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Engineering thermal stability of l‐ asparaginase by in vitro directed evolution
Author(s) -
Kotzia Georgia A.,
Labrou Nikolaos E.
Publication year - 2009
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2009.06910.x
Subject(s) - thermostability , directed evolution , enzyme , saturated mutagenesis , mutant , protein engineering , wild type , mutagenesis , chemistry , thermal stability , biochemistry , microbiology and biotechnology , biology , gene , organic chemistry
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 ( T m ) differs by 10 °C. A thermostable variant of the E. chrysamthemi enzyme was identified that contained a single point mutation (Asp133Val). The T m of this variant was 55.8 °C, whereas the wild‐type enzyme has a T m 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.