Purification, biochemical characterization, and molecular cloning of cellulase from Bacillus licheniformis strain Z9 isolated from soil

Zainab E Elsababty; Samir H Abdel-Aziz; Atef M Ibrahim; Adel A Guirgis; Ghada E Dawwam

doi:10.1186/s43141-022-00317-4

Purification, biochemical characterization, and molecular cloning of cellulase from Bacillus licheniformis strain Z9 isolated from soil

J Genet Eng Biotechnol. 2022 Feb 22;20(1):34. doi: 10.1186/s43141-022-00317-4.

Authors

Zainab E Elsababty¹, Samir H Abdel-Aziz¹, Atef M Ibrahim², Adel A Guirgis², Ghada E Dawwam³

Affiliations

¹ Botany and Microbiology Department, Faculty of Science, Benha University, Benha, 13518, Egypt.
² Microbial Biotechnology Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), University of Sadat City, P.O. Box 79, Menoufia, Egypt.
³ Botany and Microbiology Department, Faculty of Science, Benha University, Benha, 13518, Egypt. ghada.ibrahem@fsc.bu.edu.eg.

Abstract

Background: Cellulose is the most prevalent biomass and renewable energy source in nature. The hydrolysis of cellulosic biomass to glucose units is essential for the economic exploitation of this natural resource. Cellulase enzyme, which is largely generated by bacteria and fungus, is commonly used to degrade cellulose. Cellulases are used in a variety of industries, including bioethanol manufacturing, textiles, detergents, drugs, food, and paper. As part of our quest to find an efficient biocatalyst for the hydrolysis of cellulosic biomass, we describe the amplification, cloning, and sequencing of cellulase (cel9z) from Bacillus licheniformis strain Z9, as well as the characterization of the resulting enzyme.

Results: Cellulase was partially purified from B. licheniformis strain Z9 using (NH₄)₂SO₄ precipitation and Sephadex G-100 gel column chromatography with 356.5 U/mg specific activity, 2.1-purification fold, and 3.07 % yield. The nucleotide sequence of the cellulase gene was deposited to the GenBank, B. licheniformis strain Z9 cellulase (cel9z) gene, under accession number MK814929. This corresponds to 1453 nucleotides gene and encodes for a protein composed of 484 amino acids. Comparison of deduced amino acids sequence to other related cellulases showed that the enzyme cel9z can be classified as a glycoside hydrolase family 9. SDS-PAGE analysis of the purified enzyme revealed that the molecular mass was 54.5 kDa. The optimal enzyme activity was observed at pH 7.4 and 30 °C. The enzyme was found to be strongly inhibited by Mg²⁺ and Na⁺, whereas strongly activated by Fe³⁺, Cu²⁺, and Ca²⁺.

Conclusions: B. licheniformis strain Z9 and its cellulase gene can be further utilized for recombinant production of cellulases for industrial application.

Keywords: Bacillus licheniformis; Biochemical characterization; Cellulases; Cloning; Purification.