(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) > cellular organisms: NE > Bacteria: NE > Terrabacteria group: NE > Actinobacteria [phylum]: NE > Actinobacteria [class]: NE > Streptosporangiales: NE > Nocardiopsaceae: NE > Thermobifida: NE > Thermobifida alba: NE
LegendThis sequence has been compared to family alignement (MSA) red => minority aminoacid blue => majority aminoacid color intensity => conservation rate title => sequence position(MSA position)aminoacid rate Catalytic site Catalytic site in the MSA MSVTTPRREASLLSRAVAVAAAAAATVALAAPAQAANPYERGPNPTESML EARSGPFSVSEERASRLGADGFGGGTIYYPRENNTYGAIAISPGYTGTQS SIAWLGERIASHGFVVIAIDTNTTLDQPDSRARQLNAALDYMLTDASSSV RNRIDASRLAVMGHSMGGGGTLRLASQRPDLKAAIPLTPWHLNKSWRDIT VPTLIIGADLDTIAPVSSHSEPFYNSIPSSTDKAYLELNNATHFAPNITN KTIGMYSVAWLKRFVDEDTRYTQFLCPGPRTGLLSDVDEYRSTCPF
References
2 moreTitle: 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
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: Gene structure and comparative study of two different plastic-degrading esterases fromRoseateles depolymeransstrain TB-87 Ahmad A, Tsutsui A, Iijim S, Suzuki T, Shaha AA, Nakajima-kambe T Ref: Polymer Degradation and Stability, 164:109, 2019 : PubMed
The structures of two genes fromRoseateles depolymeransstrain TB-87 encoding the esterases Est-H andEst-L, which can degrade aliphatic-aromatic copolyesters, were annotated. Two open reading frames(ORFs) consisting of 1083 bp and 870 bp nucleotides, corresponding toest-Handest-L, encoding enzymesof 290 and 289 amino acids, respectively, were predicted. In addition, another ORF consisting of 735 bpencoding a chaperone-like protein (Est-Ch) of 244 amino acids was identified in the intergenic region ofest-Handest-L. The presence of a promoter region upstream ofest-Hand the absence of a terminatorregion downstream of the ORF and vice versa forest-Ch, suggests thatest-Handest-Chare poly-cistronically expressed. A homology search for Est-H and Est-L revealed homology with plastic degradingenzymes, such as esterases and cutinases, while Est-Ch showed homology with a bacterial lipasechaperone. As consensus lipase sequences (-Gly-His-Ser-Met-Gly-) were observed in these enzymes, Est-H and Est-L were hypothesized to be hydrolases with serine (Ser) in their active center. Three-dimensional structures of Est-H and Est-L without their putative signal sequences were constructedusing Est119 fromThermobifida albastrain AHK119 as the template; the structures and positions of thecatalytic triad (Ser, Asp, His) active centers were similar to those of Est119. A mutant strain in which theannotated esterase-encoding genes were disrupted using a homologous recombination method lost theability to form a clear zone on poly(butylene succinate-co-adipate (PBSA) emulsion-overlaid nutrientagar plates. Characterization of the esterases from strain TB-87 could contribute to the development ofnovel biodegradable plastics with unique properties such as recyclable monomers
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
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.
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: 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
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: Gene structure and comparative study of two different plastic-degrading esterases fromRoseateles depolymeransstrain TB-87 Ahmad A, Tsutsui A, Iijim S, Suzuki T, Shaha AA, Nakajima-kambe T Ref: Polymer Degradation and Stability, 164:109, 2019 : PubMed
The structures of two genes fromRoseateles depolymeransstrain TB-87 encoding the esterases Est-H andEst-L, which can degrade aliphatic-aromatic copolyesters, were annotated. Two open reading frames(ORFs) consisting of 1083 bp and 870 bp nucleotides, corresponding toest-Handest-L, encoding enzymesof 290 and 289 amino acids, respectively, were predicted. In addition, another ORF consisting of 735 bpencoding a chaperone-like protein (Est-Ch) of 244 amino acids was identified in the intergenic region ofest-Handest-L. The presence of a promoter region upstream ofest-Hand the absence of a terminatorregion downstream of the ORF and vice versa forest-Ch, suggests thatest-Handest-Chare poly-cistronically expressed. A homology search for Est-H and Est-L revealed homology with plastic degradingenzymes, such as esterases and cutinases, while Est-Ch showed homology with a bacterial lipasechaperone. As consensus lipase sequences (-Gly-His-Ser-Met-Gly-) were observed in these enzymes, Est-H and Est-L were hypothesized to be hydrolases with serine (Ser) in their active center. Three-dimensional structures of Est-H and Est-L without their putative signal sequences were constructedusing Est119 fromThermobifida albastrain AHK119 as the template; the structures and positions of thecatalytic triad (Ser, Asp, His) active centers were similar to those of Est119. A mutant strain in which theannotated esterase-encoding genes were disrupted using a homologous recombination method lost theability to form a clear zone on poly(butylene succinate-co-adipate (PBSA) emulsion-overlaid nutrientagar plates. Characterization of the esterases from strain TB-87 could contribute to the development ofnovel biodegradable plastics with unique properties such as recyclable monomers
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: 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
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.
Thermobifida alba Esterase TaEst119