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1Institute of Molecular Genetics, Russian Academy of Science, Kurchatov Sq. 46, 123 182 Moscow, Russia
2Institute for Microbiology, Technical University of München, Arcisstr. 21, D-80290 München, Germany
ABSTRACT
The nucleotide sequence of clone pTT26 (3786 bp), containing the gene for 1,3-β-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-β-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 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-1, respectively) and Km values of 2.8 and 2.2 mg ml-1, respectively. Limited activity on 1,3-1,4-β-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)-β-glucanase (EC 3.2.1.6). The optimum temperature of the enzymes was 95 ° (73 kDa) and 85 ° (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 °, where 57% of the initial activity remained after 30 min (82% at 95°). Thus, LamA is the most thermostable 1,3-β-glucanase described to date.
Author for correspondence: Wolfgang H. Schwarz. Tel. + 49 89 2892 2302. Fax: -49 89 2892 2360. e-mail: Wolfgang.schwarz@mikro.bioiogie.tu-muenchen.de
Present address: Institute for Microbiology, Technical University of München, Arcisstr. 21, D-80290 München, Germany.
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