Biochemical and genetic analysis of a cutinase-type polyesterase from a thermophilic Thermobifida alba AHK119

Abstract

Recombinant polyesterase (Est119) from Thermobifida alba AHK119 was purified by two chromatography steps. The final protein was observed as a single band in SDS-PAGE, and the specific activity of Est119 for p-nitrophenyl butyrate was 2.30 u/mg. Purified Est119 was active with aliphatic and aliphatic-co-aromatic polyesters. Kinetic data indicated that p-nitrophenyl butyrate (pNPB) or hexanoate was the best substrate for Est119 among p-nitrophenyl acyl esters. Calcium was required for full activity and thermostability of Est119, which was stable at 50 °C for 16 h. Three-dimensional modeling and biochemical characterization showed that Est119 is a typical cutinase-type enzyme that has the compact ternary structure of an α/β-hydrolase. Random and site-directed mutagenesis of wild-type Est119 resulted in improved activity with increased hydrophobic interaction between the antiparallel first and second β-sheets (A68V had the greatest effect). Introduction of a proline residue (S219P) in a predicted substrate-docking loop increased the thermostability. The specific activity of the A68V/S219P mutant on pNPB was increased by more than 50-fold over the wild type. The mutant was further activated by 2.6-fold (299 u/mg) with 300 mM Ca(2+) and was stable up to 60 °C with 150 mM Ca(2+). Another identical gene was located in tandem in the upstream of est119.

Effect of hydrolysis products on the enzymatic degradation of polyethylene terephthalate nanoparticles by a polyester hydrolase from Thermobifida fusca

“…It can therefore be assumed that the inhibition was caused by the ester bonds of MHET and BHET occupying the substrate binding site of TfCut2. In a previous study, EG has been shown to inhibit the hydrolysis of the model ester p-nitrophenyl butyrate by the polyester hydrolase Est119 from Thermobifida alba, an enzyme homologous to TfCut2 [44]. However, the binding site of EG in Est119 was assumed not to be directly involved in the ester bond hydrolysis [45].…”

Synthetic Polyester-Hydrolyzing Enzymes From Thermophilic Actinomycetes

“…The genes of several synthetic polyester hydrolases within the abH05 family have shown to code for an N-terminal Gram-positive signal peptide containing 29-58 amino acid residues (Chertkov et al, 2011;Dresler, van den Heuvel, Muller, & Deckwer, 2006;Hegde & Veeranki, 2013;Hu et al, 2010;Kleeberg et al, 1998Kleeberg et al, , 2005Lykidis et al, 2007;Sulaiman et al, 2012;Thumarat et al, 2012), further confirming the identity of these enzymes as secreted proteins with mature protein sequences containing 258-262 amino acid residues. Most of the enzymes from the Thermobifida genus are produced as homologous isoenzymes with a high sequence identity of 92-95% for the mature proteins without the signal peptides (Chen et al, 2008;Hegde & Veeranki, 2013;Herrero Acero et al, 2011;Hu et al, 2010;Thumarat et al, 2012). The other polyester hydrolases in this family also show a sequence similarity of more than 60% (Table 7.1).…”

“…Actinomycetes that produce polyester hydrolases Synthetic polyester hydrolases have been detected in various moderate thermophilic actinomycetes, including Thermobifida fusca (Alisch et al, 2004;Alisch-Mark, Herrmann, & Zimmermann, 2006;Billig, Oeser, Birkemeyer, & Zimmermann, 2010;Chen et al, 2008;Feuerhack et al, 2008;Gouda, Kleeberg, van den Heuvel, Muller, & Deckwer, 2002;Hegde & Veeranki, 2013;Herrero Acero et al, 2011;Kleeberg, Hetz, Kroppenstedt, Muller, & Deckwer, 1998;Kleeberg, Welzel, Vandenheuvel, Mü ller, & Deckwer, 2005;Oeser et al, 2010), T. alba (Hu, Thumarat, Zhang, Tang, & Kawai, 2010;Thumarat, Nakamura, Kawabata, Suzuki, & Kawai, 2012), T. cellulosilytica , T. halotolerans , and Thermomonospora curvata . Actinomycetes that produce polyester hydrolases Synthetic polyester hydrolases have been detected in various moderate thermophilic actinomycetes, including Thermobifida fusca (Alisch et al, 2004;Alisch-Mark, Herrmann, & Zimmermann, 2006;Billig, Oeser, Birkemeyer, & Zimmermann, 2010;Chen et al, 2008;Feuerhack et al, 2008;Gouda, Kleeberg, van den Heuvel, Muller, & Deckwer, 2002;Hegde & Veeranki, 2013;Herrero Acero et al, 2011;Kleeberg, Hetz, Kroppenstedt, Muller, & Deckwer, 1998;Kleeberg, Welzel, Vandenheuvel, Mü ller, & Deckwer, 2005;Oeser et al, 2010), T. alba (Hu, Thumarat, Zhang, Tang, & Kawai, 2010;Thumarat, Nakamura, Kawabata, Suzuki, & Kawai, 2012), T. cellulosilytica , T. halotolerans , and Thermomonospora curvata .…”

“…A genetic modification of the translation initiation region or a change in the number of signal peptide cleavage sites (Chen, Li, Hong, Chen, & Wu, 2011) has been carried out with the E. coli recombinant expression system using the PelB signal peptide. Some of the polyester hydrolases from Thermobifida have also been expressed in E. coli without any signal peptide for protein secretion (Hu et al, 2010;Su et al, 2013;Thumarat et al, 2012). The addition of 100 mM glycine and 1 mM of the surfactant sodium taurodeoxycholate facilitated the release of periplasmically located hydrolases into the extracellular space and simplified the subsequent purification from the culture medium .…”

“…As a result, an increased yield of secreted polyester hydrolases was obtained in the extracellular fraction compared to a construct without these modifications. In contrast, the active recombinant Est119 from T. alba AHK119 was extracted exclusively from the intracellular fraction of E. coli harboring a pQE80L plasmid (Qiagen) (Hu et al, 2010;Thumarat et al, 2012). Some of the polyester hydrolases from Thermobifida have also been expressed in E. coli without any signal peptide for protein secretion (Hu et al, 2010;Su et al, 2013;Thumarat et al, 2012).…”