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Comparison of polyester-degrading cutinases from genus thermobifida
Author(s)
Kawai, Fusako
Thumarat, Uschara
Kitadokoro, Kengo
Waku, Tomonori
Tada, Tomoko
Tanaka, Naoki
Kawabata, Takeshi
Date Issued
January 1, 2013
Type
Conference Paper
Abstract
Several specis of genus Thermobifida are known to possess two tandem cutinases, which have high identities and similarities with each other. Thermobifida alba AHK119 is a moderately thermophilic actinomycete isolated from compost. The strain possesses two tandem cutinase genes (estl and est 119), which were expressed with pQE80L in E. coli Rosetta-gami B (DE3) as soluble active proteins with 6xhis at the C-terminal. Recombinant enzymes showed wide substrate specificity toward aliphatic and aliphatic-aromatic polyesters. They share 95% identity and 98% similarity with each other, but differ in activity and thermostability. Divalent cations, especially calcium ion, affected a lot both activity and thermostability of two enzymes. We have constructed mutant enzymes by random and site-directed mutagenesis to improve activity and thermostability. The tertiary structure of Estll9 was analyzed by X-ray crystallography at 1.68 Å resolution. Based on crystalline structure and 3D modeling, we elucidated the difference of activity and thermostability in two enzymes and suggested amino acids relevant to activity and thermostability. Calcium ion was bound to the surface carboxyl and hydroxyl groups of Estll9 on a loop region. Ca2+-bound Estl displayed higher Tm values than Estl in the absence of Ca2+, but no different CD spectra were found in Estl with and without Ca2+. In this report, we will compare two recombinant enzymes and elucidate their reaction mechanisms on polyesters. © 2013 American Chemical Society.
Citation
ACS Symposium Series, 1144, 111-120, 2013