Gene_locus Report for: 9acto-f7ix06 Thermobifida alba Esterase TaEst119Relationship
(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) There are 3 a/b hydrolases in Thermobifida alba view in a: Table
Carb_B_Bacteria : 9actn-a0a0e4fhy0Thermobifida alba. Carboxylic ester hydrolase. Polyesterase-lipase-cutinase : 9acto-d4q9n1Thermobifida alba Esterase est1 TaEST1
Sequence
References 5 more
Kawai F Ref: Catalysts, 11:206, 2021 : PubMed ESTHER: Kawai_2021_Catalysts_11_206 PubMedSearch: Kawai 2021 Catalysts 11 206 Gene_locus related to this paper: 9acto-f7ix06, sacvd-c7mve8 Abstract This short paper reviews two groups of enzymes designated as polyethylene terephthalate (PET) hydrolases: one consists of thermophilic cutinases from thermophilic microorganisms (actinomycetes and a fungus) and the other consists of mesophilic cutinases, the representative of which is IsPETase from a mesophilic bacterium. From the viewpoint that PET hydrolysis requires a high temperature close to the glass transition temperature (65-70 degC in water) of PET, mesophilic cutinases are not suitable for use in the enzymatic recycling of PET since their degradation level is one to three orders of magnitude lower than that of thermophilic cutinases. Many studies have attempted to increase the thermostability of IsPETase by introducing mutations, but even with these modifications, the mesophilic cutinase does not reach the same level of degradation as thermophilic cutinases. In addition, this kind of trial contradicts the claim that IsPETase works at ambient temperature. As plastic pollution is an urgent environmental issue, scientists must focus on feasible thermophilic enzymes for the enzymatic processing of disposed PET, rather than on mesophilic cutinases. Thermophilic and mesophilic cutinases must be evaluated precisely and comparatively, based on their features that enable them to hydrolyze PET, with the aim of enzymatic PET disposal. The level of thermophilic cutinases has already reached their optimal level in PET biorecycling. The optimal level may be reached through the processing of PET waste, by amorphization and micronization into readily hydrolysable forms and the improvement of PET hydrolases by engineering higher degradation ability and low-cost production. Here I summarize the critical points in the evaluation of PET hydrolases and discuss the biorecycling of PET Title: Structural insights into the unique polylactate-degrading mechanism of Thermobifida alba cutinase Kitadokoro K, Kakara M, Matsui S, Osokoshi R, Thumarat U, Kawai F, Kamitani S Ref: Febs J, 286:2087, 2019 : PubMed ESTHER: Kitadokoro_2019_FEBS.J_286_2087 PubMedSearch: Kitadokoro 2019 FEBS.J 286 2087 PubMedID: 30761732 Gene_locus related to this paper: 9acto-f7ix06 Inhibitor(s) related to this paper: Lactic-acid Abstract Cutinases are enzymes known to degrade polyester-type plastics. Est119, a plastic-degrading type of cutinase from Thermobifida alba AHK119 (herein called Ta_cut), shows a broad substrate specificity toward polyesters, and can degrade substrates including polylactic acid (PLA). However, the PLA-degrading mechanism of cutinases is still poorly understood. Here, we report the structure complexes of cutinase with ethyl lactate (EL), the constitutional unit. From this complex structure, the electron density maps clearly showed one lactate (LAC) and one EL occupying different positions in the active site cleft. The binding mode of EL is assumed to show a figure prior to reaction and LAC is an after-reaction product. These complex structures demonstrate the role of active site residues in the esterase reaction and substrate recognition. The complex structures were compared with other documented complex structures of cutinases and with the structure of PETase from Ideonella sakaiensis. The amino acid residues involved in substrate interaction are highly conserved among these enzymes. Thus, mapping the precise interactions in the Ta_cut and EL complex will pave the way for understanding the plastic-degrading mechanism of cutinases and suggest ways of creating more potent enzymes by structural protein engineering. Title: Diversity of polyester-degrading bacteria in compost and molecular analysis of a thermoactive esterase from Thermobifida alba AHK119 Hu X, Thumarat U, Zhang X, Tang M, Kawai F Ref: Applied Microbiology & Biotechnology, 87:771, 2010 : PubMed ESTHER: Hu_2010_Appl.Microbiol.Biotechnol_87_771 PubMedSearch: Hu 2010 Appl.Microbiol.Biotechnol 87 771 PubMedID: 20393707 Gene_locus related to this paper: 9acto-d4q9n1, 9acto-f7ix06 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. 5 less
Erickson E, Gado JE, Avilan L, Bratti F, Brizendine RK, Cox PA, Gill R, Graham R, Kim DJ and McGeehan JE <11 more author(s)> Erickson E, Gado JE, Avilan L, Bratti F, Brizendine RK, Cox PA, Gill R, Graham R, Kim DJ, Konig G, Michener WE, Poudel S, Ramirez KJ, Shakespeare TJ, Zahn M, Boyd ES, Payne CM, Dubois JL, Pickford AR, Beckham GT, McGeehan JE (- 11) Ref: Nat Commun, 13:7850, 2022 : PubMed ESTHER: Erickson_2022_Nat.Commun_13_7850 PubMedSearch: Erickson 2022 Nat.Commun 13 7850 PubMedID: 36543766 Gene_locus related to this paper: 9arch-PETcan211, 9cren-PETcan204, 9cyan-305pEE028, 9bact-102Pee006, 9chlr-7QJM202, 9bact-a0a656d8b6, 9actn-a0a1t4kk94, 9burk-PET11, 9bact-c3ryl0, thecs-711Erick, 9actn-RII04304, 9actn-h6wx58, thecd-d1a9g5, thecd-d1a2h1, 9acto-d4q9n1, 9acto-f7ix06, 9gamm-a0a3l8bw54, 9actn-a0a0n0my27, 9burk-a0a1e4lw26, 9actn-Alr407, 9gamm-a0a3l8bdt3, 9gamm-a0a2k9lit3, 9bact-g9by57, bacsu-pnbae, thefu-q6a0i4, 9actn-a0a0n0ney5, 9pseu-a0a1i6nu60, thefu-q6a0i3, 9actn-a0a147kjy8, 9actn-e9upm2 Abstract Enzymatic deconstruction of poly(ethylene terephthalate) (PET) is under intense investigation, given the ability of hydrolase enzymes to depolymerize PET to its constituent monomers near the polymer glass transition temperature. To date, reported PET hydrolases have been sourced from a relatively narrow sequence space. Here, we identify additional PET-active biocatalysts from natural diversity by using bioinformatics and machine learning to mine 74 putative thermotolerant PET hydrolases. We successfully express, purify, and assay 51 enzymes from seven distinct phylogenetic groups; observing PET hydrolysis activity on amorphous PET film from 37 enzymes in reactions spanning pH from 4.5-9.0 and temperatures from 30-70 degreesC. We conduct PET hydrolysis time-course reactions with the best-performing enzymes, where we observe differences in substrate selectivity as function of PET morphology. We employed X-ray crystallography and AlphaFold to examine the enzyme architectures of all 74 candidates, revealing protein folds and accessory domains not previously associated with PET deconstruction. Overall, this study expands the number and diversity of thermotolerant scaffolds for enzymatic PET deconstruction. Title: The Current State of Research on PET Hydrolyzing Enzymes Available for Biorecycling Kawai F Ref: Catalysts, 11:206, 2021 : PubMed ESTHER: Kawai_2021_Catalysts_11_206 PubMedSearch: Kawai 2021 Catalysts 11 206 Gene_locus related to this paper: 9acto-f7ix06, sacvd-c7mve8 Abstract This short paper reviews two groups of enzymes designated as polyethylene terephthalate (PET) hydrolases: one consists of thermophilic cutinases from thermophilic microorganisms (actinomycetes and a fungus) and the other consists of mesophilic cutinases, the representative of which is IsPETase from a mesophilic bacterium. From the viewpoint that PET hydrolysis requires a high temperature close to the glass transition temperature (65-70 degC in water) of PET, mesophilic cutinases are not suitable for use in the enzymatic recycling of PET since their degradation level is one to three orders of magnitude lower than that of thermophilic cutinases. Many studies have attempted to increase the thermostability of IsPETase by introducing mutations, but even with these modifications, the mesophilic cutinase does not reach the same level of degradation as thermophilic cutinases. In addition, this kind of trial contradicts the claim that IsPETase works at ambient temperature. As plastic pollution is an urgent environmental issue, scientists must focus on feasible thermophilic enzymes for the enzymatic processing of disposed PET, rather than on mesophilic cutinases. Thermophilic and mesophilic cutinases must be evaluated precisely and comparatively, based on their features that enable them to hydrolyze PET, with the aim of enzymatic PET disposal. The level of thermophilic cutinases has already reached their optimal level in PET biorecycling. The optimal level may be reached through the processing of PET waste, by amorphization and micronization into readily hydrolysable forms and the improvement of PET hydrolases by engineering higher degradation ability and low-cost production. Here I summarize the critical points in the evaluation of PET hydrolases and discuss the biorecycling of PET Title: Perspectives on the Role of Enzymatic Biocatalysis for the Degradation of Plastic PET Magalhaes RP, Cunha JM, Sousa SF Ref: Int J Mol Sci, 22:11257, 2021 : PubMed ESTHER: Magalhaes_2021_Int.J.Mol.Sci_22_11257 PubMedSearch: Magalhaes 2021 Int.J.Mol.Sci 22 11257 PubMedID: 34681915 Gene_locus related to this paper: 9entr-EsEstB, 9bact-a0a2h5z9r5, 9psed-peh, 9actn-h6wx58, idesa-mheth, idesa-peth, thecd-d1a2h1, humin-cut, 9acto-d4q9n1, 9acto-f7ix06, 9bact-g9by57, bacsu-lip, bacsu-pnbae, canar-LipB, thela-m4tp71, psemy-a4y035, thefu-1831, thefu-q6a0i4, strsw-c9zcr8, fusox-CUT, sacvd-c7mve8 Abstract Plastics are highly durable and widely used materials. Current methodologies of plastic degradation, elimination, and recycling are flawed. In recent years, biodegradation (the usage of microorganisms for material recycling) has grown as a valid alternative to previously used methods. The evolution of bioengineering techniques and the discovery of novel microorganisms and enzymes with degradation ability have been key. One of the most produced plastics is PET, a long chain polymer of terephthalic acid (TPA) and ethylene glycol (EG) repeating monomers. Many enzymes with PET degradation activity have been discovered, characterized, and engineered in the last few years. However, classification and integrated knowledge of these enzymes are not trivial. Therefore, in this work we present a summary of currently known PET degrading enzymes, focusing on their structural and activity characteristics, and summarizing engineering efforts to improve activity. Although several high potential enzymes have been discovered, further efforts to improve activity and thermal stability are necessary. Title: Structural insights into the unique polylactate-degrading mechanism of Thermobifida alba cutinase Kitadokoro K, Kakara M, Matsui S, Osokoshi R, Thumarat U, Kawai F, Kamitani S Ref: Febs J, 286:2087, 2019 : PubMed ESTHER: Kitadokoro_2019_FEBS.J_286_2087 PubMedSearch: Kitadokoro 2019 FEBS.J 286 2087 PubMedID: 30761732 Gene_locus related to this paper: 9acto-f7ix06 Inhibitor(s) related to this paper: Lactic-acid Abstract Cutinases are enzymes known to degrade polyester-type plastics. Est119, a plastic-degrading type of cutinase from Thermobifida alba AHK119 (herein called Ta_cut), shows a broad substrate specificity toward polyesters, and can degrade substrates including polylactic acid (PLA). However, the PLA-degrading mechanism of cutinases is still poorly understood. Here, we report the structure complexes of cutinase with ethyl lactate (EL), the constitutional unit. From this complex structure, the electron density maps clearly showed one lactate (LAC) and one EL occupying different positions in the active site cleft. The binding mode of EL is assumed to show a figure prior to reaction and LAC is an after-reaction product. These complex structures demonstrate the role of active site residues in the esterase reaction and substrate recognition. The complex structures were compared with other documented complex structures of cutinases and with the structure of PETase from Ideonella sakaiensis. The amino acid residues involved in substrate interaction are highly conserved among these enzymes. Thus, mapping the precise interactions in the Ta_cut and EL complex will pave the way for understanding the plastic-degrading mechanism of cutinases and suggest ways of creating more potent enzymes by structural protein engineering. Title: Comparison of genetic structures and biochemical properties of tandem cutinase-type polyesterases from Thermobifida alba AHK119 Thumarat U, Kawabata T, Nakajima M, Nakajima H, Sugiyama A, Yazaki K, Tada T, Waku T, Tanaka N, Kawai F Ref: J Biosci Bioeng, 120:491, 2015 : PubMed ESTHER: Thumarat_2015_J.Biosci.Bioeng_120_491 PubMedSearch: Thumarat 2015 J.Biosci.Bioeng 120 491 PubMedID: 25910960 Gene_locus related to this paper: 9acto-d4q9n1, 9acto-f7ix06 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 degrees C and even at 65 degrees 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. Title: Crystal structure of cutinase Est119 from Thermobifida alba AHK119 that can degrade modified polyethylene terephthalate at 1.76A resolution Kitadokoro K, Thumarat U, Nakamura R, Nishimura K, Karatani H, Suzuki H, Kawai F Ref: Polymer Degradation and Stability, 97:771, 2012 : PubMed ESTHER: Kitadokoro_2012_Polym.Degrad.Stab_97_771 PubMedSearch: Kitadokoro 2012 Polym.Degrad.Stab 97 771 Gene_locus related to this paper: 9acto-f7ix06 Abstract We determined the crystal structure of a cutinase from Thermobifida alba AHK119 (Est119) at a resolution of 1.76A. The overall structure of Est119 displays a typical alpha/beta-hydrolase fold consisting of a central twisted beta-sheet of nine beta-strands that are flanked by nine alpha-helices on both sides. The refined model contains two monomers in the asymmetric unit that form a dimer interface; a polyethylene glycol fragment is bound in the interface. Polyethylene glycol-binding site on the protein may suggest a glycol-binding site. A putative polymer-recognizing groove is observed to continue through the catalytic pocket. Water molecules are bound to hydrophilic amino acids along the groove, indicating the alternating pattern of polar and hydrophobic residues. Title: Biochemical and genetic analysis of a cutinase-type polyesterase from a thermophilic Thermobifida alba AHK119 Thumarat U, Nakamura R, Kawabata T, Suzuki H, Kawai F Ref: Applied Microbiology & Biotechnology, 95:419, 2012 : PubMed ESTHER: Thumarat_2012_Appl.Microbiol.Biotechnol_95_419 PubMedSearch: Thumarat 2012 Appl.Microbiol.Biotechnol 95 419 PubMedID: 22183084 Gene_locus related to this paper: 9acto-f7ix06 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 degrees 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 alpha/beta-hydrolase. Random and site-directed mutagenesis of wild-type Est119 resulted in improved activity with increased hydrophobic interaction between the antiparallel first and second beta-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 degrees C with 150 mM Ca(2+). Another identical gene was located in tandem in the upstream of est119. Title: Diversity of polyester-degrading bacteria in compost and molecular analysis of a thermoactive esterase from Thermobifida alba AHK119 Hu X, Thumarat U, Zhang X, Tang M, Kawai F Ref: Applied Microbiology & Biotechnology, 87:771, 2010 : PubMed ESTHER: Hu_2010_Appl.Microbiol.Biotechnol_87_771 PubMedSearch: Hu 2010 Appl.Microbiol.Biotechnol 87 771 PubMedID: 20393707 Gene_locus related to this paper: 9acto-d4q9n1, 9acto-f7ix06 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. Other Papers | ||||||||||||||||||||||||||||||||
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