Purification and characterization of a new hyperthermostable, allosamidin-insensitive and denaturation-resistant chitinase from the hyperthermophilic archaeon Thermococcus chitonophagus

Abstract.

A new chitinase (1,4-β-D- N-acetyl-glucosaminidase, EC 3.2.1.14) was detected and purified to homogeneity in its native form from the chitinolytic enzyme system of the extremely thermophilic archaeon Thermococcus chitonophagus. This is the first nonrecombinant chitinase purified and characterized from archaea and also constitutes the first case of a membrane-associated chitinase isolated from archaea. The enzyme is a monomer with an apparent molecular weight of 70 kDa [therefore named chitinase 70 (Chi70)] and pI of 5.9; it is hydrophobic and appears to be associated with the outer side of the cell membrane. Chi70 is optimally active at 70°C and pH 7.0 and exhibits remarkable thermostability, maintaining 50% activity even after 1 h at 120°C, and therefore the enzyme is the most thermostable chitinase so far isolated. The enzyme was not inhibited by allosamidin, the natural inhibitor of chitinolytic activity, and was also resistant to denaturation by urea and SDS. On the other hand, guanidine hydrochloride significantly reduced enzymatic activity, indicating that, apart from the hydrophobic interactions, ion pairs located on the surface of the protein could be playing an important role in maintaining the protein's fold and enzyme activity. Chi70 showed broad substrate specificity for several chitinous substrates and derivatives. The lowest K _m and highest K _cat values were found for pNP(NAG)₂ as substrate and were determined to be 0.14 mM and 23 min^–1, respectively. The hydrolysis pattern was similar for oligomers and polymers, with N,N′-diacetylchitobiose [(NAG)₂] being the final, major hydrolysis product. Chi70 was classified as an endochitinase due to its ability to release chitobiose from colloidal chitin. Additionally, the enzyme presented considerable cellulolytic activity. Analysis of the NH₂-terminal amino acid sequence showed no detectable homology with other known sequences, suggesting that Chi70 is a new protein.