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. 1998 Jan;64(1):62-7.
doi: 10.1128/AEM.64.1.62-67.1998.

Purification and Properties of a Polyester Polyurethane-Degrading Enzyme from Comamonas acidovorans TB-35

Y Akutsu  1 , T Nakajima-Kambe, N Nomura, T Nakahara
Affiliations

Purification and Properties of a Polyester Polyurethane-Degrading Enzyme from Comamonas acidovorans TB-35

Y Akutsu et al. Appl Environ Microbiol. 1998 Jan.

Abstract

A polyester polyurethane (PUR)-degrading enzyme, PUR esterase, derived from Comamonas acidovorans TB-35, a bacterium that utilizes polyester PUR as the sole carbon source, was purified until it showed a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This enzyme was bound to the cell surface and was extracted by addition of 0.2% N,N-bis(3-d-gluconamidopropyl)deoxycholamide (deoxy-BIGCHAP). The results of gel filtration and SDS-PAGE showed that the PUR esterase was a monomer with a molecular mass of about 62,000 Da. This enzyme, which is a kind of esterase, degraded solid polyester PUR, with diethylene glycol and adipic acid released as the degradation products. The optimum pH for this enzyme was 6.5, and the optimum temperature was 45 degrees C. PUR degradation by the PUR esterase was strongly inhibited by the addition of 0.04% deoxy-BIGCHAP. On the other hand, deoxy-BIGCHAP did not inhibit the activity when p-nitrophenyl acetate, a water-soluble compound, was used as a substrate. These observations indicated that this enzyme degrades PUR in a two-step reaction: hydrophobic adsorption to the PUR surface and hydrolysis of the ester bond of PUR.

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Figures

FIG. 1
FIG. 1
Structure of PUR.
FIG. 2
FIG. 2
SDS-PAGE of the purified cell-bound esterase. Lane 1, molecular mass markers; lane 2, purified PUR esterase.
FIG. 3
FIG. 3
Time course of PUR degradation by purified PUR esterase. The reaction conditions are described in Materials and Methods.
FIG. 4
FIG. 4
Scanning electron micrograph of PUR. (A) Undegraded control; (B and C) PUR after degradation by 0.02 and 0.2 U of PUR esterase, respectively, for 24 h.
FIG. 5
FIG. 5
Effect of deoxy-BIGCHAP on the activity of the PUR esterase. The substrates used were PUR (○) and p-nitrophenyl acetate (•). The PUR degradation activity was estimated as the amount of diethylene glycol derived from PUR, and the p-nitrophenyl acetate degradation activity was estimated as the amount of p-nitrophenol derived from p-nitrophenyl acetate. The reaction conditions for each substrate are described in Materials and Methods.
FIG. 6
FIG. 6
Effect of PUR esterase concentration on PUR degradation.
FIG. 7
FIG. 7
Kinetic models of the surface binding and hydrolysis of PHA by PHA depolymerase (A) (17) and of PUR by the PUR esterase (B).
See this image and copyright information in PMC

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