Cloning and expression of a novel phytase gene (phyMS) from Mycobacterium smegmatis
Phytase, also known as phytate-degrading enzyme, catalyzes the hydrolysis of phytate (inositol hexakisphosphate) with sequential release of phosphate and lower inositol phosphate. We report here a new plasmid construct designated as pMSuia from pBAD-TOPO that harbors a 1.1 kb phytase gene (phyMS)...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Scientific Research Publishing Inc.
2014
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| Subjects: | |
| Online Access: | http://irep.iium.edu.my/47160/ http://irep.iium.edu.my/47160/ http://irep.iium.edu.my/47160/ http://irep.iium.edu.my/47160/1/Paper_-_Nuge_et_al_2014_%28Adv_in_Enzyme_Res%29.pdf |
| Summary: | Phytase, also known as phytate-degrading enzyme, catalyzes the hydrolysis of phytate (inositol
hexakisphosphate) with sequential release of phosphate and lower inositol phosphate. We report
here a new plasmid construct designated as pMSuia from pBAD-TOPO that harbors a 1.1 kb phytase
gene (phyMS) from Mycobacterium smegmatis, and expression as well as characterization of
the purified recombinant M. smegmatis phytase. DNA sequencing analysis and multiple alignment
exercise indicated that the M. smegmatis phytase is different from both known acid and alkaline
phytases. The active ~45 kDa recombinant enzyme was expressed and confirmed by enzyme assay
and Western blot analyses. Ni-NTA affinity purified recombinant M. smegmatis phytase exhibited
specific activity of 233.51 U/mg, optimal pH of 3 and 7 and optimal temperature of 60˚C. The purified
enzyme retains almost 30% of the initial activity after incubation at 90˚C for 60 min. The enzyme
showed broad substrate specificity with Km and Vmax of the recombinant enzyme for sodium
phytate substrate of 0.105 ± 0.016 mM and 26.93 ± 1.21 mM min−1, respectively. |
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