Low-Temperature Lipase from Psychrotrophic Pseudomonas sp. Strain KB700A (original) (raw)
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Production, partial purification and characterisation of lipases from Pseudomonas fragi CRDA 037
Process Biochemistry, 1997
Psychrotrophic Pseudomonas ADT3 (NCBI GenBank Acc.no.JX914667) is capable of growth on lipid as the sole carbon source. In this paper, we report the purification and characterization of an extracellular lipase from psychrotroph, isolated from soil sample of Ny-Alesund, Svalbard, Arctic region. The Pseudomonas ADT3 isolate produces lipase enzyme in the extracellular minimal media with only 1% olive oil. The lipase was purified from the concentrated culture supernatant. The crude enzyme was partially purified by saturated ammonium sulphate precipitation followed by extensive dialysis. Enzyme activity was found to be induced 6-folds in presence of 1.2 mM lead ion but strongly inhibited by heavy metals Hg 2+ as well as EDTA and β-mercaptoethanol. The purified lipase has activity at two pH optima of pH i.e. pH 3.5 and 8.5. Optimum temperature for lipase activity was recorded at 22cC. The purified active fraction of lipase exhibits specific activity of 527.8 U/mg. The Vmax and Km was 144.93 U/mg/min and 0.260 mM respectively determined using Lineweaver-Burk plot. Zymogram analysis revealed prominent lipase band at 13.9 kDa range in the 80% saturated ammonium sulphate purified enzyme fraction.
Brazilian Journal of Microbiology
Cold-active lipases are presently employed extensively in the detergent, chemical intermediate, fine chemical, food, and pharmaceutical industries. Seven cold-adaptive bacteria were isolated from the Mediterranean Sea near Alexandria, Egypt, and tested for their ability to produce cold-active lipase, with the highest activity at 10 °C. The most potent isolate was Pseudomonas sp. A6. To determine the most important variables, the bacterium was exposed to a necessary medium component and environmental factor screening using a single factor-at-a-time approach, followed by a multifactorial Plackett-Burman design strategy. After purification and characterization, the optimal activity levels for the cold-active lipase were figured out. Inoculation of Pseudomonas A6 under near optimum conditions using medium consisting of (g/L) peptone 7.14; soybean oil 7.5% (v/v); K2HPO4, 0.4; MgSO4, 0.1; glucose 2; pH 8; and temperature 10 °C led to a maximum lipase activity anticipated to be 23.36 U/mL....
Cloning and Expression of a Novel Lipase Gene From Pseudomonas fluorescens B52
Molecular Biotechnology, 2005
A novel lipase gene (lipB52) was isolated directly from the genomic DNA of Pseudomonas fluorescens B52 with the genome-walking method, an effective method for isolating lipase gene from bacteria. There was an open reading frame (ORF) of 1854 bp, which encoded 617 amino acids. The lipase gene (lipB52) was cloned into expression vector pPIC9K and successfully integrated into a heterologous fungal host, Pichia pastoris KM71, and the recombinant Pichia pastoris were screened with a high throughput method. The recombinant was induced by methanol to secrete active lipase into the culture medium. The recombinant lipase LipB52 was also purified and characterized. The optimum temperature for the purified lipase LipB52 was 40°C at pH 8.0. It exhibited better thermostability and pH stability than its homologs.
Characterization of a lipase from a newly isolated Pseudomonas sp
Iranian journal of microbiology, 2013
Lipases are valuable biocatalysts which are widely used in the detergent, food, dairy and pharmaceutical industries. The aims of the present study included the isolation of a lipase-producer from industrial zones and the partial characterization of the enzyme. A number of bacteria were isolated from sites related to the oil industries. An isolate forming a halo zone in a selective medium (TW agar) was then selected and grown on a medium suitable for the production of lipase. The isolate was subsequently identified by the 16S rRNA sequencing method, and its enzyme activity was measured by a spectrophotometer using pNPP as a substrate. The selected isolate was identified by the molecular method as Pseudomonas sp. Its extracellular lipase activity was 41.5 ± 1.4 U/ml, and the high affinity of this enzyme for the substrate was indicated by the kinetic parameters of Km and Vm, which were estimated by the the Lineweaver-Burk plot as 0.77 mM and 49.5 U/ml, respectively. Activation energy o...
Psychrophiles are particularly important due to flexible structure of their enzyme and tremendous potential for use in low temperature processes. A potential lipase producing novel psychrophilic bacteria G3 was isolated from rice rhizosphere of Nainital, India. The microscopic study showed it was gram negative, aerobic, rod shaped and motile organism. It was able to grow at 4ºC to 37ºC and at wide range of pH 5.0 to 10.0. On solid media colonies were round, smooth, viscous and light yellow in colour. 16S rDNA sequencing and bacterial identification was done. The obtained nucleotide sequence (1388 bp) compared with the NCBI databases through BLAST. The sequence showed 100% similarity with Pseudomonas vancouverensis- A-18 (HQ202824.1) and lowest e-value, so based on the identity unknown bacterium was identified as Pseudomonas vancouverensis. It was able to produce maximum extracellular lipase at its optimum growth conditions i.e. 48 h incubation period at 10°C and 8.0 pH.
Biochemical properties of cloned lipases from the Pseudomonas family
Biochimica et biophysica acta, 1995
Three Pseudomonas lipases, representing three subfamilies, were analysed for pH optima, destabilization by EGTA and surfactants, phospholipase and cholesterolesterase side activities. All the Pseudomonas lipases tested showed alkaline pH optima. The Pseudomonas cepacia and the P. pseudoalcaligenes lipases were totally inhibited by EGTA at pH 9, and the latter was also fully inhibited at pH 7. The lipase from P. mendocina was not inhibited by EGTA at any of the pH values tested. These findings indicate that a calcium binding site exists in some of the Pseudomonas lipases. The P. pseudoalcaligenes, P. cepacia and P. mendocina lipases were inhibited by the anionic surfactant SDS at concentrations between 0.01-0.5 mg/ml. The P. pseudoalcaligenes and P. cepacia lipases were not inhibited by the nonionic surfactant Brij35 in concentration up to 1 mg/ml, whereas the lipase from P. mendocina was inhibited at 0.1 mg/ml. The P. pseudoalcaligenes and P. cepacia lipases were found to possess hi...
Characterization of the lipA gene encoding the major lipase from Pseudomonas aeruginosa strain IGB83
Applied Microbiology and Biotechnology, 2001
The lipases produced by Pseudomonas have a wide range of potential biotechnological applications. Pseudomonas aeruginosa IGB83 was isolated as a highly lipolytic strain which produced a thermotolerant and alkaline lipase. In the present work, we have characterized the P. aeruginosa IGB83 gene (lipA) encoding this enzyme. We describe the construction of a lipA mutant and report on the effect of two carbon sources on lipase expression.
2005
Psuedomonas aeruginosa MTCC 2488 produced lipase on Rhodamine B agar plates containing olive oil. Extra-cellular lipase activity was analyzed spectrophotometrically using Tween-20 as well as olive oil as substrate. The semipurified enzyme, precipitated by 30% saturated ammonium sulphate, showed 20.79 fold increase in specific activity (U/mg) and reduction in carbohydrate content to 1.7% as compared to the crude enzyme. The enzyme hydrolyzed Tween-20 and -40 better than Tween-60 and -80. Lipase has been found to be thermostable with maximum activity at 55-60C but marked decrease was observed above this temperature. Ca seemed to play an important role in the thermostability as 97% of enzyme activity was retained after 2 hr incubation at 65C and 1hr incubation at 70C in presence of 10 mM CaCl2. However, thermostability of the enzyme was decreased considerably in presence of 5 mM EDTA, confirming the enzyme to be a metalloprotein. Lipase has been found to be stable in presence of 30% ac...
Applied and Environmental Microbiology
An extracellular lipase from the low-water-tolerant bacterium P. aeruginosa YS-7 was produced, purified, and characterized with respect to its functional properties in aqueous solutions and organic solvents. The enzyme was partially released from the cells during fermentation in defined medium with 5% (wt/vol) soybean oil. Approximately one-half of the total culture activity remained in solution after removal of cells. More than 95% of the activity was found in culture supernatant after mild detergent treatment (10 mM sodium deoxycholate) or after shifting the carbon source during the fermentation from triglyceride to a free fatty acid. The enzyme was recovered from an acetone precipitate of the whole culture and purified by hydrophobic interaction chromatography, yielding a preparation having a specific activity of about 1,300 mumol of fatty acid mg-1 h-1. The lipase (molecular size, approximately 40 kDa) hydrolyzes a variety of fatty acid esters and has an optimum pH of about 7. T...