Limits...
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.

Show MeSH
Molecular ellipticity value measurement for L2 mature lipase. Secondary structure prediction of L2 lipase was done by the CD spectra at 220 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f9-ijms-12-02917: Molecular ellipticity value measurement for L2 mature lipase. Secondary structure prediction of L2 lipase was done by the CD spectra at 220 nm.

Mentions: One of the most successful applications of CD in characterizing a protein depends upon the remarkable sensitivity of the far-UV to the backbone conformation of proteins to reflect the secondary content of the protein [22]. CD measurements have been widely used to follow the equilibrium between helical structures and unordered conformations [23]. Polypeptide conformations that determine protein secondary structures give rise to circular dichroism spectra [24]. Measuring the CD spectra of L2 lipase allowed rapid determination of the secondary structure content of L2 lipase. The purified L2 lipase was first diluted to a concentration of 0.2mg/ml before subjected to CD and its far UV spectra 190–240 nm were obtained (Figure 9). L2 lipase was determined structurally to be 38.6% α-helix, 2.2% ß-sheet, 23.6% ß-turn and 35.6% random coil.


A newly isolated thermostable lipase from Bacillus sp.

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

Molecular ellipticity value measurement for L2 mature lipase. Secondary structure prediction of L2 lipase was done by the CD spectra at 220 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f9-ijms-12-02917: Molecular ellipticity value measurement for L2 mature lipase. Secondary structure prediction of L2 lipase was done by the CD spectra at 220 nm.
Mentions: One of the most successful applications of CD in characterizing a protein depends upon the remarkable sensitivity of the far-UV to the backbone conformation of proteins to reflect the secondary content of the protein [22]. CD measurements have been widely used to follow the equilibrium between helical structures and unordered conformations [23]. Polypeptide conformations that determine protein secondary structures give rise to circular dichroism spectra [24]. Measuring the CD spectra of L2 lipase allowed rapid determination of the secondary structure content of L2 lipase. The purified L2 lipase was first diluted to a concentration of 0.2mg/ml before subjected to CD and its far UV spectra 190–240 nm were obtained (Figure 9). L2 lipase was determined structurally to be 38.6% α-helix, 2.2% ß-sheet, 23.6% ß-turn and 35.6% random coil.

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