Expression and characterization of a cold-adapted lipase from an antarctic Pseudomonas sp.
Eleven strains of unknown microbial isolates obtained from Antarctica were screened for extracellular lipolytic activity. Eight isolates showed positive results on Tributyrin, Rhodamine B and Triolein agar plates. A single isolate (designated as strain AMS8) that demonstrated the highest lipase a...
| Main Author: | |
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| Format: | Thesis |
| Language: | English |
| Published: |
2014
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/90649/1/FBSB%202014%2044%20-%20IR.pdf |
| _version_ | 1796982360609128448 |
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| author | Ganasen, Menega |
| author_facet | Ganasen, Menega |
| author_sort | Ganasen, Menega |
| collection | UPM |
| description | Eleven strains of unknown microbial isolates obtained from Antarctica were screened
for extracellular lipolytic activity. Eight isolates showed positive results on Tributyrin,
Rhodamine B and Triolein agar plates. A single isolate (designated as strain AMS8)
that demonstrated the highest lipase activity (0.056 U/mL) was finally used in the
current research. It was identified as Pseudomonas sp. based on its morphological study
and 16S rDNA analysis.
A lipase gene (lipAMS8) was amplified from strain AMS8 via polymerase chain
reaction (PCR) amplification. The open reading frame (ORF) of LipAMS8 is 1431 bp
in length coding for 476 amino acids. LipAMS8 lacks an N-terminal signal peptide and
contains a glycine- and aspartate-rich nanopeptide sequence at the C-terminus. The
catalytic triad of LipAMS8 was predicted as Ser-207, Asp-255 and His-313, based on
multiple sequence alignment.
Both soluble and insoluble protein of LipAMS8 was expressed in Escherichia coli. The
ORF of LipAMS8 was expressed using pTrcHis TOPO TA, pET-32b(+) and pGEX-
4T1, which are under the control of trc, T7lac and tac promoters, respectively. An
optimum expression level for pTrcHis/lipAMS8, pET32b/lipAMS8 and pGEX/lipAMS8
under constant expression conditions was 0.346 U/mL, 6.066 U/mL and 1.533 U/mL,
respectively. An improved lipase expression (9.493 U/mL) was attained using pET-
32b(+) vector in E. coli BL21(DE3) expressed at 15°C for 8 hours, induced with 0.05
mM isopropyl β-D thiogalactoside (IPTG) at E. coli growth optimal density of 0.5.
Only a small amount of lipAMS8 was expressed in soluble form. A huge amount of
expressed proteins were in the form of inclusion bodies or insoluble proteins. The
inclusion bodies were solubilized by means of urea, a strong denaturating agent and are
then refolded via single step dilution. The level of expression obtained from insoluble protein (41.84 U/mL) was almost four times higher compared to the soluble protein
(9.493 U/mL).
Crude enzyme obtained from intracellular inclusion bodies expression was then
purified. The His-tagged recombinant LipAMS8 was purified with 23.0% total
recovery and an average purification factor of 9.7. The purified LipAMS8 migrated as
a single band with a molecular weight approximately 65 kDa during sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
LipAMS8 was highly active at 30°C and pH 10. It retained almost 19 and 68% of its
relative activity at 0 and 10°C, respectively. The half-life of LipAMS8 was 4 and 2
hours at 30 and 40°C, respectively. The lipase was stable over a broad range of pH (pH
6 to 12). LipAMS8 showed enhancement effect in its relative activity under the
presence of Li+, Na+, K+, Rb+ and Cs+ after 30 minutes treatment. Nonetheless, the
enhancement effect decreases as the metallic character increases from Li+ to Cs+. As
for divalent metals, a lower concentration (1 mM) of Mg2+ and Ca2+ gave an
enhancement effect to the LipAMS8 activity after 30 minutes treatment. Heavy metal
ions such as Cu2+, Fe3+ and Zn2+ inhibited LipAMS8 activity. As for the organic
solvent, methanol, ethanol and xylene had almost no effect on lipase activity, whereas
β-mercaptoethanol, pyridine, 1-butanol, iso-amylalcohol, propylacetate and 1-propanol
exhibited an inhibitory effect. The LipAMS8 demonstrated high stability in the
presence of dimethylsulfoxide, isooctane, octane, n-decane, n-tridecane, n-tetradecane
and n-hexadecane.
In conclusion, a new cold-adapted lipase was successfully isolated and its nucleotide
sequence was deposited at gene bank under the accession number HQ162821. It
exhibited stability and activity at broad range of pH, elevated temperatures and also in
the presence of certain metal ions and organic solvents. These unique properties of
LipAMS8 will provide considerable potential for many biotechnological and industrial
applications. |
| first_indexed | 2024-03-06T10:50:53Z |
| format | Thesis |
| id | upm.eprints-90649 |
| institution | Universiti Putra Malaysia |
| language | English |
| last_indexed | 2024-03-06T10:50:53Z |
| publishDate | 2014 |
| record_format | dspace |
| spelling | upm.eprints-906492021-08-27T01:14:33Z http://psasir.upm.edu.my/id/eprint/90649/ Expression and characterization of a cold-adapted lipase from an antarctic Pseudomonas sp. Ganasen, Menega Eleven strains of unknown microbial isolates obtained from Antarctica were screened for extracellular lipolytic activity. Eight isolates showed positive results on Tributyrin, Rhodamine B and Triolein agar plates. A single isolate (designated as strain AMS8) that demonstrated the highest lipase activity (0.056 U/mL) was finally used in the current research. It was identified as Pseudomonas sp. based on its morphological study and 16S rDNA analysis. A lipase gene (lipAMS8) was amplified from strain AMS8 via polymerase chain reaction (PCR) amplification. The open reading frame (ORF) of LipAMS8 is 1431 bp in length coding for 476 amino acids. LipAMS8 lacks an N-terminal signal peptide and contains a glycine- and aspartate-rich nanopeptide sequence at the C-terminus. The catalytic triad of LipAMS8 was predicted as Ser-207, Asp-255 and His-313, based on multiple sequence alignment. Both soluble and insoluble protein of LipAMS8 was expressed in Escherichia coli. The ORF of LipAMS8 was expressed using pTrcHis TOPO TA, pET-32b(+) and pGEX- 4T1, which are under the control of trc, T7lac and tac promoters, respectively. An optimum expression level for pTrcHis/lipAMS8, pET32b/lipAMS8 and pGEX/lipAMS8 under constant expression conditions was 0.346 U/mL, 6.066 U/mL and 1.533 U/mL, respectively. An improved lipase expression (9.493 U/mL) was attained using pET- 32b(+) vector in E. coli BL21(DE3) expressed at 15°C for 8 hours, induced with 0.05 mM isopropyl β-D thiogalactoside (IPTG) at E. coli growth optimal density of 0.5. Only a small amount of lipAMS8 was expressed in soluble form. A huge amount of expressed proteins were in the form of inclusion bodies or insoluble proteins. The inclusion bodies were solubilized by means of urea, a strong denaturating agent and are then refolded via single step dilution. The level of expression obtained from insoluble protein (41.84 U/mL) was almost four times higher compared to the soluble protein (9.493 U/mL). Crude enzyme obtained from intracellular inclusion bodies expression was then purified. The His-tagged recombinant LipAMS8 was purified with 23.0% total recovery and an average purification factor of 9.7. The purified LipAMS8 migrated as a single band with a molecular weight approximately 65 kDa during sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). LipAMS8 was highly active at 30°C and pH 10. It retained almost 19 and 68% of its relative activity at 0 and 10°C, respectively. The half-life of LipAMS8 was 4 and 2 hours at 30 and 40°C, respectively. The lipase was stable over a broad range of pH (pH 6 to 12). LipAMS8 showed enhancement effect in its relative activity under the presence of Li+, Na+, K+, Rb+ and Cs+ after 30 minutes treatment. Nonetheless, the enhancement effect decreases as the metallic character increases from Li+ to Cs+. As for divalent metals, a lower concentration (1 mM) of Mg2+ and Ca2+ gave an enhancement effect to the LipAMS8 activity after 30 minutes treatment. Heavy metal ions such as Cu2+, Fe3+ and Zn2+ inhibited LipAMS8 activity. As for the organic solvent, methanol, ethanol and xylene had almost no effect on lipase activity, whereas β-mercaptoethanol, pyridine, 1-butanol, iso-amylalcohol, propylacetate and 1-propanol exhibited an inhibitory effect. The LipAMS8 demonstrated high stability in the presence of dimethylsulfoxide, isooctane, octane, n-decane, n-tridecane, n-tetradecane and n-hexadecane. In conclusion, a new cold-adapted lipase was successfully isolated and its nucleotide sequence was deposited at gene bank under the accession number HQ162821. It exhibited stability and activity at broad range of pH, elevated temperatures and also in the presence of certain metal ions and organic solvents. These unique properties of LipAMS8 will provide considerable potential for many biotechnological and industrial applications. 2014-06 Thesis NonPeerReviewed text en http://psasir.upm.edu.my/id/eprint/90649/1/FBSB%202014%2044%20-%20IR.pdf Ganasen, Menega (2014) Expression and characterization of a cold-adapted lipase from an antarctic Pseudomonas sp. Masters thesis, Universiti Putra Malaysia. Lipase Pseudomonas Enzymes - Purification |
| spellingShingle | Lipase Pseudomonas Enzymes - Purification Ganasen, Menega Expression and characterization of a cold-adapted lipase from an antarctic Pseudomonas sp. |
| title | Expression and characterization of a cold-adapted lipase from an antarctic Pseudomonas sp. |
| title_full | Expression and characterization of a cold-adapted lipase from an antarctic Pseudomonas sp. |
| title_fullStr | Expression and characterization of a cold-adapted lipase from an antarctic Pseudomonas sp. |
| title_full_unstemmed | Expression and characterization of a cold-adapted lipase from an antarctic Pseudomonas sp. |
| title_short | Expression and characterization of a cold-adapted lipase from an antarctic Pseudomonas sp. |
| title_sort | expression and characterization of a cold adapted lipase from an antarctic pseudomonas sp |
| topic | Lipase Pseudomonas Enzymes - Purification |
| url | http://psasir.upm.edu.my/id/eprint/90649/1/FBSB%202014%2044%20-%20IR.pdf |
| work_keys_str_mv | AT ganasenmenega expressionandcharacterizationofacoldadaptedlipasefromanantarcticpseudomonassp |