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Purification and characterization of an eggshell membrane decomposing protease from Pseudomonas aeruginosa strain
Minyi Cheng1, Shinji Takenaka, Shunsuke Aoki
1Department of Applied Biological Chemistry, Graduate School of Agricultural Science, Kobe University, Rokko, Kobe, Japan.
Abstract:
A bacterial strain, ME-4, isolated from farm soil and identified as Pseudomonas aeruginosa, grew well on a medium containing eggshell membrane (ESM). P. aeruginosa strain ME-4 decomposed the ESM by producing an extracellular protease able to solubilize it. The protease was purified to homogeneity from culture supernatant by fractionation with (NH(4))(2)SO(4), as well as CM52 cellulose and DE52 cellulose column chromatography, with a final yield of 47%. The molecular mass of the enzyme was 33 kDa. The isolated enzyme was a metalloprotease and was strongly inhibited by EDTA, o-phenanthroline, and phosphoramidon. The enzyme inhibited by these reagents was reactivated in the presence of several metal ions. The enzyme acted on various proteins and showed higher activity with collagen than collagenase from Clostridium histolyticum. Results of assays with the FRETS combinatorial libraries revealed that the enzyme preferred Ser at the P1 position and Lys at the P2 position. It also preferred hydrophobic amino acid residues at the P1' and P2' positions. The enzyme showed a much higher solubilization activity with the ESM substrate than commercially obtained enzymes. The enzyme decomposed ESM to produce water-soluble peptides, Val-Leu-Pro-Pro and (X)-Val-Pro-Pro, and a free amino acid, tryptophan.
Insights
Pseudomonas aeruginosa strain ME-4 secretes a metalloprotease that effectively decomposes eggshell membrane (ESM). This purified enzyme shows high activity against collagen and ESM, yielding valuable peptides and amino acids.
Area of Science:
- Microbiology
- Biochemistry
- Enzymology
Background:
- Eggshell membrane (ESM) is a rich biopolymer with potential applications.
- Decomposition of ESM requires specific enzymes, such as proteases.
- Pseudomonas aeruginosa is known for its diverse metabolic capabilities.
Purpose of the Study:
- To isolate and characterize a novel protease from Pseudomonas aeruginosa ME-4 capable of decomposing ESM.
- To evaluate the enzyme's efficiency and substrate specificity for potential biotechnological applications.
Main Methods:
- Isolation and identification of bacterial strain ME-4 (Pseudomonas aeruginosa).
- Purification of extracellular protease using (NH(4))(2)SO(4) fractionation and ion-exchange chromatography.
- Enzyme characterization including molecular mass determination (33 kDa), metalloprotease identification, and substrate specificity assays.
- Analysis of ESM decomposition products using peptide and amino acid analysis.
Main Results:
- A 33 kDa metalloprotease was purified to homogeneity from Pseudomonas aeruginosa ME-4 culture supernatant.
- The enzyme exhibited strong inhibition by EDTA and reactivation by metal ions, confirming its metalloprotease nature.
- It demonstrated higher activity against collagen than Clostridium histolyticum collagenase and efficiently solubilized ESM.
- Substrate specificity assays indicated a preference for Ser (P1) and Lys (P2), and hydrophobic residues at P1'/P2' positions.
- ESM decomposition yielded water-soluble peptides (Val-Leu-Pro-Pro, (X)-Val-Pro-Pro) and tryptophan.
Conclusions:
- Pseudomonas aeruginosa ME-4 produces a potent metalloprotease effective in decomposing eggshell membrane.
- This enzyme exhibits unique substrate specificity and superior solubilization activity compared to commercial enzymes.
- The findings suggest potential applications for this protease in biomaterial processing and waste valorization.
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