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A newly isolated thermostable lipase from Bacillus sp.

Shariff FM, Rahman RN, Basri M, Salleh AB - Int J Mol Sci (2011)

Bottom Line: Recombinant lipase was successfully overexpressed with a 178-fold increase in activity compared to crude native L2 lipase.The purified lipase was found to be reactive at a temperature range of 55-80 °C and at a pH of 6-10.The optimum activity was found to be at 70 °C and pH 9.

View Article: PubMed Central - PubMed

Affiliation: Enzyme and Microbial Technology Research, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia; E-Mails: ferrol2506@gmail.com (F.M.S.); abubakar@biotech.upm.edu.my (A.B.S.).

ABSTRACT
A thermophilic lipolytic bacterium identified as Bacillus sp. L2 via 16S rDNA was previously isolated from a hot spring in Perak, Malaysia. Bacillus sp. L2 was confirmed to be in Group 5 of bacterial classification, a phylogenically and phenotypically coherent group of thermophilic bacilli displaying very high similarity among their 16S rRNA sequences (98.5-99.2%). Polymerase chain reaction (PCR) cloning of L2 lipase gene was conducted by using five different primers. Sequence analysis of the L2 lipase gene revealed an open reading frame (ORF) of 1251 bp that codes for 417 amino acids. The signal peptides consist of 28 amino acids. The mature protein is made of 388 amino acid residues. Recombinant lipase was successfully overexpressed with a 178-fold increase in activity compared to crude native L2 lipase. The recombinant L2 lipase (43.2 kDa) was purified to homogeneity in a single chromatography step. The purified lipase was found to be reactive at a temperature range of 55-80 °C and at a pH of 6-10. The L2 lipase had a melting temperature (Tm) of 59.04 °C when analyzed by circular dichroism (CD) spectroscopy studies. The optimum activity was found to be at 70 °C and pH 9. Lipase L2 was strongly inhibited by ethylenediaminetetraacetic acid (EDTA) (100%), whereas phenylmethylsulfonyl fluoride (PMSF), pepstatin-A, 2-mercaptoethanol and dithiothreitol (DTT) inhibited the enzyme by over 40%. The CD spectra of secondary structure analysis showed that the L2 lipase structure contained 38.6% α-helices, 2.2% ß-strands, 23.6% turns and 35.6% random conformations.

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Homology lineup of lipases from several bacteria species.Note: All the lipases were labeled based on their accession numbers submitted to the GenBank. Geobacillus sp. T1 lipase (AY 260764); Bacillus sp. L2 lipase (AY 855077); B. thermoleoverans ID-1 lipase (AF 134840); B. stearothermophilus P1 lipase (AF 237623); B. stearothermophilus LI lipase (JW 0068); B. cereus ATCC 10987 lipase (NP 978931); Clostridium tetani lipase (NP 781602); Staphylococcus aureus subsp. aureus MRSA252 lipase (YP 042090); Burkholderia pseudomallei K96243 lipase (YP 111747) and Pseudomonas putida lipase (NP 744233).
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f2-ijms-12-02917: Homology lineup of lipases from several bacteria species.Note: All the lipases were labeled based on their accession numbers submitted to the GenBank. Geobacillus sp. T1 lipase (AY 260764); Bacillus sp. L2 lipase (AY 855077); B. thermoleoverans ID-1 lipase (AF 134840); B. stearothermophilus P1 lipase (AF 237623); B. stearothermophilus LI lipase (JW 0068); B. cereus ATCC 10987 lipase (NP 978931); Clostridium tetani lipase (NP 781602); Staphylococcus aureus subsp. aureus MRSA252 lipase (YP 042090); Burkholderia pseudomallei K96243 lipase (YP 111747) and Pseudomonas putida lipase (NP 744233).

Mentions: The plasmid harboring the L2 gene was sequenced and shown to contain a 1.2 kb sequence coding for lipase. The complete sequence of the ORF of the L2 lipase gene was submitted to GenBank and was assigned accession number AY855077. The amino acid composition within the ORF was determined by the ProtParam Tool of the ExPASy Molecular Biology server [6]. The molecular mass and the isoelectric point (pI) were predicted to be 46.31 kDa and 6.36, respectively. The putative signal peptide cleavage site of Bacillus sp. L2 lipase was found to be located between Ala-28 and Ala-29, based on the rules for signal peptide sequences and based on predictions from the SignalP V2.0 Web server [7]. In all lipases, a catalytic triad of Ser, His and Asp (or Glu in a few lipases) is present [8]. Thus, the predicted catalytic triad of Bacillus sp. L2 lipase should be formed by His-42, Ser-14 and Asp-345 according to comparisons with the His, Ser and Asp catalytic triad of other Bacillus lipases (Figure 2).


A newly isolated thermostable lipase from Bacillus sp.

Shariff FM, Rahman RN, Basri M, Salleh AB - Int J Mol Sci (2011)

Homology lineup of lipases from several bacteria species.Note: All the lipases were labeled based on their accession numbers submitted to the GenBank. Geobacillus sp. T1 lipase (AY 260764); Bacillus sp. L2 lipase (AY 855077); B. thermoleoverans ID-1 lipase (AF 134840); B. stearothermophilus P1 lipase (AF 237623); B. stearothermophilus LI lipase (JW 0068); B. cereus ATCC 10987 lipase (NP 978931); Clostridium tetani lipase (NP 781602); Staphylococcus aureus subsp. aureus MRSA252 lipase (YP 042090); Burkholderia pseudomallei K96243 lipase (YP 111747) and Pseudomonas putida lipase (NP 744233).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3116164&req=5

f2-ijms-12-02917: Homology lineup of lipases from several bacteria species.Note: All the lipases were labeled based on their accession numbers submitted to the GenBank. Geobacillus sp. T1 lipase (AY 260764); Bacillus sp. L2 lipase (AY 855077); B. thermoleoverans ID-1 lipase (AF 134840); B. stearothermophilus P1 lipase (AF 237623); B. stearothermophilus LI lipase (JW 0068); B. cereus ATCC 10987 lipase (NP 978931); Clostridium tetani lipase (NP 781602); Staphylococcus aureus subsp. aureus MRSA252 lipase (YP 042090); Burkholderia pseudomallei K96243 lipase (YP 111747) and Pseudomonas putida lipase (NP 744233).
Mentions: The plasmid harboring the L2 gene was sequenced and shown to contain a 1.2 kb sequence coding for lipase. The complete sequence of the ORF of the L2 lipase gene was submitted to GenBank and was assigned accession number AY855077. The amino acid composition within the ORF was determined by the ProtParam Tool of the ExPASy Molecular Biology server [6]. The molecular mass and the isoelectric point (pI) were predicted to be 46.31 kDa and 6.36, respectively. The putative signal peptide cleavage site of Bacillus sp. L2 lipase was found to be located between Ala-28 and Ala-29, based on the rules for signal peptide sequences and based on predictions from the SignalP V2.0 Web server [7]. In all lipases, a catalytic triad of Ser, His and Asp (or Glu in a few lipases) is present [8]. Thus, the predicted catalytic triad of Bacillus sp. L2 lipase should be formed by His-42, Ser-14 and Asp-345 according to comparisons with the His, Ser and Asp catalytic triad of other Bacillus lipases (Figure 2).

Bottom Line: Recombinant lipase was successfully overexpressed with a 178-fold increase in activity compared to crude native L2 lipase.The purified lipase was found to be reactive at a temperature range of 55-80 °C and at a pH of 6-10.The optimum activity was found to be at 70 °C and pH 9.

View Article: PubMed Central - PubMed

Affiliation: Enzyme and Microbial Technology Research, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia; E-Mails: ferrol2506@gmail.com (F.M.S.); abubakar@biotech.upm.edu.my (A.B.S.).

ABSTRACT
A thermophilic lipolytic bacterium identified as Bacillus sp. L2 via 16S rDNA was previously isolated from a hot spring in Perak, Malaysia. Bacillus sp. L2 was confirmed to be in Group 5 of bacterial classification, a phylogenically and phenotypically coherent group of thermophilic bacilli displaying very high similarity among their 16S rRNA sequences (98.5-99.2%). Polymerase chain reaction (PCR) cloning of L2 lipase gene was conducted by using five different primers. Sequence analysis of the L2 lipase gene revealed an open reading frame (ORF) of 1251 bp that codes for 417 amino acids. The signal peptides consist of 28 amino acids. The mature protein is made of 388 amino acid residues. Recombinant lipase was successfully overexpressed with a 178-fold increase in activity compared to crude native L2 lipase. The recombinant L2 lipase (43.2 kDa) was purified to homogeneity in a single chromatography step. The purified lipase was found to be reactive at a temperature range of 55-80 °C and at a pH of 6-10. The L2 lipase had a melting temperature (Tm) of 59.04 °C when analyzed by circular dichroism (CD) spectroscopy studies. The optimum activity was found to be at 70 °C and pH 9. Lipase L2 was strongly inhibited by ethylenediaminetetraacetic acid (EDTA) (100%), whereas phenylmethylsulfonyl fluoride (PMSF), pepstatin-A, 2-mercaptoethanol and dithiothreitol (DTT) inhibited the enzyme by over 40%. The CD spectra of secondary structure analysis showed that the L2 lipase structure contained 38.6% α-helices, 2.2% ß-strands, 23.6% turns and 35.6% random conformations.

Show MeSH
Related in: MedlinePlus