THERMOTOGA NEOPOLITANA BGLB GENE, UPSTREAM OF LAMA, ENCODES A HIGHLY THERMOSTABLE BETA-GLUCOSIDASE THAT IS A LAMINARIBIASE

The gene for thermostable 1,3-beta-glucosidase BglB was cloned from th e chromosome of Thermotoga neapolitana and its primary sequence was de termined. The purified recombinant beta-glucosidase B had a monomer mo lecular mass of 81 kDa in accordance with the amino acid sequence pred icted from the nucleotide sequence of clone pTT51, It was a member of glycosylhydrolase family 3 and belonged to enzyme class EC 3.2.1.21. b eta-Glucosidase B had a specific activity of 255 U mg(-1) on 4-nitroph enyl(PNP)-beta-glucoside at the optima of pH (5.5) and temperature (90 degrees C), and K-m values of 0.1, 10 and 50 mM for PNP-beta-glucosid e, laminaribiose and cellobiose, respectively. The gene bglB was locat ed immediately upstream of the laminarinase gene lamA. Both genes were transcribed from the same DNA strand and were not separated by a pali ndromic transcription terminator. The two purified enzymes 1,3-beta-gl ucosidase BglB (laminaribiase) and 1,3-beta-glucanase LamA (laminarina se) were together capable of completely degrading laminarin to glucose .