Purification and characterization of an extracellular medium-chain length polyhydroxyalkanoate depolymerase from Thermus thermophilus HB8

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• 作者：Christos P. Papaneophytou^a ; Ekaterini E. Velali^a ; Anastasia A. Pantazaki ; ^a ; ^{natasa@chem.auth.gr}
• 关键词：Medium-chain length polyhydroxyalkanoates (mcl-PHAs) ; Extracellular PHA depolymerase ; Thermus thermophilus HB8 ; Biodegradation
• 刊名：Polymer Degradation and Stability
• 出版年：2011
• 出版时间：April 2011
• 年：2011
• 卷：96
• 期：4
• 页码：670-678
• 全文大小：1445 K

文摘

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.