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11 pages, 3542 KiB

Open AccessArticle

The Catalytic Mechanism of Key Enzymes Involved in the Synthesis of Ergothioneine in Lentinula edodes

by Zheng Li, Jianjun Ding, Wen Huang, Yinbing Bian, Xi Feng and Ying Liu

Molecules 2024, 29(24), 6005; https://doi.org/10.3390/molecules29246005 - 20 Dec 2024

Viewed by 1084

Abstract

C-S lyase is a crucial enzyme responsible for the formation of sulfur-containing flavor compounds in Lentinula edodes. We investigated the involvement of C-S lyase in the synthesis of ergothioneine (EGT) in L. edodes, a high-producing edible mushroom. Through experimental and computational [...] Read more.

C-S lyase is a crucial enzyme responsible for the formation of sulfur-containing flavor compounds in Lentinula edodes. We investigated the involvement of C-S lyase in the synthesis of ergothioneine (EGT) in L. edodes, a high-producing edible mushroom. Through experimental and computational approaches, we identified Lecsl2, a C-S lyase, as a key enzyme involved in EGT synthesis in L. edodes. We characterized the enzymatic catalytic mechanism of Egt1 and Egt2, the two enzymes primarily catalyzing EGT synthesis in fungi. The results showed that Egt1 interacted with His, SAM, and Cys to form the intermediate product Her-sul, while Egt2, a PLP-dependent enzyme, cleaved the C-S bond on Her-sul to produce EGT. However, our findings suggested that Egt2 in L. edodes might not form a covalent bond with PLP, unlike the previously reported catalytic mechanism of Egt2 involving covalent catalysis. The study provided new insights into the synthesis pathway of EGT in L. edodes and highlighted the need for further investigation into the catalytic mechanism of Egt2 in this species. Full article

(This article belongs to the Special Issue Modern Trends of Biocatalysis in Organic Chemistry and Enzyme Engineering–2nd Edition)

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11 pages, 1527 KiB

Open AccessArticle

Enhancement of the Thermostability of Microbacterium Esterase by Combinatorial Rational Design

by Wenyu Peng, Xiaomei Wu, Baodi Ma and Yi Xu

Molecules 2024, 29(24), 5839; https://doi.org/10.3390/molecules29245839 - 11 Dec 2024

Cited by 1 | Viewed by 739

Abstract

The esterase EstSIT01 from Microbacterium can catalyze the asymmetric hydrolysis of meso-dimethyl ester to produce the crucial chiral intermediate (4S, 5R)-hemimethyl ester for d-biotin synthesis. Despite its high yields and stereoselectivity, the low thermostability of EstSIT01 limits [...] Read more.

The esterase EstSIT01 from Microbacterium can catalyze the asymmetric hydrolysis of meso-dimethyl ester to produce the crucial chiral intermediate (4S, 5R)-hemimethyl ester for d-biotin synthesis. Despite its high yields and stereoselectivity, the low thermostability of EstSIT01 limits its practical application. Herein, two kinds of rational strategies were combined to enhance the thermostability of EstSIT01. Based on the Surface Residue Substitution (SRS) method, two variants (G215A and G316A) with improved thermostability and one mutant (G293A) with superior activity were identified from nine candidates. According to the Consensus Mutation method, two mutants (E301P and A332P) with enhanced thermostability were identified from six candidates. However, the combined mutation failed to yield mutants surpassing the best single mutant, E301P, in terms of thermostability. The combined mutant E301P/G215A and E301P/G215A/G293A exhibited a slight enhancement in enzyme activity relative to E301P, while also exhibiting improved thermostability compared to the wild-type EstSIT01. Compared with the wild-type esterase, the thermal inactivation half-lives (t_1/2) of mutant E301P were enhanced 1.4-fold, 2.4-fold and 1.8-fold at 45 °C, 55 °C, and 65 °C, respectively. The optimal reaction temperature and pH for mutant E301P remained consistent with those of the wild type, at 40 °C and 10.0, respectively. The K_m of E301P was 0.22 ± 0.03 mM and the k_cat was 5.1 ± 0.28 s⁻¹. Further analysis indicated that the free energies of G215A, G293A and E301P were decreased by 0.91, 0.308 and 1.1049 kcal/mol, respectively, compared to the wild-type EstSIT01. The interaction analysis revealed that the substitution of glutamic acid with proline at position 301 enhanced the hydrophobic interactions within the protein. The decreased free energies and the increased hydrophobic interactions were well correlated with the enhanced stability in these mutants. Full article

(This article belongs to the Special Issue Modern Trends of Biocatalysis in Organic Chemistry and Enzyme Engineering–2nd Edition)

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10 pages, 1132 KiB

Open AccessArticle

Asymmetric Sulfoxidations Catalyzed by Bacterial Flavin-Containing Monooxygenases

by Gonzalo de Gonzalo, Juan M. Coto-Cid, Nikola Lončar and Marco W. Fraaije

Molecules 2024, 29(15), 3474; https://doi.org/10.3390/molecules29153474 - 25 Jul 2024

Cited by 2 | Viewed by 1418

Abstract

Flavin-containing monooxygenase from Methylophaga sp. (mFMO) was previously discovered to be a valuable biocatalyst used to convert small amines, such as trimethylamine, and various indoles. As FMOs are also known to act on sulfides, we explored mFMO and some mutants [...] Read more.

Flavin-containing monooxygenase from Methylophaga sp. (mFMO) was previously discovered to be a valuable biocatalyst used to convert small amines, such as trimethylamine, and various indoles. As FMOs are also known to act on sulfides, we explored mFMO and some mutants thereof for their ability to convert prochiral aromatic sulfides. We included a newly identified thermostable FMO obtained from the bacterium Nitrincola lacisaponensis (NiFMO). The FMOs were found to be active with most tested sulfides, forming chiral sulfoxides with moderate-to-high enantioselectivity. Each enzyme variant exhibited a different enantioselective behavior. This shows that small changes in the substrate binding pocket of mFMO influence selectivity, representing a tunable biocatalyst for enantioselective sulfoxidations. Full article

(This article belongs to the Special Issue Modern Trends of Biocatalysis in Organic Chemistry and Enzyme Engineering–2nd Edition)

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16 pages, 5642 KiB

Open AccessArticle

Functionalized Ionic Liquids-Modified Metal–Organic Framework Material Boosted the Enzymatic Performance of Lipase

by Liran Ji, Wei Zhang, Yifei Zhang, Binbin Nian and Yi Hu

Molecules 2024, 29(10), 2381; https://doi.org/10.3390/molecules29102381 - 18 May 2024

Cited by 3 | Viewed by 1893

Abstract

The development of immobilized enzymes with high activity and stability is critical. Metal–organic frameworks (MOFs) have attracted much academic and industrial interest in the field of enzyme immobilization due to their unique properties. In this study, the amino-functionalized ionic liquid (NIL)-modified metal–organic framework [...] Read more.

The development of immobilized enzymes with high activity and stability is critical. Metal–organic frameworks (MOFs) have attracted much academic and industrial interest in the field of enzyme immobilization due to their unique properties. In this study, the amino-functionalized ionic liquid (NIL)-modified metal–organic framework (UiO-66-NH₂) was prepared to immobilize Candida rugosa lipase (CRL), using dialdehyde starch (DAS) as the cross-linker. The results of the Fourier transform infrared (FT-IR) spectra, X-ray powder diffraction (XRD), and scanning electronic microscopy (SEM) confirmed that the NIL was successfully grafted to UiO-66-NH₂. The CRL immobilized on NIL-modified UiO-66-NH₂ (UiO-66-NH₂-NIL-DAS@CRL) exhibited satisfactory activity recovery (79.33%), stability, reusability, and excellent organic solvent tolerance. The research results indicated that ionic liquid-modified UiO-66-NH₂ had practical potential for application in enzyme immobilization. Full article

(This article belongs to the Special Issue Modern Trends of Biocatalysis in Organic Chemistry and Enzyme Engineering–2nd Edition)

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13 pages, 2833 KiB

Open AccessArticle

A Simple Screening and Optimization Bioprocess for Long-Chain Peptide Catalysts Applied to Asymmetric Aldol Reaction

by Shulin Wang, Haidong Teng, Lan Wang, Pengcheng Li, Xinghao Yuan, Xi Sang, Jianping Wu, Lirong Yang and Gang Xu

Molecules 2023, 28(19), 6985; https://doi.org/10.3390/molecules28196985 - 9 Oct 2023

Viewed by 1816

Abstract

Peptides have demonstrated their efficacy as catalysts in asymmetric aldol reactions. But the constraints inherent in chemical synthesis have imposed limitations on the viability of long-chain peptide catalysts. A noticeable dearth of tools has impeded the swift and effective screening of peptide catalysts [...] Read more.

Peptides have demonstrated their efficacy as catalysts in asymmetric aldol reactions. But the constraints inherent in chemical synthesis have imposed limitations on the viability of long-chain peptide catalysts. A noticeable dearth of tools has impeded the swift and effective screening of peptide catalysts using biological methods. To address this, we introduce a straightforward bioprocess for the screening of peptide catalysts for asymmetric aldol reactions. We synthesized several peptides through this method and obtained a 15-amino acid peptide. This peptide exhibited asymmetric aldol catalytic activity, achieving 77% ee in DMSO solvent and 63% ee with over an 80.8% yield in DMSO mixed with a pH 9.0 buffer solution. The successful application of our innovative approach not only represents an advancement but also paves the way for currently unexplored research avenues. Full article

(This article belongs to the Special Issue Modern Trends of Biocatalysis in Organic Chemistry and Enzyme Engineering–2nd Edition)

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Review

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33 pages, 6152 KiB

Open AccessReview

A Comprehensive Guide to Enzyme Immobilization: All You Need to Know

by Marina Simona Robescu and Teodora Bavaro

Molecules 2025, 30(4), 939; https://doi.org/10.3390/molecules30040939 - 18 Feb 2025

Cited by 3 | Viewed by 2777

Abstract

Enzyme immobilization plays a critical role in enhancing the efficiency and sustainability of biocatalysis, addressing key challenges such as limited enzyme stability, short shelf life, and difficulties in recovery and recycling, which are pivotal for green chemistry and industrial applications. Classical approaches, including [...] Read more.

Enzyme immobilization plays a critical role in enhancing the efficiency and sustainability of biocatalysis, addressing key challenges such as limited enzyme stability, short shelf life, and difficulties in recovery and recycling, which are pivotal for green chemistry and industrial applications. Classical approaches, including adsorption, entrapment, encapsulation, and covalent bonding, as well as advanced site-specific methods that integrate enzyme engineering and bio-orthogonal chemistry, were discussed. These techniques enable precise control over enzyme orientation and interaction with carriers, optimizing catalytic activity and reusability. Key findings highlight the impact of immobilization on improving enzyme performance under various operational conditions and its role in reducing process costs through enhanced stability and recyclability. The review presents numerous practical applications of immobilized enzymes, including their use in the pharmaceutical industry for drug synthesis, in the food sector for dairy processing, and in environmental biotechnology for wastewater treatment and dye degradation. Despite the significant advantages, challenges such as activity loss due to conformational changes and mass transfer limitations remain, necessitating tailored immobilization protocols for specific applications. The integration of immobilization with modern biotechnological advancements, such as site-directed mutagenesis and recombinant DNA technology, offers a promising pathway for developing robust, efficient, and sustainable biocatalytic systems. This comprehensive guide aims to support researchers and industries in selecting and optimizing immobilization techniques for diverse applications in pharmaceuticals, food processing, and fine chemicals. Full article

(This article belongs to the Special Issue Modern Trends of Biocatalysis in Organic Chemistry and Enzyme Engineering–2nd Edition)

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23 pages, 2093 KiB

Open AccessReview

Solvent Tolerance Improvement of Lipases Enhanced Their Applications: State of the Art

by Mei Chen, Tongtong Jin, Binbin Nian and Wenjun Cheng

Molecules 2024, 29(11), 2444; https://doi.org/10.3390/molecules29112444 - 22 May 2024

Cited by 7 | Viewed by 2109

Abstract

Lipases, crucial catalysts in biochemical synthesis, find extensive applications across industries such as food, medicine, and cosmetics. The efficiency of lipase-catalyzed reactions is significantly influenced by the choice of solvents. Polar organic solvents often result in a decrease, or even loss, of lipase [...] Read more.

Lipases, crucial catalysts in biochemical synthesis, find extensive applications across industries such as food, medicine, and cosmetics. The efficiency of lipase-catalyzed reactions is significantly influenced by the choice of solvents. Polar organic solvents often result in a decrease, or even loss, of lipase activity. Conversely, nonpolar organic solvents induce excessive rigidity in lipases, thereby affecting their activity. While the advent of new solvents like ionic liquids and deep eutectic solvents has somewhat improved the activity and stability of lipases, it fails to address the fundamental issue of lipases’ poor solvent tolerance. Hence, the rational design of lipases for enhanced solvent tolerance can significantly boost their industrial performance. This review provides a comprehensive summary of the structural characteristics and properties of lipases in various solvent systems and emphasizes various strategies of protein engineering for non-aqueous media to improve lipases’ solvent tolerance. This study provides a theoretical foundation for further enhancing the solvent tolerance and industrial properties of lipases. Full article

(This article belongs to the Special Issue Modern Trends of Biocatalysis in Organic Chemistry and Enzyme Engineering–2nd Edition)

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17 pages, 574 KiB

Open AccessReview

Bioremediation of Hazardous Pollutants Using Enzyme-Immobilized Reactors

by Hiroshi Yamaguchi and Masaya Miyazaki

Molecules 2024, 29(9), 2021; https://doi.org/10.3390/molecules29092021 - 27 Apr 2024

Cited by 5 | Viewed by 3420

Abstract

Bioremediation uses the degradation abilities of microorganisms and other organisms to remove harmful pollutants that pollute the natural environment, helping return it to a natural state that is free of harmful substances. Organism-derived enzymes can degrade and eliminate a variety of pollutants and [...] Read more.

Bioremediation uses the degradation abilities of microorganisms and other organisms to remove harmful pollutants that pollute the natural environment, helping return it to a natural state that is free of harmful substances. Organism-derived enzymes can degrade and eliminate a variety of pollutants and transform them into non-toxic forms; as such, they are expected to be used in bioremediation. However, since enzymes are proteins, the low operational stability and catalytic efficiency of free enzyme-based degradation systems need improvement. Enzyme immobilization methods are often used to overcome these challenges. Several enzyme immobilization methods have been applied to improve operational stability and reduce remediation costs. Herein, we review recent advancements in immobilized enzymes for bioremediation and summarize the methods for preparing immobilized enzymes for use as catalysts and in pollutant degradation systems. Additionally, the advantages, limitations, and future perspectives of immobilized enzymes in bioremediation are discussed. Full article

(This article belongs to the Special Issue Modern Trends of Biocatalysis in Organic Chemistry and Enzyme Engineering–2nd Edition)

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