Several species of genus Thermobifida possess two tandem polyesterase genes, 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 (est1 and est119), 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 Ca ion, affected a lot both activity and thermostability of both enzymes. We have constructed mutant enzymes by random and site-directed mutagenesis to improve activity and thermostability. The tertiary structure of Est119 was analyzed by X-ray crystallography at 1.68 � 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 Est119. Ca-bound Est1 displayed higher Tm values than Est1, but no  differenct CD spectra from Est1. In this report, we will compare two recombinant enzymes and elucidate their reaction mechanisms on polyesters. References:U. Thumarat, R. Nakamura, T. Kawabata, H. Suzuki and F. Kawai, Appl. Microbiol. Biotechnol., published online on December 20, 2011 (DOI 10.007/s00253-011-3781-6).K. Kitadokoro, U. Thumarat, R. Nakamura, K. Nishimura, H. Karatani, H. Suzuki and F. Kawai, Polym. Degr. Stabil., available online at http://dx.doi.org/10.1016/j.polymdegradstab.2012.02.003 (DOI 10.1016/j.polymdegradstab.2012.02.003).

Comparison of two polyester-degrading enzymes from <i>Thermobifida alba</i> AHK119

244th National Meeting (2012)

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Comparison of two polyester-degrading enzymes from Thermobifida alba AHK119