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Purification and characterization of 5′-phosphodiesterase from germinated barley

Paper ID Volume ID Publish Year Pages File Format Full-Text
36216 45124 2006 4 PDF Available
Title
Purification and characterization of 5′-phosphodiesterase from germinated barley
Abstract

5′-Phosphodiesterase (5′-PDE) was produced from germinated barley. It was observed that phosphomonoesterase (PME) was a major contaminating enzyme produced simultaneously with 5′-PDE. Purification was carried out by centrifugation, ultrafiltration (UF) through 20-kDa membrane followed by ammonium sulphate precipitation and heat treatment. These steps could not separate 5′-PDE from PME. Effect of heating at 60 °C to 90 °C for 15 min showed simultaneous loss of both 5′-PDE and PME. Both the enzymes showed an optimum pH of 5.0, and optimum temperature of 60 °C. Km of 0.33 mM and 0.26 mM were found for 5′-PDE and PME, respectively. Their corresponding Vmax were 0.123 μm/min and 0.232 μm/min, respectively. Attempts to separate 5′-PDE and PME by different chromatographic techniques were unsuccessful indicating 5′-PDE and PME activities to be associated with each other. Native gel electrophoresis showed a single band on the gel indicating both the activities to be from the single enzyme. This completely purified enzyme was further utilized for the hydrolysis of RNA to verify whether it hydrolyzed both RNA and 5′-nucleotides. Detection of 5′-nucleotides was carried out by high performance thin layer chromatography (HPTLC). Bands of nucleotides and nucleosides were clearly seen in the RNA hydrolysate sample on the HPTLC plate, reconfirming the promiscuous nature of the enzyme.

Keywords
Catalytic promiscuity; Chromatography; 5′-Phosphodiesterase; Phosphomonoesterase; Nucleotides; Barley
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Purification and characterization of 5′-phosphodiesterase from germinated barley
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 41, Issue 8, August 2006, Pages 1899–1902
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us