极端嗜热古菌Pyrococcus horikoshii几丁二糖脱乙酰酶的克隆、表达及性质研究

利用PCR扩增技术从极端嗜热古菌Pyrococcus horikoshii中得到预测为几丁二糖脱乙酰酶的基因(Dacph,PH0499),将其克隆入表达质粒pET15b,并在E.coliBL21_codonPlus(DE3)_RIL中表达获得可溶的Dacph重组蛋白(31.6kDa),TLC分析证明Dacph能够脱去N_乙酰氨基葡萄糖及几丁二糖的一个乙酰基,并与氨基葡萄糖苷酶(BglAPh)共同作用水解几丁二糖生成氨基葡萄糖,从而被命名为一种几丁二糖脱乙酰酶。与Pyrococcus horikoshii中外切氨基葡萄糖苷酶等共同作用,Dacph可能在嗜热球古菌独特的几丁质降解途径中起重要作用。

Cloning, expression and biochemical characterization of a novel diacetylchitobiose deacetylase from the hyperthermophilic archaeon Pyrococcus horikoshii

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).