Summary auto-generated
Researchers sequenced the lamA gene from the thermophilic bacterium Thermotoga neapolitana and characterized the recombinant laminarinase (1,3-β-glucanase) enzyme it encodes. The 646-amino acid protein (73 kDa) contains a central catalytic domain belonging to glycosyl hydrolase family 16, flanked by two substrate-binding domains. When expressed in Escherichia coli, the enzyme was purified in two forms: a 73 kDa full-length protein and a processed 52 kDa variant. Both exhibited extremely high specific activity toward laminarin (2600-3100 U/mg) and degraded the substrate via an endoglucanase mechanism, producing glucose, laminaribiose, and laminariose. The 73 kDa enzyme demonstrated exceptional thermostability, retaining 57% activity after 30 minutes at 100°C and showing optimal activity at 95°C (pH 6.2-6.3). Limited activity on lichenan (1,3-1,4-β-glucan) confirmed classification as an endo-1,3(4)-β-glucanase. This represents the most thermostable 1,3-β-glucanase characterized to date, with potential applications in biotechnology.
Key findings
- LamA is a 646-amino acid endo-1,3-β-glucanase with a central family 16 glycosyl hydrolase catalytic domain flanked by two substrate-binding domains
- The recombinant enzyme purified as both a 73 kDa full-length protein and a 52 kDa processed form, with specific activities of 3100 and 2600 U/mg respectively
- The 73 kDa enzyme exhibits exceptional thermostability, retaining 57% activity at 100°C for 30 minutes and showing optimal activity at 95°C
- LamA degrades laminarin endolytically producing glucose, laminaribiose, and laminariose as end products, with minor activity on lichenan
- This enzyme is the most thermostable 1,3-β-glucanase described, surpassing previously characterized enzymes from Clostridium thermocellum and Rhodothermus marinus
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Abstract
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.