(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) > cellular organisms: NE > Bacteria: NE > Proteobacteria: NE > Gammaproteobacteria: NE > Pseudomonadales: NE > Pseudomonadaceae: NE > Pseudomonas: NE > Pseudomonas pertucinogena group: NE > Pseudomonas aestusnigri: 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 MPFNKKSVLALCGAGALLFSMSALANNPAPTDPGDSGGGSAYQRGPDPSV SFLEADRGQYSVRSSRVSSLVSGFGGGTIYYPTGTTGTMGAVVVIPGFVS AESSIDWWGPKLASYGFVVMTIDTNTGFDQPPSRARQINNALDYLVSQNS RSSSPVRGMIDTNRLGVIGWSMGGGGTLRVASEGRIKAAIPLAPWDTTSY YASRSQAPTLIFACESDVIAPVLQHASPFYNSLPSSIDKAFVEINGGSHY CGNGGSIYNDVLSRFGVSWMKLHLDEDSRYKQFLCGPNHTSDSQISDYRG NCPY
References
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.
Biodegradation of synthetic polymers, in particular polyethylene terephthalate (PET), is of great importance, since environmental pollution with PET and other plastics has become a severe global problem. Here, we report on the polyester degrading ability of a novel carboxylic ester hydrolase identified in the genome of the marine hydrocarbonoclastic bacterium Pseudomonas aestusnigri VGXO14T. The enzyme, designated PE-H, belongs to the type IIa family of PET hydrolytic enzymes as indicated by amino acid sequence homology. It was produced in Escherichia coli, purified and its crystal structure was solved at 1.09 A resolution representing the first structure of a type IIa PET hydrolytic enzyme. The structure shows a typical alpha/beta-hydrolase fold and high structural homology to known polyester hydrolases. PET hydrolysis was detected at 30C with amorphous PET film (PETa), but not with PET film from a commercial PET bottle (PETb). A rational mutagenesis study to improve the PET degrading potential of PE-H yielded variant PE-H (Y250S) which showed improved activity, ultimately also allowing the hydrolysis of PETb. The crystal structure of this variant solved at 1.35 A resolution allowed to rationalize the improvement of enzymatic activity. A PET oligomer binding model was proposed by molecular docking computations. Our results indicate a significant potential of the marine bacterium P. aestusnigri for PET degradation.
Biocatalysis has emerged as an important tool in synthetic organic chemistry enabling the chemical industry to execute reactions with high regio- or enantioselectivity and under usually mild reaction conditions while avoiding toxic waste. Target substrates and products of reactions catalyzed by carboxylic ester hydrolases are often poorly water soluble and require organic solvents, whereas enzymes are evolved by nature to be active in cells, i.e., in aqueous rather than organic solvents. Therefore, biocatalysts that withstand organic solvents are urgently needed. Current strategies to identify such enzymes rely on laborious tests carried out by incubation in different organic solvents and determination of residual activity. Here, we describe a simple assay useful for screening large libraries of carboxylic ester hydrolases for resistance and activity in water-miscible organic solvents. We have screened a set of 26 enzymes, most of them identified in this study, with four different water-miscible organic solvents. The triglyceride tributyrin was used as a substrate, and fatty acids released by enzymatic hydrolysis were detected by a pH shift indicated by the indicator dye nitrazine yellow. With this strategy, we succeeded in identifying a novel highly organic-solvent-tolerant esterase from Pseudomonas aestusnigri In addition, the newly identified enzymes were tested with sterically demanding substrates, which are common in pharmaceutical intermediates, and two enzymes from Alcanivorax borkumensis were identified which outcompeted the gold standard ester hydrolase CalB from Candida antarctica IMPORTANCE Major challenges hampering biotechnological applications of esterases include the requirement to accept nonnatural and chemically demanding substrates and the tolerance of the enzymes toward organic solvents which are often required to solubilize such substrates. We describe here a high-throughput screening strategy to identify novel organic-solvent-tolerant carboxylic ester hydrolases (CEs). Among these enzymes, CEs active against water-insoluble bulky substrates were identified. Our results thus contribute to fostering the identification and biotechnological application of CEs.
Pseudomonas aestusnigri alpha/beta hydrolase Polyester hydrolase PE-H PaPETase
