TAILIEUCHUNG - Báo cáo khoa học: Engineering thermal stability of L-asparaginase by in vitro directed evolution

l-Asparaginase (EC ) catalyses the hydrolysis ofl-Asn, producingl-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. | Engineering thermal stability of L-asparaginase by in vitro directed evolution Georgia A. Kotzia and Nikolaos E. Labrou Laboratory of Enzyme Technology Department of AgriculturalBiotechnology AgriculturalUniversity of Athens Greece Keywords directed evolution enzyme engineering leukaemia saturation mutagenesis thermal stability Correspondence N. E. Labrou Laboratory of Enzyme Technology Department of Agricultural Biotechnology AgriculturalUniversity of Athens Iera Odos 75 11855 Athens Greece Fax 30 210 5294308 Tel 30 210 5294308 E-mail lambrou@ Received 19 September 2008 revised 2 December 2008 accepted 19 January 2009 doi L-Asparaginase EC 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 C whereas the wild-type enzyme has a Tm of C. At 50 C the half-life values for the wild-type and mutant enzymes were and 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 .

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