Search results for: enzyme multipoint covalent attachment

Authors: Shohei Matsuo, Saki Mikai, Hiroshi Morita

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Objectives: Shochu is a popular Japanese distilled spirits. In the production of shochu, the filamentous fungus Aspergillus kawachii has traditionally been used. A. kawachii produces two types of starch hydrolytic enzymes, α-amylase (enzymatic liquefaction) and glucoamylase (enzymatic saccharification). Liquid culture system is a relatively easy microorganism to ferment with relatively low cost of production compared for solid culture. In liquid culture system, acid-unstable α-amylase (α-A) was produced abundantly, but, acid-stable α-amylase (Aα-A) was not produced. Since there is high enzyme productivity, most in shochu brewing have been adopted by a solid culture method. In this study, therefore, we investigated production of Aα-A in liquid culture system. Materials and methods: Microorganism Aspergillus kawachii NBRC 4308 was used. The mold was cultured at 30 °C for 7~14 d to allow formation of conidiospores on slant agar medium. Liquid Culture System: A. kawachii was cultured in a 100 ml of following altered SLS medium: 1.0 g of rice flour, 0.1 g of K2HPO4, 0.1 g of KCl, 0.6 g of tryptone, 0.05 g of MgSO4・7H2O, 0.001 g of FeSO4・7H2O, 0.0003 g of ZnSO4・7H2O, 0.021 g of CaCl2, 0.33 of citric acid (pH 3.0). The pH of the medium was adjusted to the designated value with 10 % HCl solution. The cultivation was shaking at 30 °C and 200 rpm for 72 h. It was filtered to obtain a crude enzyme solution. Aα-A assay: The crude enzyme solution was analyzed. An acid-stable α-amylase activity was carried out using an α-amylase assay kit (Kikkoman Corporation, Noda, Japan). It was conducted after adding 9 ml of 100 mM acetate buffer (pH 3.0) to 1 ml of the culture product supernatant and acid treatment at 37°C for 1 h. One unit of a-amylase activity was defined as the amount of enzyme that yielded 1 mmol of 2-chloro-4-nitrophenyl 6-azide-6-deoxy-b-maltopentaoside (CNP) per minute. Results and Conclusion: We experimented with co-culture of A. kawachii and lactobacillus in order to get control of pH in altered SLS medium. However, high production of acid-stable α-amylase was not obtained. We experimented with yoghurt or milk made an addition to liquid culture. The result indicated that high production of acid-stable α-amylase (964 U/g-substrate) was obtained when milk made an addition to liquid culture. Phosphate concentration in the liquid medium was a major cause of increased acid-stable α-amylase activity. In liquid culture, acid-stable α-amylase activity was enhanced by milk, but Fats and oils in the milk were oxidized. In addition, Tryptone is not approved as a food additive in Japan. Thus, alter SLS medium added to skim milk excepting for the fats and oils in the milk instead of tryptone. The result indicated that high production of acid-stable α-amylase was obtained with the same effect as milk.

Keywords: acid-stable α-amylase, liquid culture, milk, shochu

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Authors: JaeYoon Lee, Gi-Hoon Yang, JongHan Ha, MyungGu Yeo, SeungHyun Ahn, Hyeongjin Lee, HoJun Jeon, YongBok Kim, Minseong Kim, GeunHyung Kim

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One of the important factors in tissue engineering is to design optimal biomedical scaffolds, which can be governed by topographical surface characteristics, such as size, shape, and direction. Of these properties, we focused on the effects of nano- to micro-sized hierarchical surface. To fabricate the hierarchical surface structure on poly(ε-caprolactone) (PCL) film, we employed a micro-casting technique by pressing the mold and nano-etching technique using a modified plasma process. The micro-sized topography of PCL film was controlled by sizes of the micro structures on lotus leaf. Also, the nano-sized topography and hydrophilicity of PCL film were controlled by a modified plasma process. After the plasma treatment, the hydrophobic property of the PCL film was significantly changed into hydrophilic property, and the nano-sized structure was well developed. The surface properties of the modified PCL film were investigated in terms of initial cell morphology, attachment, and proliferation using osteoblast-like-cells (MG63). In particular, initial cell attachment, proliferation and osteogenic differentiation in the hierarchical structure were enhanced dramatically compared to those of the smooth surface. We believe that these results are because of a synergistic effect between the hierarchical structure and the reactive functional groups due to the plasma process. Based on the results presented here, we propose a new biomimetic surface model that maybe useful for effectively regenerating hard tissues.

Keywords: hierarchical surface, lotus leaf, nano-etching, plasma treatment

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Authors: Vijay R. Patil, Sathiyanarayanan Lohidasan, K. R. Mahadik

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Gastrointestinal stability of andrographolide was evaluated in vitro in simulated gastric (SGF) and intestinal (SIF) fluids using a validated HPLC-PDA method. The method was validated using a 5μm ThermoHypersil GOLD C18column (250 mm × 4.0 mm) and mobile phase consisting of water: acetonitrile; 70: 30 (v/v) delivered isocratically at a flow rate of 1 mL/min with UV detection at 228 nm. Andrographolide in pure form and extract Andrographis paniculata was incubated at 37°C in an incubator shaker in USP simulated gastric and intestinal fluids with and without enzymes. Systematic protocol as per FDA Guidance System was followed for stability study and samples were assayed at 0, 15, 30 and 60 min intervals for gastric and at 0, 15, 30, 60 min, 1, 2 and 3 h for intestinal stability study. Also, the stability study was performed up to 24 h to see the degradation pattern in SGF and SIF (with enzyme and without enzyme). The developed method was found to be accurate, precise and robust. Andrographolide was found to be stable in SGF (pH ∼ 1.2) for 1h and SIF (pH 6.8) up to 3 h. The relative difference (RD) of amount of drug added and found at all time points was found to be < 3%. The present study suggests that drug loss in the gastrointestinal tract takes place may be by membrane permeation rather than a degradation process.

Keywords: andrographolide, Andrographis paniculata, in vitro, stability, gastric, Intestinal HPLC-PDA

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Authors: Dileep Kumar Verma, Sunil Kumar Lal

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Influenza still remains one of the most challenging diseases posing significant threat to public health causing seasonal epidemics and pandemics. Influenza A Virus (IAV) surface protein hemagglutinin is known to play an important role in viral attachment to the host sialic acid receptors and concentrate in lipid rafts for efficient viral fusion. Selective nature of Influenza A virus to utilize rafts micro-domain for efficient virus assembly and budding has been explored in depth. However, the detailed mechanism of IAV binding to host cell membrane and entry into the host remains elusive. In the present study we investigated the role of lipid rafts in early life cycle events of IAV. Role of host lipid rafts was studied using raft disruption method by extraction of cholesterol by Methyl-β-Cyclodextrin. Using GM1, a well-known lipid raft marker, we were able to observe co-localization of IAV on lipid rafts on the host cell membrane. This experiment suggests a direct involvement of lipid rafts in the initiation of the IAV life cycle. Upon disruption of lipid rafts by Methyl-b-cyclodextrin, we observed a significant reduction in IAV binding on the host cell surface indicating a significant decrease in virus attachment to coherent membrane rafts. Our results provide proof that host lipid rafts and their constituents play an important role in the adsorption of IAV. This study opens a new avenues in IAV virus-host interactions to combat infection at a very early steps of the viral lifecycle.

Keywords: lipid raft, adsorption, cholesterol, methyl-β-cyclodextrin, GM1

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Authors: P. Singh, R. M. Banik

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Artificial neural network (ANN) was employed to optimize assay parameters viz., time, temperature, pH of reaction mixture, enzyme volume and substrate concentration of L-glutaminase from Bacillus cereus MTCC 1305. ANN model showed high value of coefficient of determination (0.9999), low value of root mean square error (0.6697) and low value of absolute average deviation. A multilayer perceptron neural network trained with an error back-propagation algorithm was incorporated for developing a predictive model and its topology was obtained as 5-3-1 after applying Levenberg Marquardt (LM) training algorithm. The predicted activity of L-glutaminase was obtained as 633.7349 U/l by considering optimum assay parameters, viz., pH of reaction mixture (7.5), reaction time (20 minutes), incubation temperature (35˚C), substrate concentration (40mM), and enzyme volume (0.5ml). The predicted data was verified by running experiment at simulated optimum assay condition and activity was obtained as 634.00 U/l. The application of ANN model for optimization of assay conditions improved the activity of L-glutaminase by 1.499 fold.

Keywords: Bacillus cereus, L-glutaminase, assay parameters, artificial neural network

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Authors: Mostafa Heidari

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Nitrogen fertilization has played a significant role in increasing crop yield, and solving problems of hunger and malnutrition worldwide. However, excessive of heavy metals such as arsenic can interfere on growth and reduced grain yield. In order to investigate the effects of different concentrations of arsenic and nitrogen fertilizer on photosynthetic pigments and antioxidant enzyme activities in safflower (cv. Goldasht), a factorial plot experiment as randomized complete block design with three replication was conducted in university of Zabol. Arsenic treatment included: A1= control or 0, A2=30, A3=60 and A4=90 mg. kg-1 soil from the Na2HASO4 source and three nitrogen levels including W1=75, W2=150 and W3=225 kg.ha-1 from urea source. Results showed that, arsenic had a significant effect on the activity of antioxidant enzymes. By increasing arsenic levels from A1 to A4, the activity of ascorbate peroxidase (APX) and gayacol peroxidase (GPX) increased and catalase (CAT) was decreased. In this study, arsenic had no significant on chlorophyll a, b and cartoneid content. Nitrogen and interaction between arsenic and nitrogen treatment, except APX, had significant effect on CAT and GPX. The highest GPX activity was obtained at A4N3 treatment. Nitrogen increased the content of chlorophyll a, b and cartoneid.

Keywords: arsenic, physiological parameters, oxidative enzymes, nitrogen

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Authors: Heri Hermansyah, Annisa Kurnia, A. Vania Anisya, Adi Surjosatyo, Yopi Sunarya, Rita Arbianti, Tania Surya Utami

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Lipase is one of biocatalyst which is applied commercially for the process in industries, such as bioenergy, food, and pharmaceutical industry. Nowadays, biocatalysts are preferred in industries because they work in mild condition, high specificity, and reduce energy consumption (high pressure and temperature). But, the usage of lipase for industry scale is limited by economic reason due to the high price of lipase and difficulty of the separation system. Immobilization of lipase is one of the solutions to maintain the activity of lipase and reduce separation system in the process. Therefore, we conduct a study about lipase immobilization with the adsorption-cross linking method using glutaraldehyde because this method produces high enzyme loading and stability. Lipase is immobilized on different kind of resin with the various functional group. Highest enzyme loading (76.69%) was achieved by lipase immobilized on anion macroporous which have anion functional group (OH^‑). However, highest activity (24,69 U/g support) through olive oil emulsion method was achieved by lipase immobilized on anion macroporous-chitosan which have amino (NH₂) and anion (OH^-) functional group. In addition, it also success to produce biodiesel until reach yield 50,6% through interesterification reaction and after 4 cycles stable 63.9% relative with initial yield. While for Aspergillus, niger lipase immobilized on anion macroporous-kitosan have unit activity 22,84 U/g resin and yield biodiesel higher than commercial lipase (69,1%) and after 4 cycles stable reach 70.6% relative from initial yield. This shows that optimum functional group on support for immobilization with adsorption-cross linking is the support that contains amino (NH₂) and anion (OH^-) functional group because they can react with glutaraldehyde and binding with enzyme prevent desorption of lipase from support through binding lipase with a functional group on support.

Keywords: adsorption-cross linking, immobilization, lipase, resin

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Authors: Muralisankar Thirunavukkarasu, Saravana Bhavan Periyakali, Santhanam Perumal

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The present study was performed to assess the effects of dietary copper (Cu) on growth, biochemical constituents, digestive enzyme activities, enzymatic antioxidant and metabolic enzymes of the freshwater prawn, Macrobrachium rosenbergii post larvae (PL). The Cu was supplemented at 0, 10, 20, 40, 60 and 80 mg kg-1 with the basal diets. Cu supplemented diets were fed to M. rosenbergii PL for a period of 90 days. At the end of the feeding experiment, 40 mg kg-1 Cu supplemented feeds fed PL showed significant (P < 0.05) improvement in survival, growth, digestive enzyme activities and concentrations of biochemical constituents. However, PL fed with 60 to 80 mg Cu kg-1 showed negative performance. Activities of enzymatic antioxidants, metabolic enzymes and lipid peroxidation in the muscle and hepatopancreas showed insignificant alterations (P > 0.05) up to 40 mg kg-1 Cu supplemented feeds fed PL. Whereas, 60 and 80 mg of Cu kg-1 supplemented feeds fed PL showed significant alterations on these antioxidants and metabolic enzymes levels. It indicates that beyond 40 mg Cu kg-1 diets were produced some toxic to M. rosenbergii PL. Therefore, the present study suggests that 40 mg Cu kg-1 can be supplemented in the diets of M. rosenbergii PL for regulating better survival and growth.

Keywords: antioxidants, biochemical constituents, copper, growth, Macrobrachium rosenbergii

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Authors: Joanna Cabaj, Sylwia Baluta, Karol Malecha, Kamila Drzozga

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Catecholamines are the principal neurotransmitters that mediate a variety of the central nervous system functions, such as motor control, cognition, emotion, memory processing, and endocrine modulation. Dysfunctions in catecholamine neurotransmission are induced in some neurologic and neuropsychiatric diseases. Changeable neurotransmitters level in biological fluids can be a marker of several neurological disorders. Because of its significance in analytical techniques and diagnostics, sensitive and selective detection of neurotransmitters is increasingly attracting a lot of attention in different areas of bio-analysis or biomedical research. Recently, fluorescent techniques for detection of catecholamines have attracted interests due to their reasonable cost, convenient control, as well as maneuverability in biological environments. Nevertheless, with the observed need for a sensitive and selective catecholamines sensor, the development of a convenient method for this neurotransmitter is still at its basic level. The manipulation of nanostructured materials in conjunction with biological molecules has led to the development of a new class of hybrid modified biosensors in which both enhancement of charge transport and biological activity preservation may be obtained. Immobilization of biomaterials on electrode surfaces is the crucial step in fabricating electrochemical as well as optical biosensors and bioelectronic devices. Continuing systematic investigation in the manufacturing of enzyme–conducting sensitive systems, here is presented a convenient fluorescence sensing strategy for catecholamines detection based on FRET (fluorescence resonance energy transfer) phenomena observed for, i.e., complexes of Fe²⁺ and epinephrine. The biosensor was constructed using low temperature co-fired ceramics technology (LTCC). This sensing system used the catalytical oxidation of catecholamines and quench of the strong luminescence of obtained complexes due to FRET. The detection process was based on the oxidation of substrate in the presence of the enzyme–laccase/tyrosinase.

Keywords: biosensor, conducting polymer, enzyme, FRET, LTCC

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Authors: Kjellgren Maria, Lilliehorn Sara, Markström Urban

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This article describes the counselling practice of school social workers (SSWs) with individual children. SSWs work in the school system’s pupil health team, whose primary task is health promotion and prevention. The work of SSWs is about helping children and adolescents who, for various reasons, suffer from mental ill-health, school absenteeism, or stress that make them unable to achieve their intended goals. SSWs preferably meet these children in individual counselling sessions. The aim of this article is to describe and analyse SSWs’ experience of counselling with children and to examine the characteristics of counselling practice. The data collection was conducted through four semi-structured focus group interviews with a total of 22 SSWs in four different regions in Sweden. SSWs provide counselling to children in order to bring about improved feelings or behavioural changes. It can be noted that SSWs put emphasis on both the counselling process and the alliance with the child. The interviews showed a common practice among SSWs regarding the structure of the counselling sessions, with certain steps and approaches being employed. However, the specific interventions differed and were characterised by an eclectic standpoint in which SSWs utilise a broad repertoire of therapeutic schools and techniques. Furthermore, a relational perspective emerged as a most prominent focus for the SSWs by re-emerging throughout the material. We believe that SSWs could benefit from theoretical perspectives on ‘contextual model’ and ‘attachment theory’ as ‘models of the mind’. Being emotionally close to the child and being able to follow their development requires a lot from SSWs, as both professional caregivers and as “safe havens”.

Keywords: school social conselling, school social workers, contextual model, attachment thory

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Authors: Amarjyoti Das Mahapatra, Umesh Kumar, Bhaskar Datta

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A pseudo peptide can mimic the biological or structural properties of natural peptides. They have become an increasing attention in medicinal chemistry because of their interesting advantages like more bioavailability and less biodegradation than compare to the physiologically active native peptides which increase their therapeutic applications. Many biologically active compounds contain urea as functional groups, and they have improved pharmacokinetic properties because of their bioavailability and metabolic stability. Recently we have reported a single-step synthesis of sulfonyl urea and sulfonyltriuret from sulfonyl chloride and sodium cyanate. But the yield of sulfonyltriuret was less around 40-60% because of the formation of other products like sulfonamide and sulfonylureas. In the present work, we mainly focused on the selective synthesis of sulfonyltriuret using tetrabutylammonium cyanate and sulfonyl chloride. More precisely, we are interested in the controlled synthesis of oligomeric urea mainly sulfonyltriuret as a new class of pseudo peptide and their application as protease modulators. The distinctive architecture of these molecules in the form of their pseudo-peptide backbone offers promise as a potential pharmacophore. The synthesized molecules have been screened on trypsin enzyme, and we observed that these molecules are the efficient modulator of trypsin enzyme.

Keywords: pseudo peptide, pharmacophore, sulfonyltriuret, trypsin

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Authors: Z. Honarvar, M. Farhoodi, M. R. Khani, S. Shojaee-Aliabadi

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Recently, edible films and coating have attracted much attention in food industry due to their environmentally friendly nature and safety in direct contact with food. However edible films have relatively weak mechanical properties and high water vapor permeability. Therefore, the aim of the study was to develop bilayer carboxymethyl cellulose (CMC) coated polypropylene (PP) films to increase mechanical properties and water vapor resistance of each pure CMC or PP films. To modify the surface properties of PE for better attachment of CMC coating layer to PP the atmospheric cold plasma treatment was used. Then the PP surface changes were evaluated by contact angle, AFM, and ATR-FTIR. Furthermore, the physical, mechanical, optical and microstructure characteristics of plasma-treated and untreated films were analyzed. ATR-FTIR results showed that plasma treatment created oxygen-containing groups on PP surface leading to an increase in hydrophilic properties of PP surface. Moreover, a decrease in water contact angle (from 88.92° to 52.15°) and an increase of roughness were observed on PP film surface indicating good adhesion between hydrophilic CMC and hydrophobic PP. Furthermore, plasma pre-treatment improved the tensile strength of CMC coated-PP films from 58.19 to 61.82. Water vapor permeability of plasma treated bilayer film was lower in comparison with untreated film. Therefore, cold plasma treatment has potential to improve attachment of CMC coating to PP layer, leading to enhanced water barrier and mechanical properties of CMC coated polypropylene as food packaging in which also CMC is in contact with food.

Keywords: carboxymethyl cellulose film, cold plasma, Polypropylene, surface properties

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Authors: Donna M. Morrison, Lambert Chester, Coretta A. N. Samuels, David R. Ledoux

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A survey was conducted in the five rice-growing regions in Guyana to determine the presence of aflatoxins in multiple fractions of rice in June/October 2015 growing season. The fractions were paddy, steamed paddy, cargo rice, white rice and parboiled rice. Samples were analyzed by High Performance Liquid Chromatography. A subset of the samples was further analyzed by enzyme-linked immunosorbent assay (ELISA) for concurrence. All analyses were conducted at the University of Missouri, USA. Of the 186 samples tested, 16 had aflatoxin concentrations greater than 20 ppb the recommended limit for aflatoxins in food according to the United States Food and Drug Administration. An additional three samples had aflatoxin B₁ concentrations greater than the European Union Commission maximum levels for aflatoxin B₁ in rice at 5 µg/kg and total aflatoxins (B₁, B₂, G₁ and G₂) at 10 µg/kg. The survey indicates that there is no widespread aflatoxin problem in rice in Guyana. The incidence of aflatoxins appears to be localized.

Keywords: aflatoxin, enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), rice fractions

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Authors: Lorena C. Cintra, Izadora M. De Oliveira, Amanda G. Fernandes, Francieli Colussi, Rosália S. A. Jesuíno, Fabrícia P. Faria, Cirano J. Ulhoa

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Xylan is the main component of hemicellulose and for its complete degradation is required cooperative action of a system consisting of several enzymes including endo-xylanases (XYN), β-xylosidases (XYL) and α-L-arabinofuranosidases (ABF). The recombinant hemicellulolytic enzymes an endoxylanase (HXYN2), β-xylosidase (HXYLA), and an α-L-arabinofuranosidase (ABF3) were used in hydrolysis tests. These three enzymes are produced by filamentous fungi and were expressed heterologously and produced in Pichia pastoris previously. The aim of this work was to evaluate the effect of recombinant hemicellulolytic enzymes on the enzymatic hydrolysis of sugarcane bagasse (SCB). The interaction between the three recombinant enzymes during SCB pre-treated by steam explosion hydrolysis was performed with different concentrations of HXYN2, HXYLA and ABF3 in different ratios in according to a central composite rotational design (CCRD) 23, including six axial points and six central points, totaling 20 assays. The influence of the factors was assessed by analyzing the main effects and interaction between the factors, calculated using Statistica 8.0 software (StatSoft Inc. Tulsa, OK, USA). The Pareto chart was constructed with this software and showed the values of the Student’s t test for each recombinant enzyme. It was considered as response variable the quantification of reducing sugars by DNS (mg/mL). The Pareto chart showed that the recombinant enzyme ABF3 exerted more significant effect during SCB hydrolysis, with higher concentrations and with the lowest concentration of this enzyme. It was performed analysis of variance according to Fisher method (ANOVA). In ANOVA for the release of reducing sugars (mg/ml) as the variable response, the concentration of ABF3 showed significance during hydrolysis SCB. The result obtained by ANOVA, is in accordance with those presented in the analysis method based on the statistical Student's t (Pareto chart). The degradation of the central chain of xylan by HXYN2 and HXYLA was more strongly influenced by ABF3 action. A model was obtained, and it describes the performance of the interaction of all three enzymes for the release of reducing sugars, and can be used to better explain the results of the statistical analysis. The formulation capable of releasing the higher levels of reducing sugars had the following concentrations: HXYN2 with 600 U/g of substrate, HXYLA with 11.5 U.g-1 and ABF3 with 0.32 U.g-1. In conclusion, the recombinant enzyme that has a more significant effect during SCB hydrolysis was ABF3. It is noteworthy that the xylan present in the SCB is arabinoglucoronoxylan, due to this fact debranching enzymes are important to allow access of enzymes that act on the central chain.

Keywords: experimental design, hydrolysis, recombinant enzymes, sugar cane bagasse

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Authors: Surekha Rathod

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Sickle cell disease is a vascular disorder characterized by chronic, ongoing organ damage that is punctuated by episodes of acutely painful vascular complications.1 It is the most common genetic blood disorder in the United States, with about 2000 infants being identified through routine blood screenings annually, and an estimated 104,000-138,000 affected individuals living in the United States. Approximately 0.3%-1.3% of African American are affected by Sickle Cell Diseases (SCD).3 The aim of this paper is to present oral health status of patients with SCD. A total of 200 subjects of both sexes in the age group 18- 40 years were included in this study. The subjects were examined and the following indices were recorded • Oral hygiene index – Simplified (OHI-S). • Probing depths (PD). • Clinical Attachment Levels (CAL). • Gingival Index - Loe and Sillness. • Turesky Gillmore Glickman Modification of the Quigley Hein Plaque Index. (1970) • DMFT index. • Sickle Cell Disease Severity Index. A total of 1478 patients were screened of which 200 subjects were found to be diagnosed with SCD by electrophoresis. The study thus, included 200 subjects (111 females & 89 males) diagnosed with Sickle Cell Disease in the age group of 18-40 years. The probing pocket depths (PPD) were measured in millimeters. 36% had PPD in the range of 2-4mm, 48% had PPD in the range of 4-6mm while 16% had PPD of more than 6mm. Similar results were obtained for the Clinical Attachment Levels (CAL). 29.5 % subjects had CAL 2-4mm, 44.5% had 4-6mm & 26% had CAL 6mm & above. We can thus conclude that although oral health is not a priority for patients with SCD, it is supported by increased plaque accumulation. Because of the chronic anemic state of the patients with SCD, they should be encouraged to pay strict attention to oral hygiene instructions and practice.

Keywords: chronic, genetic, oral, sickle cell disease, vascular

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Authors: Daniela N. Correa-Llantén, Sebastián A. Muñoz-Ibacache, Mathilde Maire, Jenny M. Blamey

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The biosynthesis of nanoparticles by microorganisms, on the contrary to chemical synthesis, is an environmentally-friendly process which has low energy requirements. In this investigation, we used the microorganism Geobacillus wiegelii, strain GWE1, an aerobic thermophile belonging to genus Geobacillus, isolated from a drying oven. This microorganism has the ability to reduce selenite evidenced by the change of color from colorless to red in the culture. Elemental analysis and composition of the particles were verified using transmission electron microscopy and energy-dispersive X-ray analysis. The nanoparticles have a defined spherical shape and a selenium elemental state. Previous experiments showed that the presence of the whole microorganism for the reduction of selenite was not necessary. The results strongly suggested that an intracellular NADPH/NADH-dependent reductase mediates selenium nanoparticles synthesis under aerobic conditions. The enzyme was purified and identified by mass spectroscopy MALDI-TOF TOF technique. The enzyme is a 1-pyrroline-5-carboxylate dehydrogenase. Histograms of nanoparticles sizes were obtained. Size distribution ranged from 40-160 nm, where 70% of nanoparticles have less than 100 nm in size. Spectroscopic analysis showed that the nanoparticles are composed of elemental selenium. To analyse the effect of pH in size and morphology of nanoparticles, the synthesis of them was carried out at different pHs (4.0, 5.0, 6.0, 7.0, 8.0). For thermostability studies samples were incubated at different temperatures (60, 80 and 100 ºC) for 1 h and 3 h. The size of all nanoparticles was less than 100 nm at pH 4.0; over 50% of nanoparticles have less than 100 nm at pH 5.0; at pH 6.0 and 8.0 over 90% of nanoparticles have less than 100 nm in size. At neutral pH (7.0) nanoparticles reach a size around 120 nm and only 20% of them were less than 100 nm. When looking at temperature effect, nanoparticles did not show a significant difference in size when they were incubated between 0 and 3 h at 60 ºC. Meanwhile at 80 °C the nanoparticles suspension lost its homogeneity. A change in size was observed from 0 h of incubation at 80ºC, observing a size range between 40-160 nm, with 20% of them over 100 nm. Meanwhile after 3 h of incubation at size range changed to 60-180 nm with 50% of them over 100 nm. At 100 °C the nanoparticles aggregate forming nanorod structures. In conclusion, these results indicate that is possible to modulate size and shape of biologically synthesized nanoparticles by modulating pH and temperature.

Keywords: genus Geobacillus, NADPH/NADH-dependent reductase, selenium nanoparticles, biosynthesis

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Authors: Gokhan Zengin, Cengiz Sarikurkcu, Abdurrahman Aktumsek, Ramazan Ceylan

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The present study was designated to characterize the essential oil from S. galatica (SGEOs) and evaluate its antioxidant and enzyme inhibitory activities. Antioxidant capacity were tested different methods including free radical scavenging (DPPH, ABTS and NO), reducing power (FRAP and CUPRAC), metal chelating and phosphomolybdenum. Inhibitory activities were analyzed on acetylcholiesterase, butrylcholinesterase, α-amylase and α-glucosidase. SGEOs were chemically analyzed and identified by gas chromatography (GC) and gas chromatography/mass spectrophotometry (GC/MS). 23 components, representing 98.1% of SGEOs were identified. Monoterpene hydrocarbons (74.1%), especially α- (23.0%) and β-pinene (32.2%), were the main constituents in SGEOs. The main sesquiterpene hydrocarbons were β-caryophyllene (16.9%), Germacrene-D (1.2%) and Caryophyllene oxide (1.2%), respectively. Generally, SGEOs has shown moderate free radical, reducing power, metal chelating and enzyme inhibitory activities. These activities related to chemical profile in SGEOs. Our findings supported that the possible utility of SGEOs is a source of natural agents for food, cosmetics or pharmaceutical industries.

Keywords: sideritis galatica, antioxidant, monoterpenes, cholinesterase, anti-diabetic

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Authors: Rajeev Ravindran, Amit K. Jaiswal

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Lignocellulose is the largest reservoir of inexpensive, renewable source of carbon. It is composed of lignin, cellulose and hemicellulose. Cellulose and hemicellulose is composed of reducing sugars glucose, xylose and several other monosaccharides which can be metabolised by microorganisms to produce several value added products such as biofuels, enzymes, aminoacids etc. Enzymatic treatment of lignocellulose leads to the release of monosaccharides such as glucose and xylose. However, factors such as the presence of lignin, crystalline cellulose, acetyl groups, pectin etc. contributes to recalcitrance restricting the effective enzymatic hydrolysis of cellulose and hemicellulose. In order to overcome these problems, pre-treatment of lignocellulose is generally carried out which essentially facilitate better degradation of lignocellulose. A range of pre-treatment strategy is commonly employed based on its mode of action viz. physical, chemical, biological and physico-chemical. However, existing pretreatment strategies result in lower sugar yield and formation of inhibitory compounds. In order to overcome these problems, we proposes a novel pre-treatment, which utilises the superior oxidising capacity of alkaline potassium permanganate assisted by ultra-sonication to break the covalent bonds in spent coffee waste to remove recalcitrant compounds such as lignin. The pre-treatment was conducted for 30 minutes using 2% (w/v) potassium permanganate at room temperature with solid to liquid ratio of 1:10. The pre-treated spent coffee waste (SCW) was subjected to enzymatic hydrolysis using enzymes cellulase and hemicellulase. Shake flask experiments were conducted with a working volume of 50mL buffer containing 1% substrate. The results showed that the novel pre-treatment strategy yielded 7 g/L of reducing sugar as compared to 3.71 g/L obtained from biomass that had undergone dilute acid hydrolysis after 24 hours. From the results obtained it is fairly certain that ultrasonication assists the oxidation of recalcitrant components in lignocellulose by potassium permanganate. Enzyme hydrolysis studies suggest that ultrasound assisted alkaline potassium permanganate pre-treatment is far superior over treatment by dilute acid. Furthermore, SEM, XRD and FTIR were carried out to analyse the effect of the new pre-treatment strategy on structure and crystallinity of pre-treated spent coffee wastes. This novel one-step pre-treatment strategy was implemented under mild conditions and exhibited high efficiency in the enzymatic hydrolysis of spent coffee waste. Further study and scale up is in progress in order to realise future industrial applications.

Keywords: spent coffee waste, alkaline potassium permanganate, ultra-sonication, physical characterisation

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Authors: Xiran Wang, Johanna Trinkl, Thomas Hoffmann, Wilfried Schwab

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Malonyltransferases (MATs) are enzymes that play a key role in the biosynthesis of secondary metabolites in plants, such as flavonoids and anthocyanins. As a kind of flavonoid-rich fruit, strawberries are an ideal model to study MATs. From Goodberry metabolome data, in the hybrid generation of 2 strawberries various, Fragaria × ananassa cv. 'Senga Sengana' and 'Candonga', we found the malonylated flavonoid concentration is significantly higher in 'Senga Sengana' compared with 'Candonga'. Therefore, we aimed to identify and characterize the malonyltransferases responsible for the different malonylated flavonoid concentrations in two different strawberry cultivars. In this study, we have found 6 MATs via genome mapping, metabolome analysis, gene cloning, and enzyme assay from strawberries, which catalyzed the malonylation of flavonoid substrates: quercetin-3-glucoside, kaempferol-3-glucoside, pelargonidin-3-glucoside, and cyanidin-3-glucoside. All four compounds reacted with FaMATs to varying degrees. These MATs have important implication into strawberries’ flavonoid biosynthesis, and also provide insights into insights into flavonoid biosynthesis, potential applications in agriculture, plant science, and pharmacy, and information on the regulation of secondary metabolism in plants.

Keywords: malonyltransferase, strawberry, flavonoid biosynthesis, enzyme assay

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Authors: Tiantian Min, Chuanxiang Cheng, Jin Yue

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The growth and reproduction of microorganisms in food packaging can cause food decay and foodborne diseases, which pose a serious threat to the health of consumers and even cause serious economic losses. Active food packaging containing antibacterial bioactive compounds is a promising strategy for extending the shelf life of products and maintaining the food quality, as well as reducing the food waste. However, most active packaging can only act as slow-release effect for antimicrobials, which causes the release rate of antimicrobials not match the growth rate of microorganisms. Stimuli-responsive active packaging materials based on biopolymeric substrates and bioactive substances that respond to some biological and non-biological trigger factors provide more opportunities for fresh food preservation. The biological stimuli factors such as relative humidity, pH and enzyme existed in the exudate secreted by microorganisms have been expected to design food packaging materials. These stimuli-responsive materials achieved accurate release or delivery of bioactive substances at specific time and appropriate dose. Recently, metal-organic-frameworks (MOFs) nanoparticles become attractive carriers to enhance the efficiency of bioactive compounds or drugs. Cellulose nanofibrils have been widely applied for film substrates due to their biodegradability and biocompatibility. The abundant hydroxyl groups in cellulose can be oxidized to carboxyl groups by TEMPO, making it easier to anchoring MOFs and to be further modification. In this study, a pH and enzyme dual-responsive CAR@ZIF-8/TOCNF/PE film was fabricated by in-situ growth of ZIF-8 nanoparticles onto TEMPO-oxidized cellulose (TOCNF) film and further coated with pectin (PE) for stabilization and controlled release of carvacrol (CAR). The enzyme triggered release of CAR was achieved owing to the degradation of pectin by pectinase secreted by microorganisms. Similarly, the pH-responsive release of CAR was attributed to the unique skeleton degradation of ZIF-8, further accelerating the release of CAR from the topological structure of ZIF-8. The composite film performed excellent crystallinity and adsorb ability confirmed by X-ray diffraction and BET analysis, and the inhibition efficiency against Escherichia coli, Staphylococcus aureus and Aspergillus niger reached more than 99%. The composite film was capable of releasing CAR when exposure to dose-dependent enzyme (0.1, 0.2, and 0.3 mg/mL) and acidic condition (pH = 5). When inoculated 10 μL of Aspergillus niger spore suspension on the equatorial position of mango and raspberries, this composite film acted as packaging pads effectively inhibited the mycelial growth and prolonged the shelf life of mango and raspberries to 7 days. Such MOF-TOCNF based film provided a targeted, controlled and sustained release of bioactive compounds for long-term antibacterial activity and preservation effect, which can also avoid the cross-contamination of fruits.

Keywords: active food packaging, controlled release, fruit preservation, in-situ growth, stimuli-responsive

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Authors: Brizita Djordjevic, Bojana Vidovic, Milica Zrnic, Uros Cakar, Ivan Stankovic, Davor Korcok, Sladjana Sobajic

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Histamine is a biogenic amine, which is formed by enzymatic decarboxylation from the amino acid histidine. It can be found in foods such as fish and fish products, meat and fermented meat products, cheese, wine and beer. The presence of histamine in these foods can indicate microbiological spoilage or poor manufacturing processes. The consumption of food containing large amounts of histamine can have toxicological consequences. In 62 food products (31 canned fish products, 19 wines and 12 cheeses) from the market of Serbia the content of histamine was determined using enzyme-linked immunosorbent assay (ELISA) test kit according to the manufacturer's instructions (Immunolab GmbH, Kassel, Germany). The detection limits of this assay were 20 µg/kg for fish and cheese and 4 µg/L for wine. The concentration of histamine varied between 0.16-207 mg/kg in canned fish products, 0.03-1.47 mg/kg in cheeses and 0.01- 0.18 mg/L in wines. In all analyzed canned fish products the results obtained for the histamine were below the limits set by European and national legislation, so they can be considered acceptable and safe for the health consumers. The levels of histamine in analyzed cheeses and wines were very low and did not pose safety concerns.

Keywords: cheese, enzyme-linked immunosorbent assay, histamine, fish products, wine

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Authors: M. O. Bayram, H. B. Gencdal, N. O. Fercan, B. Basbug

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Liquid limit, which is used as a measure of soil strength, can be detected by Casagrande and fall-cone testing methods. The two methods majorly diverge from each other in terms of operator dependency. The Casagrande method that is applied according to ASTM D4318-17 standards may give misleading results, especially if the calibration process is not performed well. To reveal the effect of calibration for drop height and amount of soil paste placement in the Casagrande cup, a series of tests were carried out by multipoint method as it is specified in the ASTM standards. The tests include the combination of 6 mm, 8 mm, 10 mm, and 12 mm drop heights and under-filled, half-filled, and full-filled Casagrande cups by kaolinite samples. It was observed that during successive tests, the drop height of the cup deteriorated; hence the device was recalibrated before and after each test to provide the accuracy of the results. Besides, the tests by under-filled and full-filled samples for higher drop heights revealed lower liquid limit values than the lower drop heights revealed. For the half-filled samples, it was clearly seen that the liquid limit values didn’t change at all as the drop height increased, and this explains the function of standard specifications.

Keywords: calibration, casagrande cup method, drop height, kaolinite, liquid limit, placing form

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Authors: Mir Hussain Nawaz, Lyudmila Nedyalkova, Haizhong Zhu, Wael M. Rabeh

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In this study, we aim to biochemically and structurally characterize the interactions of human HK2 with the mitochondria in addition to the role of its N-terminal domain in catalysis and stability of the full-length enzyme. Here, we solved the crystal structure of human HK2 in complex with glucose and glucose-6-phosphate (PDB code: 2NZT), where it is a homodimer with catalytically active N- and C-terminal domains linked by a seven-turn α-helix. Different from the inactive N-terminal domains of isozymes 1 and 3, the N- domain of HK2 not only capable to catalyze a reaction but it is responsible for the thermodynamic stabilizes of the full-length enzyme. Deletion of first α-helix of the N-domain that binds to the mitochondria altered the stability and catalytic activity of the full-length HK2. In addition, we found the linker helix between the N- and C-terminal domains to play an important role in controlling the catalytic activity of the N-terminal domain. HK2 is a major step in the regulation of glucose metabolism in cancer making it an ideal target for the development of new anticancer therapeutics. Characterizing the structural and molecular mechanisms of human HK2 and its role in cancer metabolism will accelerate the design and development of new cancer therapeutics that are safe and cancer specific.

Keywords: cancer metabolism, enzymology, drug discovery, protein stability

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Authors: Hadjer Didouh, Izzaddine Sameut Bouhaik, Mohammed Hadj Meliani

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This research delves into the intricate dynamics of biofilm adhesion on surfaces, particularly focusing on the widely employed X52 surface in oil and gas industry pipelines. Biofilms, characterized by microorganisms within a self-produced matrix, pose significant challenges due to their detrimental impact on surfaces. Our study integrates advanced molecular techniques and cutting-edge microscopy, such as scanning electron microscopy (SEM), to identify microbial communities and visually assess biofilm adhesion. Simultaneously, we concentrate on the X52 surface, utilizing impedance spectroscopy and potentiodynamic polarization to gather electrochemical responses under various conditions. In conjunction with the broader investigation, we propose a novel approach to mitigate biofilm-induced corrosion challenges. This involves environmentally friendly inhibitors derived from plants, offering a sustainable alternative to conventional chemical treatments. Our inquiry screens and selects inhibitors based on their efficacy in hindering biofilm formation and reducing corrosion rates on the X52 surface. This study contributes valuable insights into the interplay between electrochemical processes and biofilm attachment on the X52 surface. Furthermore, the outcomes of this research have broader implications for the oil and gas industry, where biofilm-related corrosion is a persistent concern. The exploration of eco-friendly inhibitors not only holds promise for corrosion control but also aligns with environmental considerations and sustainability goals. The comprehensive nature of this research aims to enhance our understanding of biofilm dynamics, provide effective strategies for corrosion mitigation, and contribute to sustainable practices in pipeline management within the oil and gas sector.

Keywords: bio-corrosion, biofilm, attachment, X52, metal/bacteria interface

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Authors: Afshin Farahbakhsh, Hoda Khodadadi

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In this paper, we compare five methods of biological fuel cell fabrication by combining a Shewanella oneidensis microbial anode and a laccase-modified air-breathing cathode. As a result of biofuel cell laccase with graphite nanofibers, carbon surface (PAMAN) on the pt/hpg electrode, graphite sheets MWCNT and with (PG) and (MWCNT) showed, respectively. Describes methods for creating controllable and reproducible bio-anodes and demonstrates the versatility of hybrid biological fuel cells. The laccase-based biocathodes prepared either with the crude extract or with the purified enzyme can provide electrochemically active and stable biomaterials. The laccase-based biocathodes prepared either with the crude extract or with the purified enzyme can provide electrochemically active and stable biomaterials. When the device was fed with transdermal extracts, containing only 30μM of glucose, the average peak power was proportionally lower (0.004mW). The result of biofuel cell with graphite nanofibers showed the enzymatic fuel cell reaches 0.5 V at open circuit voltage with both, ethanol and methanol and the maximum current density observed for E2electrode was 228.94mAcm.

Keywords: enzymatic electrode, fuel cell, immobilization, laccase

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Authors: J. Janicki, M. Rom, C. Slusarczyk, J. Fabia, M. Siika-aho, K. Marjamaa, K. Kruus, K. Langfelder, C. Steel, M. Paloheimo, T. Puranen, S. Mäkinen, D. Wawro

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The cellulose is one of the most abundant polymers in the world, however, its application in the high-end value products such as films or fibres, it triggered by the cellulose properties. The noticeable presence of hydrogen bonding reflected with partially crystalline structure makes the cellulose insoluble in common solvents and not meltable. The existing technologies, such as viscose process, suffer from environmental and economical problems, because of the risk of harmful chemicals liberation during the spinning process. The enzymatic modification of cellulose with endoglucanase makes it directly alkali soluble in NaOH solution, giving the opportunities for film and fibers formation. As the effect of enzymatic treatment, there are observed changes in crystalline structure and accompanying changes of the affinity of cellulose to water, demonstrated by water retention value. The objective of the project ELMO - Novel carbohydrate modifying enzymes for fibre modification is is to develop new enzyme products for modification of dissolving grade pulps. The aim is to increase the reactivity of dissolving grade pulps and remove residual hemicellulose. The scientific aim of this paper is to present the effect of enzymatic treatment on the crystallinity and affinity to water of cellulose pulps modified with enzymes.

Keywords: cellulose, crystallinity, WAXS, enzyme

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Authors: A. T. Ramachandra Naik, H. Shivananda Murthy, H. n. Anjanayappa

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The freshwater prawn, Macrobrachium rosenbergii is a more popular crustacean cultured widely in monoculture system in India. It has got high nutritional value in the human diet. Hence, understanding its enzymatic and body composition is important in order to judge its flesh quality. Fish oil specially derived from Indian oil sardine is a good source of highly unsaturated fatty acid and lipid source in fish/prawn diet. A 35% crude protein diet with graded levels of Sardine oil as a source of fat was incorporated at four levels viz, 2.07, 4.07, 6.07 and 8.07% maintaining a total lipid level of feed at 8.11, 10.24, 12.28 and 14.33% respectively. Diet without sardine oil (6.05% total lipid) was served as basal treatment. The giant freshwater prawn, Macrobrachium rosenbergii was used as test animal and the experiment was lost for 112 days. Significantly, higher gain in weight of prawn was recorded in the treatment with 6.07% sardine oil incorporation followed by higher specific growth rate, food conversion rate and protein efficiency ratio. The 8.07% sardine oil diet produced the highest RNA: DNA ratio in the prawn muscle. Digestive enzyme analyses in the digestive tract and mid-gut gland showed the greatest activity in prawns fed the 8.07% diet.

Keywords: digestive enzyme, fish diet, Macrobrachium rosenbergii, sardine oil

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Authors: Paula K. Novelli, Margarida M. Barros, Luciana F. Flueri

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Utilization of agricultural by-products, wheat ban and soybean bran, as substrate for solid state fermentation (SSF) was studied, aiming the achievement of different enzymes from Aspergillus sp. with distinct biological characteristics and its application and improvement on animal nutrition. Aspergillus niger and Aspergillus oryzea were studied as they showed very high yield of phytase and protease production, respectively. Phytase activity was measure using p-nitrophenilphosphate as substrate and a standard curve of p-nitrophenol, as the enzymatic activity unit was the quantity of enzyme necessary to release one μmol of p-nitrophenol. Protease activity was measure using azocasein as substrate. Activity for phytase and protease substantially increased when the different biochemical characteristics were considered in the study. Optimum pH and stability of the phytase produced by A. niger with wheat bran as substrate was between 4.0 - 5.0 and optimum temperature of activity was 37oC. Phytase fermented in soybean bran showed constant values at all pHs studied, for optimal and stability, but low production. Phytase with both substrates showed stable activity for temperatures higher than 80oC. Protease from A. niger showed very distinct behavior of optimum pH, acid for wheat bran and basic for soybean bran, respectively and optimal values of temperature and stability at 50oC. Phytase produced by A. oryzae in wheat bran had optimum pH and temperature of 9 and 37oC, respectively, but it was very unstable. On the other hand, proteases were stable at high temperatures, all pH’s studied and showed very high yield when fermented in wheat bran, however when it was fermented in soybean bran the production was very low. Subsequently the upscale production of phytase from A. niger and proteases from A. oryzae were applied as an enzyme additive in fish fed for digestibility studies. Phytases and proteases were produced with stable enzyme activity of 7,000 U.g-1 and 2,500 U.g-1, respectively. When those enzymes were applied in a plant protein based fish diet for digestibility studies, they increased protein, mineral, energy and lipids availability, showing that these new enzymes can improve animal production and performance. In conclusion, the substrate, as well as, the microorganism species can affect the biochemical character of the enzyme produced. Moreover, the production of these enzymes by SSF can be up to 90% cheaper than commercial ones produced with the same fungi species but submerged fermentation. Add to that these cheap enzymes can be easily applied as animal diet additives to improve production and performance.

Keywords: agricultural by-products, animal nutrition, enzymes production, solid state fermentation

Procedia PDF Downloads 311

Authors: Yomna Ibrahim El-Gazzar, Hussien Ibrahim El-Subbagh, Hanan Hanaa Georgey, Ghada S. Hassan Hassan

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Dihydrofolate reductase (DHFR) is an enzyme that has pivotal importance in biochemistry and medicinal chemistry. It catalyzes the reduction of dihydrofolate to tetrahydrofolate and intimately couples with thymidylate synthase. Thymidylate synthase is a crucial enzyme that catalyzes the reductive methylation of (dUMP) to (dTMP) utilizing N5, N10-methylenetetrahydrofolate as a cofactor. A new series of 2-substituted thio-quinazolin-4-one analogs was designed that possessed electron withdrawing or donating functional groups (Cl or OCH3) at position 6- or 7-, 4-methoxyphenyl function at position 3-.The thiol function is used to connect to either 1,2,4-triazole, or 1,3,4-thiadiazole via a methylene bridge. Most of the functional groups designed to be accommodated on the quinazoline ring such as thioether, alkyl to increase lipid solubility of polar compounds, a character very much needed in the nonclassical DHFR inhibitors. The target compounds were verified with spectral data and elemental analysis. DHFR inhibitions, as well as antitumor activity, were applied on three cell lines (MCF-7, CACO-2, HEPG-2).

Keywords: nonclassical antifolates, DHFR Inhibitors, antitumor activity, quinazoline ring

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Authors: M. Fatmi, B. Gueridi, Z. Zerrougui

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Some physical properties of hexahalometallate K₂XBr₆(X=Se, Pt) were computed in the zinc blend structure using generalized gradient approximation. The cell constant of K₂SeBr₆ and K₂PtBr₆ is consistent with the experiment value quoted in the literature, where the error is 0.95 % and 1 %. K₂SeBr₆ and K₂PtBr₆ present covalent bonding, high anisotropy and are ductile. The elastic constants of K₂SeBr₆ and K₂PtBr₆ are significantly smaller due to their larger reticular distances and lower Colombian forces, and then they are soft and damage tolerant. The interatomic separation is greater in K₂SeBr₆ than in K₂PtBr₆; hence the Colombian interaction in K₂PtBr₆ is greater than that of K2SeBr₆. The internal coordinate of the Br atom in K₂PtBr₆ is lower than that of the same atom in K2SeBr₆, and this can be explained by the fact that it is inversely proportional to the atom radius of Se and Pt. There are two major plasmonic processes, with intensities of 3.7 and 1.35, located around 53.5 nm and 72.8 nm for K₂SeBr₆ and K₂PtBr₆.

Keywords: hexahalometallate, band structure, morphology, absorption, band gap, absorber

Procedia PDF Downloads 75