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Enzymatic depilation of animal hide: identification of elastase (LasB) from Pseudomonas aeruginosa MCM B-327 as a depilating protease.

Pandeeti EV, Pitchika GK, Jotshi J, Nilegaonkar SS, Kanekar PP, Siddavattam D - PLoS ONE (2011)

Bottom Line: This 33 kDa protease generated a peptide mass fingerprint and de novo sequence that matched perfectly with LasB (elastase), of Pseudomonas aeruginosa.LasB heterologously over-produced and purified from Escherichia coli also exhibited high depilating activity.Moreover, reintroduction of the lasB gene to the P. aeruginosa lasB mutant via a knock-in strategy also successfully restored depilation activity thus confirming the role of LasB as the depilating enzyme.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderabad, Andhra Pradesh, India.

ABSTRACT
Conventional leather processing involving depilation of animal hide by lime and sulphide treatment generates considerable amounts of chemical waste causing severe environmental pollution. Enzymatic depilation is an environmentally friendly process and has been considered to be a viable alternative to the chemical depilation process. We isolated an extracellular protease from Pseudomonas aeruginosa strain MCM B-327 with high depilation activity using buffalo hide as a substrate. This 33 kDa protease generated a peptide mass fingerprint and de novo sequence that matched perfectly with LasB (elastase), of Pseudomonas aeruginosa. In support of this data a lasB mutant of MCM B-327 strain lacked depilatory activity and failed to produce LasB. LasB heterologously over-produced and purified from Escherichia coli also exhibited high depilating activity. Moreover, reintroduction of the lasB gene to the P. aeruginosa lasB mutant via a knock-in strategy also successfully restored depilation activity thus confirming the role of LasB as the depilating enzyme.

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Related in: MedlinePlus

Identification of depilating protease of Pseudomonas aeruginosa MCM B-327.Panel A and B represent native PAGE and the corresponding zymogram of extracellular proteins of Pseudomonas aeruginosa MCM B-327. Panel C. SDS-PAGE showing the molecular mass of the depilating protease electro-eluted from the zymogram. Lane 1 represents protein molecular mass markers. The 33 kDa depilating protease band found in lane 2 is shown with an arrow. Depilating activity shown by crude extracellular protease (a), protease electro-eluted from the zymogram (b) and a control hide kept by adding buffer instead of protease are shown in panel D.
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pone-0016742-g001: Identification of depilating protease of Pseudomonas aeruginosa MCM B-327.Panel A and B represent native PAGE and the corresponding zymogram of extracellular proteins of Pseudomonas aeruginosa MCM B-327. Panel C. SDS-PAGE showing the molecular mass of the depilating protease electro-eluted from the zymogram. Lane 1 represents protein molecular mass markers. The 33 kDa depilating protease band found in lane 2 is shown with an arrow. Depilating activity shown by crude extracellular protease (a), protease electro-eluted from the zymogram (b) and a control hide kept by adding buffer instead of protease are shown in panel D.

Mentions: The spent medium collected from a culture grown for 72 h was initially brought to various levels of saturation with ammonium sulphate. The proteins precipitated during each stage of saturation were independently tested for both protease and depilating activity. Only the protein precipitate obtained from the spent medium saturated to 60% ammonium sulphate showed the presence of a protease with depilating activity. A zymogram developed for the proteins found in this fraction revealed the existence of a single clear zone around a major protein band (Figure 1 A & B). No other clear zones were seen in the entire zymogram, indicating the existence of a single protease complex in the 60% ammonium sulphate precipitate. To gain further insights into the depilating activity the protein band tested to be protease-positive in the zymogram was electro-eluted from the native gel and was applied to a fresh buffalo hide. The electro-eluted protein gave a single band of 33 kDa on SDS-PAGE and successfully depilated animal hide providing direct evidence for the depilatory properties of the extracellular protease (Figure 1C & D).


Enzymatic depilation of animal hide: identification of elastase (LasB) from Pseudomonas aeruginosa MCM B-327 as a depilating protease.

Pandeeti EV, Pitchika GK, Jotshi J, Nilegaonkar SS, Kanekar PP, Siddavattam D - PLoS ONE (2011)

Identification of depilating protease of Pseudomonas aeruginosa MCM B-327.Panel A and B represent native PAGE and the corresponding zymogram of extracellular proteins of Pseudomonas aeruginosa MCM B-327. Panel C. SDS-PAGE showing the molecular mass of the depilating protease electro-eluted from the zymogram. Lane 1 represents protein molecular mass markers. The 33 kDa depilating protease band found in lane 2 is shown with an arrow. Depilating activity shown by crude extracellular protease (a), protease electro-eluted from the zymogram (b) and a control hide kept by adding buffer instead of protease are shown in panel D.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037957&req=5

pone-0016742-g001: Identification of depilating protease of Pseudomonas aeruginosa MCM B-327.Panel A and B represent native PAGE and the corresponding zymogram of extracellular proteins of Pseudomonas aeruginosa MCM B-327. Panel C. SDS-PAGE showing the molecular mass of the depilating protease electro-eluted from the zymogram. Lane 1 represents protein molecular mass markers. The 33 kDa depilating protease band found in lane 2 is shown with an arrow. Depilating activity shown by crude extracellular protease (a), protease electro-eluted from the zymogram (b) and a control hide kept by adding buffer instead of protease are shown in panel D.
Mentions: The spent medium collected from a culture grown for 72 h was initially brought to various levels of saturation with ammonium sulphate. The proteins precipitated during each stage of saturation were independently tested for both protease and depilating activity. Only the protein precipitate obtained from the spent medium saturated to 60% ammonium sulphate showed the presence of a protease with depilating activity. A zymogram developed for the proteins found in this fraction revealed the existence of a single clear zone around a major protein band (Figure 1 A & B). No other clear zones were seen in the entire zymogram, indicating the existence of a single protease complex in the 60% ammonium sulphate precipitate. To gain further insights into the depilating activity the protein band tested to be protease-positive in the zymogram was electro-eluted from the native gel and was applied to a fresh buffalo hide. The electro-eluted protein gave a single band of 33 kDa on SDS-PAGE and successfully depilated animal hide providing direct evidence for the depilatory properties of the extracellular protease (Figure 1C & D).

Bottom Line: This 33 kDa protease generated a peptide mass fingerprint and de novo sequence that matched perfectly with LasB (elastase), of Pseudomonas aeruginosa.LasB heterologously over-produced and purified from Escherichia coli also exhibited high depilating activity.Moreover, reintroduction of the lasB gene to the P. aeruginosa lasB mutant via a knock-in strategy also successfully restored depilation activity thus confirming the role of LasB as the depilating enzyme.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderabad, Andhra Pradesh, India.

ABSTRACT
Conventional leather processing involving depilation of animal hide by lime and sulphide treatment generates considerable amounts of chemical waste causing severe environmental pollution. Enzymatic depilation is an environmentally friendly process and has been considered to be a viable alternative to the chemical depilation process. We isolated an extracellular protease from Pseudomonas aeruginosa strain MCM B-327 with high depilation activity using buffalo hide as a substrate. This 33 kDa protease generated a peptide mass fingerprint and de novo sequence that matched perfectly with LasB (elastase), of Pseudomonas aeruginosa. In support of this data a lasB mutant of MCM B-327 strain lacked depilatory activity and failed to produce LasB. LasB heterologously over-produced and purified from Escherichia coli also exhibited high depilating activity. Moreover, reintroduction of the lasB gene to the P. aeruginosa lasB mutant via a knock-in strategy also successfully restored depilation activity thus confirming the role of LasB as the depilating enzyme.

Show MeSH
Related in: MedlinePlus