The effect of inorganic processing during malting on the enzymatic activity of wheat malt

Определяющей целью солодоращения является повышение ферментативной активности зерна. Нами предлагается способ интенсификации солодоращения пшеницы посредством применения неорганического стимулятора роста «Энерген». В исследовании использовали пшеницу Алтайской селекции трех сортов: «Алтайская 100», «Дуэт» и «Алейская». Предложенный неорганический препарат вносили при замачивании в последнюю замочную воду в количестве 0,6 г/дм и выдерживали с ним в контакте пшеницу в течение 6 ч. За данный период в ферментативной системе обработанного зерна произошли более выраженные изменения в сравнении с контрольным вариантом (необработанным зерном). К концу замачивания уровень активности ферментов опытных образцов стал выше уровня аналогичных активностей ферментов контрольных вариантов на 11,8 и 9,9 % соответственно для амилолитической и протеолитической активностей. Последующее проращивание зерна повысило ферментативную активность пшеничного солода. По окончании 7 сут данной стадии прирост амилолитической активности над активностями необработанного зерна для разных сортов составил от 31,5 до 59,0 %, протеолитической - от 97,8 до 125,4 %. При этом отмечено маловыраженное отличие показателей амилолитической и протеолитической активностей проращиваемого обработанного пшеничного солода шестых и седьмых суток ращения, что позволяет сократить продолжительность данной стадии и всего производства солода на одни сутки. Готовый пшеничный солод отличался высокой ферментативной активностью (в диапазоне для трех сортов): амилолитическая - 344,9-360,8 ед./г, протеолитическая - 324,9-257,8 ед./г, более низкой в сравнении с контрольным вариантом продолжительностью осахаривания - от 18 до 20 мин. Кроме этого, предложенный способ солодоращения позволяет использовать пшеницу с высоким содержанием белка, как, например, сорт «Алейская» с массовой долей белка 14,6 %, поскольку в процессе проращивания под стимулирующим действием неорганического препарата «Энерген» процесс протеолиза протекает более интенсивно, и в конечном солоде содержание белка снижается до 10,4 %. The defining goal of malting is to increase the enzymatic activity of grain. We propose a method for intensifying the malting of wheat through the use of an inorganic growth stimulator «Energen». The study used wheat of the Altai selection of three varieties: «Altai 100», «Duet» and «Aleyskaya». The proposed inorganic preparation was introduced during soaking into the last soak water in an amount of 0.6 g/dm and wheat was kept in contact with it for 6 hours. During this period, more pronounced changes occurred in the enzymatic system of the processed grain in comparison with the control variant (unprocessed grain). By the end of soaking, the enzyme level of the experimental samples is 11.8 and 9.9 % higher than the level of similar enzymes of the control variants, respectively, for amylolytic and proteolytic activities. The subsequent germination of grain increased the enzymatic activity of wheat malt. At the end of seven days of this stage, the increase in amylolytic activity over the activities of unprocessed grain for different varieties ranged of 31.5 to 59.0 %, proteolytic - of 97.8 to 125.4 %. At the same time, there was a little pronounced difference in the indicators of amylolytic and proteolytic activities of the germinated processed wheat malt of the sixth and seventh days of fermentation, which makes it possible to shorten the duration of this stage and the entire malt production by one day. The finished wheat malt was characterized by high enzymatic activity (in the range for three varieties): amylolytic 344.9-360.8 units /g, proteolytic 324.9-257.8 units/g, lower duration of saccharification in comparison with the control variant of 18 to 20 minutes. In addition, the proposed method of malting allows the use of wheat with a high protein content, such as the Aleyskaya variety with a mass fraction of protein of 14.6 %, since during germination under the stimulating effect of the inorganic preparation Energen, the proteolysis process proceeds more intensively, and in the final malt the protein content decreases to 10.4 %.

Shedding Lights on Crude Venom from Solitary Foraging Predatory Ant Ectatomma opaciventre: Initial Toxinological Investigation

Some species of primitive predatory ants, despite living in a colony, exercise their hunting collection strategy individually; their venom is painful, paralyzing, digestive, and lethal for their prey, yet the toxins responsible for these effects are poorly known. Ectatomma opaciventre is a previously unrecorded solitary hunting ant from the Brazilian Cerrado. To overcome this hindrance, the present study performed the in vitro enzymatic, biochemical, and biological activities of E. opaciventre to better understand the properties of this venom. Its venom showed several proteins with masses ranging from 1–116 kDa, highlighting the complexity of this venom. Compounds with high enzymatic activity were described, elucidating different enzyme classes present in the venom, with the presence of the first L-amino acid oxidase in Hymenoptera venoms being reported. Its crude venom contributes to a state of blood incoagulability, acting on primary hemostasis, inhibiting collagen-induced platelet aggregation, and operating on the fibrinolysis of loose red clots. Furthermore, the E. opaciventre venom preferentially induced cytotoxic effects on lung cancer cell lines and three different species of Leishmania. These data shed a comprehensive portrait of enzymatic components, biochemical and biological effects in vitro, opening perspectives for bio-pharmacological application of E. opaciventre venom molecules.

Lemon maturation causes anatomical and biochemical changes at the flavedo tissue level

Plants mobilize the photosynthates by three transport pathways: apoplastic, symplastic through plasmodesmata (PD), and transcellular. In flavedo of postharvest mature lemons, a high activity of cell wall-bound invertase (WI), an enzyme associated with transcellular transport of monosaccharides, has been detected. In order to elucidate whether this high enzymatic activity is related to restricted transport in the symplastic pathway with fruit maturation, the aim of the present work was to compare anatomical and biochemical parameters in peel tissues of immature and mature lemons. Anatomical structure focusing on cell walls, callose deposition, WI activity, and sucrose content were analyzed in peel tissues of immature and mature lemons. The parenchyma of flavedo tissue of immature lemons presented an elevated number of primary pit fields (PPF). These PPF, associated to PD or cell wall interruptions, had the appearance of a string of beads. However, in mature lemons, the number of PPF was scarce due to callose deposition. WI activity and apoplastic sucrose content increased significantly in flavedo of mature lemons in comparison to immature lemons. Present findings lay structural and functional bases relevant to understand differences between immature and mature lemons, which would help to design agricultural practices in pre- and post-harvest management.

Charting the N-Terminal Acetylome: A Comprehensive Map of Human NatA Substrates

N-terminal acetylation (Nt-acetylation) catalyzed by conserved N-terminal acetyltransferases or NATs embodies a modification with one of the highest stoichiometries reported for eukaryotic protein modifications to date. Comprising the catalytic N-alpha acetyltransferase (NAA) subunit NAA10 plus the ribosome anchoring regulatory subunit NAA15, NatA represents the major acetyltransferase complex with up to 50% of all mammalian proteins representing potential substrates. Largely in consequence of the essential nature of NatA and its high enzymatic activity, its experimentally confirmed mammalian substrate repertoire remained poorly charted. In this study, human NatA knockdown conditions achieving near complete depletion of NAA10 and NAA15 expression resulted in lowered Nt-acetylation of over 25% out of all putative NatA targets identified, representing an up to 10-fold increase in the reported number of substrate N-termini affected upon human NatA perturbation. Besides pointing to less efficient NatA substrates being prime targets, several putative NatE substrates were shown to be affected upon human NatA knockdown. Intriguingly, next to a lowered expression of ribosomal proteins and proteins constituting the eukaryotic 48S preinitiation complex, steady-state levels of protein N-termini additionally point to NatA Nt-acetylation deficiency directly impacting protein stability of knockdown affected targets.

Identification of FadT as a Novel Quorum Quenching Enzyme for the Degradation of Diffusible Signal Factor in Cupriavidus pinatubonensis Strain HN-2

Quorum sensing (QS) is a microbial cell–cell communication mechanism and plays an important role in bacterial infections. QS-mediated bacterial infections can be blocked through quorum quenching (QQ), which hampers signal accumulation, recognition, and communication. The pathogenicity of numerous bacteria, including Xanthomonas campestris pv. campestris (Xcc), is regulated by diffusible signal factor (DSF), a well-known fatty acid signaling molecule of QS. Cupriavidus pinatubonensis HN-2 could substantially attenuate the infection of XCC through QQ by degrading DSF. The QQ mechanism in strain HN-2, on the other hand, is yet to be known. To understand the molecular mechanism of QQ in strain HN-2, we used whole-genome sequencing and comparative genomics studies. We discovered that the fadT gene encodes acyl-CoA dehydrogenase as a novel QQ enzyme. The results of site-directed mutagenesis demonstrated the requirement of fadT gene for DSF degradation in strain HN-2. Purified FadT exhibited high enzymatic activity and outstanding stability over a broad pH and temperature range with maximal activity at pH 7.0 and 35 °C. No cofactors were required for FadT enzyme activity. The enzyme showed a strong ability to degrade DSF. Furthermore, the expression of fadT in Xcc results in a significant reduction in the pathogenicity in host plants, such as Chinese cabbage, radish, and pakchoi. Taken together, our results identified a novel DSF-degrading enzyme, FadT, in C. pinatubonensis HN-2, which suggests its potential use in the biological control of DSF-mediated pathogens.

Biotechnology of Microorganisms Growing– Fundamentals for the Development of a Litter Biodestructor

The purpose of the research work was to select the optimal conditions for the cultivation of microorganisms. As a result of the conducted research work, the modes of growing a nitrogen-fixing culture and a microorganism with high enzymatic activity were selected and worked out. At the same time, the optimal conditions for the cultivation of Azotobacter sp were determined – the temperature optimum for cell accumulation was 30°C, for increased polysaccharide production 20 °C, aeration within 5-10 l/l/min, agitator speed-150 rpm, pH value within 6.0±0.2 units, which allowed to achieve a cell titer of at least 1.0×109 CFU/ml. A cost-effective nutrient medium was selected for growing Pseudomonas sp. molasses-autolysate medium and optimal conditions for growing the culture: cultivation temperature 30-32 °C, aeration 1.0-1.5 l/l/ min, agitator speed 150-200 rpm, pH 6.8-7.2 units, sub-titration 5.0 % KOH, defoaming with adecanol, cultivation time-72 hours, which allowed to achieve a cell titer of at least 1.0×109 CFU/ml.

Two Novel, Flavin-Dependent Halogenases from the Bacterial Consortia of Botryococcus braunii Catalyze Mono- and Dibromination

Halogen substituents often lead to a profound effect on the biological activity of organic compounds. Flavin-dependent halogenases offer the possibility of regioselective halogenation at non-activated carbon atoms, while employing only halide salts and molecular oxygen. However, low enzyme activity, instability, and narrow substrate scope compromise the use of enzymatic halogenation as an economical and environmentally friendly process. To overcome these drawbacks, it is of tremendous interest to identify novel halogenases with high enzymatic activity and novel substrate scopes. Previously, Neubauer et al. developed a new hidden Markov model (pHMM) based on the PFAM tryptophan halogenase model, and identified 254 complete and partial putative flavin-dependent halogenase genes in eleven metagenomic data sets. In the present study, the pHMM was used to screen the bacterial associates of the Botryococcus braunii consortia (PRJEB21978), leading to the identification of several putative, flavin-dependent halogenase genes. Two of these new halogenase genes were found in one gene cluster of the Botryococcus braunii symbiont Sphingomonas sp. In vitro activity tests revealed that both heterologously expressed enzymes are active flavin-dependent halogenases able to halogenate indole and indole derivatives, as well as phenol derivatives, while preferring bromination over chlorination. Interestingly, SpH1 catalyses only monohalogenation, while SpH2 can catalyse both mono- and dihalogenation for some substrates.

Toxicity assessment of gold ions and gold nanoparticles to golden perch larvae (Macquaria ambigua)

Golden perch (Macquaria ambigua) is a freshwater game-fish native to central and southeast Australia. Larvae of this fish species were used in two different types of experiments to evaluate the effects of short-term exposures (up to 6 days) to aqueous gold, 5 nm gold nanoparticles (AuNPs), or 50 nm AuNPs. Relative to the control, increased gold concentrations corresponded with yolk-sac edema (swelling). Larvae exposed to 50 μM of 5 nm AuNPs had yolk-sacs that were ~1.5 times larger resulting in the appearance of bent notochords. After two days of exposure, 100% mortality was observed. Total mortalities were <25% in the other larvae–gold systems, suggesting that these larvae can quickly adapt to the presence of gold. In terms of an oxidative stress response, the larvae from all systems did not express high enzymatic activity. The state of the gold determined how much could be taken up (or immobilised) by a larva. Aqueous gold and 5 nm AuNPs easily pass through cells; therefore, larvae exposed to these forms of gold contained the highest concentrations. Scanning electron microscopy confirmed that cells comprising the epithelium and fins contained AuNPs. Aqueous gold was reduced to nanometre-scale particles within cells. Comparatively, 5 nm AuNPs appeared to be aggregated within cells forming clusters hundreds of nanometres in size. On the contrary, 50 nm AuNPs were not observed within cells but were detected within larvae by (single particle) inductively coupled plasma mass spectroscopy, suggesting that these AuNPs were probably taken up through the mouth or gills. The results of the present study demonstrate that exposure to AuNPs had adverse effects on developing golden perch larvae. Additionally, these effects were dependent on the size of the AuNPs.

Determination of Post-Harvest Biochemical Composition, Enzymatic Activities, and Oxidative Browning in 14 Apple Cultivars

Phenolic compounds in fruit provide human health benefits, and they contribute to color, taste, and the preservation of post-harvest fruit quality. Phenolic compounds also serve as modifiers of enzymatic activity, whether inhibition or stimulation. Polyphenol oxidases (PPO) and peroxidases (POD) use phenolic compounds as substrates in oxidative browning. Apple browning leads to flesh color, taste, texture, and flavor degradation, representing a drawback for the variety and its’ market appraisal. This study was conducted to investigate the process of browning in 14 apple cultivars throughout post-harvest at three-time points: immediately (T0), one hour (T1), and 24 h (T2) after apples were cut in half. Color parameters L* (lightness), a* (red/green), b* (yellow/blue) were measured, and chroma (ΔC*) and color (ΔE) were calculated to quantify differences between T0₋T1 and T1₋T2 on the fruit surface. Enzymatic activity (PPO, POD) and phenolic composition were also quantified for each cultivar. ‘Granny Smith’ and ‘Cripps Pink’ browned minimally. In contrast, ‘Fiesta’ and ‘Mondial Gala’ browned severely, reporting high enzymatic activity and quantified phenolic concentration (QPC). Phenolic compound polymerization appears to play a significant role in enzymatic inhibition. ‘Topaz’ does not fit the high QPC, PPO, and browning formula, suggesting alternative pathways that contribute to apple browning.

THE PERIORAL (JUXTAORAL) OF J. CHIVITZ ORGAN - THE UNSOLVED MYSTERY OF THE ANATOMY OF THE XXI CENTURY

The article presents the currently available survey material on the morphology, function and location of Chivitz juxtaoral organ. The findings supporting the concepts of JOO’s both secretory and mechanosensory functions, are examined. The analysis of the results of a number of studies is presented, which allows us to assert that the juxta-oral organ is not a rudimentary formation, it can be considered a normal anatomical structure, detected both in an adult and in fetuses. The location of the organ is typical near the duct of the parotid gland in the depths of the cheek tissues, medial and above the angle of the lower jaw. The presence of epithelial cell nests with glandular or squamous cell differentiation in the juxtaoral organ indicates its epithelial nature. However, immunohistochemistry with positive expression of S-100 protein, neuron-specific enolase, neural adhesion molecules and nerve growth factor receptors allows researchers to think about the neural origin of the organ. Scientists’ reflections on the function of the juxtaoral organ are also ambiguous. Data are presented in favor of both mechanoreceptor and secretory functions of the Chivitz juxtaoral organ. The discovery of Pacini’s little bodies in the juxtaoral organ suggests that the organ can perform chemosensory and barosensory functions. The high enzymatic activity of the parenchymal cells of juxtaoral organ on the example of the activity of alkaline phosphatase in the secretion of an organ, which is so unusual for the salivary glands, suggests that juxtaoral organ can be an endocrine gland and can a neuroendocrine function. Information is presented that, since juxtaoral organ is deeply immersed in soft tissues, it can mimic odontogenic tumors or perineural carcinoma invasion