Expression, refolding, and purification of active diacetylchitobiose deacetylase from Pyrococcus horikoshii.
A chitinase from the hyperthermophilic archaeon Pyrococcus furiosus degrades chitin to produce diacetylchitobiose [(GlcNAc)(2)] as the end product. To further investigate the degradation mechanism of (GlcNAc)(2) in Pyrococcus spp., we cloned the gene of PH0499 from Pyrococcus horikoshii, which encodes a protein homologous to the diacetylchitobiose deacetylase of Thermococcus kodakaraensis. The deacetylase (Ph-Dac) was overexpressed as inclusion bodies in Escherichia coli Rosetta (DE3) pLys. The insoluble inclusion body was solubilized and reactivated through a refolding procedure. After several purification steps, 40 mg of soluble, thermostable (up to 80°C) Ph-Dac was obtained from 1L of culture. The apparent molecular mass of the refolded Ph-Dac was 180 kDa, indicating Ph-Dac to be a homohexamer. The refolded Ph-Dac also exhibited deacetylase activity toward (GlcNAc)(2), and the deacetylation site was revealed to be specific to the nonreducing end residue of (GlcNAc)(2). These expression and purification systems are useful for further characterization of Ph-Dac.
National Institute of Advanced Industrial Science and Technology (AIST), Ikeda, Osaka, Japan.
Shouhei
S
Mine
Takahisa
T
Ikegami
Kazunori
K
Kawasaki
Tsutomu
T
Nakamura
Koichi
K
Uegaki
D000107:Acetylation; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002686:Chitin / Q000378:metabolism; D004187:Disaccharides / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D002479:Inclusion Bodies / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D055503:Protein Multimerization; D058849:Protein Refolding; D055550:Protein Stability; D043902:Pyrococcus horikoshii / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015854:Up-Regulation
D019843:Archaeal Proteins; D004187:Disaccharides; D011994:Recombinant Proteins; D002686:Chitin; C010913:N,N-diacetylchitobiose