HIGHLY THERMOSTABLE ENDO-1,3-BETA-GLUCANASE (LAMINARINASE) LAMA FROM THERMOTOGA-NEAPOLITANA - NUCLEOTIDE-SEQUENCE OF THE GENE AND CHARACTERIZATION OF THE RECOMBINANT GENE-PRODUCT

The nucleotide sequence of clone pTT26 (3786 bp), containing the gene for 1,3-beta-glucanase LamA (laminarinase) from Thermotoga neapolitana , was determined. It contains an ORF encoding a protein of 646 aa (73 328 Da). The central part of the protein is homologous to the complete catalytic domain of bacterial and some eukaryotic endo-1,3-beta-D-glu canases and belongs to family 16 of glycosyl hydrolases. This domain i s flanked on both sides by one copy on each side of a substrate bindin g domain homologue (family II). The recombinant laminarinase protein w as purified from Escherichia coil host cells in two forms, a 73 kDa an d a processed 52 kDa protein, both having high specific activity towar ds laminarin (3100 and 2600 U mg(-1), respectively) and K-m values of 2.8 and 2.2 mg ml(-1), respectively. Limited activity on 1,3-1,4-beta- glucan (lichenan) was detected (90 U mg(-1)). Laminarin was degraded i n an endoglucanase modus, yielding glucose, laminaribiose and -triose as end products, Thus LamA classifies as an endo-1,3(4)-beta-glucanase (EC 3.2.1.6). The optimum temperature of the enzymes was 95 degrees C (73 kDa) and 85 degrees C (52 kDa) at an optimum pH of 6.2. The super ior thermostability of the 73 kDa enzyme is demonstrated by incubation without substrate at 100 degrees C, where 57% of the initial activity remained after 30 min (82% at 95 degrees C), Thus, LamA is the most t hermostable 1,3-beta-glucanase described to date.