A 2,373-bp full-length gene (bglA49) encoding a 790-residue polypeptide (BglA49) with a calculated mass of 87.8 kDa was cloned from Serratia sp. TN49, a symbiotic bacterium isolated from the gut of longhorned beetle (Batocera horsfieldi) larvae. The deduced amino acid sequence of BglA49 showed the highest identities of 80.1% with a conceptually translated protein from Pantoea sp. At-9b (EEW02556), 38.3% with the identified glycoside hydrolase (GH) family 3 β-glucosidase from Clostridium stercorarium NCBI 11754 (CAB08072), and <15.0% with the low-temperature-active GH 3 β-glucosidases from Shewanella sp. G5 (ABL09836) and Paenibacillus sp. C7 (AAX35883). The recombinant enzyme (r-BglA49) was expressed in Escherichia coli and displayed the typical characteristics of low-temperature-active enzymes, such as low temperature optimum (showing apparent optimal activity at 35°C), activity at low temperatures (retaining ∼60% of its maximum activity at 20°C and ∼25% at 10°C). Compared with the thermophilic GH 3 β-glucosidase, r-BglA49 had fewer hydrogen bonds and salt bridges and less proline residues. These features might relate to the increased structure flexibility and higher catalytic activity at low temperatures of r-BglA49. The molecular docking study of four GH 3 β-glucosidases revealed five conserved positions contributing to substrate accommodation, among which four positions of r-BglA49 (R192, Y228, D260, and E449) were identified to be essential based on site-directed mutagenesis analysis.
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This research was supported by the China Modern Agriculture Research System (CARS-42) and the Key Program of Transgenic Plant Breeding (2008ZX08011-005) and the Agricultural Science and Technology Conversion Funds (2009GB23260444).
Junpei Zhou and Rui Zhang contributed equally to this work.
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Zhou, J., Zhang, R., Shi, P. et al. A novel low-temperature-active β-glucosidase from symbiotic Serratia sp. TN49 reveals four essential positions for substrate accommodation. Appl Microbiol Biotechnol 92, 305–315 (2011). https://doi.org/10.1007/s00253-011-3323-2
- Longhorned beetle
- Symbiotic Serratia sp.
- Low-temperature-active β-glucosidase
- Ligand docking