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

摘要

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-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 II). The recombinant laminarinase protein was purified from Escherichia coil 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(-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 in 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 superior 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 thermostable 1,3-beta-glucanase described to date.