Lipase Production and Characterization

Soil samples were screened for lipase-producing soil.
bacteria by employing various screening methods to arrive
at two best lipase-producing soil bacterial isolates named
NAULM-1, NAULM-2, NAULM-3, and NAULM-4. The
genomic DNA extracted from these soil isolates was
purified separately and used them as templates for the
amplification of 16S rDNA gene sequences of NAULM-1,
NAULM-2, NAULM-3, and NAULM-4 using Eppendorf
thermal cycler. These 16S rDNA amplicons were purified.
and subjected to automated DNA sequencing on ABI 3730xl
genetic analyzer. The subsequent forward and reverse
sequences of the 16S rDNA genes were aligned to obtain
the consensus sequences that were analyzed with
BLASTN using the NCBI GenBank database. The pairwise
multiple alignment analysis of the ten best-chosen bacterial
strain sequences with the respective NAULM-1, NAULM-
2. NAULM-3 and NAULM-4 sequences were performed using
ClustalW and the results were processed to make the
phylogenetic tree. From these results, the bacterial soil
isolates NAULM-1 was identified as Pseudomonas
pseudoalcaligenes and its NCBI gene bank Accession
number for 16s rDNA gene was obtained as KX553845
while NAULM-2, NAULM-3, and NAULM-4 were identified
as Pseudomonas aeruginosa and the respective NCBI
GenBank Accession numbers for 16S rDNA genes were
obtained as KX592865, KX570620, and KX592864.

The manuscript is about the purification and characterization of the lipase from an endophyticbacteria isolated from the stem of Withania somnifera L. Dunal. Endophytes have long been acknowledged as enzyme manufacturers for their natural requirements, more specifically as manufacturers of a sequence of enzymes needed to penetrate and colonize their plant hosts, including hydrolytic and oxidative enzymes, however, the possible use of endophytes as sources of industrial enzymes has not received much attention. The current findings suggest the potential of B. pumilus WSS5 lipase owing to promising properties such as the alkali-thermostability, organic solvent-tolerance, and broad substrate specificity that makes it a strong candidate for application in detergent formulations, biotransformation, industries, and medicine and moreover this is the first report on on the purification and characterization of lipase from bacterial endophyte isolated from the stem of Withania somnifera L.

This study reports on the microbial screening for a bio-delipidation system of lipid-rich slaughterhouse wastewater, and on the optimal conditions for lipase production and activity. In this study, swaps were collected from the poultry slaughterhouse discharge point for screening, isolation and characterisation of lipolytic microorganisms using molecular techniques. Bacillus cereus strains AB1 (BF3) and CC-1 (B3O) were identified using 16S rRNA techniques. Maximal lipase production for both strains was observed between pH 6-8 and 45-60 °C. Optimal lipase activity for BF3 and B30 was achieved at pH 8 and 60 °C, and at pH 8.83 and 45°C, respectively. After partial purification, increased activity was observed for BF3 and B30 strains. Solvents, metal ions and detergents (triclosan and trichlorocarbonilide) affected lipase activity. It was concluded that BF3 and B30 strains were suitable candidates for bio-delipidation systems.

Lipolytic enzymes from endophytic microbes have been the focus of intense and growing research in view of the applicability of such enzymes in various industrial applications and endophytic microorganisms may emerge as a potential source of lipases with distinct characteristics. The current work involved purification and characterization of the lipase from an endophytic-bacteria isolated from the stem of Withania somnifera L. Dunal. Based on the analysis of morphology and 16S rDNA sequence, the endophytic bacteria was identified as Bacillus pumilus and designated as B. pumilus WSS5. Purified lipase from the B. pumilus WSS5 was found to be of 28 KDa as revealed by SDS-PAGE with optimum activity at 37°C and pH 8. The enzyme was found to be stable over a broad pH range of 6.0–9.0 and temperature stability was in the range of 10°C to 60°C. The enzyme activity was enhanced in the presence of organic solvents like Ethyl acetate, Methanol, 2-propanol, acetone, and ethanol. The stability of...

Lipolytic enzymes from endophytic microbes have been the focus of intense and growing research in view of the applicability of such enzymes in various industrial applications and endophytic microorganisms may emerge as a potential source of lipases with distinct characteristics. The current work involved purification and characterization of the lipase from an endophytic-bacteria isolated from the stem of Withania somnifera L. Dunal. Based on the analysis of morphology and 16S rDNA sequence, the endophytic bacteria was identified as Bacillus pumilus and designated as B. pumilus WSS5. Purified lipase from the B. pumilus WSS5 was found to be of 28 KDa as revealed by SDS-PAGE with optimum activity at 37°C and pH 8. The enzyme was found to be stable over a broad pH range of 6.0–9.0 and temperature stability was in the range of 10°C to 60°C. The enzyme activity was enhanced in the presence of organic solvents like Ethyl acetate, Methanol, 2-propanol, acetone, and ethanol. The stability of...

Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3.), has attracted a lot of attention in recent years because of its diverse biotechnological applications. Lipase occurs widely in nature, but plant lipase, is significant because of its unique substrate selectivity/specificity. Lipase was isolated from the endosperm of 4-day germinated seeds of Cumcumeropsis manii using extraction buffer containing 1.95% (w/v) Tween 80. The crude enzyme was purified through a four step purification procedures of combined ammonium sulphate ((NH 4) SO 4) and cold acetone precipitated proteins, followed by dialysis and repeated gel filtration on sephadex G-200 column chromatography. This yielded enzyme with two activity peaks of lipase indicating the presence of two forms of lipase identified as acidic and alkaline lipases based on their respective pH optimum at 5.9 and 7.5. The acid lipase was found to be stable between pH 4.5 and 6.0 while the alkaline lipase was stable between 6.5 and 8.0 pH ranges. ...

Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3.), has attracted a lot of attention in recent years because of its diverse biotechnological applications. Lipase occurs widely in nature, but plant lipase, is significant because of its unique substrate selectivity/specificity. Lipase was isolated from the endosperm of 4-day germinated seeds of Cumcumeropsis manii using extraction buffer containing 1.95% (w/v) Tween 80. The crude enzyme was purified through a four step purification procedures of combined ammonium sulphate ((NH4) SO4) and cold acetone precipitated proteins, followed by dialysis and repeated gel filtration on sephadex G-200 column chromatography. This yielded enzyme with two activity peaks of lipase indicating the presence of two forms of lipase identified as acidic and alkaline lipases based on their respective pH optimum at 5.9 and 7.5. The acid lipase was found to be stable between pH 4.5 and 6.0 while the alkaline lipase was stable between 6.5 and 8.0 pH ranges. Th...

Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3.), has attracted a lot of attention in recent years because of its diverse biotechnological applications. Lipase occurs widely in nature, but plant lipase, is significant because of its unique substrate selectivity/specificity. Lipase was isolated from the endosperm of 4-day germinated seeds of Cumcumeropsis manii using extraction buffer containing 1.95% (w/v) Tween 80. The crude enzyme was purified through a four step purification procedures of combined ammonium sulphate ((NH 4) SO 4) and cold acetone precipitated proteins, followed by dialysis and repeated gel filtration on sephadex G-200 column chromatography. This yielded enzyme with two activity peaks of lipase indicating the presence of two forms of lipase identified as acidic and alkaline lipases based on their respective pH optimum at 5.9 and 7.5. The acid lipase was found to be stable between pH 4.5 and 6.0 while the alkaline lipase was stable between 6.5 and 8.0 pH ranges. ...

Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3.), has attracted a lot of attention in recent years because of its diverse biotechnological applications. Lipase occurs widely in nature, but plant lipase, is significant because of its unique substrate selectivity/specificity. Lipase was isolated from the endosperm of 4-day germinated seeds of Cumcumeropsis manii using extraction buffer containing 1.95% (w/v) Tween 80. The crude enzyme was purified through a four step purification procedures of combined ammonium sulphate ((NH 4) SO 4) and cold acetone precipitated proteins, followed by dialysis and repeated gel filtration on sephadex G-200 column chromatography. This yielded enzyme with two activity peaks of lipase indicating the presence of two forms of lipase identified as acidic and alkaline lipases based on their respective pH optimum at 5.9 and 7.5. The acid lipase was found to be stable between pH 4.5 and 6.0 while the alkaline lipase was stable between 6.5 and 8.0 pH ranges. The two lipase fractions showed optimum activities at 37 o C and were found to be stable at 45 o C after incubation for 1 hr. Ca 2+ was observed to be a very good activator and Pb 2+ a potent inhibitor of the two forms of lipases. The acid lipase was purified to 11.0 fold while the alkaline lipase showed 5.4 purification fold. The acid lipase showed a Vmax of 166.7 Unit and Km of 15.67 g L-1 while the alkaline lipase showed Vmax of 142.8 Unit and Km of 13.28 g L-1 .