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Prolidase from Xanthomonas maltophilia: purification and characterization of the enzyme
1School of Pharmaceutical Sciences, Nagasaki University, Japan.
Abstract:
Prolidase (iminodipeptidase, EC 3.4.13.9) was purified from an extract of Xanthomonas maltophilia, by ammonium sulfate fractionation and sequential chromatographies on DEAE-Toyopearl, Toyopearl HW65C, FPLC-Hiload Superdex 200 pg, and FPLC-Hitrap Q columns, which an activity recovery of 2.3%. The enzyme was the most active at pH 7.5 with Leu-Pro as substrate. It was stable between pH 6.0 and 8.5 for 60 min at 37 degrees C and retained half of activity after 60 min at 37 degrees C. The isoelectric point of the enzyme was 3.7. Its molecular weight was estimated to be 100,000 by gel filtration on FPLC-Hiload Superdex 200 and 51,000 by SDS-PAGE, suggesting that it is a dimer. It hydrolyzed dipeptides only if proline is located at the carboxyl terminal position. The enzyme was inhibited by PCMB and o-phenanthroline, and was activated by Mn2+.
Insights
This study purified prolidase (iminodipeptidase) from Xanthomonas maltophilia, detailing its optimal activity at pH 7.5 and stability. The enzyme specifically hydrolyzes dipeptides with proline at the carboxyl terminus.
Area of Science:
- Biochemistry
- Enzymology
- Microbial biochemistry
Background:
- Prolidase (iminodipeptidase, EC 3.4.13.9) plays a role in peptide hydrolysis.
- Understanding microbial enzymes is crucial for biotechnological applications.
Purpose of the Study:
- To purify and characterize prolidase from Xanthomonas maltophilia.
- To determine the enzyme's kinetic and stability properties.
Main Methods:
- Enzyme purification using ammonium sulfate fractionation and multiple chromatography techniques (DEAE-Toyopearl, Toyopearl HW65C, FPLC Superdex 200, FPLC Hitrap Q).
- Enzyme activity assays at varying pH and substrate specificity determination.
- Stability studies, isoelectric point determination, molecular weight estimation (gel filtration and SDS-PAGE).
Main Results:
- Purified prolidase with 2.3% activity recovery.
- Optimal activity at pH 7.5 with Leu-Pro substrate.
- Enzyme stability between pH 6.0-8.5 at 37°C.
- Isoelectric point of 3.7; molecular weight suggests a dimer (100,000 Da by gel filtration, 51,000 Da by SDS-PAGE).
- Hydrolyzes dipeptides with carboxyl-terminal proline; inhibited by PCMB and o-phenanthroline; activated by Mn2+.
Conclusions:
- Xanthomonas maltophilia prolidase is a dimeric enzyme with specific substrate requirements.
- Characterization provides insights into its biochemical properties and potential functions.