Purification and characterization of Pseudomonas fluorescens SIK W1 lipase expressed in Escherichia coli

Lee, Young Phil; Chung, Guk Hoon; Rhee, Jae-Sung

doi:10.1016/0005-2760(93)90200-s

Cited by 76 publications

(6 citation statements)

References 42 publications

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“…The optimal temperature and pH of LipR were slightly higher than those of LipAAc (50 o C, pH 7.0) from Acidobacteria sp. [8] and PSL (45 o C, pH 8.5) from P. fluorescens SIK W1 [18]. In addition, LipAAc from Acidobacteria sp., which is a member of moderately thermostable bacterial lipases, retained 53% and 33% of its activity after incubation at 60 o C and 70 o C, respectively, for 1 h [8].…”

Section: Discussionmentioning

confidence: 99%

“…LipR does not contain a cysteine residue. Like other lipases [1,15,18] in subfamily I.3, LipR also contains a C-terminal targeting signal region consisting of six tandem repeats of the nine-residue motif GGXGXDXUX (U, hydrophobic amino acids; X, any amino acid residue; Fig. 1, residues 319-336; 329-346; 474-491), an 18-residue amphipathic α-helix (Fig.…”

Section: Gene Cloning and Sequence Analysismentioning

confidence: 99%

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Characterizing LipR from Pseudomonas sp. R0-14 and Applying in Enrichment of Polyunsaturated Fatty Acids from Algal Oil

Xu¹,

Zhang²,

Yan³

2015

Journal of Microbiology and Biotechnology

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In this study, Pseudomonas R0-14, which was isolated from Arctic soil samples, showed a clear halo when grown on M9 medium agarose plates containing olive oil-rhodamine B as substrate, suggesting that it expressed putative lipase(s). A putative lipase gene, lipR, was cloned from R0-14 by genome walking and Touchdown PCR. lipR encodes a 562-amino-acid polypeptide showing a typical α/β hydrolase structure with a catalytic triad consisting of Ser153-Asp202-His260 and one α-helical lid (residues 103-113). A phylogenetic analysis revealed that LipR belongs to the lipase subfamily I.3. LipR was successfully expressed in Escherichia coli, purified, and biochemically characterized. Recombinant LipR exhibited its maximum activity towards p-nitrophenyl butyrate at pH 8.5 and 60°C with a Km of 0.37 mM and a kcat of 6.42 s(-1). It retained over 90% of its original activity after incubation at 50°C for 12 h. In addition, LipR was activated by Ca(2+), Mg(2+), Ba(2+), and Sr(2+), while strongly inhibited by Cu(2+), Zn(2+), Mn(2+), and ethylenediaminetetraacetic acid. Moreover, it showed a certain tolerance to organic solvents, including acetonitrile, isopropanol, acetone, methanol, and tert-butanol. When algal oil was hydrolyzed by LipR for 24 h, there was an enrichment of n-3 long-chain polyunsaturated fatty acids, including eicosapentaenoic acid (1.22%, 1.65-fold), docosapentaenoic acid (21.24%, 2.04-fold), and docosahexaenoic acid (36.98%, 1.33-fold), and even a certain amount of diacylglycerols was also produced. As a result, LipR has great prospect in industrial applications, especially in food and/or cosmetics applications.

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Section: Discussionmentioning

confidence: 99%

Section: Gene Cloning and Sequence Analysismentioning

confidence: 99%

Characterizing LipR from Pseudomonas sp. R0-14 and Applying in Enrichment of Polyunsaturated Fatty Acids from Algal Oil

Xu¹,

Zhang²,

Yan³

2015

Journal of Microbiology and Biotechnology

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“…The inhibitory activity of pancreatic lipase (PLIA) was determined using a spectrophotometric method that measures the release of p -nitrophenol using p -nitrophenyl palmitate (PNP) as substrate . Briefly, the lyophilized WSF of FM was dissolved in buffer A (50 mM TRIS-HCl, with 0.12 mM NaCl, pH 7.2) at a protein concentration of 2.5 mg/mL.…”

Section: Methodsmentioning

confidence: 99%

“…and Lactiplantibacillus spectrophotometric method that measures the release of p-nitrophenol using p-nitrophenyl palmitate (PNP) as substrate. 21 Briefly, the lyophilized WSF of FM was dissolved in buffer A (50 mM TRIS-HCl, with 0.12 mM NaCl, pH 7.2) at a protein concentration of 2.5 mg/mL. The microplate reader was set at 37 °C and 162 μL of buffer B (75 mM TRIS-HCl, pH 8.5), 10 μL of PNP solution (10 mM in acetonitrile and dissolved in ethanol to a final concentration of 3.33 mM of PNP), 16 μL of sample solution, and 12 μL of enzyme solution (5 mg/mL) were added.…”

Section: ■ Introductionmentioning

confidence: 99%

Novel Peptides in Fermented Milk with Specific Lactobacillus Strains Potential Antiobesity Effect: In Vitro and in Silico Analysis

Manzanarez-Quin

García-Romo

Beltrán-Barrientos

et al. 2023

ACS Food Sci. Technol.

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The objective was to investigate the potential antiobesity effect of fermented milks (FMs) with 11 strains of specific Lactobacillus strains isolated from artisanal Mexican cheeses. First, pancreatic lipase inhibitory activity (PLIA) was determined in all FMs. Then, the FM with the most PLIA was selected for peptide identification by high-performance liquid chromatography (HPLC)/tandem mass spectrometry (MS–MS) and for adipogenesis studies. FMs with Limosilactobacillus fermentum J20 (J20) displayed the highest (p < 0.05) PLIA (81.70 ± 2.24%) and the lowest IC50 (0.05 0.05 mg/mL) of all. Also, FM J20 inhibited lipid accumulation in a 3T3-L1 cell line. Eight novel identified peptides released by in silico digestion from the fraction with the most PLIA (p < 0.05) presented sequences that strongly bind enzyme active sites and may be responsible for lipid accumulation inhibition during adipogenesis. Thus, FM J20 may be used for the development of a dairy product with potential antiobesity effects.

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“…Lipase activity was carried out by using p-nitrophenyl palmitate (C16, p-NPP) as a substrate unless otherwise indicated [33]. The reaction mixture consisted of 0.1 mL enzyme extract (5 µg/mL), 0.8 mL of 50 mM Tris-HCl (pH 8.0), and 0.1 mL of 10 mM p-NPP dissolved in isopropyl alcohol.…”

Section: Enzyme Assaysmentioning

confidence: 99%

Characterization of Two Unique Cold-Active Lipases Derived from a Novel Deep-Sea Cold Seep Bacterium

et al. 2021

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The deep ocean microbiota has unexplored potential to provide enzymes with unique characteristics. In order to obtain cold-active lipases, bacterial strains isolated from the sediment of the deep-sea cold seep were screened, and a novel strain gcc21 exhibited a high lipase catalytic activity, even at the low temperature of 4 °C. The strain gcc21 was identified and proposed to represent a new species of Pseudomonas according to its physiological, biochemical, and genomic characteristics; it was named Pseudomonas marinensis. Two novel encoding genes for cold-active lipases (Lipase 1 and Lipase 2) were identified in the genome of strain gcc21. Genes encoding Lipase 1 and Lipase 2 were respectively cloned and overexpressed in E. coli cells, and corresponding lipases were further purified and characterized. Both Lipase 1 and Lipase 2 showed an optimal catalytic temperature at 4 °C, which is much lower than those of most reported cold-active lipases, but the activity and stability of Lipase 2 were much higher than those of Lipase 1 under different tested pHs and temperatures. In addition, Lipase 2 was more stable than Lipase 1 when treated with different metal ions, detergents, potential inhibitors, and organic solvents. In a combination of mutation and activity assays, catalytic triads of Ser, Asp, and His in Lipase 1 and Lipase 2 were demonstrated to be essential for maintaining enzyme activity. Phylogenetic analysis showed that both Lipase 1 and Lipase 2 belonged to lipase family III. Overall, our results indicate that deep-sea cold seep is a rich source for novel bacterial species that produce potentially unique cold-active enzymes.

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Purification and characterization of Pseudomonas fluorescens SIK W1 lipase expressed in Escherichia coli

Cited by 76 publications

References 42 publications

Characterizing LipR from Pseudomonas sp. R0-14 and Applying in Enrichment of Polyunsaturated Fatty Acids from Algal Oil

Characterizing LipR from Pseudomonas sp. R0-14 and Applying in Enrichment of Polyunsaturated Fatty Acids from Algal Oil

Novel Peptides in Fermented Milk with Specific Lactobacillus Strains Potential Antiobesity Effect: In Vitro and in Silico Analysis

Characterization of Two Unique Cold-Active Lipases Derived from a Novel Deep-Sea Cold Seep Bacterium

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