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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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(a) pH profile of purified recombinant L2 lipase; (b) pH stability of purified recombinant L2 lipase. Symbols used are: (▪) Acetate buffer; (▵) Potassium phosphate buffer; (♦) Tris-HCl buffer; (□) Glycine-NaOH buffer; (•) Na2HPO4/NaOH buffer.
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f4-ijms-12-02917: (a) pH profile of purified recombinant L2 lipase; (b) pH stability of purified recombinant L2 lipase. Symbols used are: (▪) Acetate buffer; (▵) Potassium phosphate buffer; (♦) Tris-HCl buffer; (□) Glycine-NaOH buffer; (•) Na2HPO4/NaOH buffer.

Mentions: The effect of pH on enzyme activity was examined in the pH range of 4.0 to 12.0 (Figure 4a). Maximal lipolytic activity towards olive oil was observed at pH 9.0. At pH 10, L2 lipase remained at 50% of its activity and the activity started to decrease tremendously when the lipase was assayed at higher pH levels. L2 lipase also seemed to have low activity at acidic to neutral pH as it only had 20% of its activity at pH 5 to pH 7. The high enzyme activity at pH 9 to 10 may be a result of the Ala for the first Gly-residue in the consensus sequence Gly-X-Ser-X-Gly [15]. The pH-stability of L2 showed that it was fairly stable (>40% of relative activity) at pHs ranging from 8.0 to 10 after being treated for 30 min at 70 °C in both Tris-HCl and Glycine-NaOH buffers (Figure 4b). L2 lipase was most stable at pH 9.0.


A newly isolated thermostable lipase from Bacillus sp.

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

(a) pH profile of purified recombinant L2 lipase; (b) pH stability of purified recombinant L2 lipase. Symbols used are: (▪) Acetate buffer; (▵) Potassium phosphate buffer; (♦) Tris-HCl buffer; (□) Glycine-NaOH buffer; (•) Na2HPO4/NaOH buffer.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4-ijms-12-02917: (a) pH profile of purified recombinant L2 lipase; (b) pH stability of purified recombinant L2 lipase. Symbols used are: (▪) Acetate buffer; (▵) Potassium phosphate buffer; (♦) Tris-HCl buffer; (□) Glycine-NaOH buffer; (•) Na2HPO4/NaOH buffer.
Mentions: The effect of pH on enzyme activity was examined in the pH range of 4.0 to 12.0 (Figure 4a). Maximal lipolytic activity towards olive oil was observed at pH 9.0. At pH 10, L2 lipase remained at 50% of its activity and the activity started to decrease tremendously when the lipase was assayed at higher pH levels. L2 lipase also seemed to have low activity at acidic to neutral pH as it only had 20% of its activity at pH 5 to pH 7. The high enzyme activity at pH 9 to 10 may be a result of the Ala for the first Gly-residue in the consensus sequence Gly-X-Ser-X-Gly [15]. The pH-stability of L2 showed that it was fairly stable (>40% of relative activity) at pHs ranging from 8.0 to 10 after being treated for 30 min at 70 °C in both Tris-HCl and Glycine-NaOH buffers (Figure 4b). L2 lipase was most stable at pH 9.0.

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