The nucleotide sequence of clone p n 2 6 (3786 bp), containing the gene for 1,3-pglucanase LamA (laminarinase) from Tnermofoga neapolitana, was determined. It contains an ORF encoding a protein of 646 aa (73328 Da). The central part of the protein is homologous to the complete catalytic domain of bacterial and some eukaryotic endo-l,3-&~-glucanases and belongs to family 16 of glycosyl hydrolases. This domain is flanked on both sides by one copy on each side of a substrate binding domain homologue (family 11). The recombinant laminarinase protein was purified from Escherichia coli host cells in two forms, a 73 kDa and a processed 52 kDa protein, both having high specific activity towards laminarin (3100 and 2600 U mg-l, respectively) and Km values of 2.8 and 2.2 mg mF, respectively. Limited activity on 1,3-1,4-/3-glucan (lichenan) was detected (90 U mg-l). Laminarin was degraded in an endoglucanase modus, yielding glucose, laminaribiose and ltriose as end products. Thus LamA classifies as an endo-l,3(4)-/3=gIucanase (EC 3.2.1.6). The optimum temperature of the enzymes was 95 "C (73 kDa) and 85 "C (52 kDa) at an optimum pH of 62. The superior thermostability of the 73 kDa enzyme is demonstrated by incubation without substrate at 100 "C, where 57% of the initial activity remained after 30 min (82% at 95 OC). Thus, LamA is the most thermostable 1,3-&glucanase described to date.

The gene for thermostable 1,3-/?-glucosidase BglB was cloned from the chromosome of Thermotoga neapolitana and its primary sequence was determined. The purified recombinant pglucosidase B had a monomer molecular mass of 81 kDa in accordance with the amino acid sequence predicted 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. pGlucosidase B had a specific activity of 255 U mg-l on Lbnitrophenyl(PNP)-pglucoside a t the optima of pH (5-5) and temperature (90 "C), and K,,, values of 0 -1 , I O and 50 mM for PNP-/?-glucoside, laminaribiose and cellobiose, respectively. The gene bglB was located immediately upstream of the laminarinase gene lamA. Both genes were transcribed from the same DNA strand and were not separated by a palindromic transcription terminator. The two purified enzymes 1,3-&glucosidase BglB (laminaribiase) and 1,3-& glucanase LamA (laminarinase) were together capable of completely degrading laminarin to glucose.

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