A novel milk-clotting enzyme from Aspergillus oryzae and A. luchuensis is an aspartic endopeptidase PepE presumed to be a vacuolar enzyme

Aspergillus oryzae RIB40 has 11 aspartic endopeptidase genes. We searched for milk-clotting enzymes based on the homology of the deduced amino acid sequence with chymosins. As a result, we identified a milk-clotting enzyme in A. oryzae. We expected other Aspergillus species to have a homologous enzyme with milk-clotting activity, and we found the most homologous aspartic endopeptidase from A. luchuensis had milk-clotting activity. Surprisingly, two enzymes were considered as vacuole enzymes according to a study on A. niger proteases. The two enzymes from A. oryzae and A. luchuensis cleaved a peptide between the 105Phe-106Met bond in κ-casein, similar to chymosin. Although both enzymes showed proteolytic activity using casein as a substrate, the optimum pH values for milk-clotting and proteolytic activities were different. Furthermore, the substrate specificities were highly restricted. Therefore, we expected that the Japanese traditional fermentation agent, koji, could be used as an enzyme source for cheese production.

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 %.

Targeted proteolytic products of τ and α-synuclein in neurodegeneration

CNS pathological inclusions comprising τ or α-synuclein (αSyn) define a spectrum of neurodegenerative diseases, and these can often present concurrently in the same individuals. The aggregation of both proteins is clearly associated with neurodegeneration and the deleterious properties of each protein is further supported by mutations in each gene (MAPT and SNCA, respectively) resulting in disease. The initiating events in most sporadic neurodegenerative diseases are still unclear but growing evidence suggests that the aberrant proteolytic cleavage of τ and αSyn results in products that can be toxic and/or initiate aggregation that can further spread by a prion-like mechanism. The accumulation of some of these cleavage products can further potentiate the progression of protein aggregation transmission and lead to their accumulation in peripheral biofluids such as cerebrospinal fluid (CSF) and blood. The future development of new tools to detect specific τ and αSyn abnormal cleavage products in peripheral biofluids could be useful biomarkers and better understand of the role of unique proteolytic activities could yield therapeutic interventions.

Proteoform Analysis of Matrix Metalloproteinase-9/Gelatinase B and Discovery of Its Citrullination in Rheumatoid Arthritis Synovial Fluids

ObjectivesTo explore posttranslational modifications (PTMs), including proteolytic activation, multimerization, complex formation and citrullination of gelatinases, in particular of gelatinase B/MMP-9, and to detect in gelatin-Sepharose affinity-purified synovial fluids, the presence of specific MMP proteoforms in relation to arthritis.MethodsLatent, activated, complexed and truncated gelatinase-A/MMP-2 and gelatinase B/MMP-9 proteoforms were detected with the use of zymography analysis to compare specific levels, with substrate conversion assays, to test net proteolytic activities and by Western blot analysis to decipher truncation variants. Citrullination was detected with enhanced sensitivity, by the use of a new monoclonal antibody against modified citrullines.ResultsAll MMP-9 and MMP-2 proteoforms were identified in archival synovial fluids with the use of zymography analysis and the levels of MMP-9 versus MMP-2 were studied in various arthritic diseases, including rheumatoid arthritis (RA). Secondly, we resolved misinterpretations of MMP-9 levels versus proteolytic activities. Thirdly, a citrullinated, truncated proteoform of MMP-9 was discovered in archival RA synovial fluid samples and its presence was corroborated as citrullinated hemopexin-less MMP-9 in a small prospective RA sample cohort.ConclusionSynovial fluids from rheumatoid arthritis contain high levels of MMP-9, including its truncated and citrullinated proteoform. The combination of MMP-9 as analyte and its PTM by citrullination could be of clinical interest, especially in the field of arthritic diseases.

Effect of Cavity Cleanser With Long-Term Antibacterial and Anti-Proteolytic Activities on Resin–Dentin Bond Stability

ObjectiveSecondary caries caused by oral microbiome dysbiosis and hybrid layer degradation are two important contributors to the poor resin–dentin bond durability. Cavity cleansers with long-term antimicrobial and anti-proteolytic activities are in demand for eliminating bacteria-induced secondary caries and preventing hybrid layers from degradation. The objectives of the present study were to examine the long-term antimicrobial effect and anti-proteolytic potential of poly(amidoamine) dendrimers with amino terminal groups (PAMAM-NH2) cavity cleanser.MethodsAdsorption tests by attenuated total reflectance–infrared (ATR-IR) spectroscopy and confocal laser scanning microscopy (CLSM) were first performed to evaluate whether the PAMAM-NH2 cavity cleanser had binding capacity to dentin surface to fulfill its relatively long-term antimicrobial and anti-proteolytic effects. For antibacterial testing, Streptococcus mutans, Actinomyces naeslundii, and Enterococcus faecalis were grown on dentin surfaces, prior to the application of cavity cleanser. Colony-forming unit (CFU) counts and live/dead bacterial staining were performed to assess antibacterial effects. Gelatinolytic activity within the hybrid layers was directly detected by in situ zymography. Adhesive permeability of bonded interface and microtensile bond strength were employed to assess whether the PAMAM-NH2 cavity cleanser adversely affected resin–dentin bonding. Finally, the cytotoxicity of PAMAM-NH2 was evaluated by the Cell Counting Kit-8 (CCK-8) assay.ResultsAdsorption tests demonstrated that the binding capacity of PAMAM-NH2 on dentin surface was much stronger than that of 2% chlorhexidine (CHX) because its binding was strong enough to resist phosphate-buffered saline (PBS) washing. Antibacterial testing indicated that PAMAM-NH2 significantly inhibited bacteria grown on the dentin discs as compared with the control group (p < 0.05), which was comparable with the antibacterial activity of 2% CHX (p > 0.05). Hybrid layers conditioned with PAMAM-NH2 showed significant decrease in gelatin activity as compared with the control group. Furthermore, PAMAM-NH2 pretreatment did not adversely affect resin–dentin bonding because it did not decrease adhesive permeability and microtensile strength. CCK-8 assay showed that PAMAM-NH2 had low cytotoxicity on human dental pulp cells (HDPCs) and L929.ConclusionsPAMAM-NH2 cavity cleanser developed in this study could provide simultaneous long-term antimicrobial and anti-proteolytic activities for eliminating secondary caries that result from a dysbiosis in the oral microbiome and for preventing hybrid layers from degradation due to its good binding capacity to dentin collagen matrix, which are crucial for the maintenance of resin–dentin bond durability.

Staurosporine-induced cleavage of apoptosis-inducing factor in human fibrosarcoma cells is independent of matrix metalloproteinase-2

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein which mediates staurosporine (STS)-induced cell death. AIF cleavage and translocation to the cytosol is thought to be calpain-1-dependent as calpain inhibitors reduced AIF proteolysis. However, many calpain inhibitors also inhibit matrix metalloproteinase-2 (MMP-2) activity, an intracellular and extracellular protease implicated in apoptosis. Here we investigated whether MMP-2 activity is affected in response to STS and if contributes to AIF cleavage. Human fibrosarcoma HT1080 cells were treated with STS (0.1 µM, 0.25-24 hr). A significant increase in cellular MMP-2 activity was seen by gelatin zymography after 6 hr STS treatment, prior to induction of cell necrosis. Western blot showed the time-dependent appearance of two forms of AIF (~60 and 45 kDa) in the cytosol which were significantly increased at 6 hr. Surprisingly, knocking down MMP-2 or inhibiting its activity with MMP-2 preferring inhibitors ARP-100 or ONO-4817, or inhibiting calpain activity with ALLM or PD150606, did not prevent the STS-induced increase in cytosolic AIF. These results show that although STS rapidly increases MMP-2 activity, the cytosolic release of AIF may be independent of the proteolytic activities of MMP-2 or calpain.

Assessment of Probiotic Potential of Lactic Acid Bacteria Isolated from Bottle Gourds (Calabash) of Milk Fermentation of Mbéré, Cameroon

Background and Aim: Lactic acid bacteria (LAB) became a field of interest by scientists in recent years due to their technological and probiotic properties. The aim of this work was to study the technological and probiotic properties of LAB isolated from the bottle gourds (calabashes)of milk fermentation, in Mbéré, Cameroun. Methods: Five different bottle gourds from milk fermentation were collected and used for LAB isolation. These LABs were characterized using conventional cultural method, the technological (such as proteolytic, lipolytic activities) and probiotic properties (including acid and bile salt tolerance, cholesterol assimilation and antioxidant activities) were assessed. Results: From these samples, 30 LABs were isolated and among them, 21 exhibited great lipolytic and proteolytic activities with the maximum values of 18 and 29 mm respectively. In addition, 10 LAB isolates showed interesting antimicrobial activity against pathogens germs tested and good tolerance ability under acid and bile salt stress after 24h of incubation. Cholesterol assimilation and antioxidant tests revealed that isolated BC4 and BC3 have the greatest activity (35 and 39 mm respectively) while, BC4 and BL4 have the greatest antioxidant activity (IC50 = 0,15 and 0,13 respectively). Conclusion: LAB isolated from the bottle gourds (calabashes) of milk fermentation, in Mbéré, Cameroon can be used to develop dairy industry and manage the cardiovascular diseases.

Transcriptome and proteome dynamics during the embryonation of Fasciola hepatica eggs

Fasciola hepatica is a global parasite of livestock which also causes a neglected zoonosis in humans. The parasite’s communication with the host during its complicated lifecycle is based on an ingenious enzymatic apparatus which includes a variety of peptidases. These enzymes are implicated in parasite migration, pathogenesis of the disease, and modification of host immune response. Although the dynamics of proteolytic machinery produced by intra-mammalian F. hepatica life stages has been previously investigated in great detail, peptidases of the eggs so far received little scientific attention. In this study, we performed a comparative RNA-seq analysis aimed at identification of peptidases expressed in F. hepatica eggs at different ages and employed mass spectrometry in order to identify and quantify peptidases translated in F. hepatica egg lysates. Finally, we measured and classified proteolytic activities in extracts from F. hepatica eggs using a library of fluorogenic substrates and peptidase class-selective inhibitors. Using these methods, we observed significant changes in the composition of peptidases during egg maturation.

Presence of Adult Companion Goats Favors the Rumen Microbial and Functional Development in Artificially Reared Kids

Newborn dairy ruminants are usually separated from their dams after birth and fed on milk replacer. This lack of contact with adult animals may hinder the rumen microbiological and physiological development. This study evaluates the effects of rearing newborn goat kids in contact with adult companions on the rumen development. Thirty-two newborn goat kids were randomly allocated to two experimental groups which were reared either in the absence (CTL) or in the presence of non-lactating adult goats (CMP) and weaned at 7 weeks of age. Blood and rumen samples were taken at 5, 7, and 9 weeks of age to evaluate blood metabolites and rumen microbial fermentation. Next-generation sequencing was carried out on rumen samples collected at 7 weeks of age. Results showed that CTL kids lacked rumen protozoa, whereas CMP kids had an abundant and complex protozoal community as well as higher methanogen abundance which positively correlated with the body weight and blood β-hydroxybutyrate as indicators of the physiological development. CMP kids also had a more diverse bacterial community (+132 ASVs) and a different structure of the bacterial and methanogen communities than CTL kids. The core rumen bacterial community in CMP animals had 53 more ASVs than that of CTL animals. Furthermore, the number of ASVs shared with the adult companions was over 4-fold higher in CMP kids than in CTL kids. Greater levels of early rumen colonizers Proteobacteria and Spirochaetes were found in CTL kids, while CMP kids had higher levels of Bacteroidetes and other less abundant taxa (Veillonellaceae, Cyanobacteria, and Selenomonas). These findings suggest that the presence of adult companions facilitated the rumen microbial development prior to weaning. This accelerated microbial development had no effect on the animal growth, but CMP animals presented higher rumen pH and butyrate (+45%) and ammonia concentrations than CTL kids, suggesting higher fibrolytic and proteolytic activities. CMP kids also had higher blood β-hydroxybutyrate (+79%) and lower blood glucose concentrations (-23%) at weaning, indicating an earlier metabolic development which could favor the transition from pre-ruminant to ruminant after the weaning process. Further research is needed to determine the effects of this intervention in more challenging farm conditions.

High-Fat Diets Modify the Proteolytic Activities of Dipeptidyl-Peptidase IV and the Regulatory Enzymes of the Renin–Angiotensin System in Cardiovascular Tissues of Adult Wistar Rats

(1) Background: The replacement of diets high in saturated fat (SAFA) with monounsaturated fatty acids (MUFA) is associated with better cardiovascular function and is related to the modulation of the activity of the local renin–angiotensin system (RAS) and the collagenase activity of dipeptidyl peptidase IV (DPP-IV). The objective of the work was to verify the capacity of different types of dietary fat on the regulatory activities of RAS and DPP-IV. (2) Methods: Male Wistar rats were fed for 24 weeks with three different diets: the standard diet (S), the standard diet supplemented with virgin olive oil (20%) (VOO), or with butter (20%) plus cholesterol (0.1%) (Bch). The proteolytic activities were determined by fluorometric methods in the soluble (sol) and membrane-bound (mb) fractions of the left ventricle and atrium, aorta, and plasma samples. (3) Results: With the VOO diet, angiotensinase values were significantly lower than with the Bch diet in the aorta (GluAP and ArgAP (mb)), ventricle (ArgAP (mb)) and atrium (CysAP (sol)). Significant decreases in DPP-IV (mb) activity occurred with the Bch diet in the atrium and aorta. The VOO diet significantly reduced the activity of the cardiac damage marker LeuAP (mb) in the ventricle and aorta, except for LeuAP (sol) in the ventricle, which was reduced with the Bch diet. (4) Conclusions: The introduction into the diet of a source rich in MUFA would have a beneficial cardiovascular effect on RAS homeostasis and cardiovascular functional stability.