Enhanced Thermostability and Enzymatic Activity of cel6A Variants from <i>Thermobifida fusca</i> by Empirical Domain Engineering
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
Affiliations
Imran Ali
Department of Biotechnology, Mirpur University of Science and Technology (MUST), Mirpur (AJK) 10250, Pakistan
Hafiz Muzzammel Rehman
Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore 54590, Pakistan
Muhammad Usman Mirza
Department of Pharmaceutical and Pharmacological Sciences, Rega Institute for Medical Research, Medicinal Chemistry, University of Leuven, B-3000 Leuven, Belgium
Muhammad Waheed Akhtar
School of Biological Sciences, University of the Punjab, Lahore 54590, Pakistan
Rehana Asghar
Department of Biotechnology, Mirpur University of Science and Technology (MUST), Mirpur (AJK) 10250, Pakistan
Muhammad Tariq
Department of Biotechnology, Mirpur University of Science and Technology (MUST), Mirpur (AJK) 10250, Pakistan
Rashid Ahmed
Department of Biotechnology, Mirpur University of Science and Technology (MUST), Mirpur (AJK) 10250, Pakistan
Fatima Tanveer
Department of Biotechnology, Forman Christian College University, Lahore 54590, Pakistan
Hina Khalid
Shaheed Zulfiqar Ali Bhutto Medical University, Islamabad 44000, Pakistan
Huda Ahmed Alghamdi
Department of Biology, College of Sciences, King Khalid University, Abha 61413, Saudi Arabia
Matheus Froeyen
Department of Pharmaceutical and Pharmacological Sciences, Rega Institute for Medical Research, Medicinal Chemistry, University of Leuven, B-3000 Leuven, Belgium
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 Thermobifida fusca 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.
