Comparative Analysis of Peripheral Alkaline Phytase Protein Structures Expressed in E. coli
Background: Degradation of phytic acid to inorganic phosphate in domestic animals’ diets requires thermostable phytase. Although Basillus subtilis phytase shows a potential to be degraded phytate complex in high temperature, the enzyme activities and yields need to be increased to make them possible...
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| Format: | Article |
| Language: | English |
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Varastegan Institute for Medical Sciences
2015-10-01
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| Series: | Reports of Biochemistry and Molecular Biology |
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| Online Access: | http://rbmb.net/article-1-66-en.pdf |
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| author | Mohammadreza Nassiri Hamid Ariannejad |
| author_facet | Mohammadreza Nassiri Hamid Ariannejad |
| author_sort | Mohammadreza Nassiri |
| collection | DOAJ |
| description | Background: Degradation of phytic acid to inorganic phosphate in domestic animals’ diets requires thermostable phytase. Although Basillus subtilis phytase shows a potential to be degraded phytate complex in high temperature, the enzyme activities and yields need to be increased to make them possible for industrial application.
Methods: The phytase gene from Bacillus subtilis DR8886 was isolated from Dig Rostam hot mineral spring in Iran and cloned into pET21(+) and pET32(+). Expression was induced with 1.5 mM IPTG and the proteins were purified.
Results: The recombinant protein affected by thioredoxin (Trx) from pET32a-PhyC was estimated to constitute about 31% of the total soluble protein in the cells; its concentration was 3.5 μg/ml, and its maximal phytase activity was 15.9 U/ml, whereas the recombinant phytase from pET21a-PhyC was estimated to comprise about 19% of the total soluble protein; its concentration was 2.2 μg/ml, and its maximal phytase activity was 69 U/ml. The molecular masses of recombinant phytase with and without Trx were about 60 kDa and 42 kDa, respectively. Zymography confirmed that the recombinant enzymes were active. Although the concentration of the alkaline phytase expressed by pET32a was approximately 59% greater than that expressed by pET21, its phytase activity was approximately 77% less.
Conclusion: This study showed that the peripheral gene (Trx) encoded by the pET32a (+) vector are the principal reason for the decrease in recombinant phytase enzyme activity. |
| first_indexed | 2024-12-12T23:24:40Z |
| format | Article |
| id | doaj.art-8035220418404ca5936f4ec323702276 |
| institution | Directory Open Access Journal |
| issn | 2322-3480 2322-3480 |
| language | English |
| last_indexed | 2024-12-12T23:24:40Z |
| publishDate | 2015-10-01 |
| publisher | Varastegan Institute for Medical Sciences |
| record_format | Article |
| series | Reports of Biochemistry and Molecular Biology |
| spelling | doaj.art-8035220418404ca5936f4ec3237022762022-12-22T00:08:09ZengVarastegan Institute for Medical SciencesReports of Biochemistry and Molecular Biology2322-34802322-34802015-10-01411018Comparative Analysis of Peripheral Alkaline Phytase Protein Structures Expressed in E. coliMohammadreza Nassiri0Hamid Ariannejad1Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran - Department of Animal Sciences, College of Agriculture, Ferdowsi University of Mashhad, Mashhad, IranDepartment of Animal Sciences, College of Agriculture, Ferdowsi University of Mashhad, Mashhad, IranBackground: Degradation of phytic acid to inorganic phosphate in domestic animals’ diets requires thermostable phytase. Although Basillus subtilis phytase shows a potential to be degraded phytate complex in high temperature, the enzyme activities and yields need to be increased to make them possible for industrial application. Methods: The phytase gene from Bacillus subtilis DR8886 was isolated from Dig Rostam hot mineral spring in Iran and cloned into pET21(+) and pET32(+). Expression was induced with 1.5 mM IPTG and the proteins were purified. Results: The recombinant protein affected by thioredoxin (Trx) from pET32a-PhyC was estimated to constitute about 31% of the total soluble protein in the cells; its concentration was 3.5 μg/ml, and its maximal phytase activity was 15.9 U/ml, whereas the recombinant phytase from pET21a-PhyC was estimated to comprise about 19% of the total soluble protein; its concentration was 2.2 μg/ml, and its maximal phytase activity was 69 U/ml. The molecular masses of recombinant phytase with and without Trx were about 60 kDa and 42 kDa, respectively. Zymography confirmed that the recombinant enzymes were active. Although the concentration of the alkaline phytase expressed by pET32a was approximately 59% greater than that expressed by pET21, its phytase activity was approximately 77% less. Conclusion: This study showed that the peripheral gene (Trx) encoded by the pET32a (+) vector are the principal reason for the decrease in recombinant phytase enzyme activity.http://rbmb.net/article-1-66-en.pdfBacillus subtilis DR8806PhyC GeneThioredoxin |
| spellingShingle | Mohammadreza Nassiri Hamid Ariannejad Comparative Analysis of Peripheral Alkaline Phytase Protein Structures Expressed in E. coli Reports of Biochemistry and Molecular Biology Bacillus subtilis DR8806 PhyC Gene Thioredoxin |
| title | Comparative Analysis of Peripheral Alkaline Phytase Protein Structures Expressed in E. coli |
| title_full | Comparative Analysis of Peripheral Alkaline Phytase Protein Structures Expressed in E. coli |
| title_fullStr | Comparative Analysis of Peripheral Alkaline Phytase Protein Structures Expressed in E. coli |
| title_full_unstemmed | Comparative Analysis of Peripheral Alkaline Phytase Protein Structures Expressed in E. coli |
| title_short | Comparative Analysis of Peripheral Alkaline Phytase Protein Structures Expressed in E. coli |
| title_sort | comparative analysis of peripheral alkaline phytase protein structures expressed in e coli |
| topic | Bacillus subtilis DR8806 PhyC Gene Thioredoxin |
| url | http://rbmb.net/article-1-66-en.pdf |
| work_keys_str_mv | AT mohammadrezanassiri comparativeanalysisofperipheralalkalinephytaseproteinstructuresexpressedinecoli AT hamidariannejad comparativeanalysisofperipheralalkalinephytaseproteinstructuresexpressedinecoli |