The Hidden Potential of Plant-Residing Bacteria: A New Lipase for Industrial UseIf you are interested in products related to the research phase in this field, please contact for further inquiries.
Endophytic bacteria, which reside within plant tissues, offer a wealth of untapped potential for biotechnological applications. These microorganisms often form symbiotic relationships with their host plants, contributing to plant health and growth. Among the many enzymes produced by endophytic bacteria, lipases stand out for their industrial relevance. Lipases, which catalyze the hydrolysis of fats and oils, are widely used in various industries, including food processing, pharmaceuticals, and detergents. The discovery of a novel lipase from Bacillus pumilus WSS5, an endophytic bacterium found in Withania somnifera, has opened new avenues for industrial applications.
Fig 1. (A) Tributyrin Agar plate showing a zone of clearance, thus indicating lipolytic activity in Bacillus pumilus WSS5 (B) Olive oil Agar plate showing a zone of clearance, thus indicating lipolytic activity in Bacillus pumilus WSS5. (Khan S. S., et al., 2023)
The isolation of Bacillus pumilus WSS5 from the medicinal plant Withania somnifera represents a significant step in the exploration of endophytic bacteria for industrial enzymes. The process involved collecting plant samples and subjecting them to rigorous surface sterilization to eliminate any external contaminants. The sterilized plant tissues were then cultured on various media to isolate endophytic bacteria. Among the eighteen bacterial endophytes isolated, strain WSS5 exhibited the highest lipase activity, making it a prime candidate for further study.
Identification of the bacterial strain was achieved through biochemical tests and 16S rDNA sequence analysis. The 16S rDNA sequence of WSS5 showed a 97.69% similarity to Bacillus pumilus, confirming its identity. This identification was further supported by phylogenetic analysis, which compared the isolate's sequence with those in the NCBI GenBank database. The results indicated that WSS5 is a strain of Bacillus pumilus, a species known for its ability to produce enzymes with industrial potential.
The purification of the lipase enzyme from Bacillus pumilus WSS5 involved a two-step process: ammonium sulfate precipitation and hydrophobic interaction chromatography. The initial precipitation step resulted in a significant increase in enzyme purity, with a specific activity of 17.3 U/mg. This was followed by chromatography on a Hiload 16/10 Phenyl-Sepharose High-Performance column, which further refined the enzyme's purity.
The purified enzyme was analyzed using SDS-PAGE, revealing a molecular weight of approximately 28 KDa. This molecular weight is comparable to lipases from other bacterial strains, indicating that the enzyme is suitable for industrial applications. Zymogram analysis, which detects enzyme activity on a non-denaturing gel, confirmed the presence of a single active lipase band, ensuring the enzyme's purity and functionality.
The characterization of the purified lipase enzyme from Bacillus pumilus WSS5 involved assessing its activity and stability under various conditions. The optimal temperature for the enzyme was found to be 37°C, with significant stability observed from 10°C to 60°C. This wide temperature range is advantageous for industrial applications, particularly in the detergent industry, where high temperatures are often used.
The enzyme's optimal pH was determined to be 8.0, classifying it as an alkaline lipase. This is particularly significant for the detergent industry, where alkaline conditions are prevalent. The lipase remained stable in a pH range of 6.0 to 9.0, further enhancing its suitability for industrial applications.
Influence of Inhibitors and Metal Ions
The effect of various inhibitors and metal ions on the lipase activity was also investigated. The enzyme was completely inactivated by phenylmethyl sulfonyl fluoride (PMSF), indicating the presence of serine residues in its active site. Other inhibitors like b-mercaptoethanol, DTT, and EDTA had minimal inhibitory effects. The enzyme retained 90-95% of its activity in the presence of metal ions such as Ca2+, Na+, and Mg2+, confirming its non-metallo nature. This independence from metal ions is a desirable trait, as it ensures the enzyme's stability and activity under varying conditions.
Effect of Surfactants and Organic Solvents
The stability of the lipase in the presence of surfactants and organic solvents was also tested. The enzyme retained more than 90% of its activity in the presence of surfactants like SDS, Triton-X100, Tween 20, and Tween 80. This stability in the presence of surfactants is crucial for its application in detergents.
The lipase's activity was also tested in various organic solvents, including acetone, ethanol, methanol, ethyl acetate, and 2-propanol. The enzyme retained high activity in these solvents, except for chloroform, where a significant decrease was observed. This tolerance to organic solvents is another valuable property, as it allows the enzyme to function in non-aqueous environments, expanding its industrial applications.
The substrate specificity of the purified lipase was evaluated using pNP esters with varying fatty acid chain lengths. The enzyme effectively hydrolyzed substrates containing fatty acids ranging from C2 to C18, showing a preference for medium-chain fatty acids (C4, C8, and C10). This broad substrate specificity is advantageous for industrial applications, as it allows the enzyme to act on a variety of substrates.
The kinetic constants of the lipase were determined using pNP-caprylate as a substrate. The Michaelis-Menten constant (Km) was found to be 1.7 mM, indicating a high affinity for the substrate. The maximum velocity (Vmax) was 166.7 µmol/min/mg, and the catalytic rate constant (kcat) was 8,335 sec^-1. These values suggest that the enzyme is highly efficient and suitable for industrial use.
One of the most promising applications of the Bacillus pumilus WSS5 lipase is in the detergent industry. To test its oil-destaining efficiency, the researchers conducted experiments using cotton fabric stained with vegetable oil. The results were striking: while plain water and water with detergent alone were ineffective in removing oil stains, the combination of water, detergent, and the purified lipase resulted in complete stain removal. This finding highlights the enzyme's potential as a detergent additive, enhancing the cleaning efficiency of commercial detergents.
The discovery of the lipase enzyme from Bacillus pumilus WSS5 represents a significant advancement in the field of industrial microbiology. This enzyme, derived from a bacterial endophyte of the medicinal plant Withania somnifera, exhibits a range of properties that make it highly suitable for industrial applications, particularly in the detergent industry. Its stability across a broad temperature and pH range, tolerance to metal ions, surfactants, and organic solvents, along with its broad substrate specificity, make it a versatile and valuable biocatalyst.
Future research may focus on the structural characteristics of the lipase and the cloning of the lipase gene in E. coli. This could provide further insights into the enzyme's mechanism of action and potentially lead to the development of more efficient and stable lipase variants. The study of endophytic bacteria and their enzymes not only expands our understanding of microbial diversity but also opens new avenues for sustainable and efficient industrial processes. The Bacillus pumilus WSS5 lipase is a prime example of the hidden potential within these microscopic inhabitants of plants, waiting to be harnessed for the benefit of modern industries.
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Reference
This article is for research use only and cannot be used for any clinical purposes.
