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Objectives Male hypogonadism is associated with higher risk of co-morbidity and premature mortality. It is, therefore, of utmost importance to identify young men who are at the highest risk of testosterone deficiency and who may benefit from preventive measures. In this context, infertile men constitute a high-risk group. The extent of testosterone replacement therapy (TRT) among infertile men, defined as men who have to undergo assisted reproduction for fatherhood, is currently unknown. Therefore, we evaluated the pattern of prescription of TRT in the years following child conception among men who have fathered children with the help of intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). Design By sourcing data from national population registries, hazard ratio (HR) for subsequent TRT was assessed for IVF and ICSI-treated men and compared to those who conceived spontaneously with age Cox regression analysis adjusted for age, educational level and previous intake of medicines for metabolic diseases. Results ICSI and IVF fathers had increased incidence of newly prescribed TRT compared to fathers conceiving spontaneously (ICSI: HR = 3.81, 95% CI = 3.09–4.69, P

Structure and hydrogen bonding interactions of bioactive oxazolidinones have been studied by means of NMR and vibrational spectroscopies and quantum chemical calculations. We have demonstrated that oxazolidinone derivatives form hydrogen bonds in solution and solid state. Conformational space search has revealed predominant conformations in solution. In low polarity solvents, such as chloroform, dimers are formed presumably by intermolecular hydrogen bonds between two oxazolidinone molecules forming the most stable complexes, which has also been found in the solid state by IR spectroscopy and crystallography. In solvents of higher polarities, like methanol and dimethylsulfoxide, intermolecular interactions with solvent molecules and in dimers are present. As expected, raising the temperature broke hydrogen bonds, which was reflected in down-field chemical shifts of corresponding resonances. On the other hand, raising the solution concentration considerably affected oxazolidinone resonances only in chloroform, corroborating the formation of oxazolidinone dimers. These results may help in better understanding oxazolidinone structure, properties and interactions when designing new bioactive compounds and pharmaceutical products.

Aim: To evaluate hepatitis B virus (HBV) and hepatitis C virus (HCV) among individuals with HIV/AIDS in Bulgaria diagnosed between 2010 and 2015. Materials & methods: A total of 1158 individuals were diagnosed with HIV/AIDS during the study period. Different transmission groups were tested with ELISA and real-time PCR for HBV and HCV markers. Results: Hepatitis B surface antigen and hepatitis C virus antiboby were found in 9.3 and 23.2% of the tested. HBV DNA and HCV RNA has been found in 47.4 and 69.6%. Hepatitis B and C co-infections were predominant in multiple risk behavior groups, including people who inject drugs, men who have sex with men, prisoners and Roma individuals. Conclusion: HIV prevalence in Bulgaria is low but the rates of hepatitis B and C co-infections among these patients fall within the upper range reported in Europe.

Blood pumps are becoming increasingly important for medical devices. They are used to assist and control the blood flow and blood pressure in the patient’s body. To accurately control blood pumps, information about important hydrodynamic parameters such as blood flow rate, pressure difference and viscosity is needed. These parameters are difficult to measure online. Therefore, an accurate estimation of these parameters is crucial for the effective operation of implantable blood pumps. In this study, in vitro tests with bovine blood were conducted to collect data about the non-linear dependency of blood flow rate, flow resistance (pressure difference) and whole blood viscosity on motor current and rotation speed of a prototype blood pump. Gaussian process regression models are then used to model the non-linear mappings from motor current and rotation speed to the hydrodynamic variables of interest. The performance of the estimation is evaluated for all three variables and shows very high accuracy. For blood flow rate – correlation coefficient ([Formula: see text] = 1, root mean squared error ([Formula: see text]) = 0.31 ml min−1, maximal error ([Formula: see text]) = 9.31 ml min−1; for pressure [Formula: see text] = 1, [Formula: see text] = 0.09 mmHg, [Formula: see text] = 8.34 mmHg; and for viscosity [Formula: see text] = 1,[Formula: see text] = 0.09 mPa.s, [Formula: see text] = 0.31 mPa⋅s. The current findings suggest that this method can be employed for highly accurate online estimation of essential hydrodynamic parameters for implantable blood pumps.

Animal blood is used in mock circulations or in forensic bloodstain pattern analysis. Blood viscosity is important in these settings as it determines the driving pressure through biomedical devices and the shape of the bloodstain. However, animal blood can never exactly mimic human blood due to erythrocyte properties differing among species. This results in the species‐specific shear thinning behavior of blood suspensions, and it is therefore not enough to adjust the hematocrit of an animal blood sample to mimic the behavior of human blood over the entire range of shear rates that are present in the body. In order to optimize experiments that require animal blood, we need models to adapt the blood samples. We here offer mathematical models derived for each species using a multi linear regression approach to describe the influence of shear rate, hematocrit, and temperature on blood viscosity. Results show that pig blood cannot be recommended for experiments at low flow conditions (, Blood viscosity is crucial in the development of biomedical blood‐contacting devices, or forensic re‐enactment studies, and frequently, animal blood is used. Since the shear thinning of blood and the hematocrit‐temperature dependency of blood viscosity are species‐specific, our mathematical models derived from experimental data provide factors on how the hematocrit must be adjusted in a blood sample at hand to gain a desired viscosity value for human, pig, sheep and horse blood within a predefined shear rate range. These simple modifications allow running experimental setups at desired flow resistances.

CO2 removal via membrane oxygenators during lung protective ventilation has become a reliable clinical technique. For further optimization of oxygenators, accurate prediction of the CO2 removal rate is necessary. It can either be determined by measuring the CO2 content in the exhaust gas of the oxygenator (sweep flow-based) or using blood gas analyzer data and a CO2 solubility model (blood-based). In this study, we determined the CO2 removal rate of a prototype oxygenator utilizing both methods in in vitro trials with bovine and in vivo trials with porcine blood. While the sweep flow-based method is reliably accurate, the blood-based method depends on the accuracy of the solubility model. In this work, we quantified performances of four different solubility models by calculating the deviation of the CO2 removal rates determined by both methods. Obtained data suggest that the simplest model (Loeppky) performs better than the more complex ones (May, Siggaard-Anderson, and Zierenberg). The models of May, Siggaard-Anderson, and Zierenberg show a significantly better performance for in vitro bovine blood data than for in vivo porcine blood data. Furthermore, the suitability of the Loeppky model parameters for bovine blood (in vitro) and porcine blood (in vivo) is evaluated.

CO2 removal via membrane oxygenators during lung protective ventilation has become a reliable clinical technique. For further optimization of oxygenators, accurate prediction of the CO2 removal rate is necessary. It can either be determined by measuring the CO2 content in the exhaust gas of the oxygenator (sweep flow-based) or using blood gas analyzer data and a CO2 solubility model (blood-based). In this study, we determined the CO2 removal rate of a prototype oxygenator utilizing both methods in in vitro trials with bovine and in vivo trials with porcine blood. While the sweep flow-based method is reliably accurate, the blood-based method depends on the accuracy of the solubility model. In this work, we quantified performances of four different solubility models by calculating the deviation of the CO2 removal rates determined by both methods. Obtained data suggest that the simplest model (Loeppky) performs better than the more complex ones (May, Siggaard-Anderson, and Zierenberg). The models of May, Siggaard-Anderson, and Zierenberg show a significantly better performance for in vitro bovine blood data than for in vivo porcine blood data. Furthermore, the suitability of the Loeppky model parameters for bovine blood (in vitro) and porcine blood (in vivo) is evaluated.

Halohydrin dehalogenases (HHDHs) belong to a distinct group of enzymes catalyzing the removal of a halide ion and a proton from a vicinal halohydrin with formation of an epoxide. Moreover, these enzymes can catalyze the epoxide ring- opening reactions with a range of anionic nucleophiles affording β-substituted alcohols as products. Based on sequence similarities, they are divided into 7 phylogenetic groups: A, B, C, D, E, F and G. Activity, enantioselectivity and enantiopreference are dependent on the type of enzyme and the substrate structure. Among all, the enzyme HheC from Agrobacterium radiobacter (group C) is the most studied because of its high enantioselectivity and wide nucleophile scope. However, the major shortcoming of HheC is its narrow substrate tolerance, due to the relatively small active site. To expand the catalytic relevance of HHDHs we focus our attention to an enzyme from different organism. There are two similar enzymes in group B, one found in Corynebacterium sp. N1074 (HheB) and the other in Mycobacterium sp. GP1 (HheB2).They share high sequence identity (95%) with only 4 amino acid substitutions HheB/HheB2: F36/I36, T120/A120, C124/Y124, H125/Q125. HheB exhibits higher enantioselectivity than does HheB2. Because of that HheB2 has been previously characterised as non-enantioselective enzyme, and neglected as biocatalyst. In this work HheB2 was investigated in the ring-opening reaction on a set of 21 structurally different aliphatic and aromatic epoxides using sodium azide as nucleophile. In order to gain more insight into the difference in enantioselectivity of HheB2 and HheB, mutant HheB2-T120A was also investigated in the ring opening reactions. The screening confirmed low to moderate enantioselectivity of HheB2 towards monosubstituted epoxides, however high activity and enantioselectivity in conversion of 2, 2- disubstituted (E-values up to >200). Similar to HheC, the large enhancement of enantioselectivity is obtained when a second substituent (methyl or ethyl) is present at the chiral centre. T120A mutation has been shown to be a key mutation for increasing enantioselectivity in aliphatic epoxides, which is not the case for aromatic epoxides. The results further extend the repertoire of enantioselective HHDHs and their application in the kinetic resolution of epoxides.

Halohydrin dehalogenases (HHDHs) catalyse enantioselective formation and conversion of epoxides. They can be applied in the kinetic resolution of racemic epoxides by utilising a range of anionic nucleophiles such as azide, cyanide, cyanate or nitrite.[1, 2] The ring- opening products and remaining epoxides are versatile and important synthetic intermediates for fine chemicals. In this work HHDH was employed for the transformation of epoxides bearing fluoroaryl groups (Figure 1). Fluoroaromatic compounds are widely used as starting materials for chemical syntheses, especially pharmaceuticals and agrochemicals. Introduction of fluorine atoms into the molecule usually increases target effectiveness, biological half-life, bioabsorption etc. The growing interest in fluorinated organics makes the development of synthetic procedures leading to such compounds desirable. Kinetic resolution of a series of fluorinated styrene oxide derivatives was studied using the enzyme from Agrobacterium radiobacter AD1 (HheC) and a variant W249P.[3] A mutant HheC-W249P catalysed nucleophilic ring-opening with azide and cyanide ions with excellent enantioselectivity (Evalues up to >200) and higher activity compared to the wild- type, 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-CF3-styrene oxide were performed on preparative scale, affording (R)-2-azido-1-(4-trifluoromethylphenyl)- ethanol in 38% yield and 97% ee, and (S)-3- hydroxy-3-(4-trifluoromethylphenyl)-propionitrile in 30% yield and 98% ee.

Epichlorohydrin (ECH) is a valuable product that is commonly used as an intermediate in the preparation of synthetic rubbers, as a starting material in the production of insecticides, pharmaceuticals, etc. [1] This prominent product can be obtained through biocatalysis. Halohydrin dehalogenases (HHDHs) are adaptable and important biocatalysts that are mostly used for the industrial synthesis of enantiopure ECH. [2] The reasons why the biocatalytic approach takes precedence over the traditional one are high enantioselectivity, low production cost, extensive enzyme sources and environmentally friendly conditions of this synthesis. [3] The operational stability of an enzyme generally relates to the retention of activity when an enzyme is in use. On an industrial level, the long-term operational stability of biocatalysts is of crucial importance for deciding whether a process can be viable as opposed to the traditional approach. [4] Operational stability is necessary to determine at which process conditions enzyme can work properly. In this work the kinetic model of HheB2-T120A- catalysed synthesis of (R)-ECH from 1, 3-dichloro- 2-propanol was developed. Model validation was performed in batch reactor experiments. It was found that a substrate (1, 3-dichloro-2-propanol) deactivates the enzyme and the rate of deactivation is dependent upon its concentration. ACKNOWLEDGEMENTS: This work was supported by CATPHARMA (KK.01.1.1.04.0013). REFERENCES [1] Y. Bespalko et al., International Journal of Chemical Kinetics 2021, 53(3), 356-368. [2] Z. Findrik Blažević et al., Advanced Synthesis and Catalysis 2021, 363(2), 388-410. [3] X. J. Zhang et al., Bioresource Technology 2018, 263, 483-490. [4] G. A. Drago, T. D. Gibson, Enzyme Stability and Stabilisation: Apllications and Case Studies, Engineering and Manufacturing for Biotechnology 2002, 4, 361-376.

Halohydrin dehalogenases (HHDHs) are valuable and versatile biocatalysts. They can easily be obtained in large quantities by expression in recombinant E. coli cells1. HHDH enzymes are divided into subtypes A - G and show promising properties in stereoselective biocatalysis but are still insufficiently investigated1. HHDHs are industrially applied for the synthesis of chiral epichlorohydrin (ECH). ECH is widely used as a starting material in the production of insecticides, surfactants, adhesives, pharmaceuticals, etc1. Main disadvantages of the traditional synthesis of chiral ECH is use of environmentally unfriendly, metal catalyst (cobalt-salen complexes) and relatively low reaction yield as it is limited at 50 %. Biocatalytic methods for chiral ECH preparation are getting much attention mostly because of environmentally friendly conditions, extensive enzyme sources, high enantioselectivity and low production cost2. Several studies involving the synthesis of chiral ECH by HHDH mutants have been published3-5. We have developed kinetic model of HheB2-T120A-catalysed synthesis of (R)-ECH from 1, 3-dichloro-2-propanol. Kinetic characterisation offers better understanding of reaction and is an essential step in the development of mathematical model which ultimately helps with the optimization of the reaction conditions.

Halohydrin dehalogenases (HHDHs) are industrially relevant enzymes that show exceptional selectivity, activity, and versatility regarding accepting nucleophiles in epoxide ring-opening reactions.[1] HHDHs can be employed in the synthesis of important and versatile pharmaceutical building block (S)-2-(4- fluorophenyl)-3-hydroxypropanenitrile through kinetic resolution of rac-2-(4- fluorophenyl)oxirane.[1, 2] The major limitations of this reaction are poor substrate solubility and its susceptibility to hydrolytic decomposition. Introduction of organic phase in the aqueous reaction media can have a positive effect both on the solubility and hydrolytic stability of epoxide, leading to higher volume productivity. However, possible side effects of the organic phase introduction are enzyme activity and operational stability decrease, hence they need to be experimentally investigated and quantified. In this work, we determined the activity of wild-type HheC from Agrobacterium radiobacter in the presence of a wide solvent/buffer ratio range. As a result, correlation between solvent logP values and concentration of half-inactivation (C50) was observed, whereby C50 represents concentration of solvent that irreversibly reduces enzyme initial activity by half.[3]In addition to affecting the initial enzyme activity, the organic solvent may also affect the enzyme activity during continuous use. Hence, enzyme stability in selected solvents and at different ratios during time was monitored. Besides HheC, we examined the co-solvent activity and stability of thermostable variant ISM-4 obtained with iterative saturation mutagenesis, [4] as resistance to high temperatures is often accompanied with enhanced co-solvent activity and stability.[5] Finally, based on the experiments performed on a model system, we investigated the co-solvent operational stability of ISM-4 during (S)-2-(4-fluorophenyl)-3-hydroxypropanenitrile synthesis. We continually monitored the enzyme activity during reaction and estimated the values of enzyme deactivation rate constant (kd, min-1). References [1] Z. Findrik Blažević ; N. Milčić ; M. Sudar ; M. Majerić Elenkov ; Adv. Synth. Catal. 2021, 363 (2), 388-410. [2] I. Dokli ; N. Milčić ; P. Marin ; M. Svetec Miklenić ; M. Sudar ; L. Tang ; Z. Findrik Blažević ; M. Majerić Elenkov ; Catal. Commun. 2021, 152, 106285. [3] V. Stepankova ; S. Bidmanova ; T. Koudelkova ; Z. Prokop ; R. Chaloupkova ; J. Damborsky ; Adv. Synth. Catal. 2013, 3 (12), 2823-2836. [4] Z. Wu ; W. Deng ; Y. Tong ; Q. Liao ; D. Xin ; H. Yu ; J. Feng ; L. Tang ; App. Microbiol. Biotechnol. 2017, 101, 3201–3211. [5] H. Arabnejad ; M. Dal Lago ; P. A. Jekel ; R. J. Floor ; A-M. W. H. Thunnissen ; A. C. T. van Scheltinga ; H. J. Wijma ; D. B. Janssen ; Protein. Eng. Des. Sel. 2017, 30 (3), 173-187.

Halohydrin dehalogenases (HHDHs) are versatile biocatalysts that facilitate the conversion between halohydrins and epoxides in both directions. Their ability to catalyze enantioselective epoxide ring-opening reactions with different nucleophiles (azide, cyanide, cyanate, thiocyanate etc.) can be used in synthesis of optically active epoxides, β- substituted alcohols and heterocyclic compounds. Further transformation (hydrolysis, reduction, intermolecular click reactions) gives rise to valuable building blocks (e. g. amino alcohols, aziridines, triazoles, oxazolidinones) in synthesis of pharmaceutical and natural compounds. [1] Propargylic epoxides and alcohols, owing to the presence of a triple bond, undergo various intermolecular and intramolecular reactions.[2, 3] Unfortunately, there are few described methods for enantioselective synthesis of these compounds, and those available require expensive or custom-made catalysts. Using propargylic epoxides as substrates of HHDHs, enantiomerically pure starting compounds for triple bond and/or nucleophile transformations, such as intramolecular click reactions, can be obtained. Therefore, the synthesis of substituted propargylic epoxides and subsequent enantioselective ring opening by halohydrin dehalogenases (HheA-N178A and HheC) was described (Figure 1). Phenyl-, tert-butyl- and cyclopentyl- substituted epoxides were synthesized from the corresponding terminal acetylenes by introduction and epoxidation of a double bond. Also, p- and m- tolyl derivatives were synthesized in a similar reaction sequence, starting from the corresponding iodotoluene and trimethylsilylacetylene. Kinetic resolution reactions in the presence of sodium azide were catalyzed by two HHDHs with opposite stereopreference. With both enzymes reactions yielded enantiomerically pure secondary azido alcohols (e.e. > 99%, E > 200). While HheA- N178A gave mostly (S)-β-azido alcohol (β : α ratio between 90:10 and 54:46), HheC yielded almost exclusively (R)-β-azido alcohol (up to to 99:1).

Synthesis of fluorinated aromatic compounds is of increasing interest in the pharmaceutical and agrochemicals industry. More environmentally friendly biocatalytic modification of simpler molecules that contain a C-F bond is an attractive alternative to the often expensive and toxic metal-catalyzed fluorination of molecules, especially when optically active substances are obtained through the employment of enantioselective enzymes. Halohydrin dehalogenases (HHDHs) are industrially relevant enantioselective enzymes that catalyse the reversible conversion of vicinal halohydrins to epoxides. HHDH enzymes have been widely used in cascade reaction systems, whereby the most famous example is the joint action with nitrilase for the commercialized multi-ton-scale synthesis of ethyl (R)-4-cyano-3- hydroxybutyrate, chiral key precursor for statin side chains. In this work, reactions of fluorine- substituted aryl oxyranes with sodium azide catalyzed by C type HHDH (HheC) were studied. Obtained enantiopure fluoroaromatic vicinal azidoalcohols and epoxides are valuable chiral building blocks with a wide range of applications in the design and development of new drugs. The application of mathematical modelling approach can be of great importance since it can greatly accelerate the development of a new process where time is a key parameter. Therefore, in this work, kinetic characterization of HheC enzymes in the presence of individual epoxides was performed to find the system with the maximum potential for high yield enantioselective conversion. Based on kinetic constants, a system for further characterization and development of a complete kinetic model was selected. Due to the low solubility of epoxides and the influence of chemical hydrolysis in aqueous medium, the application of aqueous-organic systems has been investigated. Their applicability was examined by monitoring the activity of enzyme in the presence of different concentrations of selected organic solvents.

Background. In Bulgaria the second most predominant HIV-1 strain after subtype B is CRF01_AE. Material and Methods. 270 HIV-1 polymerase (pol) CRF01_AE sequences collected between 1995–2019 were analyzed with the phylogenetic approach. To identify transmission clusters, we used two different genetic distance thresholds (d), i. e. 1.5% and 0.5%. Results. Genetic distance d=1.5% defined more distant infections in a huge cluster of 154 sequences composed mostly of people, who inject drugs (PWID), whereas when (d) was reduced to 0.5%, we determined more recent transmissions and the large cluster disintegrated into smaller ones. Different drug resistance mutations (DRM) were detected in patient’s HIV-1 pol sequences and were most common in male heterosexual (HET) single sequences. Conclusions. Our data showed repeated introduction of CRF01_AE in Bulgaria and rapid spread of the infection among PWID groups. Molecular monitoring of the epidemic among PWID communities could help reduce the spread of HIV-1 infection.

Halohydrin dehalogenases catalyze reversible conversion of epoxides to β-haloalcohols in enantioselective fashion with an extensive scope of unnatural nucleophiles.[1] Selectivity and diversity of HHDH enzymes are promising yet insufficient properties to rely solely on them for industrial application purposes. Aqueous solution as physiological medium is the most convenient concerning enzyme natural properties, but poor solubility and hydrolytic instability of hydrophobic substrates make these reactions limited and unfavourable.[2] Thus, biotransformation scale-up often requires modification of the reaction medium by introduction of organic solvents (OSs), which can minimize these problems but also decrease enzyme activity and stability. As a HHDH representative, HheC from Agrobacterium radiobacter AD1 was selected and enzyme activity was determined in the presence of a wide OSs range. Correlation between OSs properties (logP, pKa) and concentration of half-inactivation (C50) was observed. To gain insight into the enzyme behavior in an alternative medium over an extended period of time, activity during incubation with selected OSs was monitored. Besides HheC, we examined the co-solvent stability during incubation of thermostable variant ISM-4. [3] Results obtained reveal that this enzyme is a robust and powerful ally for synthesis in more harsh conditions and a promising candidate for further experimentation in the direction of industrial implementations.

Stability of enzymes has a significant impact on their use in an alternative reaction media. Presence of organic solvents or exposure to high temperatures leads to denaturation of enzymes since they have evolved to work in the cellular environment. Lyophilization is as an attractive solution for long-term storage of enzymes at ambient temperature. Also, it can be used as a method of enzyme stabilization for their application in non-aqueous media. Halohydrin dehalogeneses are enzymes that catalyse the formation and conversion of epoxides. Reactions are catalyzed in regioselective and enantioselective manner, making the enzymes attractive for biocatalysis. Enzyme from Agrobacterium radiobacter AD1 (HheC) is usually expressed in E.coli, isolated in TEMG buffer (50 mM Tris-SO4, 2 mM EDTA, 10 % glycerol and 1 mM mercaptoethanol), and usually is stored as a cell- free extract at -70 °C for at least three months without significant loss of activity. In this work, the stability of HheC is first evaluated in different buffers (TEMG, TEM and Tris-SO4) at different temperatures. With the aim of obtaining the optimal lyophilized formulation, different additives were tested for HheC stabilization. Lyophilized HheC was incubated at 50 °C to intensify the differences induced by additives. Enzyme activity is determined by following the absorbance at 310 nm.

In comparison with traditional organic synthesis methods, cascade multi-step biocatalytic reactions have many advantages. A few of them are sustainability, simple and cheaper reactor set- up, mild reaction conditions and lower environmental impact. [1, 2] Advancements in biocatalytic processes are achieved through reaction engineering. Combined kinetic and reactor modelling can significantly contribute to the choice of reactor mode, design and optimal operating conditions. [3, 4] Using modelling and simulations helps us study the effects of different variables on the process, which increase our knowledge and understanding of the system. [5] In this work, a two-step cascade reaction was studied (Figure 1). Both steps are catalysed by halohydrin dehalogenase (HheB2), mutant T120A. The first reaction was a ring closure of a substrate 1, 3-dichloro-2-propanol (DCP) to (R)- epichlorohydrin (ECH). The second reaction was ring-opening of (R)-ECH to (R)-4-chloro-3- hydroxybutyronitrile (HBN) with cyanide ion (CN-) as a nucleophile. (R)-HBN is a precursor to many valuable chemicals, such as L- carnitine, which plays a vital role in human metabolism. [6, 7] By using mathematical modelling and simulations, optimal reaction conditions were found for this system. Kinetics research has shown that the best results are achieved when both reaction steps are carried out simultaneously in one pot reaching the maximum product yield, and shifting the equilibrium to the right side. High product concentrations cannot be achieved in a batch reactor because of the substrate inhibition. However, in the fed-batch reactor concentrations of the inhibitors can be controlled which makes it an ideal solution. Funding: European Structural and Investment Funds, KK.01.1.1.04. References [1] M. Sudar, Z. Findrik Blažević, Enzyme Cascade Kinetic Modelling, Enzyme Cascade Design and Modelling, editors: S. Kara, F. Rudroff, Springer International Publishing, 2021, 91-108. [2] M. Česnik et al. Chemical Engineering Research and Design, 2019, 150, 140-152. [3] Đ. Vasić Rački et al. Applied Microbiology and Biotechnology, 2011, 91, 845-856. [4] Đ. Vasić Rački et al. Chemical and Biochemical Engineering Quarterly, 2003, 17(1), 3-14. [5] M. Sudar et al. Journal of Biotechnology, 2018, 268, 71-80. [6] T. Suzuki et al. Bioorganic & Medicinal Chemistry Letters, 1996, 6, 2581-2584. [7] Z. Findrik Blažević et al. Advanced Synthesis & Catalysis, 2021, 363(2), 388-410.

Halohydrin dehalogenases (HHDHs) are important biocatalysts that facilitate the reversible conversion between halohydrins and epoxides. Their ability to catalyze enantioselective epoxide ring- opening reactions with different nucleophiles (azide, cyanide, cyanate, thiocyanate etc.) can be used in synthesis of optically active epoxides, β- substituted alcohols and heterocyclic compounds. Further transformation (hydrolysis, reduction, intermolecular click reactions) gives rise to valuable building blocks (e. g. amino alcohols, aziridines, triazoles, oxazolidinones) in synthesis of pharmaceutical and natural compounds. [1] Propargylic epoxides and alcohols, owing to the presence of a triple bond, undergo various intermolecular and intramolecular reactions.[2, 3] Unfortunately, there are few described methods for enantioselective synthesis of these compounds, and those available require expensive or custom-made catalysts. However, halohydrin dehalogenases can be used to obtain enantiomerically pure starting compounds from racemic propargylic epoxides for triple bond and/or nucleophile transformations. Therefore, the synthesis of mono- and disubstituted propargylic epoxides and subsequent enantioselective ring opening by halohydrin dehalogenases (HheC and HheA-N178A) was described (Figure 1). Internal propargylic epoxides were synthesized from the corresponding terminal acetylenes by introduction and epoxidation of a double bond (cyclopentyl, tert-butyl and phenyl). Also, p- and m-tolyl derivatives were synthesized in a similar reaction sequence, starting from the corresponding iodotoluene and trimethylsilylacetylene. 2, 2-Disupstituted epoxides were prepared from corresponding methyl ketones. Biocatalytic kinetic resolution reactions in the presence of sodium azide were catalyzed by two HHDHs with opposite stereopreference (HHeC and HheA-N178A). With both enzymes reactions yielded enantiomerically pure secondary azido alcohols (ee > 99%, E > 200). While HheC yielded almost exclusively (R)-β-azido alcohol (up to 99:1), HheA-N178A gave mostly (S)-β-azido alcohol (β : α ratio between 90:10 and 54:46).

Halohydrin dehalogenases (HHDHs) catalyse enantioselective formation and conversion of epoxides. Their exceptional selectivity, regioselectivity, and ability to employ different nucleophiles make the enzymes attractive for biocatalysis.[1] The enzyme from Agrobacterium radiobacter AD1 (HheC) is usually expressed in E.coli, isolated in TEMG buffer (50 mM Tris-SO4, 10 mM EDTA, 10 % glycerol, and 1 mM mercaptoethanol), and usually is stored as a cell- free extract at -70 °C for at least three months without significant loss of activity.[2] Both storage (before use) and operational stability (during use) are highly relevant for biotechnological applications.[3] Lyophilization is an attractive approach for the longterm storage of enzymes at ambient temperature. It also facilitates the use of enzymes in non-aqueous media.[4] In this work, the storage stability of HheC isolated in TEMG and TEM buffer is evaluated at room temperature. With a focus on establishing the optimal lyophilized formulation, different additives were tested for enzyme stabilization during lyophilization. Optimal conditions for the lyophilization of several HHDHs have been investigated. Lyophilized HheC was incubated at 50 °C to intensify the differences induced by additives. Enzyme activity is determined by following the absorbance at 310 nm (Figure 1).

(R)-2-Azido-2-[4-(trifluoromethyl)phenyl]ethanol is a valuable and diverse building block as its structural analogues are used for the synthesis of molecules displaying antiplasmodial, antibacterial, antitubercular and beta-blocker activities1-3. Trifluoromethyl moiety is predominantly employed structural motif in design of pharmaceuticals4, but direct fluorination of the target molecule is expensive, conducted under harsh conditions with toxic reagents and represents significant synthetic challenge5. Halohydrin dehalogenase (HHDH) can be employed in modification of existing fluorinated synthons in enantiopure fashion6. We developed a kinetic mathematical model of HHDH-catalysed and azide- mediated rac-2-[4-(trifluoromethyl)phenyl]oxirane kinetic resolution to obtain (R)-2-Azido-2-[4- (trifluoromethyl)phenyl]ethanol. Mathematical modelling enables obtaining the maximal productivity over a minimal reaction time through identification of reaction bottlenecks and selection of optimal operating conditions and enzyme microenvironment7. Since mathematical modelling is the foundation for scale-up of the biocatalytic process, through process simulations we have presented different synthesis outcomes dependent on the selected reactor type and reaction conditions. Funding: Croatian Science Foundation, IP-2018-01-4493 1 Eur. J. Med. Chem. 2017, 138, 1089-1105 2 J. Nat. Prod. 2020, 83 (1), 26-35 3 J. Org. Chem. 2008, 73 (16), 6433-6436 4 Organofluorine Chemistry, Elsevier Inc., 2020 5 Drug Discover Today 2018, 23 (8), 1458-1462 6 Catal. Commun 2021, 152, 106285 7 Adv. Synth. Catal. 2021, 363 (2), 388-410

Halohydrin dehalogenases (HHDHs) belong to a distinct group of enzymes catalyzing the removal of a halide ion and a proton from a vicinal halohydrin with formation of an epoxide. Moreover, these enzymes can catalyze the epoxide ring-opening reactions with a range of anionic nucleophiles affording β-substituted alcohols as products. Based on sequence similarities, they are divided into 7 phylogenetic groups: A, B, C, D, E, F and G. Activity, enantioselectivity and enantiopreference are dependent on the type of enzyme and the substrate structure.[3] Among all, the enzyme HheC from Agrobacterium radiobacter (group C) is the most studied because of its high enantioselectivity and wide nucleophile scope. However, the major shortcoming of HheC is its narrow substrate tolerance, due to the relatively small active site. To expand the catalytic relevance of HHDHs we focus our attention to an enzyme from different organism. The HheB2 from Mycobacterium sp. GP1 (group B) has been previously characterised as non-enantioselective enzyme, and neglected as biocatalyst. In this work HheB2 was investigated in the ring-opening reaction on a set of 20 structurally different aliphatic and aromatic epoxides using sodium azide as nucleophile. The screening confirmed low to moderate enantioselectivity of HheB2 towards monosubstituted epoxides, however high activity and enantioselectivity in conversion of 2, 2-disubstituted (E-values up to >200). Similar to HheC, the large enhancement of enantioselectivity is obtained when a second substituent (methyl or ethyl) is present at the chiral centre. The results further extend the repertoire of enantioselective HHDHs and their application in the kinetic resolution of epoxides.

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Blood pumps have found applications in heart support devices, oxygenators, and dialysis systems, among others. Often, there is no room for sensors, or the sensors are simply unreliable when long-term operation is required. However, control systems rely on those hard-to-measure parameters, such as blood flow rate and pressure difference, thus their estimation takes a central role in the development process of such medical devices. The viscosity of the blood not only influences the estimation of those parameters but is often a parameter that is of great interest to both doctors and engineers. In this work, estimation methods for blood flow rate, pressure difference, and viscosity are presented using Gaussian process regression models. Different water&ndash, glycerol mixtures were used to model blood. Data was collected from a custom-built blood pump, designed for intracorporeal oxygenators in an in vitro test circuit. The estimation was performed from motor current and motor speed measurements and its accuracy was measured for: blood flow rate r2 = 0.98, root mean squared error (RMSE) = 46 mL.min&minus, 1, pressure difference r2 = 0.98, RMSE = 8.7 mmHg, and viscosity r2 = 0.98, RMSE = 0. 0.049 mPa.s. The results suggest that the presented methods can be used to accurately predict blood flow rate, pressure, and viscosity online.

Computational fluid dynamics (CFD) provides a flexible tool for investigation of separation processes within membrane hollow fiber modules. By enabling a three-dimensional and time dependent description of the corresponding transport phenomena, very detailed information about mass transfer or geometrical influences can be provided. The high level of detail comes with high computational costs, especially since species transport simulations must discretize and resolve steep gradients in the concentration polarization layer at the membrane. In contrast, flow simulations are not required to resolve these gradients. Hence, there is a large gap in the scale and complexity of computationally feasible geometries when comparing flow and species transport simulations. A method, which tries to cover the mentioned gap, is presented in the present article. It allows upscaling of the findings of species transport simulations, conducted for reduced geometries, on the geometrical scales of flow simulations. Consequently, total transmembrane transport of complete modules can be numerically predicted. The upscaling method does not require any empirical correlation to incorporate geometrical characteristics but solely depends on results acquired by CFD flow simulations. In the scope of this research, the proposed method is explained, conducted, and validated. This is done by the example of CO2 removal in a prototype hollow fiber membrane oxygenator.