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Creating lactose phosphorylase enzymes by directed evolution of cellobiose phosphorylase

Manu De Groeve (UGent) , Miet De Baere (UGent) , Lieve Hoflack (UGent) , Tom Desmet (UGent) , Erick Vandamme (UGent) and Wim Soetaert (UGent)
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Abstract
Disaccharide phosphorylases are interesting enzymes for the production of sugar phosphates from cheap starting materials and for the synthesis of novel glycosides. Cellobiose phosphorylase (CP) from Cellulomonas uda was subjected to directed evolution in order to create enzyme variants with significantly increased lactose phosphorylase (LP) activity, useful for the production of alpha-d-galactose 1-phosphate. In a first round, random mutagenesis was performed on part of the CP gene and the resultant library was selected on minimal lactose medium. One clone containing six amino acid mutations was found with increased LP activity compared with the wild-type CP enzyme. The negative and neutral mutations were eliminated by site-directed mutagenesis and the resultant enzyme variant containing two amino acid substitutions (T508A/N667T) showed more LP activity than the parent mutant. Saturation mutagenesis of the beneficial sites and screening for improved mutants allowed us to identify the T508I/N667A mutant which has 7.5 times higher specific activity on lactose than the wild-type. The kinetic parameters of the mutants were determined and showed that the increased LP activity was caused by a higher k(cat) value. This is the first report of an engineered CP with modified substrate specificity.
Keywords
cellobiose phosphorylase, directed evolution, lactose phosphorylase, screening, selection, RECOMBINANT SUCROSE PHOSPHORYLASE, THERMOANAEROBACTER-BROCKII, BIFIDOBACTERIUM-LONGUM, GLYCOSIDE HYDROLASES, ACCEPTOR SPECIFICITY, ENZYMATIC-SYNTHESIS, REACTION-MECHANISM, CELLVIBRIO-GILVUS, TRANSGLUCOSYLATION, CELLULOMONAS-UDA

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Citation

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MLA
De Groeve, Manu et al. “Creating Lactose Phosphorylase Enzymes by Directed Evolution of Cellobiose Phosphorylase.” PROTEIN ENGINEERING DESIGN & SELECTION 22.7 (2009): 393–399. Print.
APA
De Groeve, M., De Baere, M., Hoflack, L., Desmet, T., Vandamme, E., & Soetaert, W. (2009). Creating lactose phosphorylase enzymes by directed evolution of cellobiose phosphorylase. PROTEIN ENGINEERING DESIGN & SELECTION, 22(7), 393–399.
Chicago author-date
De Groeve, Manu, Miet De Baere, Lieve Hoflack, Tom Desmet, Erick Vandamme, and Wim Soetaert. 2009. “Creating Lactose Phosphorylase Enzymes by Directed Evolution of Cellobiose Phosphorylase.” Protein Engineering Design & Selection 22 (7): 393–399.
Chicago author-date (all authors)
De Groeve, Manu, Miet De Baere, Lieve Hoflack, Tom Desmet, Erick Vandamme, and Wim Soetaert. 2009. “Creating Lactose Phosphorylase Enzymes by Directed Evolution of Cellobiose Phosphorylase.” Protein Engineering Design & Selection 22 (7): 393–399.
Vancouver
1.
De Groeve M, De Baere M, Hoflack L, Desmet T, Vandamme E, Soetaert W. Creating lactose phosphorylase enzymes by directed evolution of cellobiose phosphorylase. PROTEIN ENGINEERING DESIGN & SELECTION. 2009;22(7):393–9.
IEEE
[1]
M. De Groeve, M. De Baere, L. Hoflack, T. Desmet, E. Vandamme, and W. Soetaert, “Creating lactose phosphorylase enzymes by directed evolution of cellobiose phosphorylase,” PROTEIN ENGINEERING DESIGN & SELECTION, vol. 22, no. 7, pp. 393–399, 2009.
@article{680993,
  abstract     = {Disaccharide phosphorylases are interesting enzymes for the production of sugar phosphates from cheap starting materials and for the synthesis of novel glycosides. Cellobiose phosphorylase (CP) from Cellulomonas uda was subjected to directed evolution in order to create enzyme variants with significantly increased lactose phosphorylase (LP) activity, useful for the production of alpha-d-galactose 1-phosphate. In a first round, random mutagenesis was performed on part of the CP gene and the resultant library was selected on minimal lactose medium. One clone containing six amino acid mutations was found with increased LP activity compared with the wild-type CP enzyme. The negative and neutral mutations were eliminated by site-directed mutagenesis and the resultant enzyme variant containing two amino acid substitutions (T508A/N667T) showed more LP activity than the parent mutant. Saturation mutagenesis of the beneficial sites and screening for improved mutants allowed us to identify the T508I/N667A mutant which has 7.5 times higher specific activity on lactose than the wild-type. The kinetic parameters of the mutants were determined and showed that the increased LP activity was caused by a higher k(cat) value. This is the first report of an engineered CP with modified substrate specificity.},
  author       = {De Groeve, Manu and De Baere, Miet and Hoflack, Lieve and Desmet, Tom and Vandamme, Erick and Soetaert, Wim},
  issn         = {1741-0126},
  journal      = {PROTEIN ENGINEERING DESIGN & SELECTION},
  keywords     = {cellobiose phosphorylase,directed evolution,lactose phosphorylase,screening,selection,RECOMBINANT SUCROSE PHOSPHORYLASE,THERMOANAEROBACTER-BROCKII,BIFIDOBACTERIUM-LONGUM,GLYCOSIDE HYDROLASES,ACCEPTOR SPECIFICITY,ENZYMATIC-SYNTHESIS,REACTION-MECHANISM,CELLVIBRIO-GILVUS,TRANSGLUCOSYLATION,CELLULOMONAS-UDA},
  language     = {eng},
  number       = {7},
  pages        = {393--399},
  title        = {Creating lactose phosphorylase enzymes by directed evolution of cellobiose phosphorylase},
  url          = {http://dx.doi.org/10.1093/protein/gzp017},
  volume       = {22},
  year         = {2009},
}

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