title: Comparison of genetic structures and biochemical properties of tandem cutinase-type polyesterases from Thermobifida alba AHK119.
abstract: This study described the genetic map of tandem genes (est1 and est119) encoding cutinase-type polyesterases in Thermobifida alba AHK119 and comparison of wild type and mutant enzymes of Est1 and Est119. Two genes were independently and constitutively expressed. The activity of Est1 was higher by approximately 1.6-1.7-fold than that of Est119 towards p-nitrophenyl butyrate, although both enzymes shared 95% sequence identity and 98% similarity and possessed similar 3D structures except that several amino acids in the probable substrate-docking loops were different from each other. Calcium ion enhanced the activity and the thermostability of both enzymes. Based on conserved sequences among Thermobifida cutinases, valine, proline and lysine were introduced into Est1 at Ala68, Thr253 and Met256, respectively. Among wild and mutant enzymes of Est119 and Est1, Est1 (A68V/T253P) possessed three prolines in the substrate-docking loops and displayed the highest thermostability that spotlighted the important effect of proline numbers in the loops. Est1 (A68V/T253P) was stable for 1 h below 60°C and even at 65°C, more than 70% and 50% activities were maintained after 30 and 60 min, respectively. Est1 (A68V/T253P) degraded various aliphatic and aliphatic-co-aromatic polyesters and hydrophilized an amorphous PET film. The enzyme hydrolyzed a PET trimer model compound, indicating its specificity towards an ester bond between terephthalic acid and ethylene glycol.
authors.affiliation: Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand.
authors.forename: Uschara
authors.initials: U
authors.lastname: Thumarat
authors.affiliation: Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kawabata
authors.affiliation: Department of Materials and Life Science, Graduate School of Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.
authors.forename: Maho
authors.initials: M
authors.lastname: Nakajima
authors.affiliation: Center for Fiber and Textile Science, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.
authors.forename: Hajime
authors.initials: H
authors.lastname: Nakajima
authors.affiliation: Laboratory of Plant Gene Expression, Research Institute for Sustainable Humanosphere, Kyoto University, Gokasho, Uji City, Kyoto Prefecture 611-0011, Japan.
authors.forename: Akifumi
authors.initials: A
authors.lastname: Sugiyama
authors.affiliation: Laboratory of Plant Gene Expression, Research Institute for Sustainable Humanosphere, Kyoto University, Gokasho, Uji City, Kyoto Prefecture 611-0011, Japan.
authors.forename: Kazufumi
authors.initials: K
authors.lastname: Yazaki
authors.affiliation: Department of Biomolecular Engineering, Graduate School of Science and Technology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Tada
authors.affiliation: Department of Biomolecular Engineering, Graduate School of Science and Technology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.
authors.forename: Tomonori
authors.initials: T
authors.lastname: Waku
authors.affiliation: Department of Biomolecular Engineering, Graduate School of Science and Technology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Tanaka
authors.affiliation: Center for Fiber and Textile Science, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan. Electronic address: fkawai@kit.ac.jp.
authors.forename: Fusako
authors.initials: F
authors.lastname: Kawai
mesh_terms: D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics*; D000595:Amino Acid Sequence; D002087:Butyrates / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017124:Conserved Sequence; D004795:Enzyme Stability / Q000187:drug effects; D005798:Genes, Bacterial / Q000235:genetics; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011091:Polyesters / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D002087:Butyrates; D011091:Polyesters; C033592:4-nitrophenyl butyrate; D002265:Carboxylic Ester Hydrolases; C019952:cutinase; D002118:Calcium
keywords: Ca-activated cutinase; Cutinase-type polyesterase; PET surface hydrolysis; Tandem genes regulation; Thermobifida alba AHK119; Thermostable cutinase