410.3390/biology9080214Enhanced Thermostability and Enzymatic Activity of cel6A Variants from <i>Thermobifida fusca</i> by Empirical Domain EngineeringImran Ali0Hafiz Muzzammel Rehman1Muhammad Usman Mirza2Muhammad Waheed Akhtar3Rehana Asghar4Muhammad Tariq5Rashid Ahmed6Fatima Tanveer7Hina Khalid8Huda Ahmed Alghamdi9Matheus Froeyen10Department of Biotechnology, Mirpur University of Science and Technology (MUST), Mirpur (AJK) 10250, PakistanInstitute of Biochemistry and Biotechnology, University of the Punjab, Lahore 54590, PakistanDepartment of Pharmaceutical and Pharmacological Sciences, Rega Institute for Medical Research, Medicinal Chemistry, University of Leuven, B-3000 Leuven, BelgiumSchool of Biological Sciences, University of the Punjab, Lahore 54590, PakistanDepartment of Biotechnology, Mirpur University of Science and Technology (MUST), Mirpur (AJK) 10250, PakistanDepartment of Biotechnology, Mirpur University of Science and Technology (MUST), Mirpur (AJK) 10250, PakistanDepartment of Biotechnology, Mirpur University of Science and Technology (MUST), Mirpur (AJK) 10250, PakistanDepartment of Biotechnology, Forman Christian College University, Lahore 54590, PakistanShaheed Zulfiqar Ali Bhutto Medical University, Islamabad 44000, PakistanDepartment of Biology, College of Sciences, King Khalid University, Abha 61413, Saudi ArabiaDepartment of Pharmaceutical and Pharmacological Sciences, Rega Institute for Medical Research, Medicinal Chemistry, University of Leuven, B-3000 Leuven, BelgiumCellulases are a set of lignocellulolytic enzymes, capable of producing eco-friendly low-cost renewable bioethanol. However, low stability and hydrolytic activity limit their wide-scale applicability at the industrial scale. In this work, we report the domain engineering of endoglucanase (cel6A) of <i>Thermobifida fusca</i> to improve their catalytic activity and thermal stability. Later, enzymatic activity and thermostability of the most efficient variant named as cel6A.CBC was analyzed by molecular dynamics simulations. This variant demonstrated profound activity against soluble and insoluble cellulosic substrates like filter paper, alkali-treated bagasse, regenerated amorphous cellulose (RAC), and bacterial microcrystalline cellulose. The variant cel6A.CBC showed the highest catalysis of carboxymethyl cellulose (CMC) and other related insoluble substrates at a pH of 6.0 and a temperature of 60 °C. Furthermore, a sound rationale was observed between experimental findings and molecular modeling of cel6A.CBC which revealed thermostability of cel6A.CBC at 26.85, 60.85, and 74.85 °C as well as structural flexibility at 126.85 °C. Therefore, a thermostable derivative of cel6A engineered in the present work has enhanced biological performance and can be a useful construct for the mass production of bioethanol from plant biomass.https://www.mdpi.com/2079-7737/9/8/214endoglucanase (cel6A)domain engineering<i>Thermobifida fusca</i>molecular dynamics simulationsthermostable enzymes |
| spellingShingle | Imran Ali Hafiz Muzzammel Rehman Muhammad Usman Mirza Muhammad Waheed Akhtar Rehana Asghar Muhammad Tariq Rashid Ahmed Fatima Tanveer Hina Khalid Huda Ahmed Alghamdi Matheus Froeyen Enhanced Thermostability and Enzymatic Activity of cel6A Variants from <i>Thermobifida fusca</i> by Empirical Domain Engineering Biology endoglucanase (cel6A) domain engineering <i>Thermobifida fusca</i> molecular dynamics simulations thermostable enzymes |
| title | Enhanced Thermostability and Enzymatic Activity of cel6A Variants from <i>Thermobifida fusca</i> by Empirical Domain Engineering |
| title_full | Enhanced Thermostability and Enzymatic Activity of cel6A Variants from <i>Thermobifida fusca</i> by Empirical Domain Engineering |
| title_fullStr | Enhanced Thermostability and Enzymatic Activity of cel6A Variants from <i>Thermobifida fusca</i> by Empirical Domain Engineering |
| title_full_unstemmed | Enhanced Thermostability and Enzymatic Activity of cel6A Variants from <i>Thermobifida fusca</i> by Empirical Domain Engineering |
| title_short | Enhanced Thermostability and Enzymatic Activity of cel6A Variants from <i>Thermobifida fusca</i> by Empirical Domain Engineering |
| title_sort | enhanced thermostability and enzymatic activity of cel6a variants from i thermobifida fusca i by empirical domain engineering |
| topic | endoglucanase (cel6A) domain engineering <i>Thermobifida fusca</i> molecular dynamics simulations thermostable enzymes |
| url | https://www.mdpi.com/2079-7737/9/8/214 |
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