Isolation and screening of cellulase producing bacteria isolated from soil

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.

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

Elsababty

Abdel-Aziz

Ibrahim

et al. 2022

Journal of Genetic Engineering and Biotechnology

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A thermo-tolerant cellulase enzyme produced by Bacillus amyloliquefaciens M7, an insight into synthesis, optimization, characterization, and bio-polishing activity

Fouda,

Alshallash,

Atta

et al. 2023

Green Processing and Synthesis

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In the current study, among 36 isolates, the bacterial strain M7 was selected as the highest cellulase producer and underwent traditional and molecular identification as Bacillus amyloliquefaciens M7. The productivity of the cellulase enzyme was optimized using the one-factor-at-a-time method. The optimization analysis showed that the best pH value for cellulase production was 7, in the presence of 1% bacterial inoculum size, 5 g·L−1 of carboxymethyl cellulose, 5 g·L−1 of peptone as nitrogen source, and incubation period of 24 h at a temperature of 35°C. The highest cellulase activity (64.98 U·mL−1) was obtained after optimizing conditions using BOX-Behnken Design. The maximum cellulase yield (75.53%) was obtained after precipitation by 60% ammonium sulfate, followed by purification by dialysis bag and Sephadex G-100 column chromatography. The purified cellulase enzyme was characterized by 6.38-fold enrichment, with specific activity (60.54 U·mg−1), and molecular weight of approximately 439.0 Da. The constituent of purified cellulase was 18 amino acids with high concentrations of 200 and 160 mg·L−1 for glycine and arginine, respectively. The purified cellulase enzyme was more stable and active at pH 8 and an incubation temperature of 50°C. The metal ions CaCl2, NaCl, and ZnO enhanced the activity of purified cellulase enzyme. Finally, the B. amyloliquefaciens M7-cellulase exhibits high bio-polishing activity of cotton fabrics with low weight loss (4.3%) which was attained at a maximum concentration (1%, v/v) for 90 min.

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Cellulolytic Aerobic Bacteria Isolated from Agricultural and Forest Soils: An Overview

Bautista-Cruz,

Aquino-Bolaños,

Hernández-Canseco

et al. 2024

Biology

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This review provides insights into cellulolytic bacteria present in global forest and agricultural soils over a period of 11 years. It delves into the study of soil-dwelling cellulolytic bacteria and the enzymes they produce, cellulases, which are crucial in both soil formation and the carbon cycle. Forests and agricultural activities are significant contributors to the production of lignocellulosic biomass. Forest ecosystems, which are key carbon sinks, contain 20–30% cellulose in their leaf litter. Concurrently, the agricultural sector generates approximately 998 million tons of lignocellulosic waste annually. Predominant genera include Bacillus, Pseudomonas, Stenotrophomonas, and Streptomyces in forests and Bacillus, Streptomyces, Pseudomonas, and Arthrobacter in agricultural soils. Selection of cellulolytic bacteria is based on their hydrolysis ability, using artificial cellulose media and dyes like Congo red or iodine for detection. Some studies also measure cellulolytic activity in vitro. Notably, bacterial cellulose hydrolysis capability may not align with their cellulolytic enzyme production. Enzymes such as GH1, GH3, GH5, GH6, GH8, GH9, GH10, GH12, GH26, GH44, GH45, GH48, GH51, GH74, GH124, and GH148 are crucial, particularly GH48 for crystalline cellulose degradation. Conversely, bacteria with GH5 and GH9 often fail to degrade crystalline cellulose. Accurate identification of cellulolytic bacteria necessitates comprehensive genomic analysis, supplemented by additional proteomic and transcriptomic techniques. Cellulases, known for degrading cellulose, are also significant in healthcare, food, textiles, bio-washing, bleaching, paper production, ink removal, and biotechnology, emphasizing the importance of discovering novel cellulolytic strains in soil.

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Isolation and screening of cellulase producing bacteria isolated from soil

Cited by 3 publications

References 3 publications

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

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

A thermo-tolerant cellulase enzyme produced by Bacillus amyloliquefaciens M7, an insight into synthesis, optimization, characterization, and bio-polishing activity

Cellulolytic Aerobic Bacteria Isolated from Agricultural and Forest Soils: An Overview

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