Diversity of polyester-degrading bacteria in compost and molecular analysis of a thermoactive esterase from Thermobifida alba AHK119
Diversity of polyester-degrading bacteria in compost and molecular analysis of a thermoactive esterase from Thermobifida alba AHK119
复制标题
堆肥中聚酯降解细菌的多样性以及来自Thermobifida alba AHK119的热活性酯酶的分子分析
DOI：
10.1007/s00253-010-2555-x
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发表时间：
2010-06-01
影响因子：
5
通讯作者：
Kawai, Fusako
中科院分区：
文献类型：
--
作者：
Hu, Xiaoping;Thumarat, Uschara;Kawai, Fusako
从堆肥聚酯薄膜中分离出100多株细菌，分为放线菌（4属）和芽孢杆菌（3属）两大类。在这些分离株中，当在50 ℃下在补充有聚合物颗粒的LB培养基上生长时，白色嗜热裂菌菌株AHK 119（AB 298783）显示出具有显著降解芳香族-芳香族聚酰胺膜以及减小聚合物颗粒尺寸的能力，产生对苯二甲酸。从AHK 119中克隆了酯酶基因（est 119，903 bp，分别编码34和266个氨基酸的信号肽和成熟蛋白）。Est 119序列包含保守的脂肪酶盒（-G-X-S-X-G-）和催化三联体（Ser 129、His 207和Asp 175）。此外，Tyr 59和Met 130可能形成氧阴离子空穴。从携带pQE 80 L-est 119的大肠杆菌Rosetta-gami B（DE 3）的无细胞提取物中纯化重组酶。这种酶是一种单体蛋白质。30 kDa，其在20 ° C至75 ° C（最佳范围为45至55 ° C）和5.5至7.0的pH范围（最佳pH为6.0）内具有活性。在对硝基苯基酰基酯（C2至C8）中，其优选的底物是对硝基苯基己酸酯（C6），表明该酶是酯酶而不是脂肪酶。
More than 100 bacterial strains were isolated from composted polyester films and categorized into two groups, Actinomycetes (four genera) and Bacillus (three genera). Of these isolates, Thermobifida alba strain AHK119 (AB298783) was shown to possess the ability to significantly degrade aliphatic-aromatic copolyester film as well as decreasing the polymer particle sizes when grown at 50 degrees C on LB medium supplemented with polymer particles, yielding terephthalic acid. The esterase gene (est119, 903 bp, encoding a signal peptide and a mature protein of 34 and 266 amino acids, respectively) was cloned from AHK119. The Est119 sequence contains a conserved lipase box (-G-X-S-X-G-) and a catalytic triad (Ser129, His207, and Asp175). Furthermore, Tyr59 and Met130 likely form an oxyanion hole. The recombinant enzyme was purified from cell-free extracts of Escherichia coli Rosetta-gami B (DE3) harboring pQE80L-est119. The enzyme is a monomeric protein of ca. 30 kDa, which is active from 20 degrees C to 75 degrees C (with an optimal range of 45 to 55 degrees C) and in a pH range of 5.5 to 7.0 (with an optimal pH of 6.0). Its preferred substrate among the p-nitrophenyl acyl esters (C2 to C8) is p-nitrophenyl hexanoate (C6), indicating that the enzyme is an esterase rather than a lipase.