Search results for: cross-linked enzyme aggregates

Authors: Ali Bahri Lubis

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The study of dioxygenase enzymatic mechanism on tryptophan oxidation has been a wide interest since the reaction is rate-limiting step of kynurenine pathway. In this research, observation of tryptophan oxidation through h-IDO enzyme along with synthesis of enzyme products was conducted in order to comprehend how the enzyme works on distinct substrates. UV-vis spectrophotometry, LC-MS, H-NMR and HSQC measurement were carried out to characterise enzyme product. It is found that while tryptophan was oxidised to form Nformylkynurenine (NFK) as a major product and hydroxypyrroloindole amine carboxylic acid (HPIC) in cis and trans confirmed in HSQC, N-methyl tryptophan substrate was converted to NFK and trans HPIC only. Other intriguing results showed that 5-hydroxy- tryptophan and Stryptophan was degraded to become NFK and epoxide cyclic respectively. The formation of NFK was considered through dioxygenation pathway, however HPIC was formed via monooxygenation. The epoxide cyclic—considered as intermediate compound in the mechanism— from S-tryptophan was not able to cleave the epoxide ring since bond energy of epoxide was probably much stronger. This validates the enzymatic mechanism where the intermediate compound in the enzymatic mechanism is epoxide cyclic.

Keywords: tryptophan oxidation, heme-dioxygenases, N-formylkynurenine, hydroxypyrrroloindoleamine, monooxidation

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Authors: Eleftheria Barouni, Antonia Terpou, Maria Kanellaki, Argyro Bekatorou, Athanasios A.Koutinas

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The aim of the present work was to evaluate the production of cheese using a composite filter of tubular cellulose (TC) with [a] entrapped rennin enzyme and [b] immobilized L.casei and entrapped enzyme. Tubular cellulose from sawdust was prepared after lignin removal with 1% NaOH. The biocatalysts were thermally dried at 38oC and used for milk coagulation. The effect of temperature (5,20,37 oC) of the first dried biocatalyst on the pH kinetics of milk coagulation was examined. The optimum temperature (37oC) of the first biocatalyst was used for milk coagulation with the second biocatalyst prepared by entrapment of both rennin enzyme and probiotic lactic acid bacteria in order to introduce a sour taste in cheeses. This co-biocatalyst was used for milk coagulation. Samples were studied as regards its effect on lactic acid formation and its correlation with taste test results in cheeses. For both biocatalysts samples were analyzed for total acidity and lactic acid formation by HPLC. The quality of the produced cheeses was examined through the determination of volatile compounds by SPME GC/MS analysis. Preliminary taste tests and microbiological analysis were performed and encourage us for further research regarding scale up.

Keywords: tubular cellulose, Lactobacillus casei, rennin enzyme, cheese production

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Authors: N. Hachemi, A. Nouani, A. Benchabane

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The influence of cultivation factors such as content of ammonium sulfate, glucose and water in the culture medium and particle size of dry orange waste, on their bioconversion for pectinase production was studied using complete factorial design. a polygalacturonase (PG) was isolated using ion exchange chromatography under gradient elution 0-0,5 m/l NaCl (column equilibrate with acetate buffer pH 4,5), subsequently by sephadex G75 column chromatography was applied and the molecular weight was obtained about 51,28 KDa . Purified PG enzyme exhibits a pH and temperature optima of activity at 5 and 35°C respectively. Treatment of apple juice by purified enzyme extract yielded a clear juice, which was competitive with juice yielded by pure Sigma Aldrich Aspergillus niger enzyme.

Keywords: bioconversion, orange wastes, optimization, pectinase

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Authors: Sunbul Jassim Hamodi, Muna Khalid Khudayer, Firas Muzahem Hussein

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The experiment was conducted to study the effect of supplement sources of Phytase enzyme (bacterial, fungal, enzymes mixture) using levels (250, 500, 750) FTY/ kg feed to diets compared with control on the performance for one thousand fifty broiler chicks (Ross 308) from 1day old with initial weight 39.78 gm till 42 days. The study involved 10 treatments, three replicates per treatment (35 chicks/replicate). Treatments were as follows: T1: control diet (without any addition). T2: added bacterial phytase enzyme 250FTY/ kg feed. T3: added bacterial phytase enzyme 500FTY/ kg feed. T4: added bacterial phytase enzyme 750FTY/ kg feed. T5: added fungal phytase enzyme 250FTY/ kg feed. T6: added fungal phytase enzyme 500FTY/ kg feed. T7: added fungal phytase enzyme 750FTY/ kg feed. T8 added enzymes mixture 250U/ kg feed. T9: added enzymes mixture 500U/ kg feed. T10: added enzymes mixture 750U/ kg feed. The results revealed that supplementing 750 U from enzymes mixture to broiler diet increased significantly (p <0.05) body weight compared with (250 FTY bacterial phytase/Kgfeed), (750 FTY bacterial phytase/Kg feed), (750FTY fungal phytase/Kgfeed) at 6 weeks, also supplemented different sources and levels from phytase enzyme improved a cumulative weight gain for (500 FTY bacterial phytase/Kgfeed), (250FTY fungal phytase/Kgfeed), (500FTY fungal phytase/Kgfeed), (250 Uenzymes mixture/Kgfeed), (500 Uenzymes mixture/Kgfeed) and (750 U enzymes mixture/Kgfeed) treatments compared with (750 FTY fungal phytase/Kgfeed)treatment, about accumulative feed consumption (500 FTY fungal phytase/Kgfeed) and (250 Uenzymes mixture/Kgfeed) increased significantly compared with control group and (750FTY fungal phytase/Kgfeed) during 1-6 weeks. There were significantly improved in cumulative feed conversion for (500U enzymes mixture/Kgfeed) compared with the worse feed conversion ratio that recorded in (250 FTY bacterial phytase/Kgfeed). No significant differences between treatments in internal organs relative weights, carcass cuts, dressing percentage and production index. Mortality was increased in (750FTY fungal phytase/Kgfeed) compared with other treatments.

Keywords: phytase, phytic acid, broiler, productive performance

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Authors: Pratiksha Dubey, Praveen Singh, Shantanu Sen Gupta, Anand K. Bachhawat

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5-oxoproline (pyroglutamic acid) is a cyclic lactam of glutamic acid. In the cell, it can be produced by several different pathways and is metabolized into glutamate with the help of the 5-oxoprolinase enzyme (OPLAH or OXP1). The inhibition of 5-oxoprolinase enzyme in mammals was found to result in heart failure and is thought to be a consequence of oxidative stress [1]. To analyze the consequences of 5-oxoproline accumulation more clearly, we are generating models for 5-oxoproline accumulation in yeast. The 5-oxoproline accumulation model in yeast is being developed by two different strategies. The first one is by overexpression of the mouse  -glutamylcyclotransferase enzyme. It degrades -glu-met dipeptide into 5-oxoproline and methionine taken by the cell from the medium. The second strategy is by providing high concentration of 5-oxoproline externally to the yeast cells. The intracellular 5-oxoproline levels in both models are being evaluated. In addition, the metabolic and cellular consequences are being investigated.

Keywords: 5-oxoproline, pyroglutamic acid, yeast, genetics

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Authors: Prasanthi Malapati, R. Reshma, Vijay Soni, Perumal Yogeeswari, Dharmarajan Sriram

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In the present study, we attempted to develop Mycobacterium tuberculosis (Mtb) inhibitors by exploring the pharmaceutically underexploited enzyme targets which are majorly involved in cell wall biosynthesis of mycobacteria. For this purpose, glutamate racemase (coded by MurI gene) was selected. This enzyme racemize L-glutamate to D-glutamate required for the construction of peptidoglycan in the bacterial cell wall synthesis process. Furthermore this enzyme is neither expressed nor its product, D-glutamate is normally found in mammals, and hence designing inhibitors against this enzyme will not affect the host system as well act as potential antitubercular drugs. A library of BITS in house compounds were screened against Mtb MurI enzyme. Based on docking score, interactions and synthetic feasibility one hit lead was identified. Further optimization of lead was attempted and its derivatives were synthesized. Forty eight derivatives of 2-phenylbenzo[d]oxazole and 2-phenylbenzo[d]thiazole were synthesized and evaluated for Mtb MurI inhibition study, in vitro activities against Mtb, cytotoxicity against RAW 264.7 cell line. Chemical derivatization of the lead resulted in compounds NR-1213 AND NR-1124 as the potent M. tuberculosis glutamate racemase inhibitors with IC50 of 4-5µM which are remarkable and were found to be non-cytotoxic. Molecular dynamics, dormant models and cardiotoxicity studies of the most active molecules are in process.

Keywords: cell wall biosynthesis, dormancy, glutamate racemase, tuberculosis

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Authors: Kumaran Narayanan, Pei-Sheng Liew

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This paper adapts the bacteriophage N15 protelomerase enzyme to assemble linear chromosomes as vectors for gene expression in human cells. Phage N15 has the unique ability to replicate as a linear plasmid with telomeres in E. coli during its prophage stage of life-cycle. The virus-encoded protelomerase enzyme cuts its circular genome and caps its ends to form hairpin telomeres, resulting in a linear human-chromosome-like structure in E. coli. In mammalian cells, however, no enzyme with TelN-like activities has been found. In this work, we show for the first-time transfer of the protelomerase from phage into human and mouse cells and demonstrate recapitulation of its activity in these hosts. The function of this enzyme is assayed by demonstrating cleavage of its target DNA, followed by detecting telomere formation based on its resistance to recBCD enzyme digestion. We show protelomerase expression persists for at least 60 days, which indicates limited silencing of its expression. Next, we show that an intact human β-globin gene delivered on this linear chromosome accurately retains its expression in the human cellular environment for at least 60 hours, demonstrating its stability and potential as a vector. These results demonstrate that the N15 protelomerse is able to function in mammalian cells to cut and heal DNA to create telomeres, which provides a new tool for creating novel structures by DNA resolution in these hosts.

Keywords: chromosome, beta-globin, DNA, gene expression, linear vector

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Authors: C. Joseph Abou-Fayssal, K. Philippot, R. Poli, E. Manoury, A. Riisager

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The use of soluble polymer-supported metal nanoparticles (MNPs) has received significant attention for the ease of catalyst recovery and recycling. Of particular interest are MNPs that are supported on polymers that are either soluble or form stable colloidal dispersion in water, as this allows to combine of the advantages of the aqueous biphasic protocol with the catalytical performances of MNPs. The objective is to achieve good confinement of the catalyst in the nanoreactor cores and, thus, a better catalyst recovery in order to overcome the previously witnessed MNP extraction. Inspired by previous results, we are interested in the design of polymeric nanoreactors functionalized with ligands able to solidly anchor metallic nanoparticles in order to control the activity and selectivity of the developed nanocatalysts. The nanoreactors are core-crosslinked micelles (CCM) synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. Varying the nature of the core-linked functionalities allows us to get differently stabilized metal nanoparticles and thus compare their performance in the catalyzed aqueous biphasic hydrogenation of model substrates. Particular attention is given to catalyst recyclability.

Keywords: biphasic catalysis, metal nanoparticles, polymeric nanoreactors, catalyst recovery, RAFT polymerization

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Authors: D. G. Rejon-Parra, B. Escobar-Morales, Romeli Barbosa, J. C. Cruz

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In the southeast of Yucatan Peninsula, sedimentary limestone has different degrees of compaction. Due to its recent geological formation (Quaternary) and weathering effects causing an affordable aggregate for local manufacturers of concrete. It is characterized as lightweight aggregates (average density of 2,50), susceptible to abrasion and varying porosities (water content exceeding 7,50 % of its mass, in saturated condition). In this study, local aggregates with two moisture conditions (saturated and dry), have been examined in order to compare them for optimizing the performance of concrete. It is possible that these aggregates favour a phenomenon of mass transport (self-curing by porous aggregate); influencing the water reactions to form crystalline and gel hydration products. Based on the ACI methodology, a concrete mixture of 250 kg/cm2 was designed, with portland blended cement 30R. The bond between the mortar and the coarse aggregate was characterized as physicochemical based on trials which were carefully observed during time span of 28 days. The BET technique was used to analyse the micro porosity and surface areas of contact of the different crystalline phases of the limestone. Its chemical composition and crystal structures were verified with scanning electron microscopy SEM-EDS. On the third day, the samples with saturated aggregate reached 237 kg/cm2 of resistence, nearly the design strength; while samples with dry aggregate, exceeded the design strength, with a capacity of 308 kg/cm2. Aggregates in dry conditions demand a high quantity of water in the initial mixture, causing high resistance at the early stages. In saturated conditions, the development of resistance is progressive but constant.

Keywords: concrete, internal curing, limestone aggregate, porosity

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Authors: Zaoiai Said, Makani Abdelkadir, Tafraoui Ahmed

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Concrete material suffers from a relatively low tensile strength and deformation capacity is limited. Such defects of the concrete are very fragile and sensitive to shrinkage cracking materials. The Self- Compacting Concrete (SCC) are highly fluid concretes whose implementation without vibration. This material replaces traditional vibrated concrete mainly seen techno-economic interest it presents. The SCC has several advantages which are at the origin of their development crunching. The research is therefore to conduct a comparison in terms of rheological and mechanical performance between different formulations to find the optimal dosage for rubber granulates. Through this research, we demonstrated that it is possible to make different settings SCC composition having good rheological and mechanical properties. This study also showed that the substitution of natural coarse aggregates (NA) by coarse rubber aggregates (RA) in the composition of the SCC, contributes to a slight variation of workability in the fresh state parameters still remaining in the field of SCC required by the AFGC recommendations. The experimental results show that the compressive strengths of SCC decreased slightly by substituting NA by RA. Finally, the decrease in free shrinkage is proportional to the percentage of RA incorporated into the composition of concrete. This reduction is mainly due to the improvement of the deformability of these materials.

Keywords: self-compacting concrete, coarse rubber aggregate, rheological characterization, mechanical performance, shrinkage

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Authors: Márcia Afonso, Marisa Dinis-Almeida, Cristina Fael

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Climate change, namely precipitation patterns alteration, has led to extreme conditions such as floods and droughts. In turn, excessive construction has led to the waterproofing of the soil, increasing the surface runoff and decreasing the groundwater recharge capacity. The permeable pavements used in areas with low traffic lead to a decrease in the probability of floods peaks occurrence and the sediments reduction and pollutants transport, ensuring rainwater quality improvement. This study aims to evaluate the porous asphalt performance, developed in the laboratory, with addition of cellulosic fibres. One of the main objectives of cellulosic fibres use is to stop binder drainage, preventing its loss during storage and transport. Comparing to the conventional porous asphalt the cellulosic fibres addition improved the porous asphalt performance. The cellulosic fibres allowed the bitumen content increase, enabling retention and better aggregates coating and, consequently, a greater mixture durability. With this solution, it is intended to develop better practices of resilience and adaptation to the extreme climate changes and respond to the sustainability current demands, through the eco-friendly materials use. The mix design was performed for different size aggregates (with fine aggregates – PA1 and with coarse aggregates – PA2). The percentage influence of the fibres to be used was studied. It was observed that overall, the binder drainage decreases as the cellulose fibres percentage increases. It was found that the PA2 mixture obtained most binder drainage relative to PA1 mixture, irrespective of the fibres percentage used. Subsequently, the performance was evaluated through laboratory tests of indirect tensile stiffness modulus, water sensitivity, permeability and permanent deformation. The stiffness modulus for the two mixtures groups (with and without cellulosic fibres) presented very similar values between them. For the water sensitivity test it was observed that porous asphalt containing more fine aggregates are more susceptible to the water presence than mixtures with coarse aggregates. The porous asphalt with coarse aggregates have more air voids which allow water to pass easily leading to ITSR higher values. In the permeability test was observed that asphalt porous without cellulosic fibres presented had lower permeability than asphalt porous with cellulosic fibres. The resistance to permanent deformation results indicates better behaviour of porous asphalt with cellulosic fibres, verifying a bigger rut depth in porous asphalt without cellulosic fibres. In this study, it was observed that porous asphalt with bitumen higher percentages improve the performance to permanent deformation. This fact was only possible due to the bitumen retention by the cellulosic fibres.

Keywords: binder drainage, cellulosic fibres, permanent deformation, porous asphalt

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Authors: Zainal A. Muchlisin, Muhammad Iqbal, Abdullah A. Muhammadar

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The objective of the present study was to determine the optimum dose of papain enzyme in the diet for growing, survival rate and feed efficacy of climbing perch (Anabas testudineus). The study was conducted at the Laboratory of Aquatic of Faculty of Veterinary, Syiah Kuala University from January to March 2016. The completely randomized design was used in this study. Six dosages level of papain enzyme were tested with 4 replications i.e. 0 g kg-1 of feed, 20.0 g kg-1 feed, 22.5 g kg-1 of feed, 25.0 g kg-1 of feed, 27.5 g kg-1 of feed, and 30.0 g kg-1 of feed. The experimental fish fed twice a day at feeding level of 5% for 60 days. The results showed that weight gain ranged from 2.41g to 7.37g, total length gain ranged from 0.67cm to 3.17cm, specific growth rate ranged from 1.46 % day to 3.41% day, daily growth rate ranged from 0.04 g day to 0.13 g day, feed conversion ratio ranged from 1.94 to 3.59, feed efficiency ranged from 27.99% to 51.37%, protein retention ranged from 3.38% to 28.28%, protein digestibility ranged from 50.63% to 90.38%, and survival rate ranged from 88.89% to 100%. The highest rate for all parameters was found in the dosage of 3.00% papain enzyme kg feed. The ANOVA test showed that enzyme papain gave a significant effect on the weight gain, total length gain, daily growth rate, specific growth rate, feed conversion ratio, feed efficiency, protein retention, protein digestibility, and survival rate of the climbing perch (Anabas testudieus). The best enzyme papain dosage was 3.0%.

Keywords: betok, feed conversion ratio, freshwater fish, nutrition, feeding

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Authors: Uditi Dhakad, Baldev Ram, Chaman K. Jadon, R. K. Yadav, D. L. Yadav, Pratap Singh, Shalini Meena

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A field experiment was conducted during Kharif 2021 at Agricultural Research Station, Kota, to evaluate the effect of different post-emergence herbicides applied to soybean [Glycine max (L.) Merrill] on soil enzymes activity viz. dehydrogenase, phosphatase, and urease. The soil of the experimental site was clay loam (vertisols) in texture and slightly alkaline in reaction with 7.7 pH. The soil was low in organic carbon (0.49%), medium in available nitrogen (210 kg/ha), phosphorus (23.5 P2O5 kg/ha), and high in potassium (400 K2O kg/ha) status. The results elucidated that no significant adverse effect on soil dehydrogenase, urease, and phosphatase activity was determined with the application of post-emergence herbicides over the untreated control. Two hands weeding at 20 and 40 DAS registered maximum dehydrogenase enzyme activity (0.329 μgTPF/g soil/d) closely followed by herbicides mixtures and sole herbicide while pre-emergence application of pendimethalin + imazethapyr 960 g a.i./ha and pendimethalin 1.0 kg a.i./ha significantly reduced dehydrogenase enzyme activity compared to control. Urease enzyme activity was not much affected under different weed control treatments and weedy checks. The treatments were found statistically non-significant, and values ranged between 1.16-1.25 μgNH4N/g soil/d. Phosphatase enzyme activity was also not influenced significantly due to various weed control treatments. Though maximum phosphatase enzyme activity (30.17 μgpnp/g soil/hr) was observed under two-hand weeding, followed by fomesafen + fluazifop-p-butyl 220 g a.i./ha. Herbicidal weed control measures did not influence the total bacteria, fungi, and actinomycetes population.

Keywords: dehydrogenase, phosphatase, post-emergence, soil enzymes, urease.

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Authors: Ahsan R. Khokhar, Fizza Hassan

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Alkali-Silica Reaction (ASR) has been recognized as a potential cause of concrete degradation in the world since the 1940s. In Pakistan, mega hydropower structures like dams, weirs constructed from aggregates extracted from a local riverbed exhibited different levels of alkali-silica reactivity over an extended service period. The concrete expansion potential due to such aggregates has been categorized as slow-reactive. Apart from hydropower structures, ASR existence has been identified in the concrete structural elements of a Textile Mill building which used aggregates extracted from the nearby riverbed. The original structure of the Textile Mill was erected in the 80s with the addition of a textile ‘sizing and wrapping’ hall constructed in the 90s. In the years to follow, intensive spalling was observed in the structural members of the subject hall; enough to threat to the overall stability of the building. Limitations such as incomplete building data posed hurdles during the detailed structural investigation. The paper lists observations made while assessing the extent of damage and its effect on the building hall structure. Core testing and Petrographic tests were carried out as per the ASTM standards for strength degradation analysis followed by the identifying its root cause. Results confirmed significant structural strength reduction because of ASR which necessitated the formulation of an immediate re-strengthening solution. The paper also discusses the possible tracks of rehabilitative measures which are being adapted to stabilize the structure and seize further concrete expansion.

Keywords: Alkali-Silica Reaction (ASR), concrete strength degradation, damage assessment, damage evaluation

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Authors: Rupinder Kaur, Parmjit S. Panesar, Ram S. Singh

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Whey is the lactose rich by-product of the dairy industry, having good amount of nutrient reservoir. Most abundant nutrients are lactose, soluble proteins, lipids and mineral salts. Disposing of whey by most of milk plants which do not have proper pre-treatment system is the major issue. As a result of which, there can be significant loss of potential food and energy source. Thus, whey has been explored as the substrate for the synthesis of different value added products such as enzymes. β-galactosidase is one of the important enzymes and has become the major focus of research due to its ability to catalyze both hydrolytic as well as transgalactosylation reaction simultaneously. The enzyme is widely used in dairy industry as it catalyzes the transformation of lactose to glucose and galactose, making it suitable for the lactose intolerant people. The enzyme is intracellular in both bacteria and yeast, whereas for molds, it has an extracellular location. The present work was carried to utilize the whey for the production of β-galactosidase enzyme using both yeast and fungal cultures. The yeast isolate Kluyveromyces marxianus WIG2 and various fungal strains have been used in the present study. Different disruption techniques have also been investigated for the extraction of the enzyme produced intracellularly from yeast cells. Among the different methods tested for the disruption of yeast cells, SDS-chloroform showed the maximum β-galactosidase activity. In case of the tested fungal cultures, Aureobasidium pullulans NCIM 1050, was observed to be the maximum extracellular enzyme producer.

Keywords: β-galactosidase, fungus, yeast, whey

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Authors: Jwan J. Abdullah, Greetham Darren, Gregory A, Tucker, Chenyu Du

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Solid-state fermentation (SSF) is an alternative to liquid fermentations for the production of commercially important products such as antibiotics, single cell proteins, enzymes, organic acids, or biofuels from lignocellulosic material. This paper describes the optimisation of SSF on municipal solid waste (MSW) for the production of cellulase enzyme. Production of cellulase enzymes was optimised by Trichoderma reesei or Aspergillus niger for temperature, moisture content, inoculation, and period of incubation. Also, presence of minerals, and alternative carbon and nitrogen sources. Optimisation revealed that production of cellulolytic enzymes was optimal when using Trichoderma spp at 30°C with an incubation period of 168 hours with a 60% moisture content. Crude enzymes produced from MSW, by Trichoderma were evaluated for the saccharification of MSW and compared with activity of a commercially available enzyme, results demonstrated that MSW can be used as inexpensive lignocellulosic material for the production of cellulase enzymes using Trichoderma reesei.

Keywords: SSF, enzyme hydrolysis, municipal solid waste (MSW), optimizing conditions, enzyme hydrolysis

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Authors: Pradeep Kumar, Shalinee Shukla

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Pavement plays a vital role in the socio-economic development of a country. Bituminous roads construction with conventional paving grade bitumen obtained from hot mix plant creates pollution and involves emission of greenhouse gases, also the construction of pavements at very high temperature is not feasible or desirable for high rainfall and snowfall areas. This problem of overheating can be eliminated by the construction of pavements with the usage of emulsified cold mixes which will eliminate emissions and help in the reduction of fuel requirement at mixing plant, which leads to energy conservation. Cold mix is a mixture of unheated aggregate and emulsion or cutback and filler. The primary objective of this research is to assess the volumetric mix design parameters of recycled aggregates with cold mixing technology and also to assess the impact of additives on volumetric mix characteristics. In this present study, bituminous pavement materials are reclaimed using cold mix technology, and Marshall specimens are prepared with the help of slow setting type 2 (SS-2) cationic bitumen emulsion as a binder for recycled aggregates. This technique of road construction is more environmentally friendly and can be done in adverse weather conditions.

Keywords: cold mixes, bitumen emulsion, recycled aggregates, volumetric properties

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Authors: Sukanya Devi Ramachandran, Vaishnavi Muralidharan, Kavya Chandrasekaran

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Polycaprolactone is polymer belonging to the polyester family that has noticeable characteristics of biodegradability and biocompatibility which is essential for medical applications. Polycaprolactone is produced by the ring opening polymerization of the monomer epsilon-Caprolactone (ε-CL) which is a closed ester, comprising of seven-membered ring. This process is normally catalysed by metallic components such as stannous octoate. It is difficult to remove the catalysts after the reaction, and they are also toxic to the human body. An alternate route of using enzymes as catalysts is being employed to reduce the toxicity. Lipase enzyme is a subclass of esterase that can easily attack the ester bonds of ε-CL. This research paper throws light on the extraction of lipase from germinating sunflower seeds and the activity of the biocatalyst in the polymerization of ε-CL. Germinating Sunflower seeds were crushed with fine sand in phosphate buffer of pH 6.5 into a fine paste which was centrifuged at 5000rpm for 10 minutes. The clear solution of the enzyme was tested for activity at various pH ranging from 5 to 7 and temperature ranging from 40oC to 70oC. The enzyme was active at pH6.0 and at 600C temperature. Polymerization of ε-CL was done using toluene as solvent with the catalysis of lipase enzyme, after which chloroform was added to terminate the reaction and was washed in cold methanol to obtain the polymer. The polymerization was done by varying the time from 72 hours to 6 days and tested for the molecular weight and the conversion of the monomer. The molecular weight obtained at 6 days is comparably higher. This method will be very effective, economical and eco-friendly to produce as the enzyme used can be regenerated as such at the end of the reaction and can be reused. The obtained polymers can be used for drug delivery and other medical applications.

Keywords: lipase, monomer, polycaprolactone, polymerization

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Authors: Flackson Tshuma, James Bennett, Pieter Andreas Swanepoel, Johan Labuschagne, Stephan van der Westhuizen, Francis Rayns

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Amongst other things, tillage and synthetic agrochemicals can be effective methods of seedbed preparation and pest control. Nonetheless, frequent and intensive tillage and excessive application of synthetic agrochemicals, such as herbicides and insecticides, can reduce soil microbial enzyme activity. A decline in soil microbial enzyme activity can negatively affect nutrient cycling and crop productivity. In this study, the effects of four tillage treatments; continuous mouldboard plough; shallow tine-tillage to a depth of about 75 mm; no-tillage; and tillage rotation (involving shallow tine-tillage once every four years in rotation with three years of no-tillage), and two rates of synthetic agrochemicals (standard: with regular application of synthetic agrochemicals; and reduced: fewer synthetic agrochemicals in combination with bio-chemicals/ or bio-stimulants) on soil microbial enzyme activity were investigated between 2018 and 2020 in a typical Mediterranean climate zone in South Africa. Four different bio-stimulants applied contained: Trichoderma asperellum, fulvic acid, silicic acid, and Nereocystis luetkeana extracts, respectively. The study was laid out as a complete randomised block design with four replicated blocks. Each block had 14 plots, and each plot measured 50 m x 6 m. The study aimed to assess the combined impact of tillage practices and reduced rates of synthetic agrochemical application on soil microbial enzyme activity in a dryland cropping system. It was hypothesised that the application of bio-stimulants in combination with minimum soil disturbance will lead to a greater increase in microbial enzyme activity than the effect of applying either in isolation. Six soil cores were randomly and aseptically collected from each plot for microbial enzyme activity analysis from the 0-150 mm layer of a field trial under a dryland crop rotation system in the Swartland region. The activities of four microbial enzymes, β-glucosidase, acid phosphatase, alkaline phosphatase and urease, were assessed. The enzymes are essential for the cycling of glucose, phosphorus, and nitrogen, respectively. Microbial enzyme activity generally increased with a reduction of both tillage intensity and synthetic agrochemical application. The use of the mouldboard plough led to the least (P<0.05) microbial enzyme activity relative to the reduced tillage treatments, whereas the system with bio-stimulants (reduced synthetic agrochemicals) led to the highest (P<0.05) microbial enzyme activity relative to the standard systems. The application of bio-stimulants in combination with reduced tillage, particularly no-tillage, could be beneficial for enzyme activity in a dryland farming system.

Keywords: bio-stimulants, soil microbial enzymes, synthetic agrochemicals, tillage

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Authors: Tomáš Melichar, Jiří Bydžovský, Vít Černý

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In this article our research focused on study of basic physical and mechanical parameters of polymer-cement repair materials is presented. Namely the influence of applied aggregates in combination with active admixture is specially considered. New formulas which were exposed in ambient with temperature even to 1000°C were suggested. Subsequently densities and strength characteristics including their changes were evaluated. Selected samples were analyzed using electron microscope. The positive influence of porous aggregates based on sintered ash was definitely demonstrated. Further it was found than in terms of thermal resistance the effective micro silica amount represents 5% to 7.5% of cement weight.

Keywords: aggregate, ash, high, lightweight, microsilica, mortar, polymer-cement, repair, temperature

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Authors: Aleksandra Łochowicz, Daria Świętochowska, Loredano Pollegioni, Nazim Ocal, Franck Charmantray, Laurence Hecquet, Katarzyna Szymańska

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Multienzymatic cascade reactions are nowadays widely used in pharmaceutical, chemical and cosmetics industries to produce high valuable compounds. They can be carried out in two ways, step by step and one-pot. If two or more enzymes are in the same reaction vessel is necessary to work out the compromise to run the reaction in optimal conditions for each enzyme. So far most of the reports of multienzymatic cascades concern on usage of free enzymes. Unfortunately using free enzymes as catalysts of reactions accomplish high cost. What is more, free enzymes are soluble in solvents which makes reuse impossible. To overcome this obstacle enzymes can be immobilized what provides heterogeneity of biocatalyst that enables reuse and easy separation of the enzyme from solvents and reaction products. Usually, immobilization increase also the thermal and operational stability of enzyme. The advantages of using immobilized multienzymes are enhanced enzyme stability, improved cascade enzymatic activity via substrate channeling, and ease of recovery for reuse. The one-pot immobilized multienzymatic cascade can be carried out in mixed or coimmobilized type. When biocatalysts are coimmobilized on the same carrier the are in close contact to each other which increase the reaction rate and catalytic efficiency, and eliminate the lag time. However, in this type providing the optimal conditions both in the process of immobilization and cascade reaction for each enzyme is complicated. Herein, we examined immobilization of 3 enzymes: D-amino acid oxidase from Rhodotorula gracilis, commercially available catalase and transketolase from Geobacillus stearothermophilus. As a support we used silica monoliths with hierarchical structure of pores. Then we checked their stability and reusability in one-pot cascade of L-erythrulose and hydroxypuryvate acid synthesis.

Keywords: biocatalysts, enzyme immobilization, multienzymatic reaction, silica carriers

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Authors: Homa Torabizadeh, Mohaddeseh Mikani

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Inulinase from Aspergillus niger was covalently immobilized on magnetic nanoparticles (MNPs/Fe₃O₄) covered with soy protein isolate (SPI/Fe₃O₄) functionalized by bovine serum albumin (BSA) nanoparticles. MNPs are promising enzyme carriers because they separate easily under external magnetic fields and have enhanced immobilized enzyme reusability. As MNPs aggregate simply, surface coating strategy was employed. SPI functionalized by BSA was a suitable candidate for nanomagnetite coating due to its superior biocompatibility and hydrophilicity. Fe₃O₄@SPI-BSA nanoparticles were synthesized as a novel carrier with narrow particle size distribution. Step by step fabrication monitoring of Fe₃O₄@SPI-BSA nanoparticles was performed using field emission scanning electron microscopy and dynamic light scattering. The results illustrated that nanomagnetite with the spherical morphology was well monodispersed with the diameter of about 35 nm. The average size of the SPI-BSA nanoparticles was 80 to 90 nm, and their zeta potential was around −34 mV. Finally, the mean diameter of fabricated Fe₃O₄@SPI-BSA NPs was less than 120 nm. Inulinase enzyme from Aspergillus niger was covalently immobilized through gluteraldehyde on Fe₃O₄@SPI-BSA nanoparticles successfully. Fourier transform infrared spectra and field emission scanning electron microscopy images provided sufficient proof for the enzyme immobilization on the nanoparticles with 80% enzyme loading.

Keywords: high fructose syrup, inulinase immobilization, functionalized magnetic nanoparticles, soy protein isolate

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Authors: Jung-En Kuan, Whei-Fen Wu

Abstract:

In this study, a symbiotic bacteria from yellow mealworm's (Tenebrio molitor) mid-gut was isolated with characteristics of growth on minimal-tributyrin medium. After a PCR-amplification of its 16s rDNA, the resultant nucleotide sequences were then analyzed by schemes of the phylogeny trees. Accordingly, it was designated as Pseudomonas nitroreducens D-01. Next, by searching the lipolytic enzymes in its protein data bank, one of those potential lipolytic α/β hydrolases was identified, again using PCR-amplification and nucleotide-sequencing methods. To construct an expression of this lipolytic gene in plasmids, the target-gene primers were then designed, carrying the C-terminal his-tag sequences. Using the vector pET21a, a recombinant lipolytic hydrolase D gene with his-tag nucleotides was successfully cloned into it, of which the lipolytic D gene is under a control of the T7 promoter. After transformation of the resultant plasmids into Eescherichia coli BL21 (DE3), an IPTG inducer was used for the induction of the recombinant proteins. The protein products were then purified by metal-ion affinity column, and the purified proteins were found capable of forming a clear zone on tributyrin agar plate. Shortly, its enzyme activities were determined by degradation of p-nitrophenyl ester(s), and the substantial yellow end-product, p-nitrophenol, was measured at O.D.405 nm. Specifically, this lipolytic enzyme efficiently targets p-nitrophenyl butyrate. As well, it shows the most reactive activities at 40°C, pH 8 in potassium phosphate buffer. In thermal stability assays, the activities of this enzyme dramatically drop when the temperature is above 50°C. In metal ion assays, MgCl₂ and NH₄Cl induce the enzyme activities while MnSO₄, NiSO₄, CaCl₂, ZnSO₄, CoCl₂, CuSO₄, FeSO₄, and FeCl₃ reduce its activities. Besides, NaCl has no effects on its enzyme activities. Most organic solvents decrease the activities of this enzyme, such as hexane, methanol, ethanol, acetone, isopropanol, chloroform, and ethyl acetate. However, its enzyme activities increase when DMSO exists. All the surfactants like Triton X-100, Tween 80, Tween 20, and Brij35 decrease its lipolytic activities. Using Lineweaver-Burk double reciprocal methods, the function of the enzyme kinetics were determined such as Km = 0.488 (mM), Vmax = 0.0644 (mM/min), and kcat = 3.01x10³ (s⁻¹), as well the total efficiency of kcat/Km is 6.17 x10³ (mM⁻¹/s⁻¹). Afterwards, based on the phylogenetic analyses, this lipolytic protein is classified to type IV lipase by its homologous conserved region in this lipase family.

Keywords: enzyme, esterase, lipotic hydrolase, type IV

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Authors: Khalida Boutemak, Nasssima Benali, Nadji Moulai-Mostefa

Abstract:

Effect of enzyme on the yield and chemical composition of Artemisia campestris essential oil is reported in the present study. It was demonstrated that enzyme facilitated the extraction of essential oil with increase in oil yield and did not affect any noticeable change in flavour profile of the volatile oil. Essential oil was tested for antibacterial activity using Escherichia coli; which was extremely sensitive against control with the largest inhibition (29mm), whereas Staphylococcus aureus was the most sensitive against essential oil obtained from enzymatic pre-treatment with the largest inhibition zone (25mm). The antioxidant activity of the essential oil with hemicellulase pre-treatment (EO2) and control sample (EO1) was determined through reducing power. It was significantly lower than the standard drug (vitamin C) in this order: vitamin C˃EO2˃EO1.

Keywords: Artemisia campestris, enzyme pre-treatment, hemicellulase, antibacterial activity, antioxidant activity

Procedia PDF Downloads 299

Authors: Itti Bist, Snehasis Bhakta, Di Jiang, Tia E. Keyes, Aaron Martin, Robert J. Forster, James F. Rusling

Abstract:

DNA damage from metabolites of lipophilic drugs and pollutants, generated by enzymes, represents a major toxicity pathway in humans. These metabolites can react with DNA to form either 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG), which is the oxidative product of DNA or covalent DNA adducts, both of which are genotoxic and hence considered important biomarkers to detect cancer in humans. Therefore, detecting reactions of metabolites with DNA is an effective approach for the safety assessment of new chemicals and drugs. Here we describe a novel electrochemiluminescent (ECL) sensor array which can detect DNA oxidation and chemical DNA damage in a single array, facilitating a more accurate diagnostic tool for genotoxicity screening. Layer-by-layer assembly of DNA and enzyme are assembled on the pyrolytic graphite array which is housed in a microfluidic device for sequential detection of two type of the DNA damages. Multiple enzyme reactions are run on test compounds using the array, generating toxic metabolites in situ. These metabolites react with DNA in the films to cause DNA oxidation and chemical DNA damage which are detected by ECL generating osmium compound and ruthenium polymer, respectively. The method is further validated by the formation of 8-oxodG and DNA adduct using similar films of DNA/enzyme on magnetic bead biocolloid reactors, hydrolyzing the DNA, and analyzing by liquid chromatography-mass spectrometry (LC-MS). Hence, this combined DNA/enzyme array/LC-MS approach can efficiently explore metabolic genotoxic pathways for drugs and environmental chemicals.

Keywords: biosensor, electrochemiluminescence, DNA damage, microfluidic array

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Authors: D. Sarkar, M. Pal, A. K. Sarkar

Abstract:

Asphaltic concrete for pavement construction in India are produced by using crushed stone, gravels etc. as aggregate. In north-Eastern region of India, there is a scarcity o f stone aggregate. Therefore the road engineers are always in search of an optional material as aggregate which can replace the regularly used material. The purpose of this work was to evaluate the utilization of substandard or marginal aggregates in flexible pavement construction. The investigation was undertaken to evaluate the effects of using lower quality aggregates such as over burnt brick aggregate on the preparation of asphalt concrete for flexible pavements. The scope of this work included a review of available literature and existing data, a laboratory evaluation organized to determine the effects of marginal aggregates and potential techniques to upgrade these substandard materials, and a laboratory evaluation of these upgraded marginal aggregate asphalt mixtures. Over burnt brick aggregates are water susceptible and can leads to moisture damage. Moisture damage is the progressive loss of functionality of the material owing to loss of the adhesion bond between the asphalt binder and the aggregate surface. Hence, zycosoil as an anti striping additive were evaluated in this study. This study summarizes the results of the laboratory evaluation carried out to investigate the properties of asphalt concrete prepared with zycosoil modified over burnt brick aggregate. Marshall specimen were prepared with stone aggregate, zycosoil modified stone aggregate, over burnt brick aggregate and zycosoil modified over burnt brick aggregate. Results show that addition of zycosoil with stone aggregate increased stability by 6% and addition of zycosoil with over burnt brick aggregate increased stability by 30%.

Keywords: asphalt concrete, over burnt brick aggregate, marshall stability, zycosoil

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Authors: Amtul Jamil Sami

Abstract:

Apis mellifera is an insect of immense economic importance lives on rich carbohydrate diet including cellulose, nectar, honey and pollen. The carbohydrate metabolism in A mellifera has not been understood fully, as there are no data available, on the functional expression of cellulase gene. The cellulose hydrolyzing enzyme is required for the digestion of pollen cellulose wall, to release the important nutrients (amino acids, minerals, vitamins etc.) from the pollen. A dissection of Apis genome had revealed that there is one gene present for the expression of endo-beta-1,4-glucanase, for cellulose hydrolysis. In the presented work, functional expression of endo-beta-1,4 glucanase gene is reported. Total soluble proteins of the honey bee were isolated and were tested cellulose hydrolyzing enzyme activity, using carboxy-methyl cellulose, as a substrate. A mellifera proteins were able to hydrolyze carboxy-methyl cellulose, confirming its endo- type mode of action. Endo beta-1,4 glucanase enzyme was only present in the gut tissues, no activity was detected in the salivary glands. The pH optima of the enzyme were in the acidic pH range of 4-5-5-0, indicating its metabolic role in the acidic stomach of A mellifera. The reported enzyme is unique, as endo-beta- 1,4 glucanase was able to generate non reducing sugar, as an end product. The results presented, are supportive to the information that the honey bee is capable of producing its novel endo-beta-1,4 glucanase. Further it could be helpful, in understanding, the carbohydrate metabolism in A mellifera.

Keywords: honey bees, Endo-beta 1, 4- glucanase, Apis mellifera, functional expression

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Authors: A. K. Jain, M. C. Paliwal

Abstract:

The production of cement leads to the emission of large amounts of carbon-dioxide gas into the atmosphere which is a major contributor for the greenhouse effect and the global warming; hence it is mandatory either to quest for another material or partly replace it with some other material. According to the practical demonstrations and reports, Egg Shell Powder (ESP) can be used as a binding material for different field applications as it contains some of the properties of lime. It can partially replace the cement and further; it can be used in different proportion for enhancing the performance of cement. It can be used as a first-class alternative, for material reuse and waste recycling practices. Eggshell is calcium rich and analogous to limestone in chemical composition. Therefore, use of eggshell waste for partial replacement of cement in concrete is feasible. Different studies reveal that plasticity index of the soil can be improved by adding eggshell wastes in all the clay soil and it has wider application in construction projects including earth canals and earthen dams. The scarcity of aggregates is also increasing nowadays. Utilization of industrial waste or secondary materials is increasing in different construction applications. Coconut shell was successfully used in the construction industry for partial or full replacement for coarse aggregates. The use of coconut shell gives advantage of using waste material to partially replace the coarse aggregate. Studies carried on coconut shell indicate that it can partially replace the aggregate. It has good strength and modulus properties along with the advantage of high lignin content. It absorbs relatively low moisture due to its low cellulose content. In the paper, study carried out on eggshell powder and coconut shell will be discussed. Optimum proportions of these materials to be used for partial replacement of cement and aggregate will also be discussed.

Keywords: greenhouse, egg shell powder, binding material, aggregates, coconut shell, coarse aggregates

Procedia PDF Downloads 219

Authors: Nadjoua Bourmatte, Hacène Houari

Abstract:

Glass has been indispensable to men’s life due to its properties, including pliability to take any shape with ease, bright surface, resistance to abrasion, reasonable safety and durability. Waste glass creates serious environmental problems, mainly due to the inconsistency of waste glass streams. With increasing environmental pressure to reduce solid waste and to recycle as much as possible, the concrete industry has adopted a number of methods to achieve this goal. The object of this research work is to study the effect of using recycled glass waste, as a partial replacement of fine aggregate, on the fresh and hardened properties of concrete. Recycled glass was used to replace fine aggregate in proportions of 0%, 25% and 50%. We could observe that the Glass waste aggregates are lighter than natural aggregates and they show a very low water absorption. The experimental results showed that the slump flow increased with the increase of recycled glass content. On the other hand, the compressive strength and tensile strength of recycled glass mixtures decreased with the increase in the recycled glass content. The results showed that recycled glass aggregate can successfully be used with limited level for producing concrete. The standard sand was substituted with aggregates based on glass waste for manufacturing mortars, Mortar based on glass shows a compressive strength and low bending with a 1/2 ratio with control mortar strength.

Keywords: concrete, environment, glass waste, recycling

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Authors: Kanchan Vishwakarma, Shivesh Sharma, Nitin Kumar

Abstract:

Soil is incoherent matter on Earth’s surface having organic and mineral content. The spatial variation of 4 soil enzyme activities and microbial biomass were assessed for two seasons’ viz. monsoon and winter along the latitudinal gradient in North-central India as the area of this study is fettered with respect to national status. The study was facilitated to encompass the effect of climate change, enzyme activity and biomass on nutrient cycling. Top soils were sampled from 4 sites in North-India. There were significant correlations found between organic C, N & P wrt to latitude gradient in two seasons. This distribution of enzyme activities and microbial biomass was consequence of alterations in temperature and moisture of soil because of which soil properties change along the latitude transect.

Keywords: latitude gradient, microbial biomass, moisture, soil, organic carbon, temperature

Procedia PDF Downloads 364