Bioprocessing of polyester waste has emerged as a promising tool in the quest for a cyclic plastic economy. One key step is the enzymatic breakdown of the polymer, and this entails a complicated pathway with substrates, intermediates, and products of variable size and solubility. We have elucidated this pathway for poly(ethylene terephthalate) (PET) and four enzymes. Specifically, we combined different kinetic measurements and a novel stochastic model and found that the ability to hydrolyze internal bonds in the polymer (endo-lytic activity) was a key parameter for overall enzyme performance. Endo-lytic activity promoted the release of soluble PET fragments with two or three aromatic rings, which, in turn, were broken down with remarkable efficiency (k cat /K M values of about 10 5 M À 1 s À 1 ) in the aqueous bulk. This meant that approximatly 70 % of the final, monoaromatic products were formed via soluble di-or triaromatic intermediates.
The rate response of poly(ethylene terephthalate) (PET)-hydrolases to increased substrate crystallinity (X C ) of PET manifests as a rate-lowering effect that varies significantly for different enzymes. Herein, we report the influence of X C on the product release rate of six thermostable PET-hydrolases. All enzyme reactions displayed a distinctive lag phase until measurable product formation occurred. The duration of the lag phase increased with X C . The recently discovered PET-hydrolase PHL7 worked efficiently on "amorphous" PET disks (X C � 10 %), but this enzyme was extremely sensitive to increased X C , whereas the enzymes LCC ICCG , LCC, and DuraPETase had higher tolerance to increases in X C and had activity on PET disks having X C of 24.4 %. Microscopy revealed that the X C -tolerant hydrolases generated smooth and more uniform substrate surface erosion than PHL7 during reaction. Structural and molecular dynamics analysis of the PET-hydrolyzing enzymes disclosed that surface electrostatics and enzyme flexibility may account for the observed differences.
Bioprocessing of polyester waste has emerged as a promising tool in the quest for a cyclic plastic economy. One key step is the enzymatic breakdown of the polymer, and this entails a complicated pathway with substrates, intermediates, and products of variable size and solubility. We have elucidated this pathway for poly(ethylene terephthalate) (PET) and four enzymes. Specifically, we combined different kinetic measurements and a novel stochastic model, and found that the ability to hydrolyze internal bonds in the polymer (endo-lytic activity) was a key parameter for overall enzyme performance. Endo-lytic activity promoted the release of soluble PET fragments with two or three aromatic rings, which, in turn, were broken down with remarkable efficiency (kcat/KM-values of about 105 M-1s-1) in the aqueous bulk. This meant that about 70% of the final, monoaromatic products was formed via soluble di- or tri-aromatic intermediates.
Fatty acid hydratases (FAHs) catalyze regio‐ and stereo‐selective hydration of unsaturated fatty acids to produce hydroxy fatty acids. Fatty acid hydratase‐1 (FA‐HY1) from Lactobacillus Acidophilus is the most promiscuous and regiodiverse FAH identified so far. Here, we engineered binding site residues of FA‐HY1 (S393, S395, S218 and P380) by semi‐rational protein engineering to alter regioselectivity. Although it was not possible to obtain a completely new type of regioselectivity with our mutant libraries, a significant shift of regioselectivity was observed towards cis‐5, cis‐8, cis‐11, cis‐14, cis‐17‐eicosapentaenoic acid (EPA). We identified mutants (S393/S395 mutants) with excellent regioselectivity, generating a single hydroxy fatty acid product from EPA (15‐OH product), which is advantageous from application perspective. This result is impressive given that wild‐type FA‐HY1 produces a mixture of 12‐OH and 15‐OH products at 63 : 37 ratio (12‐OH : 15‐OH). Moreover, our results indicate that native FA‐HY1 is at its limit in terms of promiscuity and regiospecificity, thus it may not be possible to diversify its product portfolio with active site engineering. This behavior of FA‐HY1 is unlike its orthologue, fatty acid hydratase‐2 (FA‐HY2; 58 % sequence identity to FA‐HY1), which has been shown earlier to exhibit significant promiscuity and regioselectivity changes by a few active site mutations. Our reverse engineering from FA‐HY1 to FA‐HY2 further demonstrates this conclusion.
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