This study explores halohydrin dehalogenase (HHDH) variant ISM-4 for the synthesis of enantioenriched fluorinated β-hydroxy nitrile, focusing on the reaction engineering perspective for the ...enhancement of process metrics. Detailed kinetic assessments, enzyme affinities, inhibitions, and deactivation dependency are integrated into a mathematical model, providing insights into ISM-4 limitations and optimal conditions for (S)-3-(4-fluorophenyl)-3-hydroxypropanenitrile’s synthesis. By strategically feeding the substrate in a fed-batch or a repetitive-batch reactor, substantial improvements are achieved compared to the batch reactor, yielding the 75 and 145 mM desired product with ee > 90 and 80%, respectively. Additionally, findings from in silico simulations guided the selection of process conditions for a reaction on a 100 mL scale in a rotating-bed reactor with an immobilized catalyst, resulting in the 24.7 mM product (Y = 72%, ee 92%). This study highlights the important role of a reaction engineering approach in enhancing HHDH-catalyzed synthesis for scalable production of valuable enantioenriched building blocks.
Kinetic resolution of a series of fluorinated styrene oxide derivatives was studied using halohydrin dehalogenase. A mutant HheC-W249P catalysed nucleophilic ring-opening with azide and cyanide ions ...with excellent enantioselectivity (E-values up to >200), which gives access to various enantiopure β-substituted alcohols and epoxides. It was found that the enzyme tolerates substrates in concentrations over 50 mM. However, different side reactions were observed at elevated concentrations and with prolonged reaction time. The biocatalytic azidolysis and cyanolysis of racemic 4-trifluoromethylstyrene oxide were performed on preparative scale, affording (R)-2-azido-1-(4-trifluoromethyl-phenyl)-ethanol in 38% yield and 97% ee, and (S)-3-hydroxy-3-(4-trifluoromethyl-phenyl)-propionitrile in 30% yield and 98% ee.
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•Halohydrin dehalogenase catalyses transformation of fluorinated aryl epoxides.•Enantioselective kinetic resolution was achieved (E-value up to >200).•Preparative azidolysis and cyanolysis reactions were performed at 20–50 mM scale.•Under elevated concentrations substrates were sensitive to spontaneous hydrolysis.
Biokataliza u doba zelene revolucije Milčić, Nevena; Findrik Blažević, Zvjezdana; Sudar, Martina ...
Kemija u industriji; časopis kemičara i tehnologa Jugoslavije,
05/2024, Letnik:
73, Številka:
5-6
Journal Article
Recenzirano
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U dobu zelene revolucije sve više raste svijest o potrebi razvoja održivijih alternativa tradicionalnim industrijskim procesima. Gotovo svi industrijski procesi upotrebljavaju katalizatore koji su ...nerazgradivi i mogu biti štetni za okoliš. S druge strane, biokatalizatori su enzimi – molekule evoluirale u fiziološkom okruženju koje su u potpunosti biorazgradive, a u blagim reakcijskim uvjetima fiziološkog pH, temperature i tlaka okoline pokazuju najveću katalitičku aktivnost i učinkovitost. Biokatalizu ljudi primjenjuju još od starog vijeka u proizvodnji hrane poput sira, kiselog tijesta, piva, vina i octa bez znanja o pozadini procesa koji se odvija, a u posljednjih nekoliko desetljeća porasla je i njezina primjena u industriji, posebice u proizvodnji lijekova i kemikalija. Prepoznate su sve prednosti koje imaju biokatalizatori, poput njihove velike selektivnosti, velike specifičnosti prema supstratu i biorazgradivosti, te postaje sve jasnije da biokataliza može pomoći ispunjenju ciljeva održivog razvoja i implementaciji zelene kemije u industrijske procese s maksimalnim iskorištenjem resursa uz minimalno stvaranje otpada. Iako biokataliza ispunjava gotovo sva načela zelene kemije i potencijal biokatalizatora eksponencijalno raste razvojem inovacija i tehnološkog napretka, posebno u područjima biotehnologije i molekularne biologije, sama prisutnost biokatalizatora u kemijskom procesu ne podrazumijeva održivost procesa. Ekološku prihvatljivost, ali i ekonomsku isplativost, procesa je potrebno dokazati što ranije tijekom njegova razvoja praćenjem raznih procesnih pokazatelja kako bi se uštedjeli i vrijeme i novac.
In the era of the Green Revolution, awareness of the necessity to develop more sustainable alternatives to traditional industrial processes is growing. Nearly all industrial processes utilise catalysts that are nondegradable and potentially harmful to the environment. On the other hand, biocatalysts are enzymes – molecules evolved in a physiological environment – that are entirely biodegradable. Under mild reaction conditions such as physiological pH, room temperature, and pressure, they exhibit their highest catalytic activity and efficiency. Biocatalysis has been utilised since ancient times in the production of food items such as cheese, sourdough, beer, wine, and vinegar, without the knowledge of the background of the process. Over the last few decades, its industrial application has also increased, especially in the production of medicines and chemicals. All the advantages of biocatalysts, such as their high selectivity, high specificity toward substrates, and biodegradability, have been acknowledged. It is becoming increasingly evident that biocatalysis represents one of the key means to accomplish Sustainable Development Goals and to implement green chemistry in industrial processes, maximising the use of resources while minimising waste generation. Even though biocatalysis aligns with almost all green chemistry principles, and the potential of biocatalysts is experiencing exponential growth through the evolution of innovations and technological advancements, the mere presence of a biocatalyst in a chemical process does not imply sustainability of the process. The environmental acceptability, as well as the economic viability of the process need to be demonstrated as early as possible in its development by monitoring various metrics to save both time and money.
Fitoremedijacija – pregled stanja i perspektiva Milčić, Nevena; Findrik Blažević, Zvjezdana; Vuković Domanovac, Marija
Kemija u industriji,
10/2019, Letnik:
68, Številka:
9-10
Journal Article
Recenzirano
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Remedijacija tala onečišćenih kompleksnim mješavinama organskih tvari i teških metala jedan je od najvećih izazova obnavljanja okoliša. Fitoremedijacija je naziv za skup postupaka koji upotrebljavaju ...biljke, njihove enzime i prisutne mikroorganizme iz zone korijenja za izolaciju, transport, detoksikaciju i mineralizaciju ksenobiotika, čime se smanjuje njihova koncentracija, pokretljivost ili toksični učinci. Fitoekstrakcija, fitostabilizacija, fitovolatizacija, fitorazgradnja i rizorazgradnja imaju velik potencijal za nedestruktivnu remedijaciju tala, što pokazuju brojna istraživanja u laboratorijskom mjerilu. Kako bi fitoremedijacija postala pouzdana tehnologija za širok spektar primjena u većem mjerilu, potrebno je ulagati resurse u nova istraživanja s ciljem boljeg razumijevanja procesa u cjelini, posebice na genetičkoj i biokemijskoj razini.
Remediation of soils contaminated with complex mixtures of organic compounds and heavy metals is one of the greatest challenges of environmental renewal. Phytoremediation is the name for a set of techniques that employ plants, their enzymes, and associated microorganisms in the root zone for isolation, transport, detoxification, and mineralization of xenobiotics in the soil, thereby reducing their concentration, mobility or toxic effects. Phytoextraction, phytostabilization, phytovolatization, phytodegradation, and rhizodegradation have a great potential for non-destructive remediation of soils as shown by numerous laboratory-scale studies. In order for phytoremediation to become a reliable technology for a wide range of applications at a larger scale, resources need to be invested in a new research with an aim to better understand the process as a whole, especially at the genetic and biochemical levels.
“Sustavska biokataliza” (engl.
systems biocatalysis
), odnosno provedba kaskadnih reakcija koje oponašaju stanične metaboličke puteve danas se sve češće primjenjuje. Kaskadne reakcije imaju brojne ...prednosti nad tradicionalnim kemijskim postupcima, međutim, za uspješnu optimizaciju i prenošenje takvih kompleksnih sustava u veće, industrijsko mjerilo potrebno je primijeniti reakcijsko inženjerstvo. U ovom preglednom radu navedeni su primjeri uspješne primjene matematičkog modeliranja na razvoj enzimskih kaskadnih reakcija koji pokazuju važnost i potencijal te metodologije.
Today, systems biocatalysis,
i.e.
, the implementation of cascade reactions that mimic cellular metabolic pathways, is increasingly being used. Cascade reactions have numerous advantages over traditional chemical processes; however, in order to successfully optimize and transfer such complex systems to a larger, industrial scale, it is necessary to apply reaction engineering. This review paper provides examples of the successful application of mathematical modelling in development of enzymatic cascade reactions that demonstrate the importance and potential of this methodology.
At the moment, there are approx. 100 published papers investigating halohydrin dehalogenases from different aspects; enzymology, molecular biology and reactions they can catalyse. Unquestionably, ...these enzymes are of great importance and hold an immense potential due to the wide spectrum of different compounds that can be synthesized by their action. These compounds, such as chiral epoxides, β‐substituted alcohols, oxazolidinones etc., significantly enrich the chemist's toolbox and, moreover, open the possibility for the synthesis of even more complex compounds. Still, there are many unknowns, and it is the purpose of this work to demonstrate the possibilities and bottlenecks, in scientific sense, that could further help in broadening the applicative potential of these fascinating enzymes.
Biocatalytic transformations in organic synthesis often require the use of organic solvents to improve substrate solubility and promote the product formation. Halohydrin dehalogenases (HHDHs) are ...enzymes that catalyze the formation and conversion of epoxides, important synthetic class of compounds that are often sparingly soluble in water and prone to hydrolysis. In this study, the activity, stability, and enantioselectivity of HHDH from
Agrobacterium radiobacter
AD1 (HheC) in form of cell-free extract were evaluated in various aqueous-organic media. A correlation was discovered between the enzyme activity in the ring-closure reaction and logP of the solvent. Knowledge of such a relationship makes biocatalysis with organic solvents more predictable, which may reduce the need to experiment with a variety of solvents in the future. The results revealed a high enzyme compatibility with hydrophobic solvents (e.g.,
n
-heptane) in terms of activity and stability. Regarding the HHDH applicability in an organic medium, inhibitions by a number of solvents (e.g., THF, toluene, chloroform) proved to be a more challenging problem than the protein stability, especially in the ring-opening reaction, thus suggesting which solvents should be avoided. In addition, solvent tolerance of the thermostable variant ISM-4 was also evaluated, revealing increased stability and to a lesser extent enantioselectivity compared to the wild-type. This is the first time such a systematic analysis has been reported, giving insight into the behavior of HHDHs in nonconventional media and opening new opportunities for the future biocatalytic applications.
Key points
•
HheC performs better in the presence of hydrophobic than hydrophilic solvents.
•
Enzyme activity in the PNSHH ring-closure reaction is a function of the logP.
•
Thermostability of ISM-4 variant is accompanied by superior solvent tolerance.
Graphical Abstract
Although the application of organic solvents in biocatalysis is well explored, in‐depth understanding of the interactions of solvent with proteins, in particular oligomeric ones, is still scant. ...Understanding these interactions is essential in tailoring enzymes for industrially relevant catalysis in nonaqueous media. In our study, the homotetrameric enzyme halohydrin dehalogenase (HHDH) from Agrobacterium radiobacter AD1 (HheC) was investigated, as a model system, in DMSO/water solvent mixtures. DMSO, the most commonly used co‐solvent for biocatalytic transformations, was found to act as a mixed‐type inhibitor with a prevalent competitive contribution. Even 5 % (v/v) DMSO inhibits the activity of HheC by half. Molecular dynamics (MD) simulations showed that DMSO keeps close to Ser‐Tyr catalytic residues forming alternate H‐bonds with them. Stability measurements paired with differential scanning calorimetry, dynamic light scattering methods and MD studies revealed that HheC maintains its structural integrity with as much as 30 % (v/v) DMSO.
Pros and cons considered: Halohydrin dehalogenases (HHDHs) are versatile enzymes employed in the synthesis of chiral building blocks. To increase substrate solubility, DMSO stands out as the most commonly used co‐solvent. Here we found that DMSO acts as an inhibitor of the enzyme HheC. To understand the observed phenomena at the molecular level, in‐depth experimental studies were performed in combination with molecular dynamics simulations.
Invited for the cover of this issue are Zlatko Brkljača, Maja Majerić Elenkov and co‐workers at the Ruđer Bošković Institute and University of Zagreb. The image depicts the enzyme halohydrin ...dehalogenase HheC, which is made up of four identical subunits, with marked catalytic residues and volumetric maps of water and DMSO in the active site. Read the full text of the article at 10.1002/chem.202201923.
“The first comprehensive study of the mechanisms by which DMSO, the most commonly used co‐solvent in the halohydrin dehalogenase‐catalysed reactions, influences the activity and stability of the enzyme HheC.” This and more about the story behind the front cover can be found in the article at 10.1002/chem.202201923).
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