Search results for: enzyme production from fungi

Authors: B. Atika, S. Lehmann, E. Wimmer

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The defense strategy is very common in the insect world. Defensive substances play a wide variety of functions for beetles, such as repellents, toxicants, insecticides, and antimicrobics. Beetles react to predators, invaders, and parasitic microbes with the release of toxic and repellent substances. Defensive substances are directed against a large array of potential target organisms or may function for boiling bombardment or as surfactants. Usually, Coleoptera biosynthesize and store their defensive compounds in a complex secretory organ, known as odoriferous defensive stink glands. The red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae), uses these glands to produce antimicrobial p-benzoquinones and 1-alkenes. In the past, the morphology of stink gland has been studied in detail in tenebrionid beetles; however, very little is known about the genes that are involved in the production of gland secretion. In this study, we studied a subset of genes that are essential for the benzoquinone production in red flour beetle. In the first phase, we selected 74 potential candidate genes from a genome-wide RNA interference (RNAi) knockdown screen named 'iBeetle.' All these 74 candidate genes were functionally characterized by RNAi-mediated gene knockdown. Therefore, they were selected for a subsequent gas chromatography-mass spectrometry (GC-MS) analysis of secretion volatiles in respective RNAi knockdown glands. 33 of them were observed to alter the phenotype of stink gland. In the GC-MS analysis, 7 candidate genes were noted to display a strongly altered gland, in terms of secretion color and chemical composition, upon knockdown, showing their key role in the biosynthesis of gland secretion. Morphologically altered stink glands were found for odorant receptor and protein kinase superfamily. Subsequent GC-MS analysis of secretion volatiles revealed reduced benzoquinone levels in LIM domain, PDZ domain, PBP/GOBP family knockdowns and a complete lack of benzoquinones in the knockdown of sulfatase-modifying factor enzyme 1, sulfate transporter family. Based on stink gland transcriptome data, we analyzed the function of sulfatase-modifying factor enzyme 1 and sulfate transporter family via RNAi-mediated gene knockdowns, GC-MS, in situ hybridization, and enzymatic activity assays. Morphologically altered stink glands were noted in knockdown of both these genes. Furthermore, GC-MS analysis of secretion volatiles showed a complete lack of benzoquinones in the knockdown of these two genes. In situ hybridization showed that these two genes are expressed around the vesicle of certain subgroup of secretory stink gland cells. Enzymatic activity assays on stink gland tissue showed that these genes are involved in p-benzoquinone biosynthesis. These results suggest that sulfatase-modifying factor enzyme 1 and sulfate transporter family play a role specifically in benzoquinone biosynthesis in red flour beetles.

Keywords: Red Flour Beetle, defensive stink gland, benzoquinones, sulfate transporter, sulfatase-modifying factor enzyme 1

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Authors: Laref Nora, Guessas Bettache

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Lactic acid bacteria (LAB) have a major potential to be used in biopreservation methods because they are safe to consume (GRAS: generally regarded as safe) and they naturally occurring microflora of many foods. The preservative action of LAB is due to several antimicrobial metabolites, including lactic acid, acetic acid, hydrogen peroxide, bacteriocins, carbon dioxide, diacetyl, and reuterin. Several studies have focused on the antifungal activity compounds from natural sources for biopreservation in alternatives to chemical use. LAB has an antifungal activity which may inhibit food spoilage fungi. Lactobacillus strains isolated from silage prepared in our laboratory by fermentation of grass in anaerobic condition were screened for antifungal activity with overlay assay against Aspergillus spp. The antifungal compounds were originated from organic acids; inhibitory activity did not change after treatment with proteolytic enzymes. Lactobacillus strains were able also to inhibit Trichoderma spp, Penicillium spp, Fusarium roseum, and Stemphylim spp by confrontation assay. The inhibitory activity could be detected against the mould Aspergillus spp in the apricot juice but not in a bakery product. These antifungal compounds have the potential to be used as food biopreservation to inhibit conidia germination, and mycelia growth of spoilage fungi depending on food type, pH of food especially in heat, and cold processed foods.

Keywords: lactic acid bacteria, Lactobacillus, Aspergillus, antifungal activity

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Authors: Özlem Ateş Sönmezoğlu, Begüm Terzi, Ahmet Yıldırım, Ramazan Özbey

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The basic criteria which determine durum wheat quality is its suitability for pasta processing that is pasta making quality. Bright yellow color is a desired property in pasta products. Durum wheat pasta making quality is affected by grain pigment content and oxidative enzymes which affect adversely bright yellow color. Of the oxidative enzymes, lipoxygenase LOX is the most effective one on oxidative bleaching of yellow pigments in durum wheat products. Thus, wheat cultivars that are high in yellow pigments but low in LOX enzyme activity should be preferred for the production of pasta with high color quality. The aim of this study was to reduce lipoxygenase activities of the backcross durum wheat lines that were previously improved for their protein quality. For this purpose, two advanced lines with different parents (TMB2 and TMB3) were used recurrent parents. Also, Gediz-75 wheat with low LOX enzyme activity was used as the gene source. In all of the generations, backcrossed plants carrying the targeted gene region (Lpx-B1.1) were selected using SSR markers by marker assisted selection method. As a result, the study will be completed in three years instead of six years required in a classical backcross breeding study, leading to the development of high-quality candidate varieties. This research has been financially supported by TÜBİTAK (Project No: 112T910).

Keywords: durum wheat, lipoxygenase, LOX, Lpx-B1.1, MAS, Triticum durum

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Authors: Alireza Naseri, Susan L. Holdt, Charlotte Jacobsen

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In 2014, the global amount of carrageenan production was 60,000 ton with a value of US$ 626 million. From this number, it can be estimated that the total dried seaweed consumption for this production was at least 300,000 ton/year. The protein content of these types of seaweed is 5 – 25%. If just half of this total amount of protein could be extracted, 18,000 ton/year of a high-value protein product would be obtained. The overall aim of this study was to develop a technology that will ensure further utilization of the seaweed that is used only as raw materials for carrageenan production as single extraction at present. More specifically, proteins should be extracted from the seaweed either before or after extraction of carrageenan with focus on maintaining the quality of carrageenan as a main product. Different mechanical, chemical and enzymatic technologies were evaluated. The optimized process was implemented in lab scale and based on its results; the new experiments were done a pilot and larger scale. In order to calculate the efficiency of the new upstream multi-extraction process, protein content was tested before and after extraction. After this step, the extraction of carrageenan was done and carrageenan content and the effect of extraction on yield were evaluated. The functionality and quality of carrageenan were measured based on rheological parameters. The results showed that by using the new multi-extraction process (submitted patent); it is possible to extract almost 50% of total protein without any negative impact on the carrageenan quality. Moreover, compared to the routine carrageenan extraction process, the new multi-extraction process could increase the yield of carrageenan and the rheological properties such as gel strength in the final carrageenan had a promising improvement. The extracted protein has initially been screened as a plant protein source in typical food applications. Further work will be carried out in order to improve properties such as color, solubility, and taste.

Keywords: carrageenan, extraction, protein, seaweed

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Authors: Lintle Mohase, Ninikoe Lebusa, Mpho Stephen Mafa

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Wheat is an economically important cereal crop. However, the changing climatic conditions that intensify drought in production areas, and additional pest infestation, such as the Russian wheat aphid (RWA, Diuraphis noxia), severely hamper its production. Drought and pest management require an additional water supply through irrigation and applying inorganic nutrients (including silicon) as alternative strategies to mitigate the stress effects. Therefore, other approaches are needed to enhance wheat productivity during drought stress and aphid abundance. Two wheat cultivars were raised under greenhouse conditions, exposed to drought stress, and treated with silicon before infestation with the South African RWA biotype 2 (RWASA2). The morphological evaluations showed that severe drought or a combination of drought and infestation significantly reduced the plant height of wheat cultivars. Silicon treatment did not alleviate the growth reduction. The biochemical responses were measured using spectrophotometric assays with specific substrates. An evaluation of the enzyme activities associated with oxidative stress and defence responses indicated that drought stress increased NADPH oxidase activity, while silicon treatment significantly reduced it in drought-stressed and infested plants. At 48 and 72 hours sampling periods, a combination of silicon, drought and infestation treatment significantly increased peroxidase activity compared to drought and infestation treatment. The treatment also increased β-1,3-glucanase activity 72 hours after infestation. In addition, silicon and drought treatment increased glucose but reduced sucrose accumulation. Furthermore, silicon, drought, and infestation treatment combinations reduced the sucrose content. Finally, silicon significantly increased the trehalose content under severe drought and infestation, evident at 48 and 72-hour sampling periods. Our findings shed light on silicon’s ability to induce protective biochemical responses during drought and aphid infestation.

Keywords: drought, enzyme activity, silicon, soluble sugars, Russian wheat aphid, wheat

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Authors: Tamar Khardziani, Violeta Berikashvili, Mariam Rusitashvili, Eva Kachlishvili, Vladimir Elisashvili, Mikheil Asatiani

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Last years, the search for natural sources of novel and effective antifungal substances became a scientific and technological challenge. In the present research, thirty basidiomycetes isolated from various ecological niches of Georgia and belonging to different taxonomic groups were screened for their antifungal activities against pathogenic fungi such as Aspergillus, Fusarium, and Guignardia bidwellii. Among mushroom tested, several potential producers of antifungal substances have been revealed, such as Schizophyllum commune, Lentinula edodes, Ganoderma abietinum, Fomes fomentarius, Hericium erinaceus, and Trametes versicolor. For mushroom cultivation and expression of antifungal potential, submerged and solid-state fermentations of different plant raw materials were performed and various approaches and strategies have been exploited. Sch. commune appeared as a most promising producer of antifungal compounds. It was established that among different agro-industrial wastes, the presence of mandarin juice production waste in a nutrient medium, causing the significant increase of antifungal activity Sch. commune (growth inhibition: Aspergillus – 59 %, Fusarium – 55 %, G. bidwellii – 78 %, after 3, 2 and 4 days of cultivation, respectively). Besides this, Sch. commune demonstrate similar antifungal activities in the presence of glucose, glycerol, maltose, mannitol, and xylose, and growth inhibition of Fusarium ranged in 41 % - 49 % during 6 days of cultivation. Inhibition of Aspergillus growth inhibition varied in 27 % - 36 %, and inhibition of G. bidwellii was in the range 49 % - 61 %, respectively. Sch. commune under solid-state fermentation of mandarin peels at 13 days of cultivation demonstrates powerful growth inhibition of pathogenic fungi (growth inhibition: Aspergillus – 50 %, Fusarium – 61 %, G. bidwellii – 68 %, after 3, 4, and 4 days of cultivation, respectively) as well as at 20 days old mushroom (growth inhibition: Aspergillus – 41 %, Fusarium – 54 %, G. bidwellii – 66 %, after 3 days of cultivation). It was established that Sch. commune was effective as a producer of antifungal compounds in submerged as well as in solid-state fermentation. Finally, performed study confirms that the higher basidiomycetes possess antifungal potential, which strongly depends on the physiological factors of growth. Acknowledgments: The work was implemented with the financial support of fundamental science project FR-19-3719 by the Shota Rustaveli National Science Foundation of Georgia.

Keywords: antifungal potential, higher basidiomycetes, pathogenic fungi, submerged and solid-state fermentation

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Authors: F. Boukli Hacene, W. Soufi, S. Ghalem

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Glaucoma disease is a progressive degenerative optic neuropathy, with irreversible visual field deficits and high eye pressure being one of the risk factors. Sulfonamides are carbonic anhydrase-II inhibitors that aim to decrease the secretion of aqueous humor by direct inhibition of this enzyme at the level of the ciliary processes. These drugs present undesirable effects that are difficult to accept by the patient. In our study, we are interested in the inhibition of carbonic anhydrase-II by different natural ligands (curcumin analogues) using molecular modeling methods using molecular operating environment (MOE) software to predict their interaction with this enzyme.

Keywords: carbonic anhydrase-II, curcumin analogues, drug research, molecular modeling

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Authors: Ali Bahri Lubis

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Tryptophan oxidation by Heme-dioxygenase enzyme is the initial rate-limiting step in the kynurenine pathway, which leads to the formation of NADH and dangerous metabolites, implicating several severe diseases such as Parkinson’s Disease, Huntington's Disease, poliomyelitis and cataract. This oxidation, generally, allows tryptophan to convert to N-Formylkynurenine (NFK). Observing the catalytic mechanism of Heme dioxygenase in tryptophan oxidation has been a debatably scientific interest since no one has yet proven the mechanism obviously. In this research we have attempted to prove mechanistic steps of tryptophan oxidation via human indoleamine dioxygenase (h-IDO) utilising various substrates: L-tryptophan, L-tryptophan (indole-ring-2-¹³C), L-fully-labelled¹³C-tryptophan, L-N-methyl-tryptophan, L-tryptophanol and 2-amino-3-(benzo(b)thiophene-3-yl) propanoic acid. All enzyme assay experiments were measured using a UV-Vis spectrophotometer, LC-MS, 1H-NMR and HSQC. We also successfully synthesised enzyme products as our control in NMR measurements. The result exhibited that all substrates produced N-formyl kynurenine (NFK), and a side, the minor product of hydroxypyrrolloindoleamine carboxylic acid (HPIC) in cis and trans isomer, except 1-methyl tryptophan only generating cis HPIC. Interestingly, L- tryptophanol was oxidised to form HPIC derivative as a major product and 5-hydroxy tryptophan was converted to NFK derivative instead without any HPIC derivative. The bizarre result of oxidation underwent in 2-amino-3-(benzo(b)thiophene-3-yl) propanoic acid, which produced epoxide cyclic. Those phenomena have been explainable in our research based on the proposed mechanism of how tryptophan is oxidised by human indoleamine dioxygenase.

Keywords: tryptophan oxidation, heme-dioxygenases, human indoleamine dioxygenases, N-formylkynurenine, hydroxypyrroloindoleamine carboxylic acid

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Authors: Muhammad Adeel Arshad, Faiz-Ul Hassan, Yanfen Cheng

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According to FAO, enteric methane (CH4) production is about 44% of all greenhouse gas emissions from the livestock sector. Ruminants produce CH4 as a result of fermentation of feed in the rumen especially from roughages which yield more CH4 per unit of biomass ingested as compared to concentrates. Efficient ruminal fermentation is not possible without abating CO2 and CH4. Methane abatement strategies are required to curb the predicted rise in emissions associated with greater ruminant production in future to meet ever increasing animal protein requirements. Ecology of ruminal methanogenesis and avenues for its mitigation can be identified through various genomic and transcriptomic techniques. Programs such as Hungate1000 and the Global Rumen Census have been launched to enhance our understanding about global ruminal microbial communities. Through Hungate1000 project, a comprehensive reference set of rumen microbial genome sequences has been developed from cultivated rumen bacteria and methanogenic archaea along with representative rumen anaerobic fungi and ciliate protozoa cultures. But still many species of rumen microbes are underrepresented especially uncultivable microbes. Lack of sequence information specific to the rumen's microbial community has inhibited efforts to use genomic data to identify specific set of species and their target genes involved in methanogenesis. Metagenomic and metatranscriptomic study of entire microbial rumen populations offer new perspectives to understand interaction of methanogens with other rumen microbes and their potential association with total gas and methane production. Deep understanding of methanogenic pathway will help to devise potentially effective strategies to abate methane production while increasing feed efficiency in ruminants.

Keywords: Genome sequences, Hungate1000, methanogens, ruminal fermentation

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Authors: R. D. Esposito, V. M. Barberá, P. García Morales, P. Dorado Rodríguez, J. Sanz, M. Fuentes, D. Planes Meseguer, M. Saceda, L. Fernández Fornos, M. P. Ventero

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Results obtained by our group on glioblastoma multiforme (GBM) primary cultures , show a dramatic potentiation of radiation effects when 2 units/ml of D-amino acid oxidase (DAO) enzyme are added, free or immobilized in magnetic nanoparticles, to irradiated samples just after the irradiation. Cell cultures were exposed to radiation doses of 7Gy and 15Gy of 6 MV photons from a clinical linear accelerator. At both doses, we observed a clear enhancing effect of radiation-induced damages due to the addition of DAO.

Keywords: D-amino Acid Oxidase (DAO) enzyme, magnetic particles, nanotechnology, radiation therapy enhancement

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Authors: Anna Zimoch-Korzycka, Dominika Kulig, Andrzej Jarmoluk

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The aim of this study was to obtain chitooligosaccharides from chitosan with better functional properties using three different enzyme preparations and compare the products of enzymatic hydrolysis. Commercially available cellulase (CL), xylanase (X) and chitosanase (CS) preparations were used to investigate hydrolytic activity on chitosan (CH) with low molecular weight and DD of 75-85%. It has been reported that CL and X have side activities of other enzymes, such as β-glucanase or β-glucosidase. CS enzyme has a foreign activity of chitinase. Each preparation was used in 1000 U of activity and in the same reaction conditions. The degree of deacetylation and molecular weight of chitosan were specified using titration and viscometric methods, respectively. The hydrolytic activity of enzymes preparations on chitosan was monitored by dynamic viscosity measurement. After 4 h reaction with stirring, solutions were filtered and chitosan oligomers were isolated by methanol solution into two fractions: precipitate (A) and supernatant (B). A Fourier-transform infrared spectroscopy was used to characterize the structural changes of chitosan oligomers fractions and initial chitosan. Furthermore, the solubility of lyophilized hydrolytic mixture (C) and two chitooligomers fractions (A, B) of each enzyme hydrolysis was assayed. The antioxidant activity of chitosan oligomers was evaluated as DPPH free radical scavenging activity. The dynamic viscosity measured after addition of enzymes preparation to the chitosan solution decreased dramatically over time in the sample with X in comparison to solution without the enzyme. For mixtures with CL and CS, lower viscosities were also recorded but not as low as the ones with X. A and B fractions were characterized by the most similar viscosity obtained by the xylanase hydrolysis and were 15 mPas and 9 mPas, respectively. Structural changes of chitosan oligomers A, B, C and their differences related with various enzyme preparations used were confirmed. Water solubility of A fractions was not possible to filter and the result was not recorded. Solubility of supernatants was approximately 95% and was higher than hydrolytic mixture. It was observed that the DPPH radical scavenging effect of A, B, C samples is the highest for X products and was approximately 13, 17, 19% respectively. In summary, a mixture of chitooligomers may be useful for the design of edible protective coatings due to the improved biophysical properties.

Keywords: cellulase, xylanase, chitosanase, chitosan, chitooligosaccharides

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Authors: F. Keller, G. Reinhart

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The efficient and economic allocation of resources is one main goal in the field of production planning and control. Nowadays, a new variable gains in importance throughout the planning process: Energy. Energy-efficiency has already been widely discussed in literature, but with a strong focus on reducing the overall amount of energy used in production. This paper provides a brief systematic approach, how energy-supply-orientation can be used for an energy-cost-efficient production planning and thus combining the idea of energy-efficiency and energy-flexibility.

Keywords: production planning, production control, energy-efficiency, energy-flexibility, energy-supply

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Authors: Wannee Sutthachaidee

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The study of the production process of coconut-shell product in Amphawa, Samutsongkram Province is objected to study the pattern of the process of coconut-shell product by focusing in the 3 main processes which are inbound logistics process, production process and outbound process. The result of the research: There were 4 main results from the study. Firstly, most of the manufacturer of coconut-shell product is usually owned by a single owner and the quantity of the finished product is quite low and the main labor group is local people. Secondly, the production process can be divided into 4 stages which are pre-production process, production process, packaging process and distribution process. Thirdly, each 3 of the logistics process of coconut shell will find process which may cause the problem to the business but the process which finds the most problem is the production process because the production process needs the skilled labor and the quantity of the labor does not match with the demand from the customers. Lastly, the factors which affect the production process of the coconut shell can be founded in almost every process of the process such as production design, packaging design, sourcing supply and distribution management.

Keywords: production process, coconut-shell product, Amphawa District, inbound logistics process

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Authors: Pooja Kumari, Anandkumar Tengli

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Aurora kinase enzyme, a protein on overexpression, leads to metastasis and is extremely important for women’s health in terms of prevention or treatment. While creating a targeted technique, the aim of the work is to design purine molecules that inhibit in aurora kinase enzyme and helps to suppress breast cancer. Purine molecules attached to an amino acid in DNA block protein synthesis or halt the replication and metastasis caused by the aurora kinase enzyme. Various protein related to the overexpression of aurora protein was docked with purine molecule using Biovia Drug Discovery, the perpetual software. Various parameters like X-ray crystallographic structure, presence of ligand, Ramachandran plot, resolution, etc., were taken into consideration for selecting the target protein. A higher negative binding scored molecule has been taken for simulation studies. According to the available research and computational analyses, purine compounds may be powerful enough to demonstrate a greater affinity for the aurora target. Despite being clinically effective now, purines were originally meant to fight breast cancer by inhibiting the aurora kinase enzyme. In in-silico studies, it is observed that purine compounds have a moderate to high potency compared to other molecules, and our research into the literature revealed that purine molecules have a lower risk of side effects. The research involves the design, synthesis, and identification of active purine molecules against breast cancer. Purines are structurally similar to the normal metabolites of adenine and guanine; hence interfere/compete with protein synthesis and suppress the abnormal proliferation of cells/tissues. As a result, purine target metastasis cells and stop the growth of kinase; purine derivatives bind with DNA and aurora protein which may stop the growth of protein or inhibits replication and stop metastasis of overexpressed aurora kinase enzyme.

Keywords: aurora kinases, in silico studies, medicinal chemistry, combination therapies, chronic cancer, clinical translation

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Authors: Yves Mann Elate Lea Mbassi, Marie Solange Evehe Bebandoue, Wilfred Fon Mbacham

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Improving the catalytic performance of enzymes has been a long-standing theme of analytical biochemistry research. Induction of peroxidase activity by metals is a common reaction in higher plants. We thought that this increase in peroxidase activity may be due, on the one hand, to the stimulation of the gene expression of these enzymes but also to a modification of their chemical reactivity following the binding of some metal ions on their active site. We tested the effect of some metal salts (MgCl₂, MnCl₂, ZnCl₂, CaCl₂ and CuSO₄) on the activity and thermostability of peroxidase A6, a thermostable peroxidase that we discovered and purified in a previous study. The chromogenic substrate used was 3,3′,5,5′-tetramethylbenzidine. Of all the metals tested for their effect on A6, only magnesium and copper had a significant effect on the activity of the enzyme at room temperature. The Mann-Whitney test shows a slight inhibitory effect of activity by the magnesium salt (P = 0.043), while the activity of the enzyme is 5 times higher in the presence of the copper salt (P = 0.002). Moreover, the thermostability of peroxidase A6 is increased when calcium and copper salts are present. The activity in the presence of CaCl₂ is 8 times higher than the residual activity of the enzyme alone after incubation at 80°C for 10 min and 35 times higher in the presence of CuSO4 under the same conditions. In addition, manganese and zinc salts slightly reduce the thermostability of the enzyme. The activity and structural stability of peroxidase A6 can clearly be activated by Cu₂+, which therefore enhance the oxidation of 3,3′,5,5′-tetramethylbenzidine, which was used in this study as a chromogenic substrate. Ca₂+ likely has a more stabilizing function for the catalytic site.

Keywords: peroxidase activity, copper ions, calcium ions, thermostability

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Authors: Muhammad Farooq Baig, Saleem Qadeer

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Background: The frequency of hepatitis C virus infection along with tuberculosis has not been widely investigated and very low statistics on rates of hepatitis C virus co-infection in tuberculosis patients. Hepatotoxicity is the major side effect of anti-tuberculosis therapy hepatitis HCVliver disease elevates the chances of hepatotoxicity up-to five folds. Objectives & Aim: To see the frequency of Hepatitis Cvirus infection amongst people with diagnosed Tuberculosis using gene X-pert technique. To evaluate the factors associated with HCVinfection in patients with MTBtuberculosis and to determine sensitivity and specificity of the tests. Study design: Comparative analytical study. Methodology: Three hundred and thirteen patients of tuberculosis diagnosed by Genexpert included while testing hepatitis C virus using immunochromotography rapid test technique, enzyme linked immunosorbent assay method and polymerase chain reaction test for confirmation. Results:Higher frequency of tuberculosis infection in males 57.8%, 42.5% between 20-39 years and 22% of hepatitis C virus infection in tuberculosis patients.The sensitivity of rapid test and enzyme-linked immunosorbent assay was 79% and 96% respectively while the specificity of rapid test and enzyme-linked immunosorbent assay was 91% and 99% respectively.

Keywords: Mycobactrium Tuberculosis, PC'R, Gene x pert, Hepatitis C virus

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Authors: Z. Mmango, U. Nwodo, L. V. Mabinya, A. I. Okoh

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Cellulose and hemicellulose account for a large portion of the world‘s plant biomass. In nature, these polysaccharides are intertwined forming complex materials that requires multiple and expensive treatment processes to free up the raw materials trapped in the matrix. Enzymatic degradation remains as the preferred technique as it is inexpensive and eco-friendly. However, the insufficiencies of enzyme battery systems in the degradation of lignocellulosic complex motivate the search for effective degrading enzymes from bacterial isolates from uncommon environment. The study aimed at the evaluation of actinomycetes isolated from saw dust samples collected from wood factory under bed. Cellulase and xylanase production was screened through organism culture on carboxyl methyl cellulose agar and Birchwood xylan. Halo zone indicating lignocellose utilization was shown by an isolate identified through 16S rRNA gene as Micrococcus luteus. The optimum condition for the production of cellulase and xylanase were incubation temperature of 25 °C, fermentation medium pH 5 and 10, agitation speed of 50 and 200 (rpm) and fermentation incubation time of 96 and 84 (h) respectively. The high cellulose and xylanase activity obtained from this isolate portends industrial relevance.

Keywords: carboxyl methyl cellulose, birchwood xylan, optimization, cellulase, xylanase, micrococcus, DNS method

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Authors: Konja Knüppel, Gerrit Meyer, Peter Nyhuis

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The increasing interconnectedness and complexity of production processes raise the susceptibility of production systems to failure. Therefore, the ability to respond quickly to failures is increasingly becoming a competitive factor. The research project "Sustainable failure management in manufacturing SMEs" is developing a methodology to identify failures in the production and select preventive and reactive measures in order to correct failures and to establish sustainable failure management systems.

Keywords: failure categorization, failure management, logistic performance, production optimization

Procedia PDF Downloads 339

Authors: D. Shehu, Z. Alias

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Glutathione S-transferases (GSTs) are family of enzymes that function in the detoxification of variety of electrophilic substrates. In the present work, we report a novel zeta-like GST (designated as KKSG9) from the biphenyl/polychlorobiphenyl degrading organism Acidovorax sp. KKS102. KKSG9 possessed low sequence similarity but similar biochemical properties to zeta class GSTs. The gene for KKSG9 was cloned, purified and biochemically characterized. Functional analysis showed that the enzyme exhibits wider substrate specificity compared to most zeta class GSTs by reacting with 1-chloro-2,4-dinitrobenzene (CDNB), p-nitrobenzyl chloride (NBC), ethacrynic acid (EA), hydrogen peroxide, and cumene hydroperoxide (CuOOH). The enzyme also displayed dehalogenation function against dichloroacetate (a common substrate for zeta class GSTs) in addition to permethrin, and dieldrin. The functional role of Tyr12 was also investigated by site-directed mutagenesis. The mutant (Y12C) displayed low catalytic activity and dehalogenation function against all the substrates when compared with the wild type. Kinetic analysis using NBC and GSH as substrates showed that the mutant (Y12C) displayed a higher affinity for NBC when compared with the wild type, however, no significant change in GSH affinity was observed. These findings suggest that the presence of tyrosine residue in the motif might represent an evolutionary trend toward improving the catalytic activity of the enzyme. The enzyme as well could be useful in the bioremediation of various types of organochlorine pollutants.

Keywords: Acidovorax sp. KKS102, bioremediation, glutathione s-transferase, site-directed mutagenesis, zeta

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Authors: Milica Carević, Katarina Banjanac, Marija ĆOrović, Ana Milivojević, Nevena Prlainović, Aleksandar Marinković, Dejan Bezbradica

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Nowadays, considering the growing awareness of functional food beneficial effects on human health, due attention is dedicated to the research in the field of obtaining new prominent products exhibiting improved physiological and physicochemical characteristics. Therefore, different approaches to valuable bioactive compounds synthesis have been proposed. β-Galactosidase, for example, although mainly utilized as hydrolytic enzyme, proved to be a promising tool for these purposes. Namely, under the particular conditions, such as high lactose concentration, elevated temperatures and low water activities, reaction of galactose moiety transfer to free hydroxyl group of the alternative acceptor (e.g. different sugars, alcohols or aromatic compounds) can generate a wide range of potentially interesting products. Up to now, galacto-oligosaccharides and lactulose have attracted the most attention due to their inherent prebiotic properties. The goal of this study was to obtain a novel product sorbitol galactoside, using the similar reaction mechanism, namely transgalactosylation reaction catalyzed by β-galactosidase from Aspergillus oryzae. By using sugar alcohol (sorbitol) as alternative acceptor, a diverse mixture of potential prebiotics is produced, enabling its more favorable functional features. Nevertheless, an introduction of alternative acceptor into the reaction mixture contributed to the complexity of reaction scheme, since several potential reaction pathways were introduced. Therefore, the thorough optimization using response surface method (RSM), in order to get an insight into different parameter (lactose concentration, sorbitol to lactose molar ratio, enzyme concentration, NaCl concentration and reaction time) influences, as well as their mutual interactions on product yield and productivity, was performed. In view of product yield maximization, the obtained model predicted optimal lactose concentration 500 mM, the molar ratio of sobitol to lactose 9, enzyme concentration 0.76 mg/ml, concentration of NaCl 0.8M, and the reaction time 7h. From the aspect of productivity, the optimum substrate molar ratio was found to be 1, while the values for other factors coincide. In order to additionally, improve enzyme efficiency and enable its reuse and potential continual application, immobilization of β-galactosidase onto tailored silica nanoparticles was performed. These non-porous fumed silica nanoparticles (FNS)were chosen on the basis of their biocompatibility and non-toxicity, as well as their advantageous mechanical and hydrodinamical properties. However, in order to achieve better compatibility between enzymes and the carrier, modifications of the silica surface using amino functional organosilane (3-aminopropyltrimethoxysilane, APTMS) were made. Obtained support with amino functional groups (AFNS) enabled high enzyme loadings and, more importantly, extremely high expressed activities, approximately 230 mg proteins/g and 2100 IU/g, respectively. Moreover, this immobilized preparation showed high affinity towards sorbitol galactoside synthesis. Therefore, the findings of this study could provided a valuable contribution to the efficient production of physiologically active galactosides in immobilized enzyme reactors.

Keywords: β-galactosidase, immobilization, silica nanoparticles, transgalactosylation

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Authors: Katie Emerson, Sara Perez-Ojalvo, Jim Komorowski, Danielle Greenberg

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The purpose of this study was to evaluate arginase activity levels in response to combinations of an inositol-stabilized arginine silicate (ASI; Nitrosigine®), L-arginine, and Inositol. Arginine acts as a vasodilator that promotes increased blood flow resulting in enhanced delivery of oxygen and nutrients to the brain and other tissues. ASI alone has been shown to improve performance on cognitive tasks. Arginase, found in human serum, catalyzes the conversion of arginine to ornithine and urea, completing the last step in the urea cycle. Decreasing arginase levels maintains arginine and results in increased nitric oxide production. This study aimed to determine the most effective combination of ASI, L-arginine and inositol for minimizing arginase levels and therefore maximize ASI’s effect on cognition. Serum was taken from untreated healthy donors by separation from clotted factors. Arginase activity of serum in the presence or absence of test products was determined (QuantiChrom™, DARG-100, Bioassay Systems, Hayward CA). The remaining ultra-filtrated serum units were harvested and used as the source for the arginase enzyme. ASI alone or combined with varied levels of Inositol were tested as follows: ASI + inositol at 0.25 g, 0.5 g, 0.75 g, or 1.00 g. L-arginine was also tested as a positive control. All tests elicited changes in arginase activity demonstrating the efficacy of the method used. Adding L-arginine to serum from untreated subjects, with or without inositol only had a mild effect. Adding inositol at all levels reduced arginase activity. Adding 0.5 g to the standardized amount of ASI led to the lowest amount of arginase activity as compared to the 0.25g 0.75g or 1.00g doses of inositol or to L-arginine alone. The outcome of this study demonstrates an interaction of the pairing of inositol with ASI on the activity of the enzyme arginase. We found that neither the maximum nor minimum amount of inositol tested in this study led to maximal arginase inhibition. Since the inhibition of arginase activity is desirable for product formulations looking to maintain arginine levels, the most effective amount of inositol was deemed preferred. Subsequent studies suggest this moderate level of inositol in combination with ASI leads to cognitive improvements including reaction time, executive function, and concentration.

Keywords: arginine, inositol, arginase, cognitive benefits

Procedia PDF Downloads 81

Authors: Namsoo Kim, So-Hee Son, Jin-Soo Maeng, Yong-Jin Cho, Chong-Tai Kim

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It has been reported that some enzymes such as trypsin and Alcalase 2.4L are tolerant to a medium high pressure of 300 MPa and preparation of protein hydrolyzates under 300 MPa was advantageous with regard to hydrolysis rate and thus production yield compared with the counterpart under ambient pressure.1,2) In this study, nine fish comprising halibut, soft shell clam and carp were hydrolyzed using Flavourzyme 500MG only, and the combination of Flavourzyme 500 mg, Alcalase 2.4 L, Marugoto E, and Protamex under 300 MPa. Then, the effects of single and multiple protease treatments were determined with respect to contents of soluble solid (SS) and soluble nitrogen, sensory attributes, electrophoretic profiles, and HPLC peak patterns of the fish hydrolyzates (FHs) from various species. The contents of SS of the FHs were quite species-specific and the hydrolyzates of halibut showed the highest SS contents. At this point, multiple protease treatment increased SS content conspicuously in all fish tested. The contents of total soluble nitrogen and TCA-soluble nitrogen were well correlated with those of SS irrespective of fish species and methods of enzyme treatment. Also, it was noticed that multiple protease treatment improved sensory attributes of the FHs considerably. Electropherograms of the FHs showed fast migrating peptide bands that had the molecular masses mostly lower than 1 kDa and this was confirmed by peptide patterns from HPLC analysis for some FHs that had good sensory quality.

Keywords: production, fish hydrolyzates, protease treatments, high pressure

Procedia PDF Downloads 251

Authors: Lela Pintarić, Iva Rezić, Ana Vrsalović Presečki

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Considering an enormous increase of the use of metal nanoparticles with the exactly defined characteristics, the main goal of this research was to found the optimal and environmental friendly method of their synthesis. The synthesis of the inorganic core-shell nanoparticles was optimized as a model. The core-shell nanoparticles are composed of the enzyme core belted with the metal ions, oxides or salts as a shell. In this research, enzyme urease was the core catalyst and the shell nanoparticle was made of silver. Silver nanoparticles are widespread utilized and some of their common uses are: as an addition to disinfectants to ensure an aseptic environment for the patients, as a surface coating for neurosurgical shunts and venous catheters, as an addition to implants, in production of socks for diabetics and athletic clothing where they improve antibacterial characteristics, etc. Characteristics of synthesized nanoparticles directly depend on of their size, so the special care during this optimization was given to the determination of the size of the synthesized nanoparticles. For the purpose of the above mentioned optimization, sixteen experiments were generated by the Design of Experiments (DoE) method and conducted under various temperatures, with different initial concentration of the silver nitrate and constant concentration of the urease of two separate manufacturers. Synthesized nanoparticles were analyzed by the Nanoparticle Tracking Analysis (NTA) method on Malvern NanoSight NS300. Results showed that the initial concentration of the silver ions does not affect the concentration of the synthesized silver nanoparticles neither their size distribution. On the other hand, temperature of the experiments has affected both of the mentioned values.

Keywords: core-shell nanoparticles, optimization, silver, urease

Procedia PDF Downloads 282

Authors: Harison Masih, Jyotsna Kiran Peter, Sundara Singh, Geetha Singh

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The present investigation deals with diversity of dermatophytes and keratinophilic fungi from different tourist spots such as Agra Fort, Akbar tomb, It-Mat-Ud-Daulah, Mariam tomb, Radha Swami Bagh, and Taj Mahal of Agra City. These fungi are medically important which causes various infections and diseases in humans and animals. The main reservoir of these pathogens are the keratinous substances that increases due to birds and animal activities in the vicinity of monuments, where thousands (5413266) annual visitors from all over the world are visiting. The soil samples were subjected to isolate the pathogenic fungi through bait technique (buffalo skin, chicken feathers, human hair and goat tail hair). Baits were spread over the soil samples and incubated at room temperature for 30-35 days and pure culture isolates were maintained in SDA medium, stored at 4°C. Highest number of visitors were (3906453) from Taj Mahal, minimum 10785 at Mariam tomb annually, the total 271 isolates were encountered from soil samples out of these 18 genera and 38 species were found in different season. Highest incidence was 4.79% frequency shown by Chrysosporium keratinophilum while least 738% frequency occurrence by Trichophyton simii in soil samples. From the present study it was concluded that the incidence of pathogenic fungal isolates were the common in tourists soil that are etiological agents of superficial mycosis. Thus, both human and animal activity seemed to play an important role in occurrence and distribution of keratinophilic and related dermatophytes at various tourist places of Agra city.

Keywords: dermatophytic fungal diversity, bait technique, visitors at tourist spots, human and animal activities, soil samples

Procedia PDF Downloads 453

Authors: Marzieh Fotovvat, Farzaneh Najafi, Ramazan Ali Khavari-Nejad, Daryush Talei, Farhad Rejali

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Salvia leriifolia Benth. is one of the valuable and perennial medicinal plants of the Lamiaceae family, geographically growing in the south and tropical regions of Khorassan and Semnan provinces in Iran. In recent years, several medicinal properties such as antimicrobial, antifungal, anti-diabetic, analgesic, and anti-inflammatory effects have been reported from this plant. The use of elicitors such as chitosan and Arbuscular mycorrhizal fungi (AMF) symbiosis are the main methods for increasing the production of secondary metabolites, growth, and physiological factors in plants. The main aim of this study was to investigate the effects of foliar spraying applications by chitosan and/or the contribution of AMF (Glomus interaradices) on some growth factors and chlorophyll content of S. leriifolia under glasshouse conditions. The sterilized seeds were germinated by placing them into a cocopeat. After one month, seedlings that were in the 2-4 leaf stage were transferred to plastic pots (garden soil and pumice at 2:1) with or without mycorrhizal fungi. Chitosan (0, 50, 100, 200, and 400 mg L-1) was sprayed four times in the fourth month of the vegetative period. The results showed that fresh leaf weight, fresh root weight, root height, and chlorophyll content could change in the plant treated with chitosan and AMF symbiosis. So that the highest chlorophyll content and fresh weight of roots and leaves were observed in the interaction of chitosan and G. interaradices. In general, by optimizing the chitosan concentration and the use of appropriate AMF symbiosis, it is possible to improve the growth and quality of the medicinal plant S. leriifolia.

Keywords: chitosan, chlorophyll, growth factors, mycorrhiza

Procedia PDF Downloads 46

Authors: M. Ali Mandegari, S. Farzad, , J. F. Görgens

Abstract:

Sugar is one of the main agricultural industries in South Africa and approximately livelihoods of one million South Africans are indirectly dependent on sugar industry which is economically struggling with some problems and should re-invent in order to ensure a long-term sustainability. Second generation biorefinery is defined as a process to use waste fibrous for the production of biofuel, chemicals animal food, and electricity. Bioethanol is by far the most widely used biofuel for transportation worldwide and many challenges in front of bioethanol production were solved. Biorefinery annexed to the existing sugar mill for production of bioethanol and electricity is proposed to sugar industry and is addressed in this study. Since flowsheet development is the key element of the bioethanol process, in this work, a biorefinery (bioethanol and electricity production) annexed to a typical South African sugar mill considering 65ton/h dry sugarcane bagasse and tops/trash as feedstock was simulated. Aspen PlusTM V8.6 was applied as simulator and realistic simulation development approach was followed to reflect the practical behaviour of the plant. Latest results of other researches considering pretreatment, hydrolysis, fermentation, enzyme production, bioethanol production and other supplementary units such as evaporation, water treatment, boiler, and steam/electricity generation units were adopted to establish a comprehensive biorefinery simulation. Steam explosion with SO2 was selected for pretreatment due to minimum inhibitor production and simultaneous saccharification and fermentation (SSF) configuration was adopted for enzymatic hydrolysis and fermentation of cellulose and hydrolyze. Bioethanol purification was simulated by two distillation columns with side stream and fuel grade bioethanol (99.5%) was achieved using molecular sieve in order to minimize the capital and operating costs. Also boiler and steam/power generation were completed using industrial design data. Results indicates that the annexed biorefinery can be self-energy sufficient when 35% of feedstock (tops/trash) bypass the biorefinery process and directly be loaded to the boiler to produce sufficient steam and power for sugar mill and biorefinery plant.

Keywords: biorefinery, self-energy sufficiency, tops/trash, bioethanol, electricity

Procedia PDF Downloads 515

Authors: Azadeh Yasari, Michael Ganzle, Stephen Strelkov, Nuanyi Liang, Jonathan Curtis, Nat N. V. Kav

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Pathogenic fungi cause significant yield losses and quality reductions to major crops including wheat, canola, and barley. Toxic metabolites produced by phytopathogenic fungi also pose significant risks to animal and human health. Extensive application of synthetic fungicides is not a sustainable solution since it poses risks to human, animal and environmental health. Unsaturated fatty acids may provide an environmentally friendly alternative because of their direct antifungal activity against phytopathogens as well as through the stimulation of plant defense pathways. The present study assessed the in vitro and in vivo efficacy of two hydroxy fatty acids, coriolic acid and ricinoleic acid, against the phytopathogens Fusarium graminearum, Pyrenophora tritici-repentis, Pyrenophora teres f. teres, Sclerotinia sclerotiorum, and Leptosphaeria maculans. Antifungal activity of coriolic acid and ricinoleic acid was evaluated using broth micro-dilution method to determine the minimum inhibitory concentration (MIC). Results indicated that both ricinoleic acid and coriolic acid showed antifungal activity against phytopathogens, with the strongest inhibitory activity against L. maculans, but the MIC varied greatly between species. An antifungal effect was observed for coriolic acid in vivo against pathogenic fungi of wheat and barley. This effect was not correlated to the in vitro activity because ricinoleic acid with equivalent in vitro antifungal activity showed no protective effect in vivo. Moreover, neither coriolic acid nor ricinoleic acid controlled fungal pathogens of canola. In conclusion, coriolic acid inhibits some phytopathogens in vivo and may have the potential to be an effective crop protection agent.

Keywords: coriolic acid, minimum inhibitory concentration, pathogenic fungi, ricinoleic acid

Procedia PDF Downloads 145

Authors: Debit Datta, Jeffrey J. Coleman, Scott A. Enebak, Lori G. Eckhardt

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Pinus taeda, commonly known as loblolly pine, is a crucial timber species native to the southeastern USA. An emerging problem has been encountered for the past few years, which is better to be known as loblolly pine needle defoliation (LPND), which is threatening the ecological health of southeastern forests and economic vitality of the region’s timber industry. Currently, more than 1000 hectares of loblolly plantations in Alabama are affected with similar symptoms and have created concern among southeast landowners and forest managers. However, it is still uncertain whether LPND results from one or the combination of several fungal pathogens. Therefore, the objectives of the study were to identify and characterize the fungi associated with LPND in the southeastern USA and document the damage being done to loblolly pine as a result of repeated defoliation. Identification of fungi was confirmed using classical morphological methods (microscopic examination of the infected needles), conventional and species-specific priming (SSPP) PCR, and ITS sequencing. To date, 17 species of fungi, either cultured from pine needles or formed fruiting bodies on pine needles, were identified based on morphology and genetic sequence data. Among them, brown-spot pathogen Lecanostica acicola has been frequently recovered from pine needles in both spring and summer. Moreover, Ophistomatoid fungi such as Leptographium procerum, L. terebrantis are associated with pine decline have also been recovered from root samples of the infected stands. Trees have been increasingly and repeatedly chlorotic and defoliated from 2019 to 2020. Based on morphological observations and molecular data, emerging loblolly pine needle defoliation is due in larger part to the brown-spot pathogen L. acoicola followed by pine decline pathogens L. procerum and L. terebrantis. Root pathogens were suspected to emerge later, and their cumulative effects contribute to the widespread mortality of the trees. It is more likely that longer wet spring and warmer temperatures are favorable to disease development and may be important in the disease ecology of LPND. Therefore, the outbreak of the disease is assumed to be expanded over a large geographical area in a changing climatic condition.

Keywords: brown-spot fungi, emerging disease, defoliation, loblolly pine

Procedia PDF Downloads 108

Authors: Aferdita Dinaku, Jonida Canaj

Abstract:

Aflatoxins are potentially toxic metabolites produced by certain kinds of fungi (molds) that are found naturally all over the world; they can contaminate food crops and pose a serious health threat to humans by mutagenic and carcinogenic effects. Several types of aflatoxin (14 or more) occur in nature. In Albanian nutrition, cereals (especially wheat and corn) are common ingredients in some traditional meals. This study aimed to investigate the presence of aflatoxins in the flour of wheat and maize that are consumed in Albania’s markets. The samples were collected randomly in different markets in Albania and detected by the ELISA method, measured in 450 nm. The concentration of total aflatoxins was analyzed by enzyme-linked immunosorbent assay (ELISA), and they were ranged between 0.05-1.09 ppb. However, the screened mycotoxin levels in the samples were lower than the maximum permissible limits of European Commission No 1881/2006 (4 μg/kg). The linearity of calibration curves was good for total aflatoxins (B1, B2, G1, G2, M1) (R²=0.99) in the concentration range 0.005-4.05 ppb. The samples were analyzed in two replicated measurements and for each sample, the standard deviation (statistical parameter) is calculated. The results showed that the flour samples are safe, but the necessity of performing such tests is necessary.

Keywords: aflatoxins, ELISA technique, food contamination, flour

Procedia PDF Downloads 118

Authors: M. Ali Mandegari, S. Farzad, J. F. Görgens

Abstract:

Sugar is one of the main agricultural industries in South Africa and approximately livelihoods of one million South Africans are indirectly dependent on sugar industry which is economically struggling with some problems and should re-invent in order to ensure a long-term sustainability. Second generation bio-refinery is defined as a process to use waste fibrous for the production of bio-fuel, chemicals animal food, and electricity. Bio-ethanol is by far the most widely used bio-fuel for transportation worldwide and many challenges in front of bio-ethanol production were solved. Bio-refinery annexed to the existing sugar mill for production of bio-ethanol and electricity is proposed to sugar industry and is addressed in this study. Since flow-sheet development is the key element of the bio-ethanol process, in this work, a bio-refinery (bio-ethanol and electricity production) annexed to a typical South African sugar mill considering 65ton/h dry sugarcane bagasse and tops/trash as feedstock was simulated. Aspen PlusTM V8.6 was applied as simulator and realistic simulation development approach was followed to reflect the practical behavior of the plant. Latest results of other researches considering pretreatment, hydrolysis, fermentation, enzyme production, bio-ethanol production and other supplementary units such as evaporation, water treatment, boiler, and steam/electricity generation units were adopted to establish a comprehensive bio-refinery simulation. Steam explosion with SO2 was selected for pretreatment due to minimum inhibitor production and simultaneous saccharification and fermentation (SSF) configuration was adopted for enzymatic hydrolysis and fermentation of cellulose and hydrolyze. Bio-ethanol purification was simulated by two distillation columns with side stream and fuel grade bio-ethanol (99.5%) was achieved using molecular sieve in order to minimize the capital and operating costs. Also boiler and steam/power generation were completed using industrial design data. Results indicates 256.6 kg bio ethanol per ton of feedstock and 31 MW surplus power were attained from bio-refinery while the process consumes 3.5, 3.38, and 0.164 (GJ/ton per ton of feedstock) hot utility, cold utility and electricity respectively. Developed simulation is a threshold of variety analyses and developments for further studies.

Keywords: bio-refinery, bagasse, tops, trash, bio-ethanol, electricity

Procedia PDF Downloads 495