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Characterization of Thermostable Cellulase from Bacillus licheniformis PANG L Isolated from the Himalayan Soil.

ABSTRACT: This study aimed to isolate, purify, and characterize a potential thermophilic cellulase-producing bacterium from the Himalayan soil. Eleven thermophilic bacteria were isolated, and the strain PANG L was found to be the most potent cellulolytic producer. Morphological, physiological, biochemical, and molecular characterization identified PANG L as Bacillus licheniformis. This is the first study on the isolation of thermostable cellulase-producing Bacillus licheniformis from the Himalayan soil. This bacterium was processed for the production of cellulase enzyme. The optimum conditions for cellulase production were achieved at 45°C after 48 h of incubation at pH 6.5 in media-containing carboxymethyl cellulose (CMC) and yeast extract as carbon and nitrogen sources, respectively, in a thermo-shaker at 100 rpm. The enzyme was partially purified by 80% ammonium sulphate precipitation followed by dialysis, resulting in a 1.52-fold purification. The optimal activity of partially purified cellulase was observed at a temperature of 60°C and pH 5. The cellulase enzyme was stable within the pH ranges of 3-5 and retained 67% of activity even at 55°C. Cellulase activity was found to be enhanced in the presence of metal ions such as Cd²⁺, Pb²⁺, and Ba²⁺. The enzyme showed the highest activity when CMC was used as a substrate, followed by cellobiose. The K_m and V_max values of the enzyme were 1.8 mg/ml and 10.92 μg/ml/min, respectively. The cellulase enzyme obtained from Bacillus licheniformis PANG L had suitable catalytic properties for use in industrial applications.

SUBMITTER: Shyaula M

PROVIDER: S-EPMC10484656 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Characterization of Thermostable Cellulase from Bacillus licheniformis PANG L Isolated from the Himalayan Soil.

Shyaula Manita M Regmi Sunil S Khadka Deegendra D Poudel Ram Chandra RC Dhakal Agni A Dhakal Agni A Koirala Devesh D Sijapati Jaishree J Singh Anjana A Maharjan Jyoti J

International journal of microbiology 20230831

This study aimed to isolate, purify, and characterize a potential thermophilic cellulase-producing bacterium from the Himalayan soil. Eleven thermophilic bacteria were isolated, and the strain PANG L was found to be the most potent cellulolytic producer. Morphological, physiological, biochemical, and molecular characterization identified PANG L as Bacillus licheniformis. This is the first study on the isolation of thermostable cellulase-producing Bacillus licheniformis from the Him ...[more]

PMID: 37692921

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Project description:ObjectivesThe data presented in this study were collected with the aim of obtaining the complete genomes of specific strains of Bacillus bacteria, namely, Bacillus licheniformis T5. This strain was chosen based on its enzymatic activities, particularly amylolytic activity. In this study, nanopore sequencing technology was employed to obtain the genome sequences of this strain. It is important to note that these data represent a focused objective within a larger research context, which involves exploring the biochemical features of promising Bacilli strains and investigating the relationship between enzymatic activity, phenotypic features, and the microorganism's genome.Data descriptionIn this study, the whole-genome sequence was obtained from one Bacillus strain, Bacillus licheniformis T5, isolated from soil samples in Kazakhstan. Sample preparation and genomic DNA library construction were performed according to the Ligation sequencing gDNA kit (SQK-LSK109) protocol and NEBNext module. The prepared library was sequenced on a MinION instrument (Oxford Nanopore Technologies nanopore sequencer with a maximum throughput of up to 30 billion nucleotides per run and no limit on read length), using a flow cell for nanopore sequencing FLO-MIN106D. The genome de novo assembly was performed using the long sequencing reads generated by MinION Oxford Nanopore platform. Finally, one circular contig was obtained harboring a length of 4,247,430 bp with 46.16% G + C content and the mean contig 428X coverage. B. licheniformis T5 genome assembly annotation revealed 5391 protein-coding sequences, 81 tRNAs, 51 repeat regions, 24 rRNAs, 3 virulence factors and 53 antibiotic resistance genes. This sequence encompasses the complete genetic information of the strain, including genes, regulatory elements, and noncoding regions. The data reveal important insights into the genetic characteristics, phenotypic traits, and enzymatic activity of this Bacillus strain. The findings of this study have particular value to researchers interested in microbial biology, biotechnology, and antimicrobial studies. The genomic sequence offers a foundation for understanding the genetic basis of traits such as endospore formation, alkaline tolerance, temperature range for growth, nutrient utilization, and enzymatic activities. These insights can contribute to the development of novel biotechnological applications, such as the production of enzymes for industrial purposes. Overall, this study provides valuable insights into the genetic characteristics, phenotypic traits, and enzymatic activities of the Bacillus licheniformis T5 strain. The acquired genomic sequences contribute to a better understanding of this strain and have implications for various research fields, such as microbiology, biotechnology, and antimicrobial studies.

Project description:BackgroundThe rising concerns about the scarcity of fossil fuels, the emission of green house gasses and air pollution by incomplete combustion of fossil fuel have also resulted in an increasing focus on the use of cellulases to perform enzymatic hydrolysis of the lignocellulosic materials for the generation of bioethanol. The aim of this study was to isolate a potential thermo-solvent tolerant cellulase producing bacterium from natural resources, and then applied for purification and characterization. The purified enzyme was to be accessible for the bioethanol production as well as industrial exploitation (discuss in our next study).ResultsIt is the first instance when thermo-solvent tolerant cellulase producing bacterium was isolated from soil sample. The culture was identified as Bacillus vallismortis RG-07 by 16S rDNA sequence analysis. Bacillus vallismortis RG-07 reported maximum cellulase production from sugarcane baggase (4105 U ml(-1)) used as agro-waste carbon source. The cellulase enzyme produced by the Bacillus sp. was purified by (NH4)2SO4 precipitation, ion exchange and gel filtration chromatography, with overall recovery of 28.8%. The molecular weight of purified cellulase was 80 kDa as revealed by SDS-PAGE and activity gel analysis. The optimum temperature and pH for enzyme activity was determined as 65°C and 7.0 and it retained 95 and 75% of activity even at 95°C, and 9.0 respectively. The enzyme activity was enhanced in the presence of organic solvents (30%) n-dodecane, iso-octane, n-decane, xylene, toluene, n-haxane, n-butanol, and cyclohexane, after prolonged incubation (7 days). The enzyme activity was also stimulated by Ca(2+), mercaptoethanol, Tween-60, and Sodium hypochloride whereas strongly inhibited by Hg. Kinetic analysis of purified enzyme showed the Km and Vmax to be 1.923 mg ml(-1) and 769.230 μg ml(-1) min(-1), respectively.ConclusionThe unique property of solvent-thermostable-alkalophilic, nature proves the potential candidature of this isolate for current mainstream biomass conversion into fuel and other industrial process.

Project description:BackgroundBacillus strains are well recognized for their inherent production of bioactive compounds that exhibit antibacterial and anticancer properties. This study aims to evaluate the antimicrobial and anticancer effects of the secondary metabolite isolated from Bacillus licheniformis and Bacillus siamensis strain.Material and methodWe developed and purified a new soil-derived Bacillus strain to study its metabolites on cancer cells and bacteria. After evaluating the antimicrobial effects of the selected strains' secondary metabolites by well diffusion, growth conditions and temperature optimised using liquid-liquid extraction, secondary metabolites isolated, and active compounds identified using GC-MS. Evaluation of PC-3 and HPrEpC cytotoxicity. AV/PI staining and comet assay assessed necrosis and apoptosis. Real-time PCR measured apoptotic gene expression. Finally, the scratch test measured cell movement.ResultsBacillus strain metabolites exhibit dual-purpose antimicrobial and anticancer properties. Bacillus licheniformis isolate 56 and S2-G12 isolate 60 demonstrated the greatest antibacterial activity. Among all Bacillus isolates, isolates 56 (Bacillus licheniformis) and 60 (Bacillus siamensis strain) had the highest antibacterial activity. Crude extracts obtained from strains 56 and 60 decreased PC-3 cell viability in a dose-dependent manner. At 200 µg/mL, the survival rate of cells treated with strain 56 and 60 crude extract was 23% and 25%, respectively (p < 0.001). The treatment of PC-3 cells with strains 56 and 60 crude extract led to considerable apoptosis (46.2% and 50.09%, respectively) compared to the control group. After treatment with the crude extract from strains 56 and 60 at an IC50 concentration, a significant number of PC-3 cells showed comet formation, indicating DNA fragmentation. Metabolites extracted from strain 56 and 60 enhanced caspase 3, caspase 8, and Bax genes expression and reduced Bcl-2 expression (p < 0.001). Cell migration was also prevented.ConclusionOur findings show that the secondary metabolites of B. licheniformis and B. siamensis have antibiotic and anticancer properties. However in vivo studies are necessary to confirm these findings.

Dataset Information

Characterization of Thermostable Cellulase from Bacillus licheniformis PANG L Isolated from the Himalayan Soil.

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Characterization of Thermostable Cellulase from <i>Bacillus licheniformis</i> PANG L Isolated from the Himalayan Soil.

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