Switched reaction specificity in polyesterases towards amide bond hydrolysis by enzyme engineering

Biundo, Antonino; Subagia, Raditya; Maurer, Michael; Ribitsch, Doris; Syrén, Per‐Olof; Guebitz, Georg M.

doi:10.1039/c9ra07519d

Cited by 24 publications

(12 citation statements)

References 55 publications

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“…Based on the control experiments, we can identify HiC (specifically, Novozyme 51032) as the only promoter of the formation of the amide product. Amide formation catalyzed by cutinases has only been reported for Fusarium solani pisi cutinase and an engineered Humicola insolens cutinase; [37][38][39][40] the amidase/protease activity of commercial Novozyme 51032 (Humicola insolens cutinase expressed in Aspergillus oryzae) has not been reported. To get insight into the protease activity of Novozyme 51032, assays using an o-nitrophenyl amide model substrate (Scheme S1) were performed in 2 M glycine or 3 M Tris buffer at different pH values (Figure S9).…”

Section: Resultsmentioning

confidence: 99%

Efficient mechano-enzymatic hydrolysis of polylactic acid under moist-solid conditions

Pérez‐Venegas

Friščić

Auclair

2022

Preprint

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Quantitative mechano-enzymatic depolymerisation of polylactic acid to lactic acid was achieved at 55°C using the Humicola insolens cutinase enzyme in moist-solid reaction mixtures. The resulting lactic acid was easily recovered, and the crude product was pure enough to be used in further synthesis of a value-added compound, a known benzimidazole-based drug precursor. The presented mechano-enzymatic depolymerisation strategy enables the closed-loop recycling of untreated polylactic acid under mild conditions, using a renewable, non-toxic catalyst and producing minimum solvent waste.

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Section: Resultsmentioning

confidence: 99%

Efficient mechano-enzymatic hydrolysis of polylactic acid under moist-solid conditions

Pérez‐Venegas

Friščić

Auclair

2022

Preprint

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“…This could also be informative for enzymatic engineering. For example, critical enzymes of microbes for biofuel production and plastic degradation might need to tolerate some diversity in their substrates [ 56 ], while the key enzymes for biosynthesis would like to exclude promiscuity and only produce one product as pure as possible. The present study provides a relatively simple guideline for enzymatic engineering, e.g., reducing salt bridges for the former to increase its flexibility to accommodate different substrates while adding salt bridges for the latter to enhance its specificity and, probably, the enzymatic efficiency.…”

Section: Resultsmentioning

confidence: 99%

Controlling the Substrate Specificity of an Enzyme through Structural Flexibility by Varying the Salt-Bridge Density

Huang

Liu

et al. 2021

Molecules

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Many enzymes, particularly in one single family, with highly conserved structures and folds exhibit rather distinct substrate specificities. The underlying mechanism remains elusive, the resolution of which is of great importance for biochemistry, biophysics, and bioengineering. Here, we performed a neutron scattering experiment and molecular dynamics (MD) simulations on two structurally similar CYP450 proteins; CYP101 primarily catalyzes one type of ligands, then CYP2C9 can catalyze a large range of substrates. We demonstrated that it is the high density of salt bridges in CYP101 that reduces its structural flexibility, which controls the ligand access channel and the fluctuation of the catalytic pocket, thus restricting its selection on substrates. Moreover, we performed MD simulations on 146 different kinds of CYP450 proteins, spanning distinct biological categories including Fungi, Archaea, Bacteria, Protista, Animalia, and Plantae, and found the above mechanism generally valid. We demonstrated that, by fine changes of chemistry (salt-bridge density), the CYP450 superfamily can vary the structural flexibility of its member proteins among different biological categories, and thus differentiate their substrate specificities to meet the specific biological needs. As this mechanism is well-controllable and easy to be implemented, we expect it to be generally applicable in future enzymatic engineering to develop proteins of desired substrate specificities.

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“…Enabling catalytic promiscuity is a meaningful approach to expand the capacity of plasticdegrading enzymes to use different plastic substrates, especially for plastics with few enzymes known to efficiently degrade them (such as PAs and PUs) [94,95]. Recently, a pioneering study reported manipulation of the Thc_Cut1 cutinase to develop its promiscuous amidase activity towards depolymerizing artificial PAs [96]. The residues possibly obstructing the interaction of water molecules with the transition state were identified for mutation, which generated a 6-15-fold higher hydrolytic activity [96].…”

Section: Trends Trends In In Biotechnology Biotechnologymentioning

confidence: 99%

“…Recently, a pioneering study reported manipulation of the Thc_Cut1 cutinase to develop its promiscuous amidase activity towards depolymerizing artificial PAs [96]. The residues possibly obstructing the interaction of water molecules with the transition state were identified for mutation, which generated a 6-15-fold higher hydrolytic activity [96].…”

Section: Trends Trends In In Biotechnology Biotechnologymentioning

confidence: 99%

Enzyme discovery and engineering for sustainable plastic recycling

Zhu

Wang

Wei

2022

Trends in Biotechnology

204

114

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The drastically increasing amount of plastic waste is causing an environmental crisis that requires innovative technologies for recycling post-consumer plastics to achieve waste valorization while meeting environmental quality goals. Biocatalytic depolymerization mediated by enzymes has emerged as an efficient and sustainable alternative for plastic treatment and recycling. A variety of plasticdegrading enzymes have been discovered from microbial sources. Meanwhile, protein engineering has been exploited to modify and optimize plastic-degrading enzymes. This review highlights the recent trends and up-to-date advances in mining novel plastic-degrading enzymes through state-of-the-art omics-based techniques and improving the enzyme catalytic efficiency and stability via various protein engineering strategies. Future research prospects and challenges are also discussed. Biocatalysis as an Emerging Solution for the Global Plastic Waste ChallengePlastic materials play a revolutionary role in the modern world, although the enormous manufacture and extensive use of plastic commodities inevitably generate an extraordinary amount of post-consumer plastic waste. Around 12 000 million metric tons of plastic waste are predicted to accumulate in landfills and the natural environment by 2050 [1]. Improper handling of plastic waste has caused a grand environmental challenge. The debris of plastic waste, especially microplastics (see Glossary), can impose hazardous effects on various organisms and eventually threaten human well-being [2-5]. In addition, the degradation resistance of plastics further escalates their adverse environmental impacts [6]. Therefore, it is urgent to develop innovative technologies for treatment and recycling of post-consumer plastics, to achieve both waste valorization and environmental protection.Enzymatic biocatalysis has gained increasing attention as an eco-friendly alternative to conventional plastic treatment and recycling methods (Box 1) [7]. To date, various microbial plastic-degrading enzymes have been discovered, representing promising biocatalyst candidates for plastic depolymerization. Considering the ubiquity of plastics in different ecosystems and the tremendous metabolic and genetic diversity of microorganisms, microbial communities in various habitats have likely evolved capabilities in plastic decomposition and utilization. The plastic-degrading enzymes identified so far might only account for a small portion of the enzymes relevant to plastic depolymerization in the environment. Therefore, it is of ever-growing interest to explore diverse environments to discover new plastic-degrading enzymes with desirable properties and functionalities. However, naturally occurring plasticdegrading enzymes are not well suited for synthetic plastic degradation in industrial applications due to poor thermostability and low catalytic activity. Particularly, synthetic plastic materials usually possess distinct physical and chemical properties (e.g., high crystallinity) that render them more resistant to...

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Switched reaction specificity in polyesterases towards amide bond hydrolysis by enzyme engineering

Abstract: The constitution of a water network for the nitrogen inversion mechanism by H-bonding can increase amide-containing substrate acceptance of polyesterases.

Cited by 24 publications

References 55 publications

Efficient mechano-enzymatic hydrolysis of polylactic acid under moist-solid conditions

Efficient mechano-enzymatic hydrolysis of polylactic acid under moist-solid conditions

Controlling the Substrate Specificity of an Enzyme through Structural Flexibility by Varying the Salt-Bridge Density

Enzyme discovery and engineering for sustainable plastic recycling

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