Liu Bo, Ni Jin-Feng, Shen Yu-Long
State Key Laboratory of Microbial Technology, University of Shandong, Jinan 250100, China.
Wei Sheng Wu Xue Bao. 2006 Apr;46(2):255-8.
Chitin is the second most abundant organic compound in nature and the degradation of this biomass is an important process in the recycling of nutrients in the environments. Several biodegradation pathway of chitin have been classified in eukaryotes and bacteria, and a unique chitin degradation pathway was proposed according to recent studies on hyperthermophilic archaeon Thermococcus kodakaraensis. In the genome of Pyrococcus horikoshii, several ORFs show high homology to the chitin-degrading related genes from T. kodakararaensis, therefore P. horikoshii is likely to have the same chitin degrading pathway as that of T. kodakaraensis. In order to further characterize the novel chitin degrading pathway in thermophilic archaea, a diacetylchitobiose deacetylase from P. horikoshii (Dacph) was studied in the present study. Dacph belongs to the LmbE-like protein family and the amino sequence is not related to the other deacetylases studied before (except that in T. kodakararaensis). The gene (Dacph, PH0499) from the hyperthermophilic archaeon P. horikoshii was amplified by polymerase chain reaction, cloned into expression vector pET15b, and expressed in E. coli BL21-codonPlus (DE3)-RIL. A soluble fraction of Dacph (31.6kDa) was obtained as shown by SDS-PAGE. TLC analysis showed that Dacph is able to deacetylate one acetyl group of GlcNAc2 and GlcNAc. By the concerted reaction with the Exo-beta-D-Glucosaminid-ase (BglAph), it is also able to convert GlcNAc2 into GlcN. It is concluded that PH0499 is a diacetylchit-obiose deacetylase. By reaction together with Exo-beta-D-Glucosaminidase in P. horikoshii, Dacph probably plays a key role in the new chitin degradation pathway in hyperthermophilic archaea (the genera Thermococcus and Pyrococcus).
几丁质是自然界中第二丰富的有机化合物，这种生物质的降解是环境中养分循环的重要过程。几丁质的几种生物降解途径已在真核生物和细菌中分类，并且根据对嗜热古菌柯达嗜热栖热菌的最新研究提出了一种独特的几丁质降解途径。在嗜热栖热菌的基因组中，几个开放阅读框与来自柯达嗜热栖热菌的几丁质降解相关基因具有高度同源性，因此嗜热栖热菌可能具有与柯达嗜热栖热菌相同的几丁质降解途径。为了进一步表征嗜热古菌中的新型几丁质降解途径，本研究对嗜热栖热菌的二乙酰壳二糖脱乙酰酶（Dacph）进行了研究。Dacph属于LmbE样蛋白家族，其氨基酸序列与之前研究的其他脱乙酰酶无关（除了柯达嗜热栖热菌中的）。通过聚合酶链反应扩增嗜热古菌嗜热栖热菌的基因（Dacph，PH0499），克隆到表达载体pET15b中，并在大肠杆菌BL21-codonPlus(DE3)-RIL中表达。如SDS-PAGE所示，获得了Dacph的可溶性部分（31.6kDa）。薄层色谱分析表明，Dacph能够使GlcNAc2和GlcNAc的一个乙酰基脱乙酰化。通过与外切β-D-氨基葡萄糖苷酶（BglAph）协同反应，它还能够将GlcNAc2转化为GlcN。得出结论，PH0499是一种二乙酰壳二糖脱乙酰酶。通过与嗜热栖热菌中的外切β-D-氨基葡萄糖苷酶一起反应，Dacph可能在嗜热古菌（嗜热栖热菌属和嗜热栖热菌属）的新几丁质降解途径中起关键作用。
