Current state and prospects of biotechnology in Central and Eastern European countries. Part II: new and preaccession EU countries(CRO, RO, B&H, SRB).

Innovation holds the potential for economic prosperity. Biotechnology (BT) has proved to be a viable vehicle for the development and utilization of technologies, which has brought not only advances to society, but also career opportunities to nation-states that have enabling conditions. In this review, we assess the current state of BT-related activities within selected new and preaccession EU countries (NPA) of CEE region namely Croatia, Romania, Bosnia and Herzegovina and Serbia, examining educational programs, research activity, enterprises, and the financing systems. The field of BT covers a broad area of activities, including medical, food and agriculture, aquaculture or marine, environmental, biofuels, bioinformatics, and many others. Under the European Commission (EC), member-states are to set their Research and Innovation Strategies for Smart Specialization (RIS3), to identify priorities or strengths in order to develop knowledge intensive economies. As the four countries highlighted in this review are in the early stages of implementing RIS3 or have not yet fully formulated, it presents an opportunity to learn from the successes and failures of those that have already received major structural funds from the EC. A critical point will be the ability of the public and private sectors' actors to align, in the implementation of RIS3 as new investment instruments emerge, and to concentrate efforts on a few select target goals, rather than distribute funding widely without respect to a long-term vision.

Stability Increase of Phenolic Acid Decarboxylase by a Combination of Protein and Solvent Engineering Unlocks Applications at Elevated Temperatures.

Enzymatic decarboxylation of biobased hydroxycinnamic acids gives access to phenolic styrenes for adhesive production. Phenolic acid decarboxylases are proficient enzymes that have been applied in aqueous systems, organic solvents, biphasic systems, and deep eutectic solvents, which makes stability a key feature. Stabilization of the enzyme would increase the total turnover number and thus reduce the energy consumption and waste accumulation associated with biocatalyst production. In this study, we used ancestral sequence reconstruction to generate thermostable decarboxylases. Investigation of a set of 16 ancestors resulted in the identification of a variant with an unfolding temperature of 78.1 °C and a half-life time of 45 h at 60 °C. Crystal structures were determined for three selected ancestors. Structural attributes were calculated to fit different regression models for predicting the thermal stability of variants that have not yet been experimentally explored. The models rely on hydrophobic clusters, salt bridges, hydrogen bonds, and surface properties and can identify more stable proteins out of a pool of candidates. Further stabilization was achieved by the application of mixtures of natural deep eutectic solvents and buffers. Our approach is a straightforward option for enhancing the industrial application of the decarboxylation process.

The role of Cys108 in Trigonopsis variabilis d-amino acid oxidase examined through chemical oxidation studies and point mutations C108S and C108D.

Oxidative modification of Trigonopsis variabilis D-amino acid oxidase in vivo is traceable as the conversion of Cys108 into a stable cysteine sulfinic acid, causing substantial loss of activity and thermostability of the enzyme. To simulate native and modified oxidase each as a microheterogeneity-resistant entity, we replaced Cys108 individually by a serine (C108S) and an aspartate (C108D), and characterized the purified variants with regard to their biochemical and kinetic properties, thermostability, and reactivity towards oxidation by hypochlorite. Tandem MS analysis of tryptic peptides derived from a hypochlorite-treated inactive preparation of recombinant wild-type oxidase showed that Cys108 was converted into cysteine sulfonic acid, mimicking the oxidative modification of native enzyme as isolated. Colorimetric titration of protein thiol groups revealed that in the presence of ammonium benzoate (0.12 mM), the two muteins were not oxidized at cysteines whereas in the wild-type enzyme, one thiol group was derivatized. Each site-directed replacement caused a conformational change in D-amino acid oxidase, detected with an assortment of probes, and resulted in a turnover number for the O2-dependent reaction with D-Met which in comparison with the corresponding wild-type value was decreased two- and threefold for C108S and C108D, respectively. Kinetic analysis of thermal denaturation at 50 degrees C was used to measure the relative contributions of partial unfolding and cofactor dissociation to the overall inactivation rate in each of the three enzymes. Unlike wild-type, C108S and C108D released the cofactor in a quasi-irreversible manner and were therefore not stabilized by external FAD against loss of activity. The results support a role of the anionic side chain of Cys108 in the fine-tuning of activity and stability of D-amino acid oxidase, explaining why C108S was a surprisingly poor mimic of the native enzyme.

Production of lactate and acetate by Lactobacillus coryniformis subsp. torquens DSM 20004(T) in comparison with Lactobacillus amylovorus DSM 20531(T).

Lactobacillus coryniformis subsp. torquens DSM20004(T) is a d-lactate producer, with a portion of the d-lactate higher than 99.9% of total lactic acid produced. Acetate was identified as the second end-product that appeared at the end of the exponential growth phase in MRS medium when glucose concentration dropped to 38.41mM (6.92g/L). The acetate production was prolonged to the stationary phase, while the concentration of d-lactate remained constant. Other end-products were not identified by HPLC method. The known metabolic pathways of glucose fermentation in lactic acid bacteria do not produce the particular combination of these two end-products, but besides lactate and acetate also formate, ethanol and CO2 are produced. For comparison, the production of lactate and acetate by a d-/l-lactate producer Lactobacillus amylovorus DSM 20531(T) was also investigated. This strain produced equimolar quantities of d- and l-lactate in the MRS medium. Acetate was produced only when initial concentration of glucose was 55.51mM (10g/L) and production started in the exponential phase when concentration of glucose dropped to 35.52mM (6.40g/L). Similar behavior was observed with the initial concentration of maltose of 29.21mM (10g/L). An unstructured mathematical model was established for the bioprocess simulation.

The effects of wild thyme (Thymus serpyllum L.) essential oil components against ochratoxin-producing Aspergilli.

The aim of this study was to determine the effects of the essential oil of Thymus serpyllum L. and of its components thymol and total phenols (total phenolic content, TPC) extracted from the plant on the growth and mycotoxin production of Aspergillus ochraceus, A. carbonarius, and A. niger. Minimal inhibitory concentration (MIC) determined for the essential oil and thymol, and selected concentration of the TPC extract inhibited fungal growth and ochratoxin A biosynthesis by more than 60 %, depending on the conditions and duration of incubation with the fungi. Essential oil showed the strongest inhibitory effect which may have been related to the synergistic or cumulative effects of its components.

Selective modification of surface-exposed thiol groups in Trigonopsis variabilis D-amino acid oxidase using poly(ethylene glycol) maleimide and its effect on activity and stability of the enzyme.

Covalent modification of purified Trigonopsis variabilis D-amino acid oxidase using maleimide-activated poly(ethylene glycol) 5000 yielded a stable bioconjugate in which three surface-exposed cysteine side chains were selectively derivatized. Compared with the native enzyme, the PEGylated variant displayed substantially (approximately 3.3-fold) slowed dissociation rate of FAD cofactor at 50 degrees C, and this caused a twofold thermostabilization of the enzyme activity. The stability under reaction conditions at 30 degrees C was also markedly enhanced in the PEG-oxidase conjugate. PEGylation did not affect steady-state kinetic parameters for oxidative deamination of D-methionine when 2,6-dichloroindophenol replaced dioxygen as the cosubstrate while it caused a ninefold decrease in substrate catalytic efficiency for the dioxygen-dependent reaction.

Thermal inactivation of D-amino acid oxidase from Trigonopsis variabilis occurs via three parallel paths of irreversible denaturation.

Trigonopsis variabilis D-amino acid oxidase (TvDAO) is a long-known flavoenzyme whose most important biocatalytic application is currently the industrial production of 7-amino-cephalosporanic acid (7-ACA) from cephalosporin C. Lacking mechanistic foundation, rational stabilization of TvDAO for improved process performance remains a problem. We report on results of thermal denaturation studies at 50 degrees C in which two purified TvDAO forms were compared: the native enzyme, and a site-specifically oxidized protein variant that had the side chain of cysteine108 converted into a sulfinic acid and lost 75% of original specific activity. Although inactivation time courses for both enzymes are fairly well described by simple single-exponential decays, the underlying denaturation mechanisms are shown by experiments and modeling to be complex. One main path leading to inactivation is FAD release, a process whose net rate is determined by the reverse association rate constant (k), which is 25-fold lower in the oxidized form of TvDAO. Cofactor dissociation is kinetically coupled to aggregation and can be blocked completely by the addition of free FAD. Aggregation is markedly attenuated in the less stable Cys108-SO(2)H-containing enzyme, suggesting that it is a step accompanying but not causing the inactivation. A second parallel path, characterized by a k-value of 0.26/h that is not dependent on protein concentration and identical for both enzymes, likely reflects thermal unfolding reactions. A third, however, slow process is the conversion of the native enzyme into the oxidized form (k < 0.03/h). The results fully explain the different stabilities of native and oxidized TvDAO and provide an inactivation mechanism-based tool for the stabilization of the soluble oxidase.

Single-site oxidation, cysteine 108 to cysteine sulfinic acid, in D-amino acid oxidase from Trigonopsis variabilis and its structural and functional consequences.

One of the primary sources of enzyme instability is protein oxidative modification triggering activity loss or denaturation. We show here that the side chain of Cys108 is the main site undergoing stress-induced oxidation in Trigonopsis variabilis d-amino acid oxidase, a flavoenzyme employed industrially for the conversion of cephalosporin C. High-resolution anion-exchange chromatography was used to separate the reduced and oxidized protein forms, which constitute, in a molar ratio of about 3:1, the active biocatalyst isolated from the yeast. Comparative analysis of their tryptic peptides by electrospray tandem mass spectrometry allowed unequivocal assignment of the modification as the oxidation of Cys108 into cysteine sulfinic acid. Cys108 is likely located on a surface-exposed protein region within the flavin adenine dinucleotide (FAD) binding domain, but remote from the active center. Its oxidized side chain was remarkably stable in solution, thus enabling the relative biochemical characterization of native and modified enzyme forms. The oxidation of Cys108 causes a global conformational response that affects the protein environment of the FAD cofactor. In comparison with the native enzyme, it results in a fourfold-decreased specific activity, reflecting a catalytic efficiency for reduction of dioxygen lowered by about the same factor, and a markedly decreased propensity to aggregate under conditions of thermal denaturation. These results open up unprecedented routes for stabilization of the oxidase and underscore the possible significance of protein chemical heterogeneity for biocatalyst function and stability.