Diversity of polyester-degrading bacteria in compost and molecular analysis of a thermoactive esterase from Thermobifida alba AHK119

Abstract

More than 100 bacterial strains were isolated from composted polyester films and categorized into two groups, Actinomycetes (four genera) and Bacillus (three genera). Of these isolates, Thermobifida alba strain AHK119 (AB298783) was shown to possess the ability to significantly degrade aliphatic-aromatic copolyester film as well as decreasing the polymer particle sizes when grown at 50 degrees C on LB medium supplemented with polymer particles, yielding terephthalic acid. The esterase gene (est119, 903 bp, encoding a signal peptide and a mature protein of 34 and 266 amino acids, respectively) was cloned from AHK119. The Est119 sequence contains a conserved lipase box (-G-X-S-X-G-) and a catalytic triad (Ser129, His207, and Asp175). Furthermore, Tyr59 and Met130 likely form an oxyanion hole. The recombinant enzyme was purified from cell-free extracts of Escherichia coli Rosetta-gami B (DE3) harboring pQE80L-est119. The enzyme is a monomeric protein of ca. 30 kDa, which is active from 20 degrees C to 75 degrees C (with an optimal range of 45 to 55 degrees C) and in a pH range of 5.5 to 7.0 (with an optimal pH of 6.0). Its preferred substrate among the p-nitrophenyl acyl esters (C2 to C8) is p-nitrophenyl hexanoate (C6), indicating that the enzyme is an esterase rather than a lipase.

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

“…Ecoflex™ is an aliphatic-co-aromatic polyester consisting of terephthalic acid, 1, 4-butane diol, adipate, and an unknown compound (Kleeberg et al 2005). Apexa™ 4027 is an aliphatic-co-aromatic polyester consisting of terephthalic acid, ethylene glycol, and an unknown compound (Hu et al 2010). Poly (L-lactic acid) (PLLA; weightaverage mol wt of 1.69×10 5 ) and poly(D-lactic acid) (PDLA; weight-average mol wt of 1.63×10 5 ) were synthesized, as described previously (Kawai et al 2011).…”

“…Escherichia coli Rosetta-gami B (DE3) with the pQE80L-est119 plasmid was used as a transformant for expression of recombinant Est119 cloned from T. alba AHK119 (Hu et al 2010). E. coli DH5α was used as a cloning host of another identical gene, and the transformants were grown at 37°C for 12-16 h in Luria-Bertani medium (LB) supplemented with 50 μg/ml ampicillin, 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and 1 mM 5-bromo-4-chloro-3-indoryl-β-D-galactopyranoside for blue-white selection.…”

“…Biological recycling is dependent on enzymes able to depolymerize plastics to monomers. Previously we cloned a gene (est119) encoding a cutinase-type enzyme from the thermophile T. alba AHK119 to preliminarily confirm its ability to degrade polyesters (Hu et al 2010).…”

“…The thermophiles Thermobifida fusca and T. alba have been isolated from composts and are potent degraders of polyesters such as aliphatic-co-aromatic polyesters (Kleeberg et al 1998;Hu et al 2008;Sinsereekul et al 2010). The genes for polyesterhydrolyzing enzymes from T. fusca and T. alba have been cloned and determined to encode serine hydrolases (Kleeberg et al 2005;Dresler et al 2006;Yang et al 2007: Chen et al 2008Hu et al 2010) that belong to the lipase/esterase family (Arpigny and Jäger 1999). The complete genome of T. fusca YX has been disclosed (Lykidis et al 2007), and the genome includes two tandem genes that encode extracellular lipases.…”

A New Esterase from Thermobifida halotolerans Hydrolyses Polyethylene Terephthalate (PET) and Polylactic Acid (PLA)

“…A BLAST search for homologous proteins listed an esterase from T.alba (Est119, Genbank BAK48590.1; [44]), two cutinases from T.cellulosilytica (Thc_Cut1 and Thc_Cut2, Genbank ADV92526 and ADV92527), the cutinase from T.alba (Tha_Cut1, Genbank ADV92526) as well as an acetylxylan esterase from T.fusca (TFAXE, Genbank ADM47605.1; [45]) as hits showing 85-87% similarity to Thh_Est (Table 1, Figure 1 and Appendix). The gene coding for Thh_Est was cloned through the NdeI/HindIII restriction sites into pET26b(+) for expression without the pelB leader sequence.…”