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.
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).
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.
Statins are an important class of drugs used to lower blood cholesterol levels and are often used to combat cardiovascular disease. In view of the importance of safe and reliable supply and ...production of statins in modern medicine and the global need for sustainable processes, various biocatalytic strategies for their synthesis have been investigated. In this work, a novel biocatalytic route to a statin side chain precursor was investigated in a one-pot cascade reaction starting from the protected alcohol
N
-(3-hydroxypropyl)-2-phenylacetamide, which is oxidized to the corresponding aldehyde in the first reaction step, and then reacts with two equivalents of acetaldehyde to form the final product
N
-(2-((2
S
,4
S
,6
S
)-4,6-dihydroxytetrahydro-2
H
-pyran-2-yl)ethyl)-2-phenylacetamide (phenylacetamide-lactol). To study this complex reaction, an enzyme reaction engineering approach was used,
i.e.
the kinetics of all reactions occurring in the cascade (including side reactions) were determined. The obtained kinetic model together with the simulations gave an insight into the system and indicated the best reactor mode for the studied reaction, which was fed-batch with acetaldehyde feed to minimize its negative effect on the enzyme activity during the reaction. The mathematical model of the process was developed and used to simulate different scenarios and to find the reaction conditions (enzyme and coenzyme concentration, substrate feed concentration and flow rate) at which the highest yield of phenylacetamide-lactol (75%) can be obtained. In the end, our goal was to show that this novel cascade route is an interesting alternative for the synthesis of the statin side chain precursor and that is why we also calculated an initial estimate of the potential value addition.
Statins are an important class of drugs used to lower blood cholesterol levels and are often used to combat cardiovascular disease.
Aldolase and transaminase coexpressed in Escherichia coli cells and lyophilized (i.e., lyophilized whole-cell biocatalyst (LWCB)) were used as biocatalysts for the one-pot cascade synthesis of ...l-homoserine with substrate cycling. The kinetic analysis of enzymes within lyophilized cells was performed to evaluate the behavior of the system. The best result among the performed fed-batch reactor experiments achieved was 640.8 mM (76.3 g L–1) of l-homoserine with a volume productivity of 2.6 g L–1 h–1. This is comparable with the results of the same cascade synthesis using cell-free extracts (CFEs) and significantly better than the reports in the literature applying fermentation technology. The approach applied here can serve as guidance for the design of microbial cells with an optimal ratio of expressed enzymes that act as biocatalysts in the cascade, resulting in lower biocatalyst cost, no need for the addition of expensive coenzymes, and enhanced enzyme stability as compared with cell-free extracts.
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•Statin chain precursor was successfully synthesized by DERA/AlDH/NOX system•Enzymes kinetic and stability studies in reactions involved in the process are given•Sequential one-pot is ...preferable strategy to avoid side reaction of DERA substrates•A one-pot system contributed to enzyme stability
Statins are a group of drugs used for lowering cholesterol levels in the human bloodstream to prevent cardiovascular diseases and its synthesis is a challenge because of chemical and stereochemical requirements. In this work, a multi-enzyme one-pot sequential strategy was employed to obtain lactonized statin side-chain precursor and was compared to the two-pot process. The enzymatic production of this valuable product is rarely examined comparing to the traditional chemical methods. The examined cascade consisted of two consecutive reactions: lactol production by the 2-deoxyribose-5-phosphate aldolase (DERA)-catalysed double aldol addition of acetaldehyde and chloroacetaldehyde and its oxidation by the NAD-dependent aldehyde dehydrogenase (AlDH). NADH oxidase (NOX) was employed for the coenzyme regeneration. The insight in all reactions of the system was performed by enzyme kinetic and stability studies. Based on these data the mathematical models were developed and validated in a batch reactor. The oxidation was carried out using both purified (two-pot) and crude lactol (one-pot) obtained in the DERA-catalysed reaction. Better process metrics and greater stability of the AlDH was observed in one-pot system, thus demonstrating an additional benefit in favor of running multi-enzymatic (cascade) processes.
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•Multi-enzymatic reaction cascade evaluation for the synthesis of iminocyclitols.•A complex system composed of seven distinct reaction and two enzyme inactivation.•Kinetic modeling ...for elucidation of reaction mechanism.•Cross-inhibition and inactivation in peroxidase/aldolase reactions.•High improvement in reaction productivity through process intensification.
The feasibility of a peroxidase–aldolase cascade reaction for the synthesis of therapeutically-valuable iminocyclitols is discussed herein. A two-enzyme system consisting of chloroperoxidase (CPO) and D-fructose-6-phosphate aldolase (FSA) was evaluated for the synthesis of a D-fagomine precursor (preFagomine) from a N-Cbz-3-aminopropanol. An in-depth, systematic, step-by-step kinetic modeling of seven reactions and two inactivation decays was proposed to elucidate the reaction mechanism, prepare suitable stabilized biocatalysts, and find the optimal conditions for its application. The model described accurately the data and predicted the outcome at different experimental conditions. The inactivation of FSA caused by CPO was identified as the main bottleneck in the reaction. A two-step reaction approach and the use of immobilized enzymes on magnetic nanoparticle clusters and functionalized agarose carriers increased the stability of FSA, with an 1839-fold higher preFagomine formation per mol of enzyme in comparison to a one-pot reaction using soluble enzymes.
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•A multi-enzyme route for the amino lactol production was proposed.•Phenylacetamide amino-protected propanal was used as the starting substrate.•Mathematical model-based optimisation ...was performed.•DERA reaction and oxidation was studied in detail.•DERA reaction and the oxidation can be carried out in one pot process.
A multi-enzyme route for the production of an atorvastatin side-chain was proposed. The approach includes the consecutive double aldol addition of acetaldehyde to the phenylacetamide amino-protected propanal by 2-deoxyribose-5-phosphate aldolase (DERA), the oxidation by ketoreductase, the lipase-catalysed acylation and amino group deprotection by penicillin G-acylase. To explore the feasibility of the process, the DERA reaction was studied into detail. Based on the kinetic and stability studies, a mathematical model was developed and validated in a batch and fed-batch reactor. The highest productivity was obtained in repetitive batch reactor (229.1 g/(L day)). The highest final product concentration of 124 g/L was obtained in fed-batch reactor. The mathematical model-based optimisation provided insight into the possibilities for process metrics improvement. Further, the second step was explored. The results showed that the DERA reaction and the oxidation reaction can be carried out as the multi-enzyme one pot process in the sequential manner.
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
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