Editorial

2 pages, 209 KiB

Open AccessEditorial

Biocatalysis: An Eco-Friendly Scenario for the Manufacturing of APIs

by Jesús Fernández-Lucas

Int. J. Mol. Sci. 2023, 24(5), 4474; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms24054474 - 24 Feb 2023

Cited by 1 | Viewed by 1132

Nowadays, the worldwide demand for Active Pharmaceutical Ingredients (APIs) requires novel, cost-effective, safe, and environmentally friendly synthetic processes [...] Full article

(This article belongs to the Special Issue Biocatalysis: An Eco-Friendly Scenario for the Manufacturing of APIs)

Research

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13 pages, 2040 KiB

Open AccessArticle

Magnetic Multi-Enzymatic System for Cladribine Manufacturing

by Guillermo Cruz, Laura Pilar Saiz, Muhammad Bilal, Lobna Eltoukhy, Christoph Loderer and Jesús Fernández-Lucas

Int. J. Mol. Sci. 2022, 23(21), 13634; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232113634 - 07 Nov 2022

Cited by 4 | Viewed by 1705

Abstract

Enzyme-mediated processes have proven to be a valuable and sustainable alternative to traditional chemical methods. In this regard, the use of multi-enzymatic systems enables the realization of complex synthetic schemes, while also introducing a number of additional advantages, including the conversion of reversible [...] Read more.

Enzyme-mediated processes have proven to be a valuable and sustainable alternative to traditional chemical methods. In this regard, the use of multi-enzymatic systems enables the realization of complex synthetic schemes, while also introducing a number of additional advantages, including the conversion of reversible reactions into irreversible processes, the partial or complete elimination of product inhibition problems, and the minimization of undesirable by-products. In addition, the immobilization of biocatalysts on magnetic supports allows for easy reusability and streamlines the downstream process. Herein we have developed a cascade system for cladribine synthesis based on the sequential action of two magnetic biocatalysts. For that purpose, purine 2′-deoxyribosyltransferase from Leishmania mexicana (LmPDT) and Escherichia coli hypoxanthine phosphoribosyltransferase (EcHPRT) were immobilized onto Ni²⁺-prechelated magnetic microspheres (MagReSyn^®NTA). Among the resulting derivatives, MLmPDT3 (activity: 11,935 IU/g_support, 63% retained activity, operational conditions: 40 °C and pH 5–7) and MEcHPRT3 (12,840 IU/g_support, 45% retained activity, operational conditions: pH 5–8 and 40–60 °C) emerge as optimal catalysts for further synthetic application. Moreover, the MLmPDT3/MEcHPRT3 system was biochemically characterized and successfully applied to the one-pot synthesis of cladribine under various conditions. This methodology not only displayed a 1.67-fold improvement in cladribine synthesis (compared to MLmPDT3), but it also implied a practically complete transformation of the undesired by-product into a high-added-value product (90% conversion of Hyp into IMP). Finally, MLmPDT3/MEcHPRT3 was reused for 16 cycles, which displayed a 75% retained activity. Full article

(This article belongs to the Special Issue Biocatalysis: An Eco-Friendly Scenario for the Manufacturing of APIs)

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15 pages, 2835 KiB

Open AccessArticle

Rational Design of a Thermostable 2′-Deoxyribosyltransferase for Nelarabine Production by Prediction of Disulfide Bond Engineering Sites

by Guillermo Cruz, Javier Acosta, Jose Miguel Mancheño, Jon Del Arco and Jesús Fernández-Lucas

Int. J. Mol. Sci. 2022, 23(19), 11806; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms231911806 - 05 Oct 2022

Cited by 5 | Viewed by 9618

Abstract

One of the major drawbacks of the industrial implementation of enzymatic processes is the low operational stability of the enzymes under tough industrial conditions. In this respect, the use of thermostable enzymes in the industry is gaining ground during the last decades. Herein, [...] Read more.

One of the major drawbacks of the industrial implementation of enzymatic processes is the low operational stability of the enzymes under tough industrial conditions. In this respect, the use of thermostable enzymes in the industry is gaining ground during the last decades. Herein, we report a structure-guided approach for the development of novel and thermostable 2′-deoxyribosyltransferases (NDTs) based on the computational design of disulfide bonds on hot spot positions. To this end, a small library of NDT variants from Lactobacillus delbrueckii (LdNDT) with introduced cysteine pairs was created. Among them, LdNDT_S104C (100% retained activity) was chosen as the most thermostable variant, displaying a six- and two-fold enhanced long-term stability when stored at 55 °C (t_1/2⁵⁵ ^°C ≈ 24 h) and 60 °C (t_1/2⁶⁰ ^°C ≈ 4 h), respectively. Moreover, the biochemical characterization revealed that LdNDT_S104C showed >60% relative activity across a broad range of temperature (30–90 °C) and pH (5–7). Finally, to study the potential application of LdNDT_S104C as an industrial catalyst, the enzymatic synthesis of nelarabine was successfully carried out under different substrate conditions (1:1 and 3:1) at different reaction times. Under these experimental conditions, the production of nelarabine was increased up to 2.8-fold (72% conversion) compared with wild-type LdNDT. Full article

(This article belongs to the Special Issue Biocatalysis: An Eco-Friendly Scenario for the Manufacturing of APIs)

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14 pages, 1949 KiB

Open AccessArticle

Immobilization of Lathyrus cicera Amine Oxidase on Magnetic Microparticles for Biocatalytic Applications

by Elisa Di Fabio, Antonia Iazzetti, Alessio Incocciati, Valentina Caseli, Giancarlo Fabrizi, Alberto Boffi, Alessandra Bonamore and Alberto Macone

Int. J. Mol. Sci. 2022, 23(12), 6529; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23126529 - 10 Jun 2022

Cited by 3 | Viewed by 1438

Abstract

Amine oxidases are enzymes belonging to the class of oxidoreductases that are widespread, from bacteria to humans. The amine oxidase from Lathyrus cicera has recently appeared in the landscape of biocatalysis, showing good potential in the green synthesis of aldehydes. This enzyme catalyzes [...] Read more.

Amine oxidases are enzymes belonging to the class of oxidoreductases that are widespread, from bacteria to humans. The amine oxidase from Lathyrus cicera has recently appeared in the landscape of biocatalysis, showing good potential in the green synthesis of aldehydes. This enzyme catalyzes the oxidative deamination of a wide range of primary amines into the corresponding aldehydes but its use as a biocatalyst is challenging due to the possible inactivation that might occur at high product concentrations. Here, we show that the enzyme’s performance can be greatly improved by immobilization on solid supports. The best results are achieved using amino-functionalized magnetic microparticles: the immobilized enzyme retains its activity, greatly improves its thermostability (4 h at 75 °C), and can be recycled up to 8 times with a set of aromatic ethylamines. After the last reaction cycle, the overall conversion is about 90% for all tested substrates, with an aldehyde production ranging between 100 and 270 mg depending on the substrate used. As a proof concept, one of the aldehydes thus produced was successfully used for the biomimetic synthesis of a non-natural benzylisoquinoline alkaloid. Full article

(This article belongs to the Special Issue Biocatalysis: An Eco-Friendly Scenario for the Manufacturing of APIs)

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19 pages, 6637 KiB

Open AccessArticle

Characteristics of Crosslinking Polymers Play Major Roles in Improving the Stability and Catalytic Properties of Immobilized Thermomyces lanuginosus Lipase

by Yuhong Mao, Zhenling Cai, Chenxi Zhou, Hangzhen Lan and Xiuyun Ye

Int. J. Mol. Sci. 2022, 23(6), 2917; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23062917 - 08 Mar 2022

Cited by 3 | Viewed by 1411

Abstract

This study aimed to improve the stability and catalytic properties of Thermomyces lanuginosus lipase (TLL) adsorbed on a hydrophobic support. At the optimized conditions (pH 5 and 25 °C without any additions), the Sips isotherm model effectively fitted the equilibrium adsorption data, indicating [...] Read more.

This study aimed to improve the stability and catalytic properties of Thermomyces lanuginosus lipase (TLL) adsorbed on a hydrophobic support. At the optimized conditions (pH 5 and 25 °C without any additions), the Sips isotherm model effectively fitted the equilibrium adsorption data, indicating a monolayer and the homogenous distribution of immobilized lipase molecules. To preserve the high specific activity of adsorbed lipase, the immobilized lipase (IL) with a moderate loading amount (approximately 40% surface coverage) was selected. Polyethylenimine (PEI) and chitosan (CS) were successfully applied as bridging units to in situ crosslink the immobilized lipase molecules in IL. At the low polymer concentration (0.5%, w/w) and with 1 h incubation, insignificant changes in average pore size were detected. Short-chain PEI and CS (MW ≤ 2 kDa) efficiently improved the lipase stability, i.e., the lipase loss decreased from 40% to <2%. Notably, CS performed much better than PEI in maintaining lipase activity. IL crosslinked with CS-2 kDa showed a two- to three-fold higher rate when hydrolyzing p-nitrophenyl butyrate and a two-fold increase in the catalytic efficiency in the esterification of hexanoic acid with butanol. These in situ crosslinking strategies offer good potential for modulating the catalytic properties of TLL for a specific reaction. Full article

(This article belongs to the Special Issue Biocatalysis: An Eco-Friendly Scenario for the Manufacturing of APIs)

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15 pages, 1946 KiB

Open AccessArticle

Comparative Analysis of Enzymatic Transglycosylation Using E. coli Nucleoside Phosphorylases: A Synthetic Concept for the Preparation of Purine Modified 2′-Deoxyribonucleosides from Ribonucleosides

by Mikhail S. Drenichev, Vladimir E. Oslovsky, Anastasia A. Zenchenko, Claudia V. Danilova, Mikhail A. Varga, Roman S. Esipov, Dmitry D. Lykoshin and Cyril S. Alexeev

Int. J. Mol. Sci. 2022, 23(5), 2795; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23052795 - 03 Mar 2022

Cited by 2 | Viewed by 1922

Abstract

A comparative analysis of the transglycosylation conditions catalyzed by E. coli nucleoside phosphorylases, leading to the formation of 2′-deoxynucleosides, was performed. We demonstrated that maximal yields of 2′-deoxynucleosides, especially modified, can be achieved under small excess of glycosyl-donor (7-methyl-2′-deoxyguanosine, thymidine) and a 4-fold [...] Read more.

A comparative analysis of the transglycosylation conditions catalyzed by E. coli nucleoside phosphorylases, leading to the formation of 2′-deoxynucleosides, was performed. We demonstrated that maximal yields of 2′-deoxynucleosides, especially modified, can be achieved under small excess of glycosyl-donor (7-methyl-2′-deoxyguanosine, thymidine) and a 4-fold lack of phosphate. A phosphate concentration less than equimolar one allows using only a slight excess of the carbohydrate residue donor nucleoside to increase the reaction’s output. A three-step methodology was elaborated for the preparative synthesis of purine-modified 2′-deoxyribonucleosides, starting from the corresponding ribonucleosides. Full article

(This article belongs to the Special Issue Biocatalysis: An Eco-Friendly Scenario for the Manufacturing of APIs)

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Review

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52 pages, 14174 KiB

Open AccessReview

Microbial Lipases and Their Potential in the Production of Pharmaceutical Building Blocks

by César A. Godoy, Juan S. Pardo-Tamayo and Oveimar Barbosa

Int. J. Mol. Sci. 2022, 23(17), 9933; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23179933 - 01 Sep 2022

Cited by 21 | Viewed by 3307

Abstract

Processes involving lipases in obtaining active pharmaceutical ingredients (APIs) are crucial to increase the sustainability of the industry. Despite their lower production cost, microbial lipases are striking for their versatile catalyzing reactions beyond their physiological role. In the context of taking advantage of [...] Read more.

Processes involving lipases in obtaining active pharmaceutical ingredients (APIs) are crucial to increase the sustainability of the industry. Despite their lower production cost, microbial lipases are striking for their versatile catalyzing reactions beyond their physiological role. In the context of taking advantage of microbial lipases in reactions for the synthesis of API building blocks, this review focuses on: (i) the structural origins of the catalytic properties of microbial lipases, including the results of techniques such as single particle monitoring (SPT) and the description of its selectivity beyond the Kazlauskas rule as the “Mirror-Image Packing” or the “Key Region(s) rule influencing enantioselectivity” (KRIE); (ii) immobilization methods given the conferred operative advantages in industrial applications and their modulating capacity of lipase properties; and (iii) a comprehensive description of microbial lipases use as a conventional or promiscuous catalyst in key reactions in the organic synthesis (Knoevenagel condensation, Morita–Baylis–Hillman (MBH) reactions, Markovnikov additions, Baeyer–Villiger oxidation, racemization, among others). Finally, this review will also focus on a research perspective necessary to increase microbial lipases application development towards a greener industry. Full article

(This article belongs to the Special Issue Biocatalysis: An Eco-Friendly Scenario for the Manufacturing of APIs)

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13 pages, 1612 KiB

Open AccessReview

Industrially Relevant Enzyme Cascades for Drug Synthesis and Their Ecological Assessment

by Regine Siedentop and Katrin Rosenthal

Int. J. Mol. Sci. 2022, 23(7), 3605; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23073605 - 25 Mar 2022

Cited by 14 | Viewed by 3588

Abstract

Environmentally friendly and sustainable processes for the production of active pharmaceutical ingredients (APIs) gain increasing attention. Biocatalytic synthesis routes with enzyme cascades support many stated green production principles, for example, the reduced need for solvents or the biodegradability of enzymes. Multi-enzyme reactions have [...] Read more.

Environmentally friendly and sustainable processes for the production of active pharmaceutical ingredients (APIs) gain increasing attention. Biocatalytic synthesis routes with enzyme cascades support many stated green production principles, for example, the reduced need for solvents or the biodegradability of enzymes. Multi-enzyme reactions have even more advantages such as the shift of the equilibrium towards the product side, no intermediate isolation, and the synthesis of complex molecules in one reaction pot. Despite the intriguing benefits, only a few enzyme cascades have been applied in the pharmaceutical industry so far. However, several new enzyme cascades are currently being developed in research that could be of great importance to the pharmaceutical industry. Here, we present multi-enzymatic reactions for API synthesis that are close to an industrial application. Their performances are comparable or exceed their chemical counterparts. A few enzyme cascades that are still in development are also introduced in this review. Economic and ecological considerations are made for some example cascades to assess their environmental friendliness and applicability. Full article

(This article belongs to the Special Issue Biocatalysis: An Eco-Friendly Scenario for the Manufacturing of APIs)

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