Cascade enzymatic synthesis of a statin side chain precursor - the role of reaction engineering in process optimization.

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-((2S,4S,6S)-4,6-dihydroxytetrahydro-2H-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.

Impact of organic solvents on the catalytic performance of halohydrin dehalogenase.

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.

Inhibitory Effect of DMSO on Halohydrin Dehalogenase: Experimental and Computational Insights into the Influence of an Organic Co-solvent on the Structural and Catalytic Properties of a Biocatalyst.

Invited for the cover of this issue are Zlatko Brkljaca, Maja Majeric Elenkov and co-workers at the Ruder Boskovic 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.

Inhibitory Effect of DMSO on Halohydrin Dehalogenase: Experimental and Computational Insights into the Influence of an Organic Co-solvent on the Structural and Catalytic Properties of a Biocatalyst.

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.

Thermostability Engineering of a Class II Pyruvate Aldolase from Escherichia coli by in Vivo Folding Interference.

The use of enzymes in industrial processes is often limited by the unavailability of biocatalysts with prolonged stability. Thermostable enzymes allow increased process temperature and thus higher substrate and product solubility, reuse of expensive biocatalysts, resistance against organic solvents, and better "evolvability" of enzymes. In this work, we have used an activity-independent method for the selection of thermostable variants of any protein in Thermus thermophilus through folding interference at high temperature of a thermostable antibiotic reporter protein at the C-terminus of a fusion protein. To generate a monomeric folding reporter, we have increased the thermostability of the moderately thermostable Hph5 variant of the hygromycin B phosphotransferase from Escherichia coli to meet the method requirements. The final Hph17 variant showed 1.5 °C higher melting temperature (T m) and 3-fold longer half-life at 65 °C compared to parental Hph5, with no changes in the steady-state kinetic parameters. Additionally, we demonstrate the validity of the reporter by stabilizing the 2-keto-3-deoxy-l-rhamnonate aldolase from E. coli (YfaU). The most thermostable multiple-mutated variants thus obtained, YfaU99 and YfaU103, showed increases of 2 and 2.9 °C in T m compared to the wild-type enzyme but severely lower retro-aldol activities (150- and 120-fold, respectively). After segregation of the mutations, the most thermostable single variant, Q107R, showed a T m 8.9 °C higher, a 16-fold improvement in half-life at 60 °C and higher operational stability than the wild-type, without substantial modification of the kinetic parameters.

A cascade reaction for the synthesis of d-fagomine precursor revisited: Kinetic insight and understanding of the system.

The synthesis of aldol adduct (3S,4R)-6-[(benzyloxycarbonyl)amino]-5,6-dideoxyhex-2-ulose, a precursor of the interesting dietary supplement, iminosugar d-fagomine, was studied in a cascade reaction with three enzymes starting from Cbz-N-3-aminopropanol. This system was studied previously using a statistical optimization method which enabled a 79 % yield of the aldol adduct with a 10 % yield of the undesired amino acid by-product. Here, a kinetic model of the cascade, including enzyme operational stability decay rate and the undesired overoxidation of the intermediate product, was developed. The validated model was instrumental in the optimization of the cascade reaction in the batch reactor. Simulations were carried out to determine the variables with the most significant impact on substrate conversion and product yield. As a result, process conditions were found that provided the aldol adduct in 92 % yield with only 0.7 % yield of the amino acid in a one-pot one-step reaction. Additionally, compared to previous work, this improved process outcome was achieved at lower concentrations of two enzymes used in the reaction. With this study the advantages are demonstrated of a modelling approach in developing complex biocatalytical processes. Mathematical models enable better understanding of the interactions of variables in the investigated system, reduce cost, experimental efforts in the lab and time necessary to obtain results since the simulations are carried out in silico.