Application of ESI FT-ICR MS to Study Kraft Lignin Modification by the Exoenzymes of the White Rot Basidiomycete Fungus TrametesHirsutaLE-BIN 072

Trameteshirsuta is a wood rotting fungus that possesses a vast array of lignin degrading enzymes, including7 laccases, 7 ligninolyticmanganese peroxidases, 9 lignin peroxidases and 2 versatile peroxidases. In this study,electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS)was used to examine kraft lignin modification by the enzymatic system of this fungus.The observed pattern of lignin modification suggested that before the 6th day of cultivation,the fungal enzymatic system tended to degrade more oxidized moleculesand, hence, less recalcitrant molecules, with the production of hard-to-modify reduced molecular species. At some point after the 6th day of cultivation,the fungal enzymatic system tended to degrade more oxidized moleculesand, hence, less recalcitrant molecules, with the production of hard-to-modify reduced molecular species. At some point after the 6th day of cultivation,the fungus started to degrade less oxidized, more recalcitrant, compounds, converting them into the more oxidized forms. The altered pattern of lignin modification enabled changes in the fungal enzymatic system. These changes were further attributed to the appearance of the particular ligninolyticmanganese peroxides enzyme(MnP7), which was added by the fungus to the mixture of enzymes that had already been secreted (VP2 and MnP5). Keywords: wood rotting fungi, kraft lignin, mass spectrometry, peroxidases

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 %.

Two Biotechnological Approaches to the Preparative Synthesis of Natural Dihydrocoumarin

In this work, we describe two different biotechnological processes that provide the natural flavour dihydrocoumarin in preparative scale. Both the presented approaches are based on the enzyme-mediated reduction of natural coumarin. The first one is a whole-cell process exploiting the reductive activity of the yeast Kluyveromyces marxianus, a Generally Recognized As Safe (GRAS) microorganism that possesses high resistance to the substrate toxicity. Differently, the second is based on the reduction of natural coumarin by nicotinamide adenine dinucleotide phosphate (NADPH) and using the Old Yellow Enzyme reductase OYE2 as catalyst. NADPH is used in catalytic amount since the co-factor regeneration is warranted employing an enzymatic system based on glucose oxidation, in turn catalysed by a further enzyme, namely glucose dehydrogenase (GDH). Both processes compare favourably over the previously reported industrial method as they work with higher coumarin concentration (up to 3 g/L for the enzymatic process) yet allowing the complete conversion of the substrate. Furthermore, the two approaches have significant differences. The microbial reduction is experimentally simple but the isolated dihydrocoumarin yield does not exceed 60%. On the contrary, the enzymatic approach requires the use of two specially prepared recombinant enzymes, however, it is more efficient, affording the product in 90% of isolated yield.

The activity of enzymes can be modified by homeopathic dilutions of their effectors

Introduction: The fungal and bacterial materials are very useful for testing the influence of low and very low doses of low molecular phenolic effectors on enzymatic system of phenoloxidases when they are incubated together in the reaction space. Aim: Searching for the model useful biological systems to study the action of diluted low molecular substances on living organisms, which is based on common physical and biochemical analytical procedures. Methods: The fungal and actinomycetal bacterial materials from laboratory cultures as a source of common phenoloxidases, laccase, peroxidase and O-demethylase as well as the pure plant peroxidase were used in experiments described earlier [1-5]. Subsequent dilutions of low molecular phenolic metabolites, appropriate for studied enzymatic systems, prepared in 75% ethanol in the proportion of 1:100 (centesimal) and dynamized by shaking in accordance with homeopathic procedures were prepared in our laboratory. During experiments with bacterial and fungal materials and a pure plant peroxidase, which were incubated together with subsequent dilutions of proper phenolic effector, different analytic methods were used including a gel (PAGE) [4] and capillary (MEKCE) electrophoresis [5], spectral and colorimetric methods [1,2,3] as well as the electron microscopy [5]. Results: In the light of presented data [1-5], the incubation of biological material with diluted phenolic effectors induces various effects on tested enzyme activity. It changed in sinusoidal manner with an gradual growth of dilution rate of tested effectors, which was distinctly visible on the diagram when the number of dilutions was localized on abscissa and biological activity on the ordinate. Exemplary results of the chosen experiments will be presented. For tested enzymes: laccase, peroxidase and O-demethylase, the distance between maximal points of enzymatic activity, shown on a sine curve, repeats more often every 10 subsequent centesimal dilutions. Along with the extension of incubation period the displacement of maximal and minimal points on curve were noticed, which revealed a dynamic aspect of studied phenomenon. Conclusions: Fungal and bacterial cells seem to be a very convenient material for studying the action of diluted metabolites on enzymatic systems because their popular presence in environment. Results of all experiments confirmed the same nature of the mentioned observations. Because other authors had similar conclusion concerning human [6,7] and plant materials [8,9], the described relations seem to be common in natural world. It could also be stressed that the therapeutic effect of homeopathic remedies could be based on the mechanism described above and it is highly probable that it leads to a normalisation of disturbed enzyme systems in the living organisms.

Evaluation of bamboo water-retting for fiber bundle extraction

Bamboo fiber bundles were successfully extracted from bamboo culms using water-retting, taking advantage of enzymes secreted by microorganisms in the retting liquid. The harvest year and place of origin of the bamboo and the source of water impacted the products of the retting process. One-month-old bamboo was decomposed completely, whereas the one-year-old sample was hardly changed after 24-day retting. Moisture regain and crystallinity varied with the different origins of the bamboo. However, all samples resulted in similar chemical structures and thermal properties. The best operational conditions for water-retting were 3-month-old bamboo from Wuxi incubated in deionized water. Enzyme activities, including cellulase, xylanase, pectinase, and ligninolytic enzymes (lignin peroxidase, manganese peroxidase, and laccase) were monitored during a 24-day retting. Manganese peroxidase was the primary enzyme used to degrade lignin, resulting in absorbance at 294 nm of UV-Vis spectra. In addition, xylanase played a leading role in hydrolyzing hemicellulose, which was consistent with the change in reducing sugar yield. In addition, variations in dissolved oxygen and pH values were also recorded, indicating the changes in bacterial strains and the enzymatic system. The wastewater from bamboo retting showed good biodegradability but a lack of nitrogen and phosphorus. Overall, a manganese peroxidase–xylanase combined enzyme-retting treatment would offer a more environmentally friendly approach for extracting bamboo fibers.

Active sites of peptide from Arg-Ser-Ser protect against oxidative stress in HepG2 cells

Peptide Arg-Ser-Ser (RSS) was derived from Lactobacillus amylolyticus co-incubated with edible <i>Dendrobium aphyllum</i>. Here, we further examined the antioxidative effects of RSS in HepG2 cells subjected to 2,2-azobis(2-methylpropanimidamidine) dihydrochloride-induced oxidative stress. RSS protected cells by eliminating the level of reactive oxygen species (ROS). The protein expression of antioxidant enzymes, Nrf2 and Keap1 determined by western blot, indicated that RSS might maintain cellular homeostasis by directly scavenging free radicals instead of by enzymatic system. Furthermore, quantum chemistry calculations and a characterization of electronic-related properties showed that the highest occupied molecular orbital energy distribution was on arginine residue. Pre-treatment with RSS with the active site methylated resulted in increased ROS levels, thereby verifying that N₂-H₃ is the active site for antioxidant activity. Our findings provide valuable insights into the antioxidant activity of RSS and a basis for developing antioxidant functional foods.

Ultrasound-Assisted Multi-Enzymatic System for the Preparation of ACE Inhibitory Peptides with Low Bitterness from Corn Gluten Meal

The promising angiotensin converting enzyme (ACE) inhibitory peptides derived from corn protein usually have strong bitterness and thus limit their use among consumers. To prepare ACE inhibitory peptides with low bitterness, two energy-efficient types of ultrasound pretreatment were introduced into the multi-enzymatic system of corn gluten meal. The results showed that Flavourzyme–Protamex sequential enzymolysis produced the peptides with high ACE inhibitory activity and the lowest bitterness compared with other enzymolysis conditions. During the optimized sequential enzymolysis, the divergent ultrasound pretreatment with a frequency of 40 kHz for 60 min exhibited higher ACE inhibitory activity (65.36%, accounting for 73.84% of the highest ACE inhibitory activity) and lower bitterness intensity of peptides, compared with an energy-gathered ultrasound. The results of the study showed that, on the one hand, divergent ultrasound pretreatment induced the highest intrinsic fluorescence of protein, with more hydrophobic amino acid residues exposed for cleavage by exopeptidases, which leads to a reduction in bitterness. On the other hand, the amino acid composition analysis proved that more Tyr, Ile, and Val moieties, instead of Leu (bitterest substance), and more peptide fractions with a molecular weight >1000 Da should be the structural features of high ACE inhibitory peptides.

Male Infertility: Pathogenetic Significance of Oxidative Stress and Antioxidant Defence (Review)

The basis of the pathogenesis of male infertility is the processes of peroxide oxidation of biological substrates, especially lipids and proteins. By destroying the sperm membrane, toxic peroxidation products reduce its motility and ability to fertilize the egg, which is determined by a decrease in the number of motile sperm in the ejaculate. These changes lead to complete or partial male infertility. The authors of the review found that is accompanied by a damaging effect on the structural and functional activity of the gonads and is manifested, in particular, by an imbalance in the hormonal background of the male body. Similar effects are characteristic of an increase in the content of reactive Nitrogen species and its metabolites, which cause nitrosative stress, which is also the cause of male hypofertility and is inseparable from the state of oxidative stress. In scientific work it is determined that the accumulation of harmful peroxidation products leads to damage and destruction of sperm DNA, reduced activity of acrosomal enzymes and mitochondrial potential of sperm, reduced overall antioxidant activity. This makes it impossible for an adequate response of the body. Multi component antioxidant defense system resists stress. It is represented by enzymatic and non-enzymatic links, which can neutralize harmful radicals and peroxidation products. It contributes to the full manifestation of reproductive function. The presence of powerful antioxidant properties of catalase, superoxide dismutase, and enzymes of the thiol-disulfide system, which form the enzymatic system of antioxidant protection, as well as selenium, zinc, copper, other trace elements, retinol, tocopherol, ascorbic acid, and vitamins as parts of the non-enzymatic system is shown. The efficiency of registration is substantiated thin biochemical shift detectors or complex methods, such as total antioxidant status of sperm or sperm plasma, mitochondrial membrane potential, etc along with simple markers of oxidative stress, such as diene conjugates, malonic dialdehyde, and metabolites of the Nitrogen Oxide cycle. Given the leading role of oxidative stress in the development of male hypofertility, the prospect of further research is the search for modern means for correction, especially among substances with pronounced redox activity

Infant colic: where is the line between norm and pathology, the role of disruption of the intestinal microbiome

Infant colic is one of the most common functional disorders of the gastrointestinal tract in children of the first year of life, worsening the condition of both the child himself and the psychological climate in the family. Infant intestinal colic is a multifactorial symptom complex that requires differential diagnosis between norm and pathology. Until now, there is no clear understanding of the criteria for the need for dietary and drug correction of this condition. The article presents an analysis of the modern literature on the problem of intestinal colic in infants The pathogenesis of functional intestinal colic is determined by a combination of reasons, including the immaturity of the central nervous system, hypothalamic-pituitary system, regulation of the intestinal innervation and the enzymatic system, a certain dependence on psychosocial factors has also been identified, the intestinal microflora has a great influence. H. Kianifar et al. (2014) in the findings of a study noted that a multiprobiotic (seven strains of probiotics and fructooligosaccharides) significantly improved colic symptoms, in particular a reduction in crying time, (82.6%) compared with placebo (35.7%) on day 7 of intake (p < 0.005). On day 30, treatment success was 87% and 46% in the synbiotics and placebo groups, respectively (p < 0.01). The results of a domestic study by I.N. Zakharova et al. (2016) multiprobiotic, which showed that after the use of a multistrain probiotic, intestinal colic remained in 3 (10%) children of the main group and in all children in the placebo group. After taking the probiotic, 80% of children showed normalization of stool, and in the placebo group, stool disorders (loose, watery stools or no stool with the need to empty after an enema) were recorded in 87% of children. Cases from clinical practice with experience of using a multistrain probiotic for colic are shown.