Dataset Information

Enhanced Thermostability and Enzymatic Activity of Cel6A Variants from Thermobifida fusca by Empirical Domain Engineering (Short Title: Domain Engineering of Cel6A).

ABSTRACT: 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.

SUBMITTER: Ali I

PROVIDER: S-EPMC7464639 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Enhanced Thermostability and Enzymatic Activity of Cel6A Variants from <i>Thermobifida fusca</i> by Empirical Domain Engineering (Short Title: Domain Engineering of Cel6A).

Ali Imran I Rehman Hafiz Muzzammel HM Mirza Muhammad Usman MU Akhtar Muhammad Waheed MW Asghar Rehana R Tariq Muhammad M Ahmed Rashid R Tanveer Fatima F Khalid Hina H Alghamdi Huda Ahmed HA Froeyen Matheus M

Biology 20200807 8

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 mole ...[more]

PMID: 32784797

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Project description:Thermostability and specific activity of enzymes are two of the most important properties for industrial biocatalysts. Here, we developed a petri dish-based double-layer high-throughput screening (HTS) strategy for rapid identification of desired mutants of polyphosphate glucokinase (PPGK) from a thermophilic actinobacterium, Thermobifida fusca YX, with both enhanced thermostability and activity. Escherichia coli colonies representing a PPGK mutant library were grown on the first-layer Phytagel-based plates, which can remain solid for 1 h, even at heat treatment temperatures of more than 100°C. The second layer that was poured on the first layer contained agarose, substrates, glucose 6-phosphate dehydrogenase (G6PDH), the redox dye tetranitroblue tetrazolium (TNBT), and phenazine methosulfate. G6PDH was able to oxidize the product from the PPGK-catalyzed reaction and generate NADH, which can be easily examined by a TNBT-based colorimetric assay. The best mutant obtained after four rounds of directed evolution had a 7,200-fold longer half-life at 55°C, 19.8°C higher midpoint of unfolding temperature (Tm ), and a nearly 3-fold enhancement in specific activities compared to those of the wild-type PPGK. The best mutant was used to produce 9.98 g/liter myo-inositol from 10 g/liter glucose, with a theoretical yield of 99.8%, along with two other hyperthermophilic enzymes at 70°C. This PPGK mutant featuring both great thermostability and high activity would be useful for ATP-free production of glucose 6-phosphate or its derived products.IMPORTANCE Polyphosphate glucokinase (PPGK) is an enzyme that transfers a terminal phosphate group from polyphosphate to glucose, producing glucose 6-phosphate. A petri dish-based double-layer high-throughput screening strategy was developed by using ultrathermostable Phytagel as the first layer instead of agar or agarose, followed by a redox dye-based assay for rapid identification of ultrathermostable PPGK mutants. The best mutant featuring both great thermostability and high activity could produce glucose 6-phosphate from glucose and polyphosphate without in vitro ATP regeneration.

Dataset Information

Enhanced Thermostability and Enzymatic Activity of Cel6A Variants from Thermobifida fusca by Empirical Domain Engineering (Short Title: Domain Engineering of Cel6A).

Publications

Enhanced Thermostability and Enzymatic Activity of Cel6A Variants from <i>Thermobifida fusca</i> by Empirical Domain Engineering (Short Title: Domain Engineering of Cel6A).

Similar Datasets

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