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Enzymatic properties and substrate specificity of the trehalose phosphorylase from Caldanaerobacter subterraneus

Jef Van der Borght (UGent) , Chao Chen (UGent) , Lieve Hoflack (UGent) , Lucas Van Renterghem, Tom Desmet (UGent) and Wim Soetaert (UGent)
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Biotechnology for a sustainable economy (Bio-Economy)
Abstract
A putative glycoside phosphorylase from Caldanaerobacter subterraneus subsp. pacificus was recombinantly expressed in E. coli, after codon optimization and chemical synthesis of the encoding gene. The enzyme was purified by His-tag chromatography and was found to be specifically active towards trehalose, with an optimal temperature of 80°C. In addition, no loss of activity could be detected after 1 h incubation at 65°C, which means that it is the most stable trehalose phosphorylase reported so far. The substrate specificity was investigated in detail by measuring the relative activity on a range of alternative acceptors, applied in the reverse synthetic reaction, and determination of the kinetic parameters for the best acceptors. These results were rationalized based on the enzyme-substrate interactions observed in a homology model with a docked ligand. The specificity for the orientation of the acceptor's hydroxyl groups was found to decrease in the order C3 > C2 > C4. This results in a particularly high activity on the monosaccharides D-fucose, D-xylose, L-arabinose and D-galactose, as well as on L-fucose. However, determination of the kinetic parameters revealed that these acceptors bind less tightly in the active site than the natural acceptor D-glucose, resulting in drastically increased Km values. Nevertheless, the enzyme's high thermostability and broad acceptor specificity makes it a valuable candidate for industrial disaccharide synthesis.
Keywords
THERMOANAEROBACTER-BROCKII, EUGLENA-GRACILIS, GEN.-NOV., PURIFICATION, ENVIRONMENTS, DISACCHARIDE, METABOLISM, PACIFICUM, SWISS-MODEL WORKSPACE

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Citation

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Chicago
Van der Borght, Jef, Chao Chen, Lieve Hoflack, Lucas Van Renterghem, Tom Desmet, and Wim Soetaert. 2011. “Enzymatic Properties and Substrate Specificity of the Trehalose Phosphorylase from Caldanaerobacter Subterraneus.” Applied and Environmental Microbiology 77 (19): 6939–6944.
APA
Van der Borght, J., Chen, C., Hoflack, L., Van Renterghem, L., Desmet, T., & Soetaert, W. (2011). Enzymatic properties and substrate specificity of the trehalose phosphorylase from Caldanaerobacter subterraneus. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 77(19), 6939–6944.
Vancouver
1.
Van der Borght J, Chen C, Hoflack L, Van Renterghem L, Desmet T, Soetaert W. Enzymatic properties and substrate specificity of the trehalose phosphorylase from Caldanaerobacter subterraneus. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. 2011;77(19):6939–44.
MLA
Van der Borght, Jef, Chao Chen, Lieve Hoflack, et al. “Enzymatic Properties and Substrate Specificity of the Trehalose Phosphorylase from Caldanaerobacter Subterraneus.” APPLIED AND ENVIRONMENTAL MICROBIOLOGY 77.19 (2011): 6939–6944. Print.
@article{1908756,
  abstract     = {A putative glycoside phosphorylase from Caldanaerobacter subterraneus subsp. pacificus was recombinantly expressed in E. coli, after codon optimization and chemical synthesis of the encoding gene. The enzyme was purified by His-tag chromatography and was found to be specifically active towards trehalose, with an optimal temperature of 80{\textdegree}C. In addition, no loss of activity could be detected after 1 h incubation at 65{\textdegree}C, which means that it is the most stable trehalose phosphorylase reported so far. The substrate specificity was investigated in detail by measuring the relative activity on a range of alternative acceptors, applied in the reverse synthetic reaction, and determination of the kinetic parameters for the best acceptors. These results were rationalized based on the enzyme-substrate interactions observed in a homology model with a docked ligand. The specificity for the orientation of the acceptor's hydroxyl groups was found to decrease in the order C3 {\textrangle} C2 {\textrangle} C4. This results in a particularly high activity on the monosaccharides D-fucose, D-xylose, L-arabinose and D-galactose, as well as on L-fucose. However, determination of the kinetic parameters revealed that these acceptors bind less tightly in the active site than the natural acceptor D-glucose, resulting in drastically increased Km values. Nevertheless, the enzyme's high thermostability and broad acceptor specificity makes it a valuable candidate for industrial disaccharide synthesis.},
  author       = {Van der Borght, Jef and Chen, Chao and Hoflack, Lieve and Van Renterghem, Lucas and Desmet, Tom and Soetaert, Wim},
  issn         = {0099-2240},
  journal      = {APPLIED AND ENVIRONMENTAL MICROBIOLOGY},
  keyword      = {THERMOANAEROBACTER-BROCKII,EUGLENA-GRACILIS,GEN.-NOV.,PURIFICATION,ENVIRONMENTS,DISACCHARIDE,METABOLISM,PACIFICUM,SWISS-MODEL WORKSPACE},
  language     = {eng},
  number       = {19},
  pages        = {6939--6944},
  title        = {Enzymatic properties and substrate specificity of the trehalose phosphorylase from Caldanaerobacter subterraneus},
  url          = {http://dx.doi.org/10.1128/AEM.05190-11},
  volume       = {77},
  year         = {2011},
}

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