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A special issue of Molecules (ISSN 1420-3049). This special issue belongs to the section "Chemical Biology".

Deadline for manuscript submissions: closed (31 May 2024) | Viewed by 13818

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Dr. Lindomar Alberto Lerin

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Department of Chemical, Pharmaceutical and Agricultural Sciences, University of Ferrara, Ferrara, Italy
Interests: biotechnological processes; enzyme; lipase; bioreactor enzymatic; optimization processes; esterification
Special Issues, Collections and Topics in MDPI journals

Dr. Daniela Remonatto

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Guest Editor

Department of Bioprocesses and Biotechnology Engineering, São Paulo State University, Araraquara, Brazil
Interests: lipases; enzymes immobilization; biotechnological processes; bioreactor enzymatic; fermentative processes

Special Issue Information

Dear Colleagues,

Biocatalysis is an exponentially growing area concerned with the biosynthesis of compounds that have aroused great interest in the market, especially the pharmaceutical, food, cosmetic, and fine chemical industries. This aligns with the growing demand for natural products and/or ingredients obtained by processes with a low environmental impact. Enzymes play a crucial role in the biosynthesis of high-added-value compounds that can be recognized as natural. In addition, they represent a powerful tool in green chemistry, minimizing energy consumption, and reducing the generation of effluents, perfectly aligning with the circular economy and sustainable development. Despite these advantages, there are many challenges regarding the application of biocatalysts. Efforts are still necessary to develop biocatalysts with high stability, longer half-life, and lower economic impact. To overcome current challenges, research in different areas of enzymology, such as the search for new enzymes with different abilities, new immobilization methods and more economical carriers, the optimization of established processes, the development of new enzymatic biosynthetic routes of biomolecules and the use of alternative substrates in reactions (e.g., agro-industrial residues), must be constant and the future looks promising.

Dr. Lindomar Alberto Lerin
Dr. Daniela Remonatto
Guest Editors

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Keywords

biocatalysis
biosynthesis
green processes
enzyme
renewable sources
immobilization
enzymatic synthesis

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Published Papers (10 papers)

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14 pages, 1135 KiB

Open AccessArticle

Synthesis and Biological Activity of Homohypotaurine Obtained by the Enzyme-Based Conversion of Homocysteine Sulfinic Acid Using Recombinant Escherichia Coli Glutamate Decarboxylase

by Mario Fontana, Aysenur Gunaydin Akyildiz, Chiara D’Alonzo, Fabio Giovannercole, Arianna Zicchi, Antonio Francioso, Elisabetta Capuozzo and Daniela De Biase

Molecules 2024, 29(17), 3985; https://doi.org/10.3390/molecules29173985 - 23 Aug 2024

Viewed by 918

Abstract

l-Homocysteine, formed from S-adenosyl methionine following demethylation and adenosine release, accumulates when the methionine recycling pathway and other pathways become impaired, thus leading to hyperhomocysteinemia, a biomarker in cardiovascular diseases, neurological/psychiatric disorders, and cancer. The partial oxidation of the l-homocysteine thiol group and its decarboxylation on C-alpha lead to the formation of l-homocysteinesulfinic acid (l-HCSA) and homohypotaurine (HHT), respectively. Both compounds are not readily available from commercial suppliers, which hinders the investigation of their biological activities. Herein, the chemical synthesis of l-HCSA, from l-homocystine, was the starting point for establishing the bio-based synthesis of HHT using recombinant Escherichia coli glutamate decarboxylase (EcGadB), an enzyme already successfully employed for the bio-based synthesis of GABA and its phosphinic analog. Prior to HHT synthesis, k_cat (33.92 ± 1.07) and K_M (38.24 ± 3.45 mM) kinetic constants were determined for l-HCSA on EcGadB. The results of our study show that the EcGadB-mediated synthesis of HHT can be achieved with good yields (i.e., 40% following enzymatic synthesis and column chromatography). Purified HHT was tested in vitro on primary human umbilical vein endothelial cells and rat cardiomyoblasts and compared to the fully oxidized analog, homotaurine (OT, also known as tramiprosate), in widespread pharmaceutical use. The results show that both cell lines display statistically significant recovery from the cytotoxic effects induced by H₂O₂ in the presence of HHT. Full article

(This article belongs to the Special Issue Enzymes in Biosynthesis and Biocatalysis)

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11 pages, 887 KiB

Open AccessArticle

Enantiocomplementary Bioreduction of 1-(Arylsulfanyl)propan-2-ones

by Emese Sándor, Pál Csuka, László Poppe and József Nagy

Molecules 2024, 29(16), 3858; https://doi.org/10.3390/molecules29163858 - 15 Aug 2024

Viewed by 681

Abstract

This study explored the enantiocomplementary bioreduction of substituted 1-(arylsulfanyl)propan-2-ones in batch mode using four wild-type yeast strains and two different recombinant alcohol dehydrogenases from Lactobacillus kefir and Rhodococcus aetherivorans. The selected yeast strains and recombinant alcohol dehydrogenases as whole-cell biocatalysts resulted in the corresponding 1-(arylsulfanyl)propan-2-ols with moderate to excellent conversions (60–99%) and high selectivities (ee > 95%). The best bioreductions—in terms of conversion (>90%) and enantiomeric excess (>99% ee)—at preparative scale resulted in the expected chiral alcohols with similar conversion and selectivity to the screening reactions. Full article

(This article belongs to the Special Issue Enzymes in Biosynthesis and Biocatalysis)

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12 pages, 2924 KiB

Open AccessArticle

Characterization of Two Glycoside Hydrolases of Family GH13 and GH57, Present in a Polysaccharide Utilization Locus (PUL) of Pontibacter sp. SGAir0037

by Hilda Hubertha Maria Bax and Edita Jurak

Molecules 2024, 29(12), 2788; https://doi.org/10.3390/molecules29122788 - 12 Jun 2024

Viewed by 855

Abstract

Glycogen, an α-glucan polymer serving as an energy storage compound in microorganisms, is synthesized through distinct pathways (GlgC-GlgA or GlgE pathway). Both pathways involve multiple enzymes, with a shared glycogen branching enzyme (GBE). GBEs play a pivotal role in establishing α-1,6-linkages within the glycogen structure. GBEs are also used for starch modification. Understanding how these enzymes work is interesting for both glycogen synthesis in microorganisms, as well as novel applications for starch modification. This study focuses on a putative enzyme GH13_9 GBE (PoGBE13), present in a polysaccharide utilization locus (PUL) of Pontibacter sp. SGAir0037, and related to the GlgE glycogen synthesis pathway. While the PUL of Pontibacter sp. SGAir0037 contains glycogen-degrading enzymes, the branching enzyme (PoGBE13) was also found due to genetic closeness. Characterization revealed that PoGBE13 functions as a typical branching enzyme, exhibiting a relatively high branching over non-branching (hydrolysis and α-1,4-transferase activity) ratio on linear maltooctadecaose (3.0 ± 0.4). Besides the GH13_9 GBE, a GH57 (PoGH57) enzyme was selected for characterization from the same PUL due to its undefined function. The combined action of both GH13 and GH57 enzymes suggested 4-α-glucanotransferase activity for PoGH57. The characterization of these unique enzymes related to a GlgE glycogen synthesis pathway provides a more profound understanding of their interactions and synergistic roles in glycogen synthesis and are potential enzymes for use in starch modification processes. Due to the structural similarity between glycogen and starch, PoGBE13 can potentially be used for starch modification with different applications, for example, in functional food ingredients. Full article

(This article belongs to the Special Issue Enzymes in Biosynthesis and Biocatalysis)

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19 pages, 3472 KiB

Open AccessArticle

Synthesis and Bioactive Properties of the Novel Coloured Compound Obtained via the Laccase-Mediated Transformation of 5-Aminosalicylic Acid

by Jolanta Polak, Marcin Grąz, Katarzyna Szałapata, Justyna Kapral-Piotrowska, Kamila Wlizło, Marcin Polak and Anna Jarosz-Wilkołazka

Molecules 2024, 29(6), 1310; https://doi.org/10.3390/molecules29061310 - 15 Mar 2024

Cited by 1 | Viewed by 936

Abstract

Biocatalysis processes based on oxidoreductases, such as fungal laccase, are important for discovering new organic compounds with broad structures and potential applications. They include bioactive compounds, which can be obtained through laccase-mediated oxidation of organic substrates having hydroxyl and/or amino groups especially, e.g., 5-aminosalicylic acid (5-ASA) is characterised for its potential for oxidation by a fungal laccase obtained from a Cerrena unicolor strain. The biotransformation process was optimised in terms of the buffer and co-solvent concentration, buffer pH value, and laccase activity. Selected crude dyes were analysed for their bioactive properties, toxicity, and suitability for the dyeing of wool fibres. The data obtained clearly indicated that a low concentration of the reaction buffer in the pH range from 5 to 6 and in the presence of 10% acetonitrile increased the rate of substrate oxidation and the amount of the product formed. The red-brown compound obtained via laccase-mediated oxidation of 5-aminosalicylic acid showed antioxidant properties and unique antimicrobial activity against Staphylococcus aureus and Staphylococcus epidermidis strains with the MIC value of 0.125 mg/mL detected for the purest dye. In addition, it was reported to have good wool fibre dyeing properties and no irritant effect after patch tests on a selected group with increased skin sensitivity. Full article

(This article belongs to the Special Issue Enzymes in Biosynthesis and Biocatalysis)

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13 pages, 641 KiB

Open AccessArticle

Selective Wine Aroma Enhancement through Enzyme Hydrolysis of Glycosidic Precursors

by José Manuel Rodríguez-Nogales, Encarnación Fernández-Fernández, Violeta Ruipérez and Josefina Vila-Crespo

Molecules 2024, 29(1), 16; https://doi.org/10.3390/molecules29010016 - 19 Dec 2023

Cited by 1 | Viewed by 1340

Abstract

Selective enhancement of wine aroma was achieved using a broad spectrum of exogenous glycosidases. Eight different enzyme preparations were added to Verdejo wine, resulting in an increase in the levels of varietal volatile compounds compared to the control wine after 15 days of treatment. The enzyme preparations studied were robust under winemaking conditions (sulfur dioxide, reducing sugars, and alcohol content), and no inhibition of β-glucosidase activity was observed. Significant differences were detected in four individual terpenes (α-terpineol, terpinen-4-ol, α-pinene, and citronellal) and benzyl alcohol in all the treated wines compared to the control wine, contributing to the final wine to varying degrees. In addition, a significant increase in the other aromatic compounds was observed, which showed different patterns depending on the enzyme preparation that was tested. The principal component analysis of the data revealed the possibility of modulating the different aromatic profiles of the final wines depending on the enzyme preparation used. Taking these results into account, enhancement of the floral, balsamic, and/or fruity notes of wines is possible by using a suitable commercial enzyme preparation. Full article

(This article belongs to the Special Issue Enzymes in Biosynthesis and Biocatalysis)

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24 pages, 2343 KiB

Open AccessArticle

Statistical Improvement of rGILCC 1 and rPOXA 1B Laccases Activity Assay Conditions Supported by Molecular Dynamics

by María P. C. Mora-Gamboa, María C. Ferrucho-Calle, Leidy D. Ardila-Leal, Lina M. Rojas-Ojeda, Johan F. Galindo, Raúl A. Poutou-Piñales, Aura M. Pedroza-Rodríguez and Balkys E. Quevedo-Hidalgo

Molecules 2023, 28(21), 7263; https://doi.org/10.3390/molecules28217263 - 25 Oct 2023

Cited by 1 | Viewed by 1202

Abstract

Laccases (E.C. 1.10.3.2) are glycoproteins widely distributed in nature. Their structural conformation includes three copper sites in their catalytic center, which are responsible for facilitating substrate oxidation, leading to the generation of H₂O instead of H₂O₂. The measurement of laccase activity (UL⁻¹) results may vary depending on the type of laccase, buffer, redox mediators, and substrates employed. The aim was to select the best conditions for rGILCC 1 and rPOXA 1B laccases activity assay. After sequential statistical assays, the molecular dynamics proved to support this process, and we aimed to accumulate valuable insights into the potential application of these enzymes for the degradation of novel substrates with negative environmental implications. Citrate buffer treatment T2 (CB T2) (pH 3.0 ± 0.2; λ_420nm, 2 mM ABTS) had the most favorable results, with 7.315 ± 0.131 UL⁻¹ for rGILCC 1 and 5291.665 ± 45.83 UL⁻¹ for rPOXA 1B. The use of citrate buffer increased the enzyme affinity for ABTS since lower K_m values occurred for both enzymes (1.49 × 10⁻² mM for rGILCC 1 and 3.72 × 10⁻² mM for rPOXA 1B) compared to those obtained in acetate buffer (5.36 × 10⁻² mM for rGILCC 1 and 1.72 mM for rPOXA 1B). The molecular dynamics of GILCC 1–ABTS and POXA 1B–ABTS showed stable behavior, with root mean square deviation (RMSD) values not exceeding 2.0 Å. Enzyme activities (rGILCC 1 and rPOXA 1B) and 3D model–ABTS interactions (GILCC 1–ABTS and POXA 1B–ABTS) were under the strong influence of pH, wavelength, ions, and ABTS concentration, supported by computational studies identifying the stabilizing residues and interactions. Integration of the experimental and computational approaches yielded a comprehensive understanding of enzyme–substrate interactions, offering potential applications in environmental substrate treatments. Full article

(This article belongs to the Special Issue Enzymes in Biosynthesis and Biocatalysis)

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18 pages, 3144 KiB

Open AccessArticle

Solvent-Free Enzymatic Synthesis of Dietary Triacylglycerols from Cottonseed Oil in a Fluidized Bed Reactor

by Daniela Remonatto, Núbia Santaella, Lindomar Alberto Lerin, Juliana Cristina Bassan, Marcel Otávio Cerri and Ariela Veloso de Paula

Molecules 2023, 28(14), 5384; https://doi.org/10.3390/molecules28145384 - 13 Jul 2023

Cited by 3 | Viewed by 1874

Abstract

The synthesis of structured lipids with nutraceutical applications, such as medium-long-medium (MLM) triacylglycerols, via modification of oils and fats represents a challenge for the food industry. This study aimed to synthesize MLM-type dietary triacylglycerols by enzymatic acidolysis of cottonseed oil and capric acid (C10) catalyzed by Lipozyme RM IM (lipase from Rhizomucor miehei) in a fluidized bed reactor (FBR). After chemical characterization of the feedstock and hydrodynamic characterization of the reactor, a 2² central composite rotatable design was used to optimize capric acid incorporation. The independent variables were cycle number (20–70) and cottonseed oil/capric acid molar ratio (1:2–1:4). The temperature was set at 45 °C. The best conditions, namely a 1:4 oil/acid molar ratio and 80 cycles (17.34 h), provided a degree of incorporation of about 40 mol%, as shown by compositional analysis of the modified oil. Lipozyme RM IM showed good operational stability (k_d = 2.72 × 10⁻⁴ h⁻¹, t_1/2 = 2545.78 h), confirming the good reuse capacity of the enzyme in the acidolysis of cottonseed oil with capric acid. It is concluded that an FBR configuration is a promising alternative for the enzymatic synthesis of MLM triacylglycerols. Full article

(This article belongs to the Special Issue Enzymes in Biosynthesis and Biocatalysis)

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16 pages, 3730 KiB

Open AccessReview

The Final Step in Molybdenum Cofactor Biosynthesis—A Historical View

by Ralf R. Mendel and Kevin D. Oliphant

Molecules 2024, 29(18), 4458; https://doi.org/10.3390/molecules29184458 - 20 Sep 2024

Viewed by 735

Abstract

Molybdenum (Mo) is an essential micronutrient across all kingdoms of life, where it functions as a key component of the active centers of molybdenum-dependent enzymes. For these enzymes to gain catalytic activity, Mo must be complexed with a pterin scaffold to form the molybdenum cofactor (Moco). The final step of Moco biosynthesis is catalyzed by the enzyme Mo-insertase. This review focuses on eukaryotic Mo-insertases, with an emphasis on those found in plants and mammals, which have been instrumental in advancing the understanding of Mo biochemistry. Additionally, a historical perspective is provided, tracing the discovery of Mo-insertase from the early 1960s to the detailed characterization of its reaction mechanism in 2021. This review also highlights key milestones in the study of Mo-insertase, including mutant characterization, gene cloning, structural elucidation at the atomic level, functional domain assignment, and the spatial organization of the enzyme within cellular protein networks. Full article

(This article belongs to the Special Issue Enzymes in Biosynthesis and Biocatalysis)

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30 pages, 2642 KiB

Open AccessReview

Advancements in Aqueous Two-Phase Systems for Enzyme Extraction, Purification, and Biotransformation

by Nikša Bekavac, Maja Benković, Tamara Jurina, Davor Valinger, Jasenka Gajdoš Kljusurić, Ana Jurinjak Tušek and Anita Šalić

Molecules 2024, 29(16), 3776; https://doi.org/10.3390/molecules29163776 - 9 Aug 2024

Viewed by 2366

Abstract

In recent years, the increasing need for energy conservation and environmental protection has driven industries to explore more efficient and sustainable processes. Liquid–liquid extraction (LLE) is a common method used in various sectors for separating components of liquid mixtures. However, the traditional use of toxic solvents poses significant health and environmental risks, prompting the shift toward green solvents. This review deals with the principles, applications, and advantages of aqueous two-phase systems (ATPS) as an alternative to conventional LLE. ATPS, which typically utilize water and nontoxic components, offer significant benefits such as high purity and single-step biomolecule extraction. This paper explores the thermodynamic principles of ATPS, factors influencing enzyme partitioning, and recent advancements in the field. Specific emphasis is placed on the use of ATPS for enzyme extraction, showcasing its potential in improving yields and purity while minimizing environmental impact. The review also highlights the role of ionic liquids and deep eutectic solvents in enhancing the efficiency of ATPS, making them viable for industrial applications. The discussion extends to the challenges of integrating ATPS into biotransformation processes, including enzyme stability and process optimization. Through comprehensive analysis, this paper aims to provide insights into the future prospects of ATPS in sustainable industrial practices and biotechnological applications. Full article

(This article belongs to the Special Issue Enzymes in Biosynthesis and Biocatalysis)

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18 pages, 25594 KiB

Open AccessReview

Advances in 4-Hydroxyphenylacetate-3-hydroxylase Monooxygenase

by Kai Yang, Qianchao Zhang, Weirui Zhao, Sheng Hu, Changjiang Lv, Jun Huang, Jiaqi Mei and Lehe Mei

Molecules 2023, 28(18), 6699; https://doi.org/10.3390/molecules28186699 - 19 Sep 2023

Cited by 1 | Viewed by 1973

Abstract

Catechols have important applications in the pharmaceutical, food, cosmetic, and functional material industries. 4-hydroxyphenylacetate-3-hydroxylase (4HPA3H), a two-component enzyme system comprising HpaB (monooxygenase) and HpaC (FAD oxidoreductase), demonstrates significant potential for catechol production because it can be easily expressed, is highly active, and exhibits ortho-hydroxylation activity toward a broad spectrum of phenol substrates. HpaB determines the ortho-hydroxylation efficiency and substrate spectrum of the enzyme; therefore, studying its structure–activity relationship, improving its properties, and developing a robust HpaB-conducting system are of significance and value; indeed, considerable efforts have been made in these areas in recent decades. Here, we review the classification, molecular structure, catalytic mechanism, primary efforts in protein engineering, and industrial applications of HpaB in catechol synthesis. Current trends in the further investigation of HpaB are also discussed. Full article

(This article belongs to the Special Issue Enzymes in Biosynthesis and Biocatalysis)

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