TY - JOUR
T1 - Purification and characterization of an extracellular medium-chain length polyhydroxyalkanoate depolymerase from Thermus thermophilus HB8
AU - Papaneophytou, Christos P.
AU - Velali, Ekaterini E.
AU - Pantazaki, Anastasia A.
PY - 2011/4
Y1 - 2011/4
N2 - During growth on medium-chain length (mcl) polyhydroxyalkanoates (PHAs), or on sodium octanoate Thermus thermophilus HB8 produces an extracellular mcl-PHA depolymerase. This enzyme was purified from the culture medium of sodium octanoate-grown cells to electrophoretic homogeneity by hydrophobic interaction chromatography using Octyl-Sepharose CL-4B and gel permeation chromatography using Sephadex G-150. The molecular mass of the purified enzyme was approximately 28 kDa. A part of the gene TTHA1605 encoding a 24.17 kDa protein was demonstrated to encode the mcl-PHA depolymerase of T. thermophilus. The primary amino-acid sequence of purified enzyme reveals similarity to all reported so far extracellular mcl-PHA depolymerases. The purified enzyme could hydrolyze mcl - PHAs and p-nitrophenyl (pNP) esters but not short chain length (scl) - PHAs. The optimum pH range was 7.5-9 and the optimum temperature was 70 °C for pNP-octanoate (pNPO) hydrolysis. The Km value for pNPO was 53.2 μM. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF) and non-ionic detergents (Tween 20, Tween 80 and Triton X-100). The results demonstrated in this study revealed that the mcl-PHA depolymerase from T. thermophilus is a distinct enzyme, which is different from those of other mcl-PHA-degrading bacteria.
AB - During growth on medium-chain length (mcl) polyhydroxyalkanoates (PHAs), or on sodium octanoate Thermus thermophilus HB8 produces an extracellular mcl-PHA depolymerase. This enzyme was purified from the culture medium of sodium octanoate-grown cells to electrophoretic homogeneity by hydrophobic interaction chromatography using Octyl-Sepharose CL-4B and gel permeation chromatography using Sephadex G-150. The molecular mass of the purified enzyme was approximately 28 kDa. A part of the gene TTHA1605 encoding a 24.17 kDa protein was demonstrated to encode the mcl-PHA depolymerase of T. thermophilus. The primary amino-acid sequence of purified enzyme reveals similarity to all reported so far extracellular mcl-PHA depolymerases. The purified enzyme could hydrolyze mcl - PHAs and p-nitrophenyl (pNP) esters but not short chain length (scl) - PHAs. The optimum pH range was 7.5-9 and the optimum temperature was 70 °C for pNP-octanoate (pNPO) hydrolysis. The Km value for pNPO was 53.2 μM. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF) and non-ionic detergents (Tween 20, Tween 80 and Triton X-100). The results demonstrated in this study revealed that the mcl-PHA depolymerase from T. thermophilus is a distinct enzyme, which is different from those of other mcl-PHA-degrading bacteria.
KW - Biodegradation
KW - Extracellular PHA depolymerase
KW - Medium-chain length polyhydroxyalkanoates (mcl-PHAs)
KW - Thermus thermophilus HB8
UR - http://www.scopus.com/inward/record.url?scp=79952190976&partnerID=8YFLogxK
U2 - 10.1016/j.polymdegradstab.2010.12.015
DO - 10.1016/j.polymdegradstab.2010.12.015
M3 - Article
AN - SCOPUS:79952190976
SN - 0141-3910
VL - 96
SP - 670
EP - 678
JO - Polymer Degradation and Stability
JF - Polymer Degradation and Stability
IS - 4
ER -