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13 pages, 2172 KB

Open AccessReview

by Ana Robles-Martín, Sergi Roda, Rubén Muñoz-Tafalla and Victor Guallar

Eng 2024, 5(1), 91-103; https://doi.org/10.3390/eng5010006 - 3 Jan 2024

Cited by 4 | Viewed by 3581

Protein engineering is the design and modification of protein structures to optimize their functions or create novel functionalities for applications in biotechnology, medicine or industry. It represents an essential scientific solution for many of the environmental and societal challenges ahead of us, such as polymer degradation. Unlike traditional chemical methods, enzyme-mediated degradation is selective and environmentally friendly and requires milder conditions. Computational methods will play a critical role in developing such solutions by enabling more efficient bioprospecting of natural polymer-degrading enzymes. They provide structural information, generate mechanistic studies, and formulate new hypotheses, facilitating the modeling and modification of these biocatalysts through enzyme engineering. The recent development of pluriZymes constitutes an example, providing a rational mechanism to integrate different biochemical processes into one single enzyme. In this review, we summarize our recent efforts in this line and introduce our early work towards polymer degradation using a pluriZyme-like technology, including our latest development in PET nanoparticle degradation. Moreover, we provide a comprehensive recipe for developing one’s own pluriZyme so that different laboratories can experiment with them and establish new limits. With modest computational resources and with help from this review, your first pluriZyme is one step closer. Full article

(This article belongs to the Section Materials Engineering)

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14 pages, 8200 KB

Open AccessArticle

Design and Characterization of In-One Protease-Esterase PluriZyme

by Laura Fernandez-Lopez, Sergi Roda, Jose L. Gonzalez-Alfonso, Francisco J. Plou, Víctor Guallar and Manuel Ferrer

Int. J. Mol. Sci. 2022, 23(21), 13337; https://doi.org/10.3390/ijms232113337 - 1 Nov 2022

Cited by 14 | Viewed by 3779

Abstract

Proteases are abundant in prokaryotic genomes (~10 per genome), but their recovery encounters expression problems, as only 1% can be produced at high levels; this value differs from that of similarly abundant esterases (1–15 per genome), 50% of which can be expressed at good levels. Here, we design a catalytically efficient artificial protease that can be easily produced. The PluriZyme EH_1AB1 with two active sites supporting the esterase activity was employed. A Leu24Cys mutation in EH_1AB1, remodelled one of the esterase sites into a proteolytic one through the incorporation of a catalytic dyad (Cys24 and His214). The resulting artificial enzyme, EH_1AB1C, efficiently hydrolysed (azo)casein at pH 6.5–8.0 and 60–70 °C. The presence of both esterase and protease activities in the same scaffold allowed the one-pot cascade synthesis (55.0 ± 0.6% conversion, 24 h) of L-histidine methyl ester from the dipeptide L-carnosine in the presence of methanol. This study demonstrates that active sites supporting proteolytic activity can be artificially introduced into an esterase scaffold to design easy-to-produce in-one protease-esterase PluriZymes for cascade reactions, namely, the synthesis of amino acid esters from dipeptides. It is also possible to design artificial proteases with good production yields, in contrast to natural proteases that are difficult to express. Full article

(This article belongs to the Special Issue Designing Enzymes with Artificially Designed Properties: Genetic and Physicochemical Tools)

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17 pages, 2198 KB

Open AccessReview

Design of Artificial Enzymes Bearing Several Active Centers: New Trends, Opportunities and Problems

by Diego Carballares, Roberto Morellon-Sterling and Roberto Fernandez-Lafuente

Int. J. Mol. Sci. 2022, 23(10), 5304; https://doi.org/10.3390/ijms23105304 - 10 May 2022

Cited by 28 | Viewed by 5683

Abstract

Harnessing enzymes which possess several catalytic activities is a topic where intense research has been carried out, mainly coupled with the development of cascade reactions. This review tries to cover the different possibilities to reach this goal: enzymes with promiscuous activities, fusion enzymes, enzymes + metal catalysts (including metal nanoparticles or site-directed attached organometallic catalyst), enzymes bearing non-canonical amino acids + metal catalysts, design of enzymes bearing a second biological but artificial active center (plurizymes) by coupling enzyme modelling and directed mutagenesis and plurizymes that have been site directed modified in both or in just one active center with an irreversible inhibitor attached to an organometallic catalyst. Some examples of cascade reactions catalyzed by the enzymes bearing several catalytic activities are also described. Finally, some foreseen problems of the use of these multi-activity enzymes are described (mainly related to the balance of the catalytic activities, necessary in many instances, or the different operational stabilities of the different catalytic activities). The design of new multi-activity enzymes (e.g., plurizymes or modified plurizymes) seems to be a topic with unarguable interest, as this may link biological and non-biological activities to establish new combo-catalysis routes. Full article

(This article belongs to the Special Issue Designing Enzymes with Artificially Designed Properties: Genetic and Physicochemical Tools)

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