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Published online before print April 25, 2008, 10.1110/ps.083492608
Protein Science (2008), 17:967-976. Published by Cold Spring Harbor Laboratory Press. Copyright © 2008 The Protein Society
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Combinatorial engineering to enhance thermostability of amylosucrase

Stéphane Emond1,2,3, Isabelle André1,2,3, Kais Jaziri1,2,3, Gabrielle Potocki-Véronèse1,2,3, Philippe Mondon4, Khalil Bouayadi4, Hakim Kharrat4, Pierre Monsan1,2,3, and Magali Remaud-Simeon1,2,3

1 Université de Toulouse, INSA, UPS, INP, LISBP, F-31077 Toulouse, France
2 INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France
3 CNRS, UMR5504, F-31400 Toulouse, France
4 MilleGen SA, Immeuble BIOSTEP, 31681 Labège Cedex, France

(RECEIVED February 13, 2008; FINAL REVISION March 26, 2008; ACCEPTED March 26, 2008)

Amylosucrase is a transglucosidase that catalyzes amylose-like polymer synthesis from sucrose substrate. About 60,000 amylosucrase variants from two libraries generated by the MutaGen random mutagenesis method were submitted to an in vivo selection procedure leading to the isolation of more than 7000 active variants. These clones were then screened for increased thermostability using an automated screening process. This experiment yielded three improved variants (two double mutants and one single mutant) showing 3.5- to 10-fold increased half-lives at 50°C compared to the wild-type enzyme. Structural analysis revealed that the main differences between wild-type amylosucrase and the most improved variant (R20C/A451T) might reside in the reorganization of salt bridges involving the surface residue R20 and the introduction of a hydrogen-bonding interaction between T451 of the B' domain and D488 of flexible loop 8. This double mutant is the most thermostable amylosucrase known to date and the only one usable at 50°C. At this temperature, amylose synthesis by this variant using high sucrose concentration (600 mM) led to the production of amylose chains twice as long as those obtained by the wild-type enzyme at 30°C.

Keywords: amylosucrase; directed evolution; random mutagenesis; high-throughput screening; thermostability; amylose synthesis


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