| dc.contributor.author |
Kotzia, GA |
en |
| dc.contributor.author |
Labrou, NE |
en |
| dc.date.accessioned |
2014-06-06T06:49:23Z |
|
| dc.date.available |
2014-06-06T06:49:23Z |
|
| dc.date.issued |
2009 |
en |
| dc.identifier.issn |
1742464X |
en |
| dc.identifier.uri |
http://dx.doi.org/10.1111/j.1742-4658.2009.06910.x |
en |
| dc.identifier.uri |
http://62.217.125.90/xmlui/handle/123456789/4561 |
|
| dc.subject |
Directed evolution |
en |
| dc.subject |
Enzyme engineering |
en |
| dc.subject |
Leukaemia |
en |
| dc.subject |
Saturation mutagenesis |
en |
| dc.subject |
Thermal stability |
en |
| dc.subject.other |
ammonia |
en |
| dc.subject.other |
asparaginase |
en |
| dc.subject.other |
asparagine |
en |
| dc.subject.other |
leucine |
en |
| dc.subject.other |
acute lymphoblastic leukemia |
en |
| dc.subject.other |
amino acid sequence |
en |
| dc.subject.other |
article |
en |
| dc.subject.other |
enzyme engineering |
en |
| dc.subject.other |
genetic screening |
en |
| dc.subject.other |
hydrolysis |
en |
| dc.subject.other |
in vitro directed evolution |
en |
| dc.subject.other |
in vitro study |
en |
| dc.subject.other |
molecular evolution |
en |
| dc.subject.other |
nonhuman |
en |
| dc.subject.other |
Pectobacterium carotovorum |
en |
| dc.subject.other |
Pectobacterium chrysanthemi |
en |
| dc.subject.other |
point mutation |
en |
| dc.subject.other |
priority journal |
en |
| dc.subject.other |
site directed mutagenesis |
en |
| dc.subject.other |
site saturation mutagenesis |
en |
| dc.subject.other |
thermostability |
en |
| dc.subject.other |
Asparaginase |
en |
| dc.subject.other |
Base Sequence |
en |
| dc.subject.other |
Cloning, Molecular |
en |
| dc.subject.other |
Directed Molecular Evolution |
en |
| dc.subject.other |
DNA Primers |
en |
| dc.subject.other |
Electrophoresis, Polyacrylamide Gel |
en |
| dc.subject.other |
Enzyme Stability |
en |
| dc.subject.other |
Hot Temperature |
en |
| dc.subject.other |
Kinetics |
en |
| dc.subject.other |
Models, Molecular |
en |
| dc.subject.other |
Mutagenesis, Site-Directed |
en |
| dc.subject.other |
Protein Conformation |
en |
| dc.subject.other |
Protein Engineering |
en |
| dc.subject.other |
Static Electricity |
en |
| dc.subject.other |
Erwinia chrysanthemi |
en |
| dc.subject.other |
Pectobacterium carotovorum |
en |
| dc.title |
Engineering thermal stability of l-asparaginase by in vitro directed evolution |
en |
| heal.type |
journalArticle |
en |
| heal.identifier.primary |
10.1111/j.1742-4658.2009.06910.x |
en |
| heal.publicationDate |
2009 |
en |
| heal.abstract |
l-Asparaginase (EC 3.5.1.1, l-ASNase) catalyses the hydrolysis of l-Asn, producing l-Asp and ammonia. This enzyme is an anti-neoplastic agent; it is used extensively in the chemotherapy of acute lymphoblastic leukaemia. In this study, we describe the use of in vitro directed evolution to create a new enzyme variant with improved thermal stability. A library of enzyme variants was created by a staggered extension process using the genes that code for the l-ASNases from Erwinia chrysanthemi and Erwinia carotovora. The amino acid sequences of the parental l-ASNases show 77% identity, but their half-inactivation temperature (Tm) differs by 10 °C. A thermostable variant of the E. chrysamthemi enzyme was identified that contained a single point mutation (Asp133Val). The Tm of this variant was 55.8 °C, whereas the wild-type enzyme has a Tm of 46.4 °C. At 50 °C, the half-life values for the wild-type and mutant enzymes were 2.7 and 159.7 h, respectively. Analysis of the electrostatic potential of the wild-type enzyme showed that Asp133 is located at a neutral region on the enzyme surface and makes a significant and unfavourable electrostatic contribution to overall stability. Site-saturation mutagenesis at position 133 was used to further analyse the contribution of this position on thermostability. Screening of a library of random Asp133 mutants confirmed that this position is indeed involved in thermostability and showed that the Asp133Leu mutation confers optimal thermostability. © 2009 FEBS. |
en |
| heal.journalName |
FEBS Journal |
en |
| dc.identifier.issue |
6 |
en |
| dc.identifier.volume |
276 |
en |
| dc.identifier.doi |
10.1111/j.1742-4658.2009.06910.x |
en |
| dc.identifier.spage |
1750 |
en |
| dc.identifier.epage |
1761 |
en |