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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Biochemistry".

Deadline for manuscript submissions: closed (31 October 2015)

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Prof. Dr. Vladimír Křen

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Institute of Microbiology, Academy of Sciences of the Czech Republic, Laboratory of Biotransformation, National Centre of Biocatalysis and Biotransformation, Videnska 1083, CZ 142 20 Praha 4, Czech Republic
Interests: biocatalysis and biotransformation; immobilized microbial cells, their use in production and biotransformation of natural products; biotransformation of natural products by enzymes and microorganisms; preparation of glycosidases of microbial origin and their use for glycosylation of natural compounds: glycoconjugates, multivalent compounds, ergot alkaloids, flavonoids, antioxidants and chemoprotectants
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Dr. Daniela Monti

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Istituto di Chimica del Riconoscimento Molecolare, C.N.R.–Via Mario Bianco 9, 20131 Milano, Italy
Interests: biocatalysis; applications of different classes of enzymes (hydrolases, phospholipases, glycosidases, glycosyltransferases, oxidoreductases) in organic synthesis; multienzymatic and chemo-enzymatic cascade systems; free and immobilized enzymes in industrial applications; discovery and characterization of novel enzymes; metagenomics; optimization of protein expression in heterologous hosts; improvement of biocatalyst performances
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear colleagues,

Systems and molecular biocatalysis are new approaches consisting of organizing enzymes in vitro to generate an artificial metabolism for synthetic purposes. Another similar approach for integrating biocatalysis into chemical procedures is cascade biocatalysis. The strategy of these new platforms involves the analysis of enzymatic systems in vivo, the development and discovery of new biocatalysts, and their assembly in vitro into novel synthetic metabolic pathways. This network aims to control the construction of metabolic pathways to efficiently synthesize valuable chemical products. Topics for this Special Issue include the discovery of new biocatalysts, the optimization of their functions (including via enzyme engineering), mutual compatibility and regulation, and the construction of a continuous flux for high efficiency. These tasks require research in various disciplines, including work concerning the molecular and engineering aspects of whole synthetic reaction sequences. Enzymes are powerful tools for chemical synthesis. This Special Issue of IJMS is interested in contributions that describe important new studies, which will help fill the enzyme toolbox for chemistry.

Major requirements for papers submitted to this Special Issue are (i) clear novelty; (ii) reproducibility; (iii) molecular bases for reactions and processes; and (iv) defined chemical reactions. Papers dealing with minor optimizations of known procedures, or which use poorly defined organisms or organisms that are unavailable to other researchers, and which contain poorly defined substrates and products, will be returned without further review.

Prof. Dr. Vladimír Křen
Dr. Daniela Monti
Guest Editor

* This issue is dedicated to the memory of Prof. Dr. Bruno Danieli, professor emeritus of organic chemistry, Università degli Studi di Milano, Italy, a founder of application of biocatalytic transformations of natural products. Prof. Danieli deceased on November 13, 2014.

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Keywords

Biocatalysts discovery
Biocatalyst development and design
Cofactor regeneration and multienzyme processes
Fundamentals of biocatalysis
New solvents and special processes for biocatalysis
Biocatalysis integration into chemical processes

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Published Papers (12 papers)

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Research

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1170 KiB

Open AccessArticle

Microwave-Assisted Synthesis of Glycoconjugates by Transgalactosylation with Recombinant Thermostable β-Glycosidase from Pyrococcus

by Manja Henze, Dorothee Merker and Lothar Elling

Int. J. Mol. Sci. 2016, 17(2), 210; https://doi.org/10.3390/ijms17020210 - 4 Feb 2016

Cited by 6 | Viewed by 7282

Abstract

The potential of the hyperthermophilic β-glycosidase from Pyrococcus woesei (DSM 3773) for the synthesis of glycosides under microwave irradiation (MWI) at low temperatures was investigated. Transgalactosylation reactions with β-N-acetyl-d-glucosamine as acceptor substrate (GlcNAc-linker-tBoc) under thermal heating (TH, 85 °C) and under MWI at 100 and 300 W resulted in the formation of (Galβ(1,4)GlcNAc-linker-tBoc) as the main product in all reactions. Most importantly, MWI at temperatures far below the temperature optimum of the hyperthermophilic glycosidase led to higher product yields with only minor amounts of side products β(1,6-linked disaccharide and trisaccharides). At high acceptor concentrations (50 mM), transgalactosylation reactions under MWI at 300 W gave similar product yields when compared to TH at 85 °C. In summary, we demonstrate that MWI is useful as a novel experimental set-up for the synthesis of defined galacto-oligosaccharides. In conclusion, glycosylation reactions under MWI at low temperatures have the potential as a general strategy for regioselective glycosylation reactions of hyperthermophilic glycosidases using heat-labile acceptor or donor substrates. Full article

(This article belongs to the Special Issue Molecular Biocatalysis)

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3900 KiB

Open AccessArticle

Enhancement of Alkaline Protease Activity and Stability via Covalent Immobilization onto Hollow Core-Mesoporous Shell Silica Nanospheres

by Abdelnasser Salah Shebl Ibrahim, Ali A. Al-Salamah, Ahmed M. El-Toni, Khalid S. Almaary, Mohamed A. El-Tayeb, Yahya B. Elbadawi and Garabed Antranikian

Int. J. Mol. Sci. 2016, 17(2), 184; https://doi.org/10.3390/ijms17020184 - 29 Jan 2016

Cited by 58 | Viewed by 7818

Abstract

The stability and reusability of soluble enzymes are of major concerns, which limit their industrial applications. Herein, alkaline protease from Bacillus sp. NPST-AK15 was immobilized onto hollow core-mesoporous shell silica (HCMSS) nanospheres. Subsequently, the properties of immobilized proteases were evaluated. Non-, ethane- and amino-functionalized HCMSS nanospheres were synthesized and characterized. NPST-AK15 was immobilized onto the synthesized nano-supports by physical and covalent immobilization approaches. However, protease immobilization by covalent attachment onto the activated HCMSS–NH₂ nanospheres showed highest immobilization yield (75.6%) and loading capacity (88.1 μg protein/mg carrier) and was applied in the further studies. In comparison to free enzyme, the covalently immobilized protease exhibited a slight shift in the optimal pH from 10.5 to 11.0, respectively. The optimum temperature for catalytic activity of both free and immobilized enzyme was seen at 60 °C. However, while the free enzyme was completely inactivated when treated at 60 °C for 1 h the immobilized enzyme still retained 63.6% of its initial activity. The immobilized protease showed higher V_max, k_cat and k_cat/K_m, than soluble enzyme by 1.6-, 1.6- and 2.4-fold, respectively. In addition, the immobilized protease affinity to the substrate increased by about 1.5-fold. Furthermore, the enzyme stability in various organic solvents was significantly enhanced upon immobilization. Interestingly, the immobilized enzyme exhibited much higher stability in several commercial detergents including OMO, Tide, Ariel, Bonux and Xra by up to 5.2-fold. Finally, the immobilized protease maintained significant catalytic efficiency for twelve consecutive reaction cycles. These results suggest the effectiveness of the developed nanobiocatalyst as a candidate for detergent formulation and peptide synthesis in non-aqueous media. Full article

(This article belongs to the Special Issue Molecular Biocatalysis)

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4715 KiB

Open AccessArticle

Tuning the Phosphoryl Donor Specificity of Dihydroxyacetone Kinase from ATP to Inorganic Polyphosphate. An Insight from Computational Studies

by Israel Sánchez-Moreno, Isabel Bordes, Raquel Castillo, José Javier Ruiz-Pernía, Vicent Moliner and Eduardo García-Junceda

Int. J. Mol. Sci. 2015, 16(11), 27835-27849; https://doi.org/10.3390/ijms161126073 - 24 Nov 2015

Cited by 12 | Viewed by 7004

Abstract

Dihydroxyacetone (DHA) kinase from Citrobacter freundii provides an easy entry for the preparation of DHA phosphate; a very important C3 building block in nature. To modify the phosphoryl donor specificity of this enzyme from ATP to inorganic polyphosphate (poly-P); a directed evolution program has been initiated. In the first cycle of evolution, the native enzyme was subjected to one round of error-prone PCR (EP-PCR) followed directly (without selection) by a round of DNA shuffling. Although the wild-type DHAK did not show activity with poly-P, after screening, sixteen mutant clones showed an activity with poly-phosphate as phosphoryl donor statistically significant. The most active mutant presented a single mutation (Glu526Lys) located in a flexible loop near of the active center. Interestingly, our theoretical studies, based on molecular dynamics simulations and hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) optimizations, suggest that this mutation has an effect on the binding of the poly-P favoring a more adequate position in the active center for the reaction to take place. Full article

(This article belongs to the Special Issue Molecular Biocatalysis)

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1573 KiB

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Alteration of the Donor/Acceptor Spectrum of the (S)-Amine Transaminase from Vibrio fluvialis

by Maika Genz, Clare Vickers, Tom Van den Bergh, Henk-Jan Joosten, Mark Dörr, Matthias Höhne and Uwe T. Bornscheuer

Int. J. Mol. Sci. 2015, 16(11), 26953-26963; https://doi.org/10.3390/ijms161126007 - 11 Nov 2015

Cited by 30 | Viewed by 7848

Abstract

To alter the amine donor/acceptor spectrum of an (S)-selective amine transaminase (ATA), a library based on the Vibrio fluvialis ATA targeting four residues close to the active site (L56, W57, R415 and L417) was created. A 3DM-derived alignment comprising fold class I pyridoxal-5′-phosphate (PLP)-dependent enzymes allowed identification of positions, which were assumed to determine substrate specificity. These positions were targeted for mutagenesis with a focused alphabet of hydrophobic amino acids to convert an amine:α-keto acid transferase into an amine:aldehyde transferase. Screening of 1200 variants revealed three hits, which showed a shifted amine donor/acceptor spectrum towards aliphatic aldehydes (mainly pentanal), as well as an altered pH profile. Interestingly, all three hits, although found independently, contained the same mutation R415L and additional W57F and L417V substitutions. Full article

(This article belongs to the Special Issue Molecular Biocatalysis)

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1138 KiB

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Cloning, Expression, and Characterization of a Thermophilic Endoglucanase, AcCel12B from Acidothermus cellulolyticus 11B

by Junling Wang, Gui Gao, Yuwei Li, Liangzhen Yang, Yanli Liang, Hanyong Jin, Weiwei Han, Yan Feng and Zuoming Zhang

Int. J. Mol. Sci. 2015, 16(10), 25080-25095; https://doi.org/10.3390/ijms161025080 - 22 Oct 2015

Cited by 30 | Viewed by 6796

Abstract

The gene ABK52392 from the thermophilic bacterium Acidothermus cellulolyticus 11B was predicted to be endoglucanase and classified into glycoside hydrolase family 12. ABK52392 encodes a protein containing a catalytic domain and a carbohydrate binding module. ABK52392 was cloned and functionally expressed in Escherichia coli. After purification by Ni-NTA agarose affinity chromatography and Q-Sepharose^® Fast Flow chromatography, the properties of the recombinant protein (AcCel12B) were characterized. AcCel12B exhibited optimal activity at pH 4.5 and 75 °C. The half-lives of AcCel12B at 60 and 70 °C were about 90 and 2 h, respectively, under acidic conditions. The specific hydrolytic activities of AcCel12B at 70 °C and pH 4.5 for sodium carboxymethylcellulose (CMC) and regenerated amorphous cellulose (RAC) were 118.3 and 104.0 U·mg⁻¹, respectively. The K_m and V_max of AcCel12B for CMC were 25.47 mg·mL⁻¹ and 131.75 U·mg⁻¹, respectively. The time course of hydrolysis for RAC was investigated by measuring reducing ends in the soluble and insoluble phases. The total hydrolysis rate rapidly decreased after the early stage of incubation and the generation of insoluble reducing ends decreased earlier than that of soluble reducing ends. High thermostability of the cellulase indicates its potential commercial significance and it could be exploited for industrial application in the future. Full article

(This article belongs to the Special Issue Molecular Biocatalysis)

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1168 KiB

Open AccessCommunication

Enzymatic Polymerization on DNA Modified Gold Nanowire for Label-Free Detection of Pathogen DNA

by Jaepil Jeong, Hyejin Kim and Jong Bum Lee

Int. J. Mol. Sci. 2015, 16(6), 13653-13660; https://doi.org/10.3390/ijms160613653 - 15 Jun 2015

Cited by 4 | Viewed by 7061

Abstract

This paper presents a label-free biosensor for the detection of single-stranded pathogen DNA through the target-enhanced gelation between gold nanowires (AuNW) and the primer DNAs branched on AuNW. The target DNA enables circularization of the linear DNA template, and the primer DNA is elongated continuously via rolling circle amplification. As a result, in the presence of the target DNA, a macroscopic hydrogel was fabricated by the entanglement of the elongated DNA with AuNWs as a scaffold fiber for effective gelation. In contrast, very small separate particles were generated in the absence of the target DNA. This label-free biosensor might be a promising tool for the detection of pathogen DNAs without any devices for further analysis. Moreover, the biosensor based on the weaving of AuNW and DNAs suggests a novel direction for the applications of AuNWs in biological engineering. Full article

(This article belongs to the Special Issue Molecular Biocatalysis)

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1471 KiB

Open AccessArticle

Regioselective Alcoholysis of Silychristin Acetates Catalyzed by Lipases

by Eva Vavříková, Paolo Gavezzotti, Kateřina Purchartová, Kateřina Fuksová, David Biedermann, Marek Kuzma, Sergio Riva and Vladimír Křen

Int. J. Mol. Sci. 2015, 16(6), 11983-11995; https://doi.org/10.3390/ijms160611983 - 26 May 2015

Cited by 6 | Viewed by 5840

Abstract

A panel of lipases was screened for the selective acetylation and alcoholysis of silychristin and silychristin peracetate, respectively. Acetylation at primary alcoholic group (C-22) of silychristin was accomplished by lipase PS (Pseudomonas cepacia) immobilized on diatomite using vinyl acetate as an acetyl donor, whereas selective deacetylation of 22-O-acetyl silychristin was accomplished by Novozym 435 in methyl tert-butyl ether/ n-butanol. Both of these reactions occurred without diastereomeric discrimination of silychristin A and B. Both of these enzymes were found to be capable to regioselective deacetylation of hexaacetyl silychristin to afford penta-, tetra- and tri-acetyl derivatives, which could be obtained as pure synthons for further selective modifications of the parent molecule. Full article

(This article belongs to the Special Issue Molecular Biocatalysis)

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Review

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1925 KiB

Open AccessReview

Radical-Mediated Enzymatic Polymerizations

by Scott R. Zavada, Tsatsral Battsengel and Timothy F. Scott

Int. J. Mol. Sci. 2016, 17(2), 195; https://doi.org/10.3390/ijms17020195 - 2 Feb 2016

Cited by 52 | Viewed by 9777

Abstract

Polymerization reactions are commonly effected by exposing monomer formulations to some initiation stimulus such as elevated temperature, light, or a chemical reactant. Increasingly, these polymerization reactions are mediated by enzymes―catalytic proteins―owing to their reaction efficiency under mild conditions as well as their environmental friendliness. The utilization of enzymes, particularly oxidases and peroxidases, for generating radicals via reduction-oxidation mechanisms is especially common for initiating radical-mediated polymerization reactions, including vinyl chain-growth polymerization, atom transfer radical polymerization, thiol–ene step-growth polymerization, and polymerization via oxidative coupling. While enzyme-mediated polymerization is useful for the production of materials intended for subsequent use, it is especially well-suited for in situ polymerizations, where the polymer is formed in the place where it will be utilized. Such polymerizations are especially useful for biomedical adhesives and for sensing applications. Full article

(This article belongs to the Special Issue Molecular Biocatalysis)

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3852 KiB

Open AccessReview

Enzymatic Kinetic Resolution of 2-Piperidineethanol for the Enantioselective Targeted and Diversity Oriented Synthesis

by Dario Perdicchia, Michael S. Christodoulou, Gaia Fumagalli, Francesco Calogero, Cristina Marucci and Daniele Passarella

Int. J. Mol. Sci. 2016, 17(1), 17; https://doi.org/10.3390/ijms17010017 - 24 Dec 2015

Cited by 7 | Viewed by 6613

Abstract

2-Piperidineethanol (1) and its corresponding N-protected aldehyde (2) were used for the synthesis of several natural and synthetic compounds. The existence of a stereocenter at position 2 of the piperidine skeleton and the presence of an easily-functionalized group, such as the alcohol, set 1 as a valuable starting material for enantioselective synthesis. Herein, are presented both synthetic and enzymatic methods for the resolution of the racemic 1, as well as an overview of synthesized natural products starting from the enantiopure 1. Full article

(This article belongs to the Special Issue Molecular Biocatalysis)

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7105 KiB

Open AccessReview

Recent Advances in Lipase-Mediated Preparation of Pharmaceuticals and Their Intermediates

by Ana Caroline Lustosa de Melo Carvalho, Thiago De Sousa Fonseca, Marcos Carlos de Mattos, Maria Da Conceição Ferreira de Oliveira, Telma Leda Gomes de Lemos, Francesco Molinari, Diego Romano and Immacolata Serra

Int. J. Mol. Sci. 2015, 16(12), 29682-29716; https://doi.org/10.3390/ijms161226191 - 11 Dec 2015

Cited by 121 | Viewed by 12246

Abstract

Biocatalysis offers an alternative approach to conventional chemical processes for the production of single-isomer chiral drugs. Lipases are one of the most used enzymes in the synthesis of enantiomerically pure intermediates. The use of this type of enzyme is mainly due to the characteristics of their regio-, chemo- and enantioselectivity in the resolution process of racemates, without the use of cofactors. Moreover, this class of enzymes has generally excellent stability in the presence of organic solvents, facilitating the solubility of the organic substrate to be modified. Further improvements and new applications have been achieved in the syntheses of biologically active compounds catalyzed by lipases. This review critically reports and discusses examples from recent literature (2007 to mid-2015), concerning the synthesis of enantiomerically pure active pharmaceutical ingredients (APIs) and their intermediates in which the key step involves the action of a lipase. Full article

(This article belongs to the Special Issue Molecular Biocatalysis)

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2389 KiB

Open AccessReview

Recombinant Lipases and Phospholipases and Their Use as Biocatalysts for Industrial Applications

by Grazia M. Borrelli and Daniela Trono

Int. J. Mol. Sci. 2015, 16(9), 20774-20840; https://doi.org/10.3390/ijms160920774 - 1 Sep 2015

Cited by 269 | Viewed by 20581

Abstract

Lipases and phospholipases are interfacial enzymes that hydrolyze hydrophobic ester linkages of triacylglycerols and phospholipids, respectively. In addition to their role as esterases, these enzymes catalyze a plethora of other reactions; indeed, lipases also catalyze esterification, transesterification and interesterification reactions, and phospholipases also show acyltransferase, transacylase and transphosphatidylation activities. Thus, lipases and phospholipases represent versatile biocatalysts that are widely used in various industrial applications, such as for biodiesels, food, nutraceuticals, oil degumming and detergents; minor applications also include bioremediation, agriculture, cosmetics, leather and paper industries. These enzymes are ubiquitous in most living organisms, across animals, plants, yeasts, fungi and bacteria. For their greater availability and their ease of production, microbial lipases and phospholipases are preferred to those derived from animals and plants. Nevertheless, traditional purification strategies from microbe cultures have a number of disadvantages, which include non-reproducibility and low yields. Moreover, native microbial enzymes are not always suitable for biocatalytic processes. The development of molecular techniques for the production of recombinant heterologous proteins in a host system has overcome these constraints, as this allows high-level protein expression and production of new redesigned enzymes with improved catalytic properties. These can meet the requirements of specific industrial process better than the native enzymes. The purpose of this review is to give an overview of the structural and functional features of lipases and phospholipases, to describe the recent advances in optimization of the production of recombinant lipases and phospholipases, and to summarize the information available relating to their major applications in industrial processes. Full article

(This article belongs to the Special Issue Molecular Biocatalysis)

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1739 KiB

Open AccessReview

Trehalose Analogues: Latest Insights in Properties and Biocatalytic Production

by Maarten Walmagh, Renfei Zhao and Tom Desmet

Int. J. Mol. Sci. 2015, 16(6), 13729-13745; https://doi.org/10.3390/ijms160613729 - 15 Jun 2015

Cited by 33 | Viewed by 11658

Abstract

Trehalose (α-d-glucopyranosyl α-d-glucopyranoside) is a non-reducing sugar with unique stabilizing properties due to its symmetrical, low energy structure consisting of two 1,1-anomerically bound glucose moieties. Many applications of this beneficial sugar have been reported in the novel food (nutricals), medical, pharmaceutical and cosmetic industries. Trehalose analogues, like lactotrehalose (α-d-glucopyranosyl α-d-galactopyranoside) or galactotrehalose (α-d-galactopyranosyl α-d-galactopyranoside), offer similar benefits as trehalose, but show additional features such as prebiotic or low-calorie sweetener due to their resistance against hydrolysis during digestion. Unfortunately, large-scale chemical production processes for trehalose analogues are not readily available at the moment due to the lack of efficient synthesis methods. Most of the procedures reported in literature suffer from low yields, elevated costs and are far from environmentally friendly. “Greener” alternatives found in the biocatalysis field, including galactosidases, trehalose phosphorylases and TreT-type trehalose synthases are suggested as primary candidates for trehalose analogue production instead. Significant progress has been made in the last decade to turn these into highly efficient biocatalysts and to broaden the variety of useful donor and acceptor sugars. In this review, we aim to provide an overview of the latest insights and future perspectives in trehalose analogue chemistry, applications and production pathways with emphasis on biocatalysis. Full article

(This article belongs to the Special Issue Molecular Biocatalysis)

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