Cloning, expression and purification of cellobiohydrolase gene from Caldicellulosiruptor bescii for efficient saccharification of plant biomass.

Autor: Aqeel A; Dr. Ikram-ul-Haq Institute of Industrial Biotechnology, Government College University Lahore, 54000, Pakistan. Electronic address: amna.aqeel45@gmail.com., Ahmed Z; Dr. Ikram-ul-Haq Institute of Industrial Biotechnology, Government College University Lahore, 54000, Pakistan., Akram F; Dr. Ikram-ul-Haq Institute of Industrial Biotechnology, Government College University Lahore, 54000, Pakistan., Abbas Q; School of Biological Sciences, University of Punjab, Lahore 54000, Pakistan., Ikram-Ul-Haq; Dr. Ikram-ul-Haq Institute of Industrial Biotechnology, Government College University Lahore, 54000, Pakistan.
Jazyk: angličtina
Zdroj: International journal of biological macromolecules [Int J Biol Macromol] 2024 Jun; Vol. 271 (Pt 2), pp. 132525. Date of Electronic Publication: 2024 May 24.
DOI: 10.1016/j.ijbiomac.2024.132525
Abstrakt: Anthropogenic activities have led to a drastic shift from natural fuels to alternative renewable energy reserves that demand heat-stable cellulases. Cellobiohydrolase is an indispensable member of cellulases that play a critical role in the degradation of cellulosic biomass. This article details the process of cloning the cellobiohydrolase gene from the thermophilic bacterium Caldicellulosiruptor bescii and expressing it in Escherichia coli (BL21) CondonPlus DE3-(RIPL) using the pET-21a(+) expression vector. Multi-alignments and structural modeling studies reveal that recombinant CbCBH contained a conserved cellulose binding domain III. The enzyme's catalytic site included Asp-372 and Glu-620, which are either involved in substrate or metal binding. The purified CbCBH, with a molecular weight of 91.8 kDa, displayed peak activity against pNPC (167.93 U/mg) at 65°C and pH 6.0. Moreover, it demonstrated remarkable stability across a broad temperature range (60-80°C) for 8 h. Additionally, the Plackett-Burman experimental model was employed to assess the saccharification of pretreated sugarcane bagasse with CbCBH, aiming to evaluate the cultivation conditions. The optimized parameters, including a pH of 6.0, a temperature of 55°C, a 24-hour incubation period, a substrate concentration of 1.5% (w/v), and enzyme activity of 120 U, resulted in an observed saccharification efficiency of 28.45%. This discovery indicates that the recombinant CbCBH holds promising potential for biofuel sector.
Competing Interests: Declaration of competing interest The authors explicitly state the absence of any competing interests related to this research. Our commitment to upholding the integrity and quality of our work is unwavering. Additionally, we affirm that there is no instance of plagiarism in this study; all references to other authors are appropriately cited in the text. This investigation is conducted with complete independence and impartiality, underscoring our dedication to scholarly rigor and ethical research practices.
(Copyright © 2024 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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