Elbanna Khaled, Lütke-Eversloh Tina, Jendrossek Dieter, Luftmann Heinrich, Steinbüchel Alexander
Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstrasse 3, 48149 Münster, Germany.
Arch Microbiol. 2004 Oct;182(2-3):212-25. doi: 10.1007/s00203-004-0715-z. Epub 2004 Aug 31.
The biodegradability of microbial polythioesters (PTEs), a novel class of biopolymers which were discovered recently and can be produced by polyhydroxyalkanoate (PHA)-accumulating bacteria, was studied. Using poly(3-hydroxybutyrate- co-3-mercaptopropionate) [poly(3HB- co-3MP)] as sole carbon source for screening, 22 new bacterial strains were isolated and characterized. Interestingly, none of the PHA-degrading bacteria was able to utilize the homopolymer poly(3MP) as a carbon source for growth or to form clear zones on poly(3MP)-containing agar plates. The extracellular PHA depolymerases from two strains ( Schlegelella thermodepolymerans, Pseudomonas indica K2) were purified to electrophoretic homogeneity and biochemically characterized. The PHA depolymerase of S. thermodepolymerans exhibited a temperate optimum of about 75 degrees C to 80 degrees C and was stable at 70 degrees C for more than 24 h. Regarding the substrate specificities of the PHA depolymerase of S. thermodepolymerans, enzyme activities decreased significantly with increasing 3MP content of the copolymer substrates. Interestingly, no activity could be detected with homoPTEs consisting only of 3MP or of 3-mercaptobutyrate. Similar results were obtained with the PHA depolymerases PhaZ2, PhaZ5 and PhaZ7 of Paucimonas lemoignei which were also investigated. The PHA depolymerase of Ps. indica K2 did not cleave any of the investigated polymers containing 3MP. Gas chromatography, infrared and (1)H-NMR spectrometry and matrix-assisted laser desorption/ionization time-of-flight analysis revealed that 3MPs containing oligomers were enriched in the water-insoluble fraction remaining after partial digestion of poly(3HB- co-3MP) by purified poly(3HB) depolymerase of S. thermodepolymerans. In contrast, 3HB was enriched in the water-soluble fraction, which also contained 3HB- co-3MP dimer obtained by partial digestion of this copolymer by the enzyme. This study clearly indicates that PHA depolymerases are specific for oxoester linkages of PHAs and that the thioester bonds of PTEs cannot be cleaved by this type of enzyme.
对微生物聚硫酯（PTEs）的生物降解性进行了研究，PTEs是一类新发现的生物聚合物，可由积累聚羟基脂肪酸酯（PHA）的细菌产生。以聚（3-羟基丁酸酯- co -3-巯基丙酸酯）[聚（3HB- co -3MP）]作为唯一碳源进行筛选，分离并鉴定了22株新的细菌菌株。有趣的是，没有一株PHA降解细菌能够利用均聚物聚（3MP）作为生长的碳源，也不能在含聚（3MP）的琼脂平板上形成透明圈。对两株菌株（嗜热解聚施氏菌、印度假单胞菌K2）的胞外PHA解聚酶进行了纯化，使其达到电泳纯，并进行了生化特性分析。嗜热解聚施氏菌的PHA解聚酶在约75℃至80℃时表现出最适温度，在70℃下稳定超过24小时。关于嗜热解聚施氏菌PHA解聚酶的底物特异性，随着共聚物底物中3MP含量的增加，酶活性显著降低。有趣的是，对于仅由3MP或3-巯基丁酸组成的均聚PTEs，未检测到活性。对勒莫因寡养单胞菌的PHA解聚酶PhaZ2、PhaZ5和PhaZ7也进行了研究，得到了类似的结果。印度假单胞菌K2的PHA解聚酶不能切割任何一种含3MP的研究聚合物。气相色谱、红外光谱、（1）H-NMR光谱和基质辅助激光解吸/电离飞行时间分析表明，在嗜热解聚施氏菌纯化的聚（3HB）解聚酶对聚（3HB- co -3MP）进行部分消化后，剩余的水不溶性部分中富含含3MP的低聚物。相反，3HB富集在水溶性部分中，该部分还包含通过该酶对该共聚物进行部分消化而获得的3HB- co -3MP二聚体。这项研究清楚地表明，PHA解聚酶对PHA的氧酯键具有特异性，而PTEs的硫酯键不能被这类酶切割。
