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Purification and properties of a serine protease from Pseudomonas matophilia
Abstract:
The extracellular protease of Pseudomonas maltophilia was partially purified by ammonium sulfate precipitation and chromatography on Sephadex G-75 and Bio-rex 70. Gel electrophoresis revealed minor impurities. The enzyme exhibited the following properties: (i) molecular weight, 35,000; (ii) A see article; 10.8; (iii) isoelectric point, 9.3; (iv) pH optimum, 10.0; (v)s20, w equal 3.47. The enzyme was rapidly inactivated by ethylenediaminetetracetate, but activity could be partially restored with divalent cations. Of those tested, Ca2+, Sr2+, Ba2+, Co2+, Cu2+, Mg2+, and Zn2+ were all effective. Both phenylmethylsulfonylfluoride and diisopropylfluorophosphate were powerful inhibitors of protease activity, but L-1-tosylamide-2-phenylethylchloromethyl ketone, iodoacetic acid, and iodoacetamide were without effect. The enzyme hydrolyzed the esters N-acetyl-L-tyrosine ethyl ester and alpha-N-benzoyl-L-arginine ethyl ester (BAEE) with Km values of 10.4 and 3.4 mM, respectively. The hydrolysis of BAEE was also inhibited by phenylarsonic acids. The kinetics of inhibition by m-nitrophenylarsonate were of the mixed type, and the K1 was 1.8 mM. The data followed a theoretical curve for a 1:1 enzyme-inhibitor complex with a dissociation constant of 1.8 mM. Inhibition by m-nitrophenylarsonate was pH dependent and followed a theoretical curve for the titration of a protonated group with a pKa of 7.0.
Insights
This study details the purification and characterization of an extracellular protease from Pseudomonas maltophilia. The enzyme shows specific inhibition patterns and catalytic activity, offering insights into microbial enzyme function.
Area of Science:
- Microbiology
- Enzymology
- Biochemistry
Background:
- Pseudomonas maltophilia produces extracellular proteases.
- Understanding these enzymes is crucial for microbial physiology and potential biotechnological applications.
Purpose of the Study:
- To purify and characterize the extracellular protease from Pseudomonas maltophilia.
- To investigate its enzymatic properties, substrate specificity, and inhibition mechanisms.
Main Methods:
- Partial purification using ammonium sulfate precipitation and Sephadex G-75/Bio-rex 70 chromatography.
- Enzyme characterization including molecular weight, isoelectric point, pH optimum, and sedimentation coefficient.
- Inhibition studies using various chemical agents and kinetic analysis.
Main Results:
- The purified protease has a molecular weight of 35,000, isoelectric point of 9.3, and pH optimum of 10.0.
- The enzyme is inactivated by ethylenediaminetetracetate but restored by divalent cations.
- It is potently inhibited by phenylmethylsulfonylfluoride and diisopropylfluorophosphate, and by phenylarsonic acids.
Conclusions:
- The Pseudomonas maltophilia extracellular protease is a serine protease with alkaline activity.
- Its activity is modulated by divalent cations and specific inhibitors, providing a basis for further functional studies.