Catalytic Stability of S-1-(4-Hydroxyphenyl)-Ethanol Dehydrogenase from Aromatoleum aromaticum.

Derived from the denitrifying bacterium Aromatoleum aromaticum EbN1 (Azoarcus sp.), the enzyme S-1-(4-hydroxyphenyl)-ethanol dehydrogenase (S-HPED) belongs to the short-chain dehydrogenase/reductase family. Using research techniques like UV-Vis spectroscopy, dynamic light scattering, thermal-shift assay and HPLC, we investigated the catalytic and structural stability of S-HPED over a wide temperature range and within the pH range of 5.5 to 9.0 under storage and reaction conditions. The relationship between aggregation and inactivation of the enzyme in various pH environments was also examined and interpreted. At pH 9.0, where the enzyme exhibited no aggregation, we characterized thermally induced enzyme inactivation. Through isothermal and multitemperature analysis of inactivation data, we identified and confirmed the first-order inactivation mechanism under these pH conditions and determined the kinetic parameters of the inactivation process. Additionally, we report the positive impact of glucose as an enzyme stabilizer, which slows down the dynamics of S-HPED inactivation over a wide range of pH and temperature and limits enzyme aggregation. Besides characterizing the stability of S-HPED, the enzyme's catalytic activity and high stereospecificity for 10 prochiral carbonyl compounds were positively verified, thus expanding the spectrum of substrates reduced by S-HPED. Our research contributes to advancing knowledge about the biocatalytic potential of this catalyst.

Bacteria at Work - Experimental and Theoretical Studies Reveal the Catalytic Mechanism of Ectoine Synthase.

Ectoine synthase (EctC) catalyses the ultimate step of ectoine biosynthesis, a kosmotropic compound produced as compatible solute by many bacteria and some archaea or eukaryotes. EctC is an Fe2+-dependent homodimeric cytoplasmic protein. Using Mössbauer spectroscopy, molecular dynamics simulations and QM/MM calculations, we determined the most likely coordination number and geometry of the Fe2+ ion and proposed a mechanism of the EctC-catalysed reaction. Most notably, we show that apart from the three amino acids binding to the iron ion (Glu57, Tyr84 and His92), one water molecule and one hydroxide ion are required as additional ligands for the reaction to occur. They fill the first coordination sphere of the Fe2+-cofactor and act as critical proton donors and acceptors during the cyclization reaction.

Inactivation and aggregation of R-specific 1-(4-hydroxyphenyl)-ethanol dehydrogenase from Aromatoleum aromaticum.

R-specific 1-(4-hydroxyphenyl)-ethanol dehydrogenase (R-HPED) is a promising biotool for stereoselective synthesis of chiral aromatic alcohols. This work focused on the evaluation of its stability under storage and in-process conditions in the pH range from 5.5 to 8.5. The relationship between the dynamics of aggregation and activity loss under various pH conditions and in the presence of glucose, serving as a stabilizer, was analysed using spectrophotometric techniques and dynamic light scattering. pH 8.5 was indicated as a representative environment in which the enzyme, despite relatively low activity, shows high stability and the highest total product yield. Based on a series of inactivation experiments, the mechanism of thermal inactivation at pH 8.5 was modelled. The irreversible first-order mechanism of R-HPED inactivation in the temperature range of 47.5-60 °C was verified by isothermal and multi-temperature evaluation of data, confirming that in the alkaline pH 8.5, R-HPED aggregation is the secondary process occurring at already inactivated protein molecules. The rate constants were from 0.029 min-1 to 0.380 min-1 for a buffer solution but they decreased to 0.011 min-1 and 0.161 min-1, respectively, when 1.5 M glucose was added as a stabilizer. The activation energy was however about 200 kJ mol-1 in both cases.

The efficient Δ1-dehydrogenation of a wide spectrum of 3-ketosteroids in a broad pH range by 3-ketosteroid dehydrogenase from Sterolibacterium denitrificans.

Cholest-4-en-3-one Δ1-dehydrogenase (AcmB) from Sterolibacterium denitrificans, a key enzyme of the central degradation pathway of cholesterol, is a protein catalyzing Δ1-dehydrogenation of a wide range of 3-ketosteroids. In this study, we demonstrate the application of AcmB in the synthesis of 1-dehydro-3-ketosteroids and investigate the influence of reaction conditions on the catalytic performance of the enzyme. The recombinant AcmB expressed in E. coli BL21(DE3)Magic exhibits a broad pH optimum and pH stability in the range of 6.5 to 9.0. The activity-based pH optimum of AcmB reaction depends on the type of electron acceptor (2,6-dichloroindophenol - DCPIP, phenazine methosulfate - PMS or potassium hexacyanoferrate - K3[Fe(CN)6]) used in the biocatalytic process yielding the best kinetic properties for the reaction with a DCPIP/PMS mixture (kcat/Km = 1.4·105 s-1·M-1 at pH 9.0) followed by DCPIP (kcat/Km = 1.0·105 s-1·M-1 at pH = 6.5) and K3[Fe(CN)6] (kcat/Km = 0.5·102 s-1·M-1 at pH = 8.0). The unique feature of AcmB is its capability to convert both testosterone derivatives (C20-C22) as well as steroids substituted at C17 (C27-C30) such as cholest-4-en-3-one or (25R)-spirost-4-en-3-one (diosgenone). Apparent steady-state kinetic parameters were determined for both groups of AcmB substrates. In a batch reactor synthesis, the solubility of water-insoluble steroids was facilitated by the addition of a solubilizer, 2-hydroxypropyl-ß-cyclodextrin, and organic co-solvent, 2-methoxyethanol. Catalytic properties characterization of AcmB was tested in fed-batch reactor set-ups, using 0.81 µM of isolated enzyme, PMS and aerobic atmosphere resulting in >99% conversion of the C17-C20 3-ketosteroids within 2 h. Finally, the whole cell E. coli system with recombinant enzyme was demonstrated as an efficient biocatalyst in the synthesis of 1-dehydro-3-ketosteroids.

Hollow silica microspheres as robust immobilization carriers.

Hollow silica microspheres provide an ideal solid support for enzyme immobilization. We tested one of the newest development, namely MATSPHERES®, a silica openwork material as a carrier for the covalent immobilization of enzymes used to synthesize bioactive compounds. Two model enzymes - ethylbenzene dehydrogenase and EL070 lipase - were considered. They belong to two different enzyme classes and catalyse reactions taking place in various environments (aqueous and non-aqueous, aerobic and anaerobic). The enzymes were immobilized by covalent bonds (via divinyl sulfone and glutaraldehyde) on new silica material. Effectiveness of immobilization processes on the spheres grafted with amine groups and on the analogues without functionalization was determined for both enzymes. Microspheres were characterized morphologically and also their mechanical stability was examined during exposure to varying physical conditions. It was shown that MATSPHERES® due to their openwork structure and relative stability under batch and flow conditions can be a competitive SBA support for enzyme immobilization and production of bioactive compounds.