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.
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.
Esterification of glucose and palmitic acid catalyzed by Novozyme 435 was carried out in three ionic liquids (IL): Cyphos 104, Cyphos 109 and bmimPF6. To our knowledge this product has not been ...obtained in these ILs from these substrates up to now. The equilibrium conversion, as well as the amount of produced ester, were the highest in bmimPF6, i.e. X = 77%, 0.115 mmol g−1 of ester. Hence, this IL was chosen for further experiments. The enzyme was reused four times at 50 and 70 °C to evaluate its stability in bmimPF6. It was found that the enzyme activity slowly decreases at both temperatures. The reaction conditions in bmimPF6 were optimized to achieve the best reaction yield using the factorial design of experiments. The highest concentration of ester that could be achieved in the investigated variable range after 48 h was 0.205 mmol g−1 at 70 °C, equimolar concentrations of substrates of 0.5 mmol g−1 and 0.5% of water. Since it takes more than 48 h to achieve equilibrium, the equilibrium ester concentration at optimal conditions is even higher, i.e. 0.254 mmol g−1. The novelty of the present work lies in the use of IL as a solvent for the synthesis of sugar fatty acid ester, and particularly in the use of palmitic acid as an acyl donor in esterification with glucose in this solvent.
•Sugar fatty acid ester was produced by enzymatic esterification in ionic liquid.•Glucose and palmitic acid were used as substrates.•Initial reaction conditions were optimized by using factorial design.•Maximum equilibrium ester yield at the optimal conditions was 0.254 mmol g−1.
“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.
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
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.
•Untypical substrate, acrylonitrile, used in enzymatic reaction.•Kinetic model of MenD-catalyzed 1,4-addition of α-ketoglutaric acid to acrylonitrile.•Yield of the product dependent on the initial ...acrylonitrile concentration.•6-Cyano-4-oxohexanoic acid synthesized on preparative scale at optimized conditions.•New product for potential new applications.
The Stetter reaction, a conjugate umpolung reaction, is well known for cyanide-catalyzed transformations of mostly aromatic aldehydes. Enzymatic Stetter reactions, however, have been largely unexplored, especially with respect to preparative transformations. We have investigated the kinetics of the MenD-catalyzed 1,4-addition of α-ketoglutaric acid to acrylonitrile which has shown that acrylonitrile, while an interesting candidate, is a poor substrate for MenD due to low affinity of the enzyme for this substrate. The kinetic model of the reaction was simplified to double substrate Michaelis–Menten kinetics where the reaction rate linearly depends on acrylonitrile concentration. Experiments at different initial concentrations of acrylonitrile under batch, repetitive batch, and fed-batch reactor conditions were carried out to validate the developed mathematical model. Thiamine diphosphate dependent MenD proved to be quite a robust enzyme; nevertheless, enzyme operational stability decay occurs in the reactor. The spontaneous reactivity of acrylonitrile towards polymerization was also taken into account during mathematical modeling. Almost quantitative conversion of acrylonitrile was achieved in all batch reactor experiments, while the yield of the desired product was dependent on initial acrylonitrile concentration (i.e., the concentration of the stabilizer additive). Using the optimized reactor parameters, it was possible to synthesize the product, 6-cyano-4-oxohexanoic acid, in a concentration of 250 mM. The highest concentration of product was achieved in a repetitive batch reactor experiment. A fed-batch reactor experiment also delivered promising results, especially regarding the short reaction time needed to achieve a 200 mM concentration of product. Hence, the enzymatic Stetter reaction with a highly reactive acceptor substrate can be performed on a preparative scale, which should enable similar transformations with acrylate, methacrylate, and methyl vinyl ketone.
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