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Cold-adapted RTX lipase from antarctic Pseudomonas sp. strain AMS8: isolation, molecular modeling and heterologous expression

A new strain of psychrophilic bacteria (designated strain AMS8) from Antarctic soil was screened for extracellular lipolytic activity and further analyzed using molecular approach. Analysis of 16S rDNA showed that strain AMS8 was similar to Pseudomonas sp. A lipase gene named lipAMS8 was successfull...

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Main Authors: Mohamad Ali, Mohd Shukuri, Ganasen, Menega, Raja Abdul Rahman, Raja Noor Zaliha, Leow, Adam Thean Chor, Salleh, Abu Bakar, Basri, Mahiran
Format: Article
Language:English
Published: Springer 2013
Online Access:http://psasir.upm.edu.my/id/eprint/28129/1/Cold.pdf
http://psasir.upm.edu.my/id/eprint/28129/
http://link.springer.com/article/10.1007/s10930-013-9488-z?view=classic
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spelling my.upm.eprints.281292016-09-28T02:38:34Z http://psasir.upm.edu.my/id/eprint/28129/ Cold-adapted RTX lipase from antarctic Pseudomonas sp. strain AMS8: isolation, molecular modeling and heterologous expression Mohamad Ali, Mohd Shukuri Ganasen, Menega Raja Abdul Rahman, Raja Noor Zaliha Leow, Adam Thean Chor Salleh, Abu Bakar Basri, Mahiran A new strain of psychrophilic bacteria (designated strain AMS8) from Antarctic soil was screened for extracellular lipolytic activity and further analyzed using molecular approach. Analysis of 16S rDNA showed that strain AMS8 was similar to Pseudomonas sp. A lipase gene named lipAMS8 was successfully isolated from strain AMS8, cloned, sequenced and overexpressed in Escherichia coli. Sequence analysis revealed that lipAMS8 consist of 1,431 bp nucleotides that encoded a polypeptide consisting of 476 amino acids. It lacked an N-terminal signal peptide and contained a glycine- and aspartate-rich nonapeptide sequence at the C-terminus, which are known to be the characteristics of repeats-in-toxin bacterial lipases. Furthermore, the substrate binding site of lipAMS8 was identified as S207, D 255 and H313, based on homology modeling and multiple sequence alignment. Crude lipase exhibited maximum activity at 20 C and retained almost 50 % of its activity at 10 C. The molecular weight of lipAMS8 was estimated to be 50 kDa via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal expression level was attained using the recombinant plasmid pET32b/BL21(DE3) expressed at 15 C for 8 h, induced by 0.1 mM isopropyl β-D thiogalactoside (IPTG) at E. coli growth optimal density of 0.5. Springer 2013 Article PeerReviewed application/pdf en http://psasir.upm.edu.my/id/eprint/28129/1/Cold.pdf Mohamad Ali, Mohd Shukuri and Ganasen, Menega and Raja Abdul Rahman, Raja Noor Zaliha and Leow, Adam Thean Chor and Salleh, Abu Bakar and Basri, Mahiran (2013) Cold-adapted RTX lipase from antarctic Pseudomonas sp. strain AMS8: isolation, molecular modeling and heterologous expression. The Protein Journal, 32 (4). pp. 317-325. ISSN 1572-3887; ESSN: 1573-4943 http://link.springer.com/article/10.1007/s10930-013-9488-z?view=classic 10.1007/s10930-013-9488-z
institution Universiti Putra Malaysia
building UPM Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Putra Malaysia
content_source UPM Institutional Repository
url_provider http://psasir.upm.edu.my/
language English
description A new strain of psychrophilic bacteria (designated strain AMS8) from Antarctic soil was screened for extracellular lipolytic activity and further analyzed using molecular approach. Analysis of 16S rDNA showed that strain AMS8 was similar to Pseudomonas sp. A lipase gene named lipAMS8 was successfully isolated from strain AMS8, cloned, sequenced and overexpressed in Escherichia coli. Sequence analysis revealed that lipAMS8 consist of 1,431 bp nucleotides that encoded a polypeptide consisting of 476 amino acids. It lacked an N-terminal signal peptide and contained a glycine- and aspartate-rich nonapeptide sequence at the C-terminus, which are known to be the characteristics of repeats-in-toxin bacterial lipases. Furthermore, the substrate binding site of lipAMS8 was identified as S207, D 255 and H313, based on homology modeling and multiple sequence alignment. Crude lipase exhibited maximum activity at 20 C and retained almost 50 % of its activity at 10 C. The molecular weight of lipAMS8 was estimated to be 50 kDa via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal expression level was attained using the recombinant plasmid pET32b/BL21(DE3) expressed at 15 C for 8 h, induced by 0.1 mM isopropyl β-D thiogalactoside (IPTG) at E. coli growth optimal density of 0.5.
format Article
author Mohamad Ali, Mohd Shukuri
Ganasen, Menega
Raja Abdul Rahman, Raja Noor Zaliha
Leow, Adam Thean Chor
Salleh, Abu Bakar
Basri, Mahiran
spellingShingle Mohamad Ali, Mohd Shukuri
Ganasen, Menega
Raja Abdul Rahman, Raja Noor Zaliha
Leow, Adam Thean Chor
Salleh, Abu Bakar
Basri, Mahiran
Cold-adapted RTX lipase from antarctic Pseudomonas sp. strain AMS8: isolation, molecular modeling and heterologous expression
author_facet Mohamad Ali, Mohd Shukuri
Ganasen, Menega
Raja Abdul Rahman, Raja Noor Zaliha
Leow, Adam Thean Chor
Salleh, Abu Bakar
Basri, Mahiran
author_sort Mohamad Ali, Mohd Shukuri
title Cold-adapted RTX lipase from antarctic Pseudomonas sp. strain AMS8: isolation, molecular modeling and heterologous expression
title_short Cold-adapted RTX lipase from antarctic Pseudomonas sp. strain AMS8: isolation, molecular modeling and heterologous expression
title_full Cold-adapted RTX lipase from antarctic Pseudomonas sp. strain AMS8: isolation, molecular modeling and heterologous expression
title_fullStr Cold-adapted RTX lipase from antarctic Pseudomonas sp. strain AMS8: isolation, molecular modeling and heterologous expression
title_full_unstemmed Cold-adapted RTX lipase from antarctic Pseudomonas sp. strain AMS8: isolation, molecular modeling and heterologous expression
title_sort cold-adapted rtx lipase from antarctic pseudomonas sp. strain ams8: isolation, molecular modeling and heterologous expression
publisher Springer
publishDate 2013
url http://psasir.upm.edu.my/id/eprint/28129/1/Cold.pdf
http://psasir.upm.edu.my/id/eprint/28129/
http://link.springer.com/article/10.1007/s10930-013-9488-z?view=classic
_version_ 1643829378253062144
score 13.211869

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