title: Molecular weight-dependent degradation of D-lactate-containing polyesters by polyhydroxyalkanoate depolymerases from Variovorax sp. C34 and Alcaligenes faecalis T1.
abstract: Polyhydroxyalkanoate depolymerase derived from Variovorax sp. C34 (PhaZVs) was identified as the first enzyme that is capable of degrading isotactic P[67 mol% (R)-lactate(LA)-co-(R)-3-hydroxybutyrate(3HB)] [P(D-LA-co-D-3HB)]. This study aimed at analyzing the monomer sequence specificity of PhaZVs for hydrolyzing P(LA-co-3HB) in comparison with a P(3HB) depolymerase from Alcaligenes faecalis T1 (PhaZAf) that did not degrade the same copolymer. Degradation of P(LA-co-3HB) by action of PhaZVs generated dimers, 3HB-3HB, 3HB-LA, LA-3HB, and LA-LA, and the monomers, suggesting that PhaZVs cleaved the linkages between LA and 3HB units and between LA units. To provide a direct evidence for the hydrolysis of these sequences, the synthetic methyl trimers, 3HB-3HB-3HB, LA-LA-3HB, LA-3HB-LA, and 3HB-LA-LA, were treated with the PhaZs. Unexpectedly, not only PhaZVs but also PhaZAf hydrolyzed all of these substrates, namely PhaZAf also cleaved LA-LA linkage. Considering the fact that both PhaZs did not degrade P[(R)-LA] (PDLA) homopolymer, the cleavage capability of LA-LA linkage by PhaZs was supposed to depend on the length of the LA-clustering region in the polymer chain. To test this hypothesis, PDLA oligomers (6 to 40 mer) were subjected to the PhaZ assay, revealing that there was an inverse relationship between molecular weight of the substrates and their hydrolysis efficiency. Moreover, PhaZVs exhibited the degrading activity toward significantly longer PDLA oligomers compared to PhaZAf. Therefore, the cleaving capability of PhaZs used here toward the D-LA-based polymers containing the LA-clustering region was strongly associated with the substrate length, rather than the monomer sequence specificity of the enzyme.
authors.affiliation: Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University, N13-W8, Kita-ku, Sapporo, 060-8628, Japan.
authors.forename: Jian
authors.initials: J
authors.lastname: Sun
authors.affiliation: Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University, N13-W8, Kita-ku, Sapporo, 060-8628, Japan.
authors.forename: Ken'ichiro
authors.initials: K
authors.lastname: Matsumoto
authors.affiliation: Department of Innovative and Engineering Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Kanagawa, 226-8502, Japan.|Bioplastic Research Team, Biomass Engineering Program Cooperation Division, RIKEN Center for Sustainable Resource Science, 2-1, Hirosawa, Wako-shi, Saitama, 351-0198, Japan.
authors.forename: Yuta
authors.initials: Y
authors.lastname: Tabata
authors.affiliation: Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University, N13-W8, Kita-ku, Sapporo, 060-8628, Japan.|CREST, JST, 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan.
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Kadoya
authors.affiliation: Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University, N13-W8, Kita-ku, Sapporo, 060-8628, Japan.|CREST, JST, 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan.
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Ooi
authors.affiliation: Department of Innovative and Engineering Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Kanagawa, 226-8502, Japan.|Bioplastic Research Team, Biomass Engineering Program Cooperation Division, RIKEN Center for Sustainable Resource Science, 2-1, Hirosawa, Wako-shi, Saitama, 351-0198, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Abe
authors.affiliation: Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University, N13-W8, Kita-ku, Sapporo, 060-8628, Japan. staguchi@eng.hokudai.ac.jp.|CREST, JST, 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan. staguchi@eng.hokudai.ac.jp.
authors.forename: Seiichi
authors.initials: S
authors.lastname: Taguchi
mesh_terms: D042481:Alcaligenes faecalis / Q000201:enzymology* / Q000378:metabolism; D001673:Biodegradation, Environmental; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D042621:Comamonadaceae / Q000201:enzymology* / Q000378:metabolism; D006868:Hydrolysis; D006885:Hydroxybutyrates / Q000378:metabolism; D019344:Lactic Acid / Q000378:metabolism; D008970:Molecular Weight; D011091:Polyesters / Q000737:chemistry / Q000378:metabolism*; D011108:Polymers / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D006885:Hydroxybutyrates; D011091:Polyesters; D011108:Polymers; D019344:Lactic Acid; C033616:poly(lactide); D002265:Carboxylic Ester Hydrolases; C077226:poly(3-hydroxyalkanoic acid) depolymerase
keywords: Biobased polyester; Biodegradable material; Biodegradation; Monomer sequence specificity; Polylactic acid; Size-dependent hydrolysis