scholarly journals Digestive Alkaline Proteases from Zosterisessor ophiocephalus, Raja clavata, and Scorpaena scrofa: Characteristics and Application in Chitin Extraction

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Rim Nasri ◽  
Islem Younes ◽  
Imen Lassoued ◽  
Sofiane Ghorbel ◽  
Olfa Ghorbel-Bellaaj ◽  
...  
Keyword(s):  
Proteolytic Enzymes ◽  
Tween 20 ◽  
Sodium Perborate ◽  
Alkaline Proteases ◽  
Shrimp Waste ◽  
Wide Range ◽  
Raja Clavata ◽  
Shrimp Wastes ◽  
Scorpaena Scrofa ◽  
Zosterisessor Ophiocephalus

The aim of this work was to study some biochemical characteristics of crude alkaline protease extracts from the viscera of goby (Zosterisessor ophiocephalus), thornback ray (Raja clavata), and scorpionfish (Scorpaena scrofa), and to investigate their applications in the deproteinization of shrimp wastes. At least four caseinolytic proteases bands were observed in zymogram of each enzyme preparation. The optimum pH for enzymatic extracts activities of Z. ophiocephalus, R. clavata, and S. scrofa were 8.0-9.0, 8.0, and 10.0, respectively. Interestingly, all the enzyme preparations were highly stable over a wide range of pH from 6.0 to 11.0. The optimum temperatures for enzyme activity were 50∘C for Z. ophiocephalus and R. clavata and 55∘C for S. scrofa crude alkaline proteases. Proteolytic enzymes showed high stability towards non-ionic surfactants (5% Tween 20, Tween 80, and Triton X-100). In addition, crude proteases of S. scrofa, R. clavata, and Z. ophiocephalus were found to be highly stable towards oxidizing agents, retaining 100%, 70%, and 66%, respectively, of their initial activity after incubation for 1 h in the presence of 1% sodium perborate. They were, however, highly affected by the anionic surfactant SDS. The crude alkaline proteases were tested for the deproteinization of shrimp waste in the preparation of chitin. All proteases were found to be effective in the deproteinization of shrimp waste. The protein removals after 3 h of hydrolysis at 45∘C with an enzyme/substrate ratio (E/S) of 10 were about 76%, 76%, and 80%, for Z. ophiocephalus, R. clavata, and S. scrofa crude proteases, respectively. These results suggest that enzymatic deproteinization of shrimp wastes by fish endogenous alkaline proteases could be applicable to the chitin production process.

scholarly journals Individual Expression of Hepatitis A Virus 3C Protease Induces Ferroptosis in Human Cells In Vitro

2021 ◽  
Vol 22 (15) ◽  
pp. 7906
Author(s):  
Alexey A. Komissarov ◽  
Maria A. Karaseva ◽  
Marina P. Roschina ◽  
Andrey V. Shubin ◽  
Nataliya A. Lunina ◽  
...  
Keyword(s):  
Cell Death ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Proteolytic Enzymes ◽  
Morphological Changes ◽  
Human Cells ◽  
Mitochondrial Potential ◽  
3C Protease ◽  
Wide Range

Regulated cell death (RCD) is a fundamental process common to nearly all living beings and essential for the development and tissue homeostasis in animals and humans. A wide range of molecules can induce RCD, including a number of viral proteolytic enzymes. To date, numerous data indicate that picornaviral 3C proteases can induce RCD. In most reported cases, these proteases induce classical caspase-dependent apoptosis. In contrast, the human hepatitis A virus 3C protease (3Cpro) has recently been shown to cause caspase-independent cell death accompanied by previously undescribed features. Here, we expressed 3Cpro in HEK293, HeLa, and A549 human cell lines to characterize 3Cpro-induced cell death morphologically and biochemically using flow cytometry and fluorescence microscopy. We found that dead cells demonstrated necrosis-like morphological changes including permeabilization of the plasma membrane, loss of mitochondrial potential, as well as mitochondria and nuclei swelling. Additionally, we showed that 3Cpro-induced cell death was efficiently blocked by ferroptosis inhibitors and was accompanied by intense lipid peroxidation. Taken together, these results indicate that 3Cpro induces ferroptosis upon its individual expression in human cells. This is the first demonstration that a proteolytic enzyme can induce ferroptosis, the recently discovered and actively studied type of RCD.

STUDYING OF ACTIVITY OF INHIBITORS OF PROTEOLYTIC ENZYMES IN PRODUCTS OF PROCESSING OF GRAIN HARICOT (PHASEOLUS VULGARIS)

2018 ◽  
Author(s):  
Н.Т. ШАМКОВА ◽  
А.М. АБДУЛХАМИД
Keyword(s):  
Phaseolus Vulgaris ◽  
Liquid Medium ◽  
Proteolytic Enzymes ◽  
Trypsin Inhibitors ◽  
Milk Whey ◽  
Fine Grinding ◽  
Bean Flour ◽  
Wide Range ◽  
Mashed Potatoes

Определено содержание ингибиторов протеолитических ферментов в фасолевой муке, в пюре из зерновой фасоли, сваренной в воде, и в пюре из зерновой фасоли, сваренной после замачивания в воде в молочной сыворотке. Обосновано использование молочной сыворотки в качестве жидкой среды для варки зерновой фасоли после замачивания. Разработана технология полуфабриката в виде фасолевого пюре, предусматривающая замачивание фасоли в воде, варку в молочной сыворотке, грубое измельчение доведенной до готовности фасоли, последующее тонкое измельчение и охлаждение. Установлено, что в фасолевом пюре активность ингибиторов трипсина значительно ниже, чем в муке из фасоли, что делает пюре более предпочтительным полуфабрикатом для производства широкого ассортимента кулинарной продукции. The content of inhibitors of proteolytic enzymes in bean flour, in puree from beans harvested in water and in puree from cereal beans welded in milk whey after soaking in water is determined. The use of whey as a liquid medium for cooking grain beans after soaking is substantiated. The technology of semi-finished product in the form of bean puree, providing for soaking beans in water, cooking in milk whey, coarse grinding of the bean brought to the ready, subsequent fine grinding and cooling is developed. It has been found that the activity of trypsin inhibitors in bean puree is much lower than in bean flour, which makes mashed potatoes a more preferred semi-finished product for the production of a wide range of culinary products.

Steps in the reactions of proteolytic enzymes with their substrates

1970 ◽  
Vol 257 (813) ◽  
pp. 105-110 ◽  
Keyword(s):  
Active Sites ◽  
Dissociation Constants ◽  
Proteolytic Enzymes ◽  
Substrate Binding ◽  
Hydrolytic Enzymes ◽  
Dimensional Structure ◽  
Chemical Information ◽  
Substrate Interaction ◽  
Enzyme Substrate ◽  
Wide Range

Kinetic experiments should be designed to answer specific questions about a reaction mechanism. The present paper is intended to show how a number of specific questions have been answered. Chymotrypsin and trypsin are mainly used to illustrate the different approaches, but many of the arguments used are equally applicable to the reactions of other hydrolytic enzymes with serine-OH or cysteine-SH at the active site. T he recognition of serine-OH and cysteine-SH as essential groups at the active sites of different hydrolytic enzymes did not rest on kinetic evidence. This was deduced from the correlation of enzyme activity with the extent of modification of specially reactive groups. The investigation of proton dissociation equilibria and the assignment of dissociation constants to groups with specified functions in substrate binding, catalysis or protein conformation was the first objective of serious kinetic studies of enzyme reactions. Steady state rate measurements over a wide range of pH showed that groups with p K 6.25 and 6.85 respectively are involved in the catalytic activity of trypsin and chymotrypsin with certain specific substrates (Hammond & Gutfreund 1955). In the case of chymotrypsin it was also shown by Hammond & Gutfreund (1955) that a group with a more alkaline pK is involved in substrate binding. This latter group was subsequently identified and its function was elucidated through the elegant experiments of Oppenheimer, Labouresse & Hess (1966). The identification of histidine as the group with p K A near neutrality, involved in the catalytic mechanism of trypsin and chymotrypsin, was subsequently confirmed by direct chemical methods by Schoelmann & Shaw (1963). Only kinetic analysis can demonstrate the involvement of proton donors or acceptors with specific properties in enzyme-substrate interaction or in catalysis. The clear identification of chemical groups capable of performing such functions is coming from the crystallographic analysis of the three-dimensional structure at the site of enzyme-substrate interaction, as illustrated in other papers presented in this discussion. Very interesting chemical information is obtained when the effect of structure on reactivity is synthesized from the composite of crystallographic and kinetic data.

scholarly journals First Report of Leaf Blight of Rice Caused by Cochliobolus lunatus in China

Plant Disease ◽  
2014 ◽  
Vol 98 (5) ◽  
pp. 686-686 ◽  
Author(s):  
L. M. Liu ◽  
S. W. Huang ◽  
L. Wang ◽  
E. Q. Hou ◽  
D. F. Xiao
Keyword(s):  
Leaf Blight ◽  
Green Water ◽  
Tween 20 ◽  
Curvularia Lunata ◽  
Conidial Suspension ◽  
First Report ◽  
Rice Plants ◽  
Rdna Its ◽  
Cochliobolus Lunatus ◽  
Wide Range

Leaf-streak symptoms were observed on rice (Oryza sativa L.) starting at the booting stage through harvest in Zhejiang Province, China, in 2012. Based on Fuyang County, only 15% of the rice fields were estimated to show these symptoms. However, incidence could be 40 to 80% when the rice got infected. Typical symptoms started as green water-soaked streaks from the tip or edge of leaf blades, similar to bacterial leaf blight caused by Xanthomonas oryzae. Infected leaves turned yellow, then eventually became wilted and dry. No bacterial streaming was observed and no bacteria were isolated. Pieces of infected leaf tissue were surface sterilized using 0.1% (v/v) mercuric chloride, rinsed with sterilized water, then placed on water agar (WA). After 2 or 3 days on WA at 28°C, only fungal growth was observed from surface sterilized tissues. Fungi were isolated, purified by single spore separation process, and subcultured to potato dextrose agar (PDA) plates. Growing on PDA, the surface of the colony was circular, fluffy, and shiny velvety-black, whereas the under surface was dark Prussian blue. Conidiophores were single or fascicled, brown to dark brown, rarely branched, multiseptate, and straight or often geniculate near the apex. Conidia were brown, smooth, fusiform, geniculate or hook-shaped, 17.5 to 28.5 × 8.5 to 14.0 μm, and 3-septate, with the third cell from the base larger and darker than the others. Molecular identification was performed by analysis of the rDNA internal transcribed spacer region (ITS1-5.8S-ITS2). The rDNA-ITS region was amplified with primer pair ITS1 and ITS4 (5), sequenced, and deposited in GenBank (Accession No. KC462186). The sequence of rDNA-ITS (KC462186) showed 100% identity with Cochliobolus lunatus R.R. Nelson & Haasis (JN943422) after BLAST. Based on the results of morphological and molecular analyses, the fungus isolated from infected leaves was identified as C. lunatus (anamorph: Curvularia lunata (Wakk.) Boedijn) (3). Pathogenicity tests were conducted three times by spraying a conidial suspension (1 × 105 spores/ml) with 0.1% (v/v) Tween 20 on 12 healthy rice plants at late tillering stage. The same number of the healthy rice plants sprayed with sterilized water with 0.1% (v/v) Tween 20 were used as control. All plants were kept at 30°C and 75 to 85% relative humidity (RH) under a 12-h light/dark rotation. About 5 to 7 days after inoculation, green water-soaked streaks began to appear on inoculated plants. From 7 to 14 days after inoculation, the lesions developed quickly and the leaves began to wilt. After 14 days, inoculated plants showed symptoms similar to those originally observed in the field, while control plants (sprayed with sterilized water) remained healthy. C. lunatus was re-isolated from all inoculated plants, and re-identified by the same methods (morphological and molecular methods) as described above, thereby satisfying Koch's postulates, and confirming C. lunatus as the cause of the disease. C. lunatus is a pathogen of a wide range of plants and is common in paddy environments. It was reported as one of the causal agents of black kernel of rice (4) and rice spikelet rot disease (SRD) (1,2). The level of incidence observed in the affected fields suggest that this disease could potentially cause major losses under favorable weather conditions if susceptible cultivars are grown. To our knowledge, this is the first report of C. lunatus causing leaf blight of rice in China. References: (1) S. W. Huang et al. Crop Prot. 30:1, 2011. (2) S. W. Huang et al. Crop Prot. 30:10, 2011. (3) D. S. Manamgoda et al. Fungal Divers. 51:3. (4) S. H. Ou. Rice diseases [M]. CABI, 1985. (5) T. J. White et al. PCR Protocols: a Guide to Methods and Application. Academic Press, San Diego, CA, 1990.

Enterococcus faecium isolated from Lombo, a Portuguese traditional meat product: characterisation of antibacterial compounds and factors affecting bacteriocin production

2012 ◽  
Vol 3 (4) ◽  
pp. 319-330 ◽  
Author(s):  
S.D. Todorov ◽  
L. Favaro ◽  
P. Gibbs ◽  
M. Vaz-Velho
Keyword(s):  
Mode Of Action ◽  
Enterococcus Faecium ◽  
Proteolytic Enzymes ◽  
Sodium Dodecyl ◽  
Meat Product ◽  
Maximum Activity ◽  
Tween 20 ◽  
Potential Candidate ◽  
Spoilage Bacteria ◽  
Faecium Strain

Strain ST211CH, identified as a strain of Enterococcus faecium, isolated from Lombo produced a bacteriocin that inhibited the growth of Enterococcus spp., Listeria spp., Klebsiella spp., Lactobacillus spp., Pseudomonas spp., Staphylococcus spp. and Streptococcus spp. The mode of action of the bacteriocin named as bacteriocin ST211Ch was bactericidal against Enterococcus faecalis ATCC19443. As determined by Tricine-SDS-PAGE, the approximate molecular mass of the bacteriocin was 8.0 kDa. Loss in antimicrobial activity was recorded after treatment with proteolytic enzymes. Maximum activity of bacteriocin ST211Ch was measured in broth cultures of E. faecium strain ST211Ch after 24 h; thereafter, the activity was reduced. Bacteriocin ST211Ch remained active after exposure to various temperatures and pHs, as well as to Triton X-100, Tween-80, Tween-20, sodium dodecyl sulfate, NaCl, urea and EDTA. Effect of media components on production of bacteriocin ST211Ch was also studied. On the basis of PCR reactions targeting different bacteriocin genes, i.e. enterocins, curvacins and sakacins, no evidences for the presence of these genes in the total DNA of E. faecium strain ST211Ch was obtained. The bacterium most probably produced a bacteriocin different from those mentioned above. Based on the antimicrobial spectrum, stability and mode of action of bacteriocin ST211CH, E. faecium strain ST211Ch might be considered as a potential candidate with beneficial properties for use in biopreservation to control food spoilage bacteria.

scholarly journals Development of a recipe for a multicomponent mixture "Solodok +" to improve the consumer properties of bakery products

ScienceRise ◽  
2021 ◽  
pp. 18-24
Author(s):  
Olena Bilyk ◽  
Yulia Bondarenko ◽  
Oksana Kochubei-Lytvynenko ◽  
Liudmyla Burchenko
Keyword(s):  
Multicomponent Mixture ◽  
Proteolytic Enzymes ◽  
Experimental Studies ◽  
Optimal Dose ◽  
Bakery Products ◽  
Milk Whey ◽  
Baked Goods ◽  
Wide Range ◽  
Scientific Results ◽  
Technological Product

The object of research is the technology of bakery products enriched with a mixture of germinated grains of wheat, corn, barley and oats. Investigated problem: The problem of using a mixture of germinated grains is the formation of a closure sticky crumb in baked goods with a mixture. The reason for this is the high activity in the mixture of amylolytic and proteolytic enzymes. The solution to the problem consists in the developed multicomponent mixture (MM) to improve the consumer properties of bakery products, the formulation of which includes 15 % of the flour mass of the germinated grain mixture. Main scientific results: On the basis of experimental studies, the formulation of the "Solodok+" multicomponent mixture has been developed. The mixture contains: chicory inulin, dry milk whey enriched with Mg and Mn, apple pectin, phosphatide concentrate, enzyme preparation Deltamalt FN-A 50 and ascorbic acid. The optimal dose of the "Solodok+" MM for bakery products is 2.5 % by weight of flour. The area of practical use of the research results: "Solodok+" MM is recommended to be used in the production of bakery products enriched with sprouted grains at enterprises of the bakery industry of various capacities. An innovative technological product: "Solodok+" MM helps not only to reduce the stickiness of the crumb, improve its porosity, increase the volume of products, but also lengthen the freshness of unpackaged products. Scope of application of the innovative technological product: Bakery products with a mixture of sprouted grains and "Solodok+" MM have increased nutritional value, high consumer properties and are intended for a wide range of consumers.

scholarly journals Analysis of the lipid extraction performance in a cascade process for Scenedesmus almeriensis biorefinery

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
I. Papachristou ◽  
S. Akaberi ◽  
A. Silve ◽  
E. Navarro-López ◽  
R. Wüstner ◽  
...  
Keyword(s):  
Enzymatic Hydrolysis ◽  
Proteolytic Enzymes ◽  
Lipid Extraction ◽  
Protein Hydrolysis ◽  
Cascade Process ◽  
Disruptive Effect ◽  
Total Lipids ◽  
Water Fraction ◽  
Wide Range ◽  
Scenedesmus Almeriensis

Abstract Background Microalgae have attracted considerable interest due to their ability to produce a wide range of valuable compounds. Pulsed Electric Fields (PEF) has been demonstrated to effectively disrupt the microalgae cells and facilitate intracellular extraction. To increase the commercial viability of microalgae, the entire biomass should be exploited with different products extracted and valorized according to the biorefinery scheme. However, demonstrations of multiple component extraction in series are very limited in literature. This study aimed to develop an effective lipid extraction protocol from wet Scenedesmus almeriensis after PEF-treatment with 1.5 MJ·kgDW−1. A cascade process, i.e., the valorization of several products in row, was tested with firstly the collection of the released carbohydrates in the water fraction, then protein enzymatic hydrolysis and finally lipid extraction. Biomass processed with high pressure homogenization (HPH) on parallel, served as benchmark. Results Lipid extraction with ethanol:hexane (1:0.41 vol/vol) offered the highest yields from the different protocols tested. PEF-treatment promoted extraction with almost 70% of total lipids extracted against 43% from untreated biomass. An incubation step after PEF-treatment, further improved the yields, up to 83% of total lipids. Increasing the solvent volume by factor 2 offered no improvement. In comparison, extraction with two other systems utilizing only ethanol at room temperature or elevated at 60 °C were ineffective with less than 30% of total lipids extracted. Regarding cascade extraction, carbohydrate release after PEF was detected albeit in low concentrations. PEF-treated samples displayed slightly better kinetics during the enzymatic protein hydrolysis compared to untreated or HPH-treated biomass. The yields from a subsequent lipid extraction were not affected after PEF but were significantly increased for untreated samples (66% of total lipids), while HPH displayed the lowest yields (~ 49% of total lipids). Conclusions PEF-treatment successfully promoted lipid extraction from S. almeriensis but only in combination with a polar:neutral co-solvent (ethanol:hexane). After enzymatic protein hydrolysis in cascade processing; however, untreated biomass displayed equal lipid yields due to the disruptive effect of the proteolytic enzymes. Therefore, the positive impact of PEF in this scheme is limited on the improved reaction kinetics exhibited during the enzymatic hydrolysis step.

scholarly journals Oral Candidiasis: A Disease of Opportunity

2020 ◽  
Vol 6 (1) ◽  
pp. 15 ◽  
Author(s):  
Taissa Vila ◽  
Ahmed S. Sultan ◽  
Daniel Montelongo-Jauregui ◽  
Mary Ann Jabra-Rizk
Keyword(s):  
Immune Response ◽  
Oral Cavity ◽  
Oral Mucosa ◽  
Virulence Factors ◽  
Proteolytic Enzymes ◽  
Oral Candidiasis ◽  
Host Susceptibility ◽  
Initial Growth ◽  
Denture Stomatitis ◽  
Wide Range

Oral candidiasis, commonly referred to as “thrush,” is an opportunistic fungal infection that commonly affects the oral mucosa. The main causative agent, Candida albicans, is a highly versatile commensal organism that is well adapted to its human host; however, changes in the host microenvironment can promote the transition from one of commensalism to pathogen. This transition is heavily reliant on an impressive repertoire of virulence factors, most notably cell surface adhesins, proteolytic enzymes, morphologic switching, and the development of drug resistance. In the oral cavity, the co-adhesion of C. albicans with bacteria is crucial for its persistence, and a wide range of synergistic interactions with various oral species were described to enhance colonization in the host. As a frequent colonizer of the oral mucosa, the host immune response in the oral cavity is oriented toward a more tolerogenic state and, therefore, local innate immune defenses play a central role in maintaining Candida in its commensal state. Specifically, in addition to preventing Candida adherence to epithelial cells, saliva is enriched with anti-candidal peptides, considered to be part of the host innate immunity. The T helper 17 (Th17)-type adaptive immune response is mainly involved in mucosal host defenses, controlling initial growth of Candida and inhibiting subsequent tissue invasion. Animal models, most notably the mouse model of oropharyngeal candidiasis and the rat model of denture stomatitis, are instrumental in our understanding of Candida virulence factors and the factors leading to host susceptibility to infections. Given the continuing rise in development of resistance to the limited number of traditional antifungal agents, novel therapeutic strategies are directed toward identifying bioactive compounds that target pathogenic mechanisms to prevent C. albicans transition from harmless commensal to pathogen.

Bacillus velezensis Identification and Recombinant Expression, Purification and Characterization of Its Alpha-Amylase

Fermentation ◽  
2021 ◽  
Vol 7 (4) ◽  
pp. 227
Author(s):  
Xiaodong Zhang ◽  
Caixia Li ◽  
Xuantong Chen ◽  
Chonlong Chio ◽  
Sarita Shrestha ◽  
...  
Keyword(s):  
Recombinant Expression ◽  
Industrial Applications ◽  
Global Market ◽  
Tween 20 ◽  
Bacillus Velezensis ◽  
Modeled Structure ◽  
Wide Range ◽  
Relative Molecular Weight ◽  
Industrial Needs ◽  
Triton X

Amylases account for about 30% of the global market of industrial enzymes, and the current amylases cannot fully meet industrial needs. This study aimed to identify a high α-amylase producing bacterium WangLB, to clone its α-amylase coding gene, and to characterize the α-amylase. Results showed that WangLB belonged to Bacillus velezensis whose α-amylase gene was 1980 bp coding 659 amino acids designated as BvAmylase. BvAmylase was a hydrophilic stable protein with a signal peptide and a theoretical pI of 5.49. The relative molecular weight of BvAmylase was 72.35 kDa, and was verified by SDS-PAGE. Its modeled structure displayed that it was a monomer composed of three domains. Its optimum temperature and pH were 70 °C and pH 6.0, respectively. It also showed high activity in a wide range of temperatures (40–75 °C) and a relatively narrow pH (5.0–7.0). It was a Ca2+-independent enzyme, whose α-amylase activity was increased by Co2+, Tween 20, and Triton X-100, and severely decreased by SDS. The Km and the Vmax of BvAmylase were 3.43 ± 0.53 and 434.19 ± 28.57 U/mg. In conclusion, the α-amylase producing bacterium WangLB was identified, and one of its α-amylases was characterized, which will be a candidate enzyme for industrial applications.

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