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 (73328 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 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 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.
Zverlov
Vladimir V
VV
Institute of Molecular Genetics, Russian Academy of Science, Kurchatov Sq. 46, 123 182 Moscow, Russia.
Volkov
Ilia Y
IY
Institute of Molecular Genetics, Russian Academy of Science, Kurchatov Sq. 46, 123 182 Moscow, Russia.
Velikodvorskaya
Tatjana V
TV
Institute of Molecular Genetics, Russian Academy of Science, Kurchatov Sq. 46, 123 182 Moscow, Russia.
Schwarz
Wolfgang H
WH
Institute for Microbiology, Technical University of München, Arcisstr. 21, D-80290 München, Germany.
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D004269:DNA, Bacterial / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D005798:Genes, Bacterial; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000235:genetics*; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology / Q000235:genetics*; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; D004693:Glucan Endo-1,3-beta-D-Glucosidase