Xylanase B (XynTB) from hyperthermophilic bacterium Thermotoga maritima MSB8 is a thermostable xylanase classified into glycoside hydrolase family 10. XynTB is most active at pH 6.0, and shows lower activity at alkaline pHs. Improvement of alkaliphily of XynTB was attempted by directed evolution. One mutant enzyme that showed slightly higher activity under high temperature and alkaline pH conditions was acquired from a newly constructed random mutant library. Protein engineering study of this mutant revealed that the amino acid substitution N92D (Asn92 was substituted by Asp) could contribute to the improvement of alkaliphily.
An open reading frame encoding a chitinase homolog (ChiN1) was found in the genome of extremely halophilic archaeon Halobacterium salinarum NRC-1. ChiN1 is a multidomain enzyme consisting of a chitin-binding domain, a polycystic kidney disease domain and a catalytic domain belonging to glycoside hydrolase family 18. chiN1 gene was successfully expressed in extremely halophilic archaeon Haloarcula japonica TR-1 by employing the promoter sequence of its cell surface glycoprotein gene. A large amount of recombinant ChiN1 was secreted into the culture supernatant. The Ha. japonica-produced ChiN1 was purified and characterized. The optimal pH and temperature of ChiN1 are pH 4.5 and 55°C, respectively. ChiN1 was most active at 1.0 M NaCl and stable over a wide range of NaCl concentration from 1.0 to 4.5 M. This is the first report on a chitinase from extremely halophilic archaeon.
A procedure for the isolation of aliphatic polyester-degrading bacteria from deep-sea environments was developed using three step high-pressure cultivation. First, the sediments collected from the deep-sea floor, the Kurile and Japan Trenches at a depth of 5000-7000 m, were cultivated with poly ε-caprolactone (PCL) films under low-temperature (4°C) and high-pressure (50MPa) conditions. Secondly, high-pressure continuous cultivation using the deep-sea baro-piezophile and thermophile isolation and cultivation (DEEPBATH) system was performed three times to accumulate piezophilic bacteria. Finally, piezophilic PCL degrading bacteria were isolated using the pressure bag method involving media with PCL granules. Using this procedure, we succeeded in isolating several piezophilic aliphatic polyester-degrading bacteria from the sediment samples of deep-trenches.