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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 (2009) PROTEIN ENGINEERING DESIGN & SELECTION. 22(7). p.393-399
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.
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
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
journal title
PROTEIN ENGINEERING DESIGN & SELECTION
Protein Eng. Des. Sel.
volume
22
issue
7
pages
393 - 399
Web of Science type
Article
Web of Science id
000267226800002
JCR category
BIOTECHNOLOGY & APPLIED MICROBIOLOGY
JCR impact factor
2.596 (2009)
JCR rank
53/150 (2009)
JCR quartile
2 (2009)
ISSN
1741-0126
DOI
10.1093/protein/gzp017
language
English
UGent publication?
yes
classification
A1
copyright statement
I have transferred the copyright for this publication to the publisher
id
680993
handle
http://hdl.handle.net/1854/LU-680993
date created
2009-06-05 16:18:01
date last changed
2009-11-17 14:27:36
@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},
  keyword      = {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},
}

Chicago
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.
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.
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.
MLA
De Groeve, Manu, Miet De Baere, Lieve Hoflack, et al. “Creating Lactose Phosphorylase Enzymes by Directed Evolution of Cellobiose Phosphorylase.” PROTEIN ENGINEERING DESIGN & SELECTION 22.7 (2009): 393–399. Print.