title: Engineering thermal stability of L-asparaginase by in vitro directed evolution.
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 (T(m)) differs by 10 degrees 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 degrees C, whereas the wild-type enzyme has a T(m) of 46.4 degrees C. At 50 degrees 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.
authors.affiliation: Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, Athens, Greece.
authors.forename: Georgia A
authors.initials: GA
authors.lastname: Kotzia
authors.forename: Nikolaos E
authors.initials: NE
authors.lastname: Labrou
mesh_terms: D001215:Asparaginase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D019020:Directed Molecular Evolution; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D015202:Protein Engineering; D055672:Static Electricity
chemical_list: D017931:DNA Primers; D001215:Asparaginase