Improvement of thermal stability via outer-loop ion pair interaction of mutated T1 lipase from Geobacillus zalihae strain T1
Mutant D311E and K344R were constructed using site-directed mutagenesis to introduce an additional ion pair at the inter-loop and the intra-loop, respectively, to determine the effect of ion pairs on the stability of T1 lipase isolated from Geobacillus zalihae. A series of purification steps was app...
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| Format: | Article |
| Language: | English |
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MDPI AG
2012
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| Online Access: | http://psasir.upm.edu.my/id/eprint/27295/ http://psasir.upm.edu.my/id/eprint/27295/1/27295.pdf |
| _version_ | 1848845806545666048 |
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| author | Ruslan, Rudzanna Raja Abdul Rahman, Raja Noor Zaliha Leow, Thean Chor Mohamad Ali, Mohd Shukuri Basri, Mahiran Salleh, Abu Bakar |
| author_facet | Ruslan, Rudzanna Raja Abdul Rahman, Raja Noor Zaliha Leow, Thean Chor Mohamad Ali, Mohd Shukuri Basri, Mahiran Salleh, Abu Bakar |
| author_sort | Ruslan, Rudzanna |
| building | UPM Institutional Repository |
| collection | Online Access |
| description | Mutant D311E and K344R were constructed using site-directed mutagenesis to introduce an additional ion pair at the inter-loop and the intra-loop, respectively, to determine the effect of ion pairs on the stability of T1 lipase isolated from Geobacillus zalihae. A series of purification steps was applied, and the pure lipases of T1, D311E and K344R were obtained. The wild-type and mutant lipases were analyzed using circular dichroism. The T(m) for T1 lipase, D311E lipase and K344R lipase were approximately 68.52 °C, 70.59 °C and 68.54 °C, respectively. Mutation at D311 increases the stability of T1 lipase and exhibited higher T(m) as compared to the wild-type and K344R. Based on the above, D311E lipase was chosen for further study. D311E lipase was successfully crystallized using the sitting drop vapor diffusion method. The crystal was diffracted at 2.1 Å using an in-house X-ray beam and belonged to the monoclinic space group C2 with the unit cell parameters a = 117.32 Å, b = 81.16 Å and c = 100.14 Å. Structural analysis showed the existence of an additional ion pair around E311 in the structure of D311E. The additional ion pair in D311E may regulate the stability of this mutant lipase at high temperatures as predicted in silico and spectroscopically. |
| first_indexed | 2025-11-15T08:52:40Z |
| format | Article |
| id | upm-27295 |
| institution | Universiti Putra Malaysia |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-15T08:52:40Z |
| publishDate | 2012 |
| publisher | MDPI AG |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | upm-272952016-09-28T05:04:50Z http://psasir.upm.edu.my/id/eprint/27295/ Improvement of thermal stability via outer-loop ion pair interaction of mutated T1 lipase from Geobacillus zalihae strain T1 Ruslan, Rudzanna Raja Abdul Rahman, Raja Noor Zaliha Leow, Thean Chor Mohamad Ali, Mohd Shukuri Basri, Mahiran Salleh, Abu Bakar Mutant D311E and K344R were constructed using site-directed mutagenesis to introduce an additional ion pair at the inter-loop and the intra-loop, respectively, to determine the effect of ion pairs on the stability of T1 lipase isolated from Geobacillus zalihae. A series of purification steps was applied, and the pure lipases of T1, D311E and K344R were obtained. The wild-type and mutant lipases were analyzed using circular dichroism. The T(m) for T1 lipase, D311E lipase and K344R lipase were approximately 68.52 °C, 70.59 °C and 68.54 °C, respectively. Mutation at D311 increases the stability of T1 lipase and exhibited higher T(m) as compared to the wild-type and K344R. Based on the above, D311E lipase was chosen for further study. D311E lipase was successfully crystallized using the sitting drop vapor diffusion method. The crystal was diffracted at 2.1 Å using an in-house X-ray beam and belonged to the monoclinic space group C2 with the unit cell parameters a = 117.32 Å, b = 81.16 Å and c = 100.14 Å. Structural analysis showed the existence of an additional ion pair around E311 in the structure of D311E. The additional ion pair in D311E may regulate the stability of this mutant lipase at high temperatures as predicted in silico and spectroscopically. MDPI AG 2012 Article PeerReviewed application/pdf en http://psasir.upm.edu.my/id/eprint/27295/1/27295.pdf Ruslan, Rudzanna and Raja Abdul Rahman, Raja Noor Zaliha and Leow, Thean Chor and Mohamad Ali, Mohd Shukuri and Basri, Mahiran and Salleh, Abu Bakar (2012) Improvement of thermal stability via outer-loop ion pair interaction of mutated T1 lipase from Geobacillus zalihae strain T1. International Journal of Molecular Sciences, 13 (1). pp. 943-960. ISSN 1422-0067 http://www.mdpi.com/1422-0067/13/1/943 10.3390/ijms13010943 |
| spellingShingle | Ruslan, Rudzanna Raja Abdul Rahman, Raja Noor Zaliha Leow, Thean Chor Mohamad Ali, Mohd Shukuri Basri, Mahiran Salleh, Abu Bakar Improvement of thermal stability via outer-loop ion pair interaction of mutated T1 lipase from Geobacillus zalihae strain T1 |
| title | Improvement of thermal stability via outer-loop ion pair interaction of mutated T1 lipase from Geobacillus zalihae strain T1 |
| title_full | Improvement of thermal stability via outer-loop ion pair interaction of mutated T1 lipase from Geobacillus zalihae strain T1 |
| title_fullStr | Improvement of thermal stability via outer-loop ion pair interaction of mutated T1 lipase from Geobacillus zalihae strain T1 |
| title_full_unstemmed | Improvement of thermal stability via outer-loop ion pair interaction of mutated T1 lipase from Geobacillus zalihae strain T1 |
| title_short | Improvement of thermal stability via outer-loop ion pair interaction of mutated T1 lipase from Geobacillus zalihae strain T1 |
| title_sort | improvement of thermal stability via outer-loop ion pair interaction of mutated t1 lipase from geobacillus zalihae strain t1 |
| url | http://psasir.upm.edu.my/id/eprint/27295/ http://psasir.upm.edu.my/id/eprint/27295/ http://psasir.upm.edu.my/id/eprint/27295/ http://psasir.upm.edu.my/id/eprint/27295/1/27295.pdf |