title: A novel Ca²⁺-activated, thermostabilized polyesterase capable of hydrolyzing polyethylene terephthalate from Saccharomonospora viridis AHK190.
abstract: Only two polyethylene glycol terephthalate (PET)-degrading enzymes have been reported, and their mechanism for the biochemical degradation of PET remains unclear. To identify a novel PET-degrading enzyme, a putative cutinase gene (cut190) was cloned from the thermophile Saccharomonospora viridis AHK190 and expressed in Escherichia coli Rosetta-gami B (DE3). Mutational analysis indicated that substitution of Ser226 with Pro and Arg228 with Ser yielded the highest activity and thermostability. The Ca(2+) ion enhanced the enzyme activity and thermostability of the wild-type and mutant Cut190. Circular dichroism suggested that the Ca(2+) changes the tertiary structure of the Cut190 (S226P/R228S), which has optimal activity at 65-75 °C and pH 6.5-8.0 in the presence of 20 % glycerol. The enzyme was stable over a pH range of 5-9 and at temperatures up to 65 °C for 24 h with 40 % activity remaining after incubation for 1 h at 70 °C. The Cut190 (S226P/R228S) efficiently hydrolyzed various aliphatic and aliphatic-co-aromatic polyester films. Furthermore, the enzyme degraded the PET film above 60 °C. Therefore, Cut190 is the novel-reported PET-degrading enzyme with the potential for industrial applications in polyester degradation, monomer recycling, and PET surface modification. Thus, the Cut190 will be a useful tool to elucidate the molecular mechanisms of the PET degradation, Ca(2+) activation, and stabilization.
authors.affiliation: Center for Nanomaterials and Devices, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto, 606-8585, Japan, fkawai@kit.ac.jp.
authors.forename: Fusako
authors.initials: F
authors.lastname: Kawai
authors.forename: Masayuki
authors.initials: M
authors.lastname: Oda
authors.forename: Tomonari
authors.initials: T
authors.lastname: Tamashiro
authors.forename: Tomonori
authors.initials: T
authors.lastname: Waku
authors.forename: Naoki
authors.initials: N
authors.lastname: Tanaka
authors.forename: Masaki
authors.initials: M
authors.lastname: Yamamoto
authors.forename: Hiroki
authors.initials: H
authors.lastname: Mizushima
authors.forename: Takuya
authors.initials: T
authors.lastname: Miyakawa
authors.forename: Masaru
authors.initials: M
authors.lastname: Tanokura
mesh_terms: D039903:Actinobacteria / Q000201:enzymology* / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D020536:Enzyme Activators / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011093:Polyethylene Terephthalates / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
chemical_list: D002413:Cations, Divalent; D020536:Enzyme Activators; D050505:Mutant Proteins; D011093:Polyethylene Terephthalates; D011994:Recombinant Proteins; D006867:Hydrolases; D002118:Calcium