Commenced in January 2007
Frequency: Monthly
Edition: International
Paper Count: 6466

Search results for: specific enzyme

6466 Effect of Ultrasound on the Hydrolysis of Soy Oil Catalyzed by 1,3-Specific Lipase Abstract

Authors: Jamal Abd Awadallak, Thiago Olinek Reinehr, Eduardo Raizer, Deise Molinari, Edson Antonio, Camila da Silva da Silva

Abstract:

The hydrolysis of soy oil catalyzed by 1,3-specific enzyme (Lecitase Ultra) in a well-stirred bioreactor was studied. Two forms of applications of the ultrasound were evaluated aiming to increase reaction rates, wherein the use of probe ultrasound associated with the use of surfactant to pre-emulsify the substrate showed the best results. Two different reaction periods were found: the first where the ultrasound has great influence on reaction rates, and the second where ultrasound influence is minimal. Studies on the time of pre-emulsification, surfactant concentration and enzyme concentration showed that the initial rate of hydrolysis depends on the interfacial area between the oil phase and the aqueous phase containing the enzyme.

Keywords: specific enzyme, free fatty acids, Hydrolysis, lecitase ultra, ultrasound

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6465 Cytotoxic Effect of Purified and Crude Hyaluronidase Enzyme on Hep G2 Cell Line

Authors: Furqan M. Kadhum, Asmaa A. Hussein, Maysaa Ch. Hatem

Abstract:

Hyaluronidase enzyme was purified from the clinical isolate Staphyloccus aureus in three purification steps, first by precipitation with 90% saturated ammonium sulfate, ion exchange chromatography on DEAE-Cellulose, and gel filtration chromatography throughout Sephacryl S-300. Specific activity of the purified enzyme was reached 930 U/mg protein with 7.4 folds of purification and 46.5% recovery. The enzyme has an average molecular weight of about 69 kDa, with an optimum pH of enzyme activity and stability at pH 7, also the optimum temperature for activity was 37oC. The enzyme was stable with full activity at a temperature ranged between 30-40 oC. Metal ions showed variable inhibitory degree with the strongest effect for Fe+3, however, the chelating and reducing agents had no or little effects. Cytotoxic studies for purified and crude hyaluronidase against cancer cell Hep G2 type at different enzyme concentrations and exposure times showed that the inhibition effect of both crude and purified enzyme increased by increasing the enzyme concentration with no change was observed at 24hr, while at 48 and 72 hrs the same inhibition rate were observed for purified enzyme and differ for the crude filtrate.

Keywords: hyaluronidase, S. aureus, metal ions, cytotoxicity

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6464 The Construction of a Probiotic Lactic Acid Bacterium Expressing Acid-Resistant Phytase Enzyme

Authors: R. Majidzadeh Heravi, M. Sankian, H. Kermanshahi, M. R. Nassiri, A. Heravi Moussavi, S. A. Lari, A. R. Varasteh

Abstract:

The use of probiotics engineered to express specific enzymes has been the subject of considerable attention in poultry industry because of increased nutrient availability and reduced cost of enzyme supplementation. Phytase enzyme is commonly added to poultry feed to improve digestibility and availability of phosphorus from plant sources. To construct a probiotic with potential of phytate degradation, phytase gene (appA) from E. coli was cloned and transformed into two probiotic bacteria Lactobacillus salivarius and Lactococcus lactis. L. salivarous showed plasmid instability, unable to express the gene. The expression of appA gene in L. lactis was analyzed by detecting specific RNA and zymography assay. Phytase enzyme was isolated from cellular extracts of recombinant L. lactis, showing a 46 kDa band upon the SDS-PAGE analysis. Zymogram also confirmed the phytase activity of the 46 kDa band corresponding to the enzyme. An enzyme activity of 4.9U/ml was obtained in cell extracts of L. lactis. The growth of native and recombinant L. lactis was similar in the presence of two concentrations of ox bile.

Keywords: Lactobacillus salivarus, Lactococcuslactis, recombinant, phytase, poultry

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6463 Medium Design and Optimization for High Β-Galactosidase Producing Microbial Strains from Dairy Waste through Fermentation

Authors: Ashish Shukla, K. P. Mishra, Pushplata Tripathi

Abstract:

This paper investigates the production and optimization of β-galactosidase enzyme using synthetic medium by isolated wild strains (S1, S2) mutated strains (M1, M2) through SSF and SmF. Among the different cell disintegration methods used, the highest specific activity was obtained when the cells were permeabilized using isoamyl alcohol. Wet lab experiments were performed to investigate the effects of carbon and nitrogen substrates present in Vogel’s medium on β-galactosidase enzyme activity using S1, S2, and M1, M2 strains through SSF. SmF experiments were performed for effects of carbon and nitrogen sources in YLK2Mg medium on β-galactosidase enzyme activity using S1, S2 and M1, M2 strains. Effect of pH on β-galactosidase enzyme production was also done using S1, S2, and M1, M2 strains. Results were found to be very appreciable in all the cases.

Keywords: β-galactosidase, cell disintegration, permeabilized, SSF, SmF

Procedia PDF Downloads 192
6462 Colorimetric Measurement of Dipeptidyl Peptidase IV (DPP IV) Activity via Peptide Capped Gold Nanoparticles

Authors: H. Aldewachi, M. Hines, M. McCulloch, N. Woodroofe, P. Gardiner

Abstract:

DPP-IV is an enzyme whose expression is affected in a variety of diseases, therefore, has been identified as possible diagnostic or prognostic marker for various tumours, immunological, inflammatory, neuroendocrine, and viral diseases. Recently, DPP-IV enzyme has been identified as a novel target for type II diabetes treatment where the enzyme is involved. There is, therefore, a need to develop sensitive and specific methods that can be easily deployed for the screening of the enzyme either as a tool for drug screening or disease marker in biological samples. A variety of assays have been introduced for the determination of DPP-IV enzyme activity using chromogenic and fluorogenic substrates, nevertheless these assays either lack the required sensitivity especially in inhibited enzyme samples or displays low water solubility implying difficulty for use in vivo samples in addition to labour and time-consuming sample preparation. In this study, novel strategies based on exploiting the high extinction coefficient of gold nanoparticles (GNPs) are investigated in order to develop fast, specific and reliable enzymatic assay by investigating synthetic peptide sequences containing a DPP IV cleavage site and coupling them to GNPs. The DPP IV could be detected by colorimetric response of peptide capped GNPs (P-GNPS) that could be monitored by a UV-visible spectrophotometer or even naked eyes, and the detection limit could reach 0.01 unit/ml. The P-GNPs, when subjected to DPP IV, showed excellent selectivity compared to other proteins (thrombin and human serum albumin) , which led to prominent colour change. This provided a simple and effective colorimetric sensor for on-site and real-time detection of DPP IV.

Keywords: gold nanoparticles, synthetic peptides, colorimetric detection, DPP-IV enzyme

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6461 Growth Performance, Survival Rate and Feed Efficacy of Climbing Perch, Anabas testudineus, Feed Experimental Diet with Several Dosages of Papain Enzyme

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

Abstract:

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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6460 The Enzyme Inhibitory Potentials of Different Extracts from Linaria genistifolia subsp. genistifolia

Authors: Gokhan Zengin, Abdurrahman Aktumsek

Abstract:

The key enzyme inhibitory theory is one of the most accepted strategies in the treatment of global health problems including Alzheimer’s Disease and Diabetes mellitus. For this reason, the enzyme inhibitory potentials of different solvent extracts from Linaria genistifolia subsp. genistifolia were investigated against cholinesterase, and tyrosinase. The in vitro enzyme inhibitory potentials were measured with a microplate reader. The acetone and methanol extracts exhibited the strongest enzyme inhibitory effects on cholinesterase. However, the water extract was only active on tyrosinase. The results suggested that Linaria genistifolia subsp. genistifolia could be considered as a source of natural enzyme inhibitors for the treatment of major health problems.

Keywords: enzyme inhibitors, cholinesterase, tyrosinase, linaria, Turkey

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6459 Expression of Fused Plasmodium falciparum Orotate Phosphoribosyltransferase and Orotidine 5'-Monophosphate Decarboxylase in Escherichia coli

Authors: Waranya Imprasittichai, Patsarawadee Paojinda, Sudaratana R. Krungkrai, Nirianne Marie Q. Palacpac, Toshihiro Horii, Jerapan Krungkrai

Abstract:

Fusion of the last two enzymes in the pyrimidine biosynthetic pathway in the inversed order by having COOH-terminal orotate phosphoribosyltransferase (OPRT) and NH2-terminal orotidine 5'-monophosphate decarboxylase (OMPDC), as OMPDC-OPRT, are described in many organisms. In this study, we constructed gene fusions of Plasmodium falciparum OMPDC-OPRT (1,836 bp) in pTrcHisA vector and expressed as an 6xHis-tag bifunctional protein in three Escherichia coli strains (BL21, Rosetta, TOP10) at 18 °C, 25 °C and 37 °C. The recombinant bifunctional protein was partially purified by Ni-Nitrilotriacetic acid-affinity chromatography. Specific activities of OPRT and OMPDC domains in the bifunctional enzyme expressed in E. coli TOP10 cells were approximately 3-4-fold higher than those in BL21 cells. There were no enzymatic activities when the construct vector expressed in Rosetta cells. Maximal expression of the fused gene was observed at 18 °C and the bifunctional enzyme had specific activities of OPRT and OMPDC domains in a ratio of 1:2. These results provide greater yields and better catalytic activities of the bifunctional OMPDC-OPRT enzyme for further purification and kinetic study.

Keywords: bifunctional enzyme, orotate phosphoribosyltransferase, orotidine 5'-monophosphate decarboxylase, plasmodium falciparum

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6458 Production of Linamarase from Lactobacillus delbrueckii NRRL B-763

Authors: Ogbonnaya Nwokoro, Florence O. Anya

Abstract:

Nutritional factors relating to the production of linamarase from Lactobacillus delbrueckii NRRL B–763 were investigated. The microorganism was cultivated in a medium containing 1% linamarin. Enzyme was produced using a variety of carbon substrates but the highest enzyme activity was detected in the presence of salicin (522 U/ml) after 48 h while the lowest yield was observed with CM cellulose (38 U/ml) after 72 h. Enzyme was not produced in the presence of cellobiose. Among a variety of nitrogen substrates tested, peptone supported maximum enzyme production (412 U/ml) after 48 h. Lowest enzyme production was observed with urea (40 U/ml). Organic nitrogen substrates generally supported higher enzyme productivity than inorganic nitrogen substrates. Enzyme activity was observed in the presence of Mn2+ (% relative activity = 216) while Hg2+ was inhibitory (% relative activity = 28). Locally-formulated media were comparable to MRS broth in supporting linamarase production by the bacterium. Higher enzyme activity was produced in media with surfactant than in media without surfactant. The enzyme may be useful in enhanced degradation of cassava cyanide.

Keywords: linamarase, locally formulated media, carbon substrates, nitrogen substrates, metal ions

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6457 Effect of Ethanol Concentration and Enzyme Pre-Treatment on Bioactive Compounds from Ginger Extract

Authors: S. Lekhavat, T. Kajsongkram, S. Sang-han

Abstract:

Dried ginger was extracted and investigated the effect of ethanol concentration and enzyme pre-treatment on its bioactive compounds in solvent extraction process. Sliced fresh gingers were dried by oven dryer at 70 °C for 24 hours and ground to powder using grinder which their size were controlled by passing through a 20-mesh sieve. In enzyme pre-treatment process, ginger powder was sprayed with 1 % (w/w) cellulase and then was incubated at 45 °C for 2 hours following by extraction process using ethanol at concentration of 0, 20, 40, 60 and 80 % (v/v), respectively. The ratio of ginger powder and ethanol are 1:9 and extracting conditions were controlled at 80 °C for 2 hours. Bioactive compounds extracted from ginger, either enzyme-treated or non enzyme-treated samples, such as total phenolic content (TPC), 6-Gingerol (6 G), 6-Shogaols (6 S) and antioxidant activity (IC50 using DPPH assay), were examined. Regardless of enzyme treatment, the results showed that 60 % ethanol provided the highest TPC (20.36 GAE mg /g. dried ginger), 6G (0.77%), 6S (0.036 %) and the lowest IC50 (625 μg/ml) compared to other ratios of ethanol. Considering the effect of enzyme on bioactive compounds and antioxidant activity, it was found that enzyme-treated sample has more 6G (0.17-0.77 %) and 6S (0.020-0.036 %) than non enzyme-treated samples (0.13-0.77 % 6G, 0.015-0.036 % 6S). However, the results showed that non enzyme-treated extracts provided higher TPC (6.76-20.36 GAE mg /g. dried ginger) and Lowest IC50 (625-1494 μg/ml ) than enzyme-treated extracts (TPC 5.36-17.50 GAE mg /g. dried ginger, IC50 793-2146 μg/ml).

Keywords: antioxidant activity, enzyme, extraction, ginger

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6456 The Modeling of Viscous Microenvironment for the Coupled Enzyme System of Bioluminescence Bacteria

Authors: Irina E. Sukovataya, Oleg S. Sutormin, Valentina A. Kratasyuk

Abstract:

Effect of viscosity of media on kinetic parameters of the coupled enzyme system NADH:FMN-oxidoreductase–luciferase was investigated with addition of organic solvents (glycerol and sucrose), because bioluminescent enzyme systems based on bacterial luciferases offer a unique and general tool for analysis of the many analytes and enzymes in the environment, research, and clinical laboratories and other fields. The possibility of stabilization and increase of activity of the coupled enzyme system NADH:FMN-oxidoreductase–luciferase activity in vicious aqueous-organic mixtures have been shown.

Keywords: coupled enzyme system of bioluminescence bacteria NAD(P)H:FMN-oxidoreductase–luciferase, glycerol, stabilization of enzymes, sucrose

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6455 Effect of a Muscarinic Antagonist Drug on Extracellular Lipase Activityof Pseudomonas aeruginosa

Authors: Zohreh Bayat, Dariush Minai-Tehrani

Abstract:

Pseudomonas aeruginosa is a Gram-negative, rode shape and aerobic bacterium that has shown to be resistance to many antibiotics. This resistance makes the bacterium very harmful in some diseases. It can also generate diseases in any part of the gastrointestinal tract from oropharynx to rectum. P. aeruginosa has become an important cause of infection, especially in patients with compromised host defense mechanisms. One of the most important reasons that make P. aeruginosa an emerging opportunistic pathogen in patients is its ability to use various compounds as carbon sources. Lipase is an enzyme that catalyzes the hydrolysis of lipids. Most lipases act at a specific position on the glycerol backbone of lipid substrate. Some lipases are expressed and secreted by pathogenic organisms during the infection. Muscarinic antagonist used as an antispasmodic and in urinary incontinence. The drug has little effect on glandular secretion or the cardiovascular system. It does have some local anesthetic properties and is used in gastrointestinal, biliary, and urinary tract spasms. Aim: In this study the inhibitory effect of a muscarinic antagonist on lipase of P. aeruginosa was investigated. Methods: P. aeruginosa was cultured in minimal salt medium with 1% olive oil as carbon source. The cells were harvested and the supernatant, which contained lipase, was used for enzyme assay. Results: Our results showed that the drug can inhibit P. aeruginosa lipase by competitive manner. In the presence of different concentrations of the drug, the Vmax (2 mmol/min/mg protein) of enzyme did not change, while the Km raised by increasing the drug concentration. The Ki (inhibition constant) and IC50 (the half maximal inhibitory concentration) value of drug was estimated to be about 30 uM and 60 uM which determined that the drug binds to enzyme with high affinity. Maximum activity of the enzyme was observed at pH 8 in the absence and presence of muscarinic antagonist, respectively. The maximum activity of lipase was observed at 600C and the enzyme became inactive at 900C. Conclusion: The muscarinic antagonist drug could inhibit lipase of P. aeruginosa and changed the kinetic parameters of the enzyme. The drug binded to enzyme with high affinity and did not chang the optimum pH of the enzyme. Temperature did not affect the binding of drug to musmuscarinic antagonist.

Keywords: Pseudomonas aeruginosa, drug, enzyme, inhibition

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6454 Quality of Low Fat Traditional Pork Sausage Containing Transglutaminase

Authors: Jiraporn Burakorn, Pran Pinthong, Supida Hutabaedya

Abstract:

Commercial traditional pork sausages (Moo Yaw) were produced by added more than 30% of pork fat for appetite customer. The pork sausages texture were softness, firmness, juiciness and smooth. If the pork sausages contained less fat, their textures were hardness, dryness and incoherence. This research investigated production of low fat traditional pork sausage containing transglutaminase for improved its sensory properties and nutritive values. The enzyme pork sausage composed of transglutaminase, soybean cake, rice bran oil and other ingredients. Consumer acceptance test was done by comparing the enzyme pork sausage with the 3 commercial pork sausage with 95 consumer. The enzyme pork sausage was accepted 92.6% and was preferred in all attributes over the 3 commercial pork sausages such as appearance, color, flavor, taste, firmness and overall liking. The enzyme pork sausage was high protein but low total calories, calories from fat, total fat, saturated fat, cholesterol and carbohydrate. The enzyme pork sausage was lower calorie (90 kcal) than the commercial reference pork sausage (150 kcal) 64%. The morphological texture of the enzyme pork sausage was smooth and consistency when analyzed by SEM.

Keywords: low fat, Moo Yaw, pork sausage, transglutaminase

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6453 Enzyme Immobilization on Functionalized Polystyrene Nanofibersfor Bioprocessing Applications

Authors: Mailin Misson, Bo Jin, Sheng Dai, Hu Zhang

Abstract:

Advances in biotechnology have witnessed a growing interest in enzyme applications for the development of green and sustainable bio processes. While known as powerful bio catalysts, enzymes are no longer of economic value when extended to large commercialization. Alternatively, immobilization technology allows enzyme recovery and continuous reuse which subsequently compensates high operating costs. Employment of enzymes on nano structured materials has been recognized as a promising approach to enhance enzyme catalytic performances. High porosity, inter connectivity and self-assembling behaviors endow nano fibers as exciting candidate for enzyme carrier in bio reactor systems. In this study, nano fibers were successfully fabricated via electro spinning system by optimizing the polymer concentration (10-30 %, w/v), applied voltage (10-30 kV) and discharge distance (11-26 cm). Microscopic images have confirmed the quality as homogeneous and good fiber alignment. The nano fibers surface was modified using strong oxidizing agent to facilitate bio molecule binding. Bovine serum albumin and β-galactosidase enzyme were employed as model bio catalysts and immobilized onto the oxidized surfaces through covalent binding. Maximum enzyme adsorption capacity of the modified nano fibers was 3000 mg/g, 3-fold higher than the unmodified counterpart (1000 mg/g). The highest immobilization yield was 80% and reached the saturation point at 2 mg/ml of enzyme concentration. The results indicate a significant increase of activity retention by the enzyme-bound modified nano fibers (80%) as compared to the nascent one (60%), signifying excellent enzyme-nano carrier bio compatibility. The immobilized enzyme was further used for the bio conversion of dairy wastes into value-added products. This study demonstrates great potential of acid-modified electrospun polystyrene nano fibers as enzyme carriers.

Keywords: immobilization, enzyme, nanocarrier, nanofibers

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6452 Production, Optimization, Characterization, and Kinetics of a Partially Purified Laccase from Pleurotus citrinopileatus and Its Application in Swift Bioremediation of Azo Dyes

Authors: Ankita Kushwaha, M. P. Singh

Abstract:

Background: In the present investigation the efficiency of laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) from Pleurotus citrinopileatus was assessed for the decolorization of azo dyes. Aim: Enzyme production, characterization and kinetics of a partially purified laccase from Pleurotus citrinopileatus were determined for its application in bioremediation of azo dyes. Methods & Results: Laccase has been partially purified by using 80% ammonium sulphate solution. Total activity, total protein, specific activity and purification fold for partially purified laccase were found to be 40.38U, 293.33mg/100ml, 0.91U/mg and 2.84, respectively. The pH and temperature optima of laccase were 5.0 and 50ºC, respectively, while the enzyme was most stable at pH 4.0 and temperature 30ºC when exposed for one hour. The Km of the partially purified laccase for substrates guaiacol, DMP (2,6-dimethoxyphenol) and syringaldazine (3,5-dimethoxy-4-hydroxybenzaldehyde azine) were 60, 95 and 26, respectively. This laccase has been tested for the use in the bioremediation of azo dyes in the absence of mediator molecules. Two dyes namely congo red and bromophenol blue were tested. Discussion: It was observed that laccase enzyme was very effective in the decolorization of these two dyes. More than 80% decolorization was observed within half an hour even in the absence of mediator and their lower Km value indicates that efficiency of the enzyme is very high. The results were promising due to quicker decolorization in the absence of mediators showing that it can be used as a valuable biocatalyst for quick bioremediation of azo dyes. Conclusion: The enzymatic properties of laccase from P. citrinopileatus should be considered for a potential environmental (biodegradation and bioremediation) or industrial applications.

Keywords: azo dyes, decolorization, laccase, P.citrinopileatus

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6451 Sensitivity, Specificity and Efficiency Real-Time PCR Using SYBR Green Method to Determine Porcine and Bovine DNA Using Specific Primer Cytochrome B Gene

Authors: Ahlam Inayatullah Badrul Munir, M. Husaini A. Rahman, Mohd Sukri Hassan

Abstract:

Real-time PCR is a molecular biology technique that is currently being widely used for halal services to differentiating between porcine and bovine DNA. The useful of technique become very important for student or workers (who works in the laboratory) to learn how the technique could be run smoothly without fail. Same concept with conventional PCR, real-time PCR also needed DNA template, primer, enzyme polymerase, dNTP, and buffer. The difference is in real-time PCR, have additional component namely fluorescent dye. The most common use of fluorescent dye in real-time PCR is SYBR green. The purpose of this study was to find out how sensitive, specific and efficient real-time PCR technique was combined with SYBR green method and specific primers of CYT b. The results showed that real-time PCR technique using SYBR Green, capable of detecting porcine and bovine DNA concentrations up to 0.0001 µl/ng. The level of efficiency for both types of DNA was 91% (90-110). Not only that in specific primer CYT b bovine primer could detect only bovine DNA, and porcine primer could detect only porcine primer. So, from the study could be concluded that real-time PCR technique that was combined with specific primer CYT b and SYBR green method, was sensitive, specific and efficient to detect porcine and bovine DNA.

Keywords: sensitivity, specificity, efficiency, real-time PCR, SYBR green, Cytochrome b, porcine DNA, bovine DNA

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6450 Comparative Electrochemical Studies of Enzyme-Based and Enzyme-less Graphene Oxide-Based Nanocomposite as Glucose Biosensor

Authors: Chetna Tyagi. G. B. V. S. Lakshmi, Ambuj Tripathi, D. K. Avasthi

Abstract:

Graphene oxide provides a good host matrix for preparing nanocomposites due to the different functional groups attached to its edges and planes. Being biocompatible, it is used in therapeutic applications. As enzyme-based biosensor requires complicated enzyme purification procedure, high fabrication cost and special storage conditions, we need enzyme-less biosensors for use even in a harsh environment like high temperature, varying pH, etc. In this work, we have prepared both enzyme-based and enzyme-less graphene oxide-based biosensors for glucose detection using glucose-oxidase as enzyme and gold nanoparticles, respectively. These samples were characterized using X-ray diffraction, UV-visible spectroscopy, scanning electron microscopy, and transmission electron microscopy to confirm the successful synthesis of the working electrodes. Electrochemical measurements were performed for both the working electrodes using a 3-electrode electrochemical cell. Cyclic voltammetry curves showed the homogeneous transfer of electron on the electrodes in the scan range between -0.2V to 0.6V. The sensing measurements were performed using differential pulse voltammetry for the glucose concentration varying from 0.01 mM to 20 mM, and sensing was improved towards glucose in the presence of gold nanoparticles. Gold nanoparticles in graphene oxide nanocomposite played an important role in sensing glucose in the absence of enzyme, glucose oxidase, as evident from these measurements. The selectivity was tested by measuring the current response of the working electrode towards glucose in the presence of the other common interfering agents like cholesterol, ascorbic acid, citric acid, and urea. The enzyme-less working electrode also showed storage stability for up to 15 weeks, making it a suitable glucose biosensor.

Keywords: electrochemical, enzyme-less, glucose, gold nanoparticles, graphene oxide, nanocomposite

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6449 In-House Enzyme Blends from Polyporus ciliatus CBS 366.74 for Enzymatic Saccharification of Pretreated Corn Stover

Authors: Joseph A. Bentil, Anders Thygesen, Lene Langea, Moses Mensah, Anne Meyer

Abstract:

The study investigated the saccharification potential of in-house enzymes produced from a white-rot basidiomycete strain, Polyporus ciliatus CBS 366.74. The in-house enzymes were produced by growing the fungus on mono and composite substrates of cocoa pod husk (CPH) and green seaweed (GS) (Ulva lactuca sp.) with and without the addition of 25mM ammonium nitrate at 4%w/v substrate concentration in submerged condition for 144 hours. The crude enzyme extracts preparations (CEE 1-5 and CEE 1-5+AN) obtained from the fungal cultivation process were sterile-filtered and used as enzyme sources for enzymatic hydrolysis of hydrothermally pretreated corn stover using a commercial cocktail enzyme, Cellic Ctec3, as benchmark. The hydrolysis was conducted at 50ᵒC with 50mM sodium acetate buffer, pH 5 based on enzyme dosages of 5 and 10 CMCase Units/g biomass at 1%w/v dry weight substrate concentration at time points of 6, 24, and 72 hours. The enzyme activity profile of the in-house enzymes varied among the growth substrates with the composite substrates (50-75% GS and AN inclusion), yielding better enzyme activities, especially endoglucanases (0.4-0.5U/mL), β-glucosidases (0.1-0.2 U/mL), and xylanases (3-10 U/mL). However, nitrogen supplementation had no significant effect on enzyme activities of crude extracts from 100% GS substituted substrates. From the enzymatic hydrolysis, it was observed that the in-house enzymes were capable of hydrolysing the pretreated corn stover at varying degrees; however, the saccharification yield was less than 10%. Consequently, theoretical glucose yield was ten times lower than Cellic Ctec3 at both dosage levels. There was no linear correlation between glucose yield and enzyme dosage for the in-house enzymes, unlike the benchmark enzyme. It is therefore recommended that the in-house enzymes are used to complement the dosage of commercial enzymes to reduce the cost of biomass saccharification.

Keywords: enzyme production, hydrolysis yield, feedstock, enzyme blend, Polyporus ciliatus

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6448 Structure, Bioinformatics Analysis and Substrate Specificity of a 6-Phospho-β-Glucosidase Glycoside Hydrolase 1 Enzyme from Bacillus licheniformis

Authors: Wayde Veldman, Ozlem T. Bishop, Igor Polikarpov

Abstract:

In bacteria, mono and disaccharides are phosphorylated during uptake into the cell via the widely used phosphoenolpyruvate (PEP)-dependent phosphotransferase transport system. As an initial step in the phosphorylated disaccharide metabolism pathway, certain glycoside hydrolase family 1 (GH1) enzymes play a crucial role in releasing phosphorylated and non-phosphorylated monosaccharides. However, structural determinants for the specificity of these enzymes still need to be clarified. GH1 enzymes are known to have a wide array of functions. According to the CAZy database, there are twenty-one different enzymatic activities in the GH1 family. Here, the structure and substrate specificity of a GH1 enzyme from Bacillus licheniformis, hereafter known as BlBglH, was investigated. The sequence of the enzyme BlBglH was compared to the sequences of other characterized GH1 enzymes using sequence alignment, sequence identity calculations, phylogenetic analysis, and motif discovery. Through these various analyses, BlBglH was found to have sequence features characteristic of the 6-phospho-β-glucosidase activity enzymes. Additionally, motif and structure comparisons of the three most commonly studied GH1 enzyme-activities revealed a shared loop amongst the different structures that consist of different sequence motifs – this loop is thought to guide specific substrates (depending on activity) towards the active-site. To further affirm BlBglH enzyme activity, molecular docking and molecular dynamics simulations were performed. Docking was carried out using 6-phospho-β-glucosidase enzyme-activity positive (p-Nitrophenyl-beta-D-glucoside-6-phosphate) and negative (p-Nitrophenyl-beta-D-galactoside-6-phosphate) control ligands, followed by 400 ns molecular dynamics simulations. The positive-control ligand maintained favourable interactions within the active site until the end of the simulation. The negative-control ligand was observed exiting the enzyme at 287 ns. Binding free energy calculations showed that the positive-control complex had a substantially more favourable binding energy compared to the negative-control complex. Jointly, the findings of this study suggest that the BlBglH enzyme possesses 6-phospho-β-glucosidase enzymatic activity.

Keywords: 6-P-β-glucosidase, glycoside hydrolase 1, molecular dynamics, sequence analysis, substrate specificity

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6447 Biological Regulation of Endogenous Enzymatic Activity of Rainbow Trout (Oncorhynchus Mykiss) with Protease Inhibitors Chickpea in Model Systems

Authors: Delgado-Meza M., Minor-Pérez H.

Abstract:

Protease is the generic name of enzymes that hydrolyze proteins. These are classified in the subgroup EC3.4.11-99X of the classification enzymes. In food technology the proteolysis is used to modify functional and nutritional properties of food, and in some cases this proteolysis may cause food spoilage. In general, seafood and rainbow trout have accelerated decomposition process once it has done its capture, due to various factors such as the endogenous enzymatic activity that can result in loss of structure, shape and firmness, besides the release of amino acid precursors of biogenic amines. Some studies suggest the use of protease inhibitors from legume as biological regulators of proteolytic activity. The enzyme inhibitors are any substance that reduces the rate of a reaction catalyzed by an enzyme. The objective of this study was to evaluate the reduction of the proteolytic activity of enzymes in extracts of rainbow trout with protease inhibitors obtained from chickpea flour. Different proportions of rainbow trout enzyme extract (75%, 50% and 25%) and extract chickpea enzyme inhibitors were evaluated. Chickpea inhibitors were obtained by mixing 5 g of flour in 30 mL of pH 7.0 phosphate buffer. The sample was centrifuged at 8000 rpm for 10 min. The supernatant was stored at -15°C. Likewise, 20 g of rainbow trout were ground in 20 mL of phosphate buffer solution at pH 7.0 and the mixture was centrifuged at 5000 rpm for 20 min. The supernatant was used for the study. In each treatment was determined the specific enzymatic activity with the technique of Kunitz, using hemoglobin as substrate for the enzymes acid fraction and casein for basic enzymes. Also biuret protein was quantified for each treatment. The results showed for fraction of basic enzymes in the treatments evaluated, that were inhibition of endogenous enzymatic activity. Inhibition values compared to control were 51.05%, 56.59% and 59.29% when the proportions of endogenous enzymes extract rainbow trout were 75%, 50% and 25% and the remaining volume used was extract with inhibitors. Treatments with acid enzymes showed no reduction in enzyme activity. In conclusion chickpea flour reduced the endogenous enzymatic activity of rainbow trout, which may favor its application to increase the half-life of this food. The authors acknowledge the funding provided by the CONACYT for the project 131998.

Keywords: rainbouw trout, enzyme inhibitors, proteolysis, enzyme activity

Procedia PDF Downloads 315
6446 Enhanced Peroxidase Production by Raoultella Species

Authors: Ayodeji O. Falade, Leonard V. Mabinya, Uchechukwu U. Nwodo, Anthony I. Okoh

Abstract:

Given the high-utility of peroxidase, its production in large amount is of utmost importance. Over the years, actinomycetes have been the major peroxidase-producing bacteria. Consequently, other classes of bacteria with peroxidase production potentials are underexplored. This study, therefore, sought to enhance peroxidase production by a Raoultella species, a new ligninolytic proteobacteria strain, by determining the optimum culture conditions (initial pH, incubation temperature and agitation speed) for peroxidase production under submerged fermentation using the classical process of one variable at a time and supplementing the fermentation medium with some lignin model and inorganic nitrogen compounds. Subsequently, the time-course assay was carried out under optimized conditions. Then, some agricultural residues were valorized for peroxidase production under solid state fermentation. Peroxidase production was optimal at initial pH 5, incubation temperature of 35 °C and agitation speed of 150 rpm with guaiacol and ammonium chloride as the best inducer and nitrogen supplement respectively. Peroxidase production by the Raoultella species was optimal at 72 h with specific productivity of 16.48 ± 0.89 U mg⁻¹. A simultaneous production of a non-peroxide dependent extracellular enzyme which suggests probable laccase production was observed with specific productivity of 13.63 ± 0.45 U mg⁻¹ while sawdust gave the best peroxidase yield under solid state fermentation. In conclusion, peroxidase production by the Raoultella species was increased by 3.40-fold.

Keywords: enzyme production, ligninolytic bacteria, peroxidase, proteobacteria

Procedia PDF Downloads 195
6445 Degradation of Poly -β- Hydroxybutyrate by Trichoderma asperellum

Authors: Nuha Mansour Alhazmi

Abstract:

Replacement of petro-based plastics by a biodegradable plastic are vastly growing process. Poly-β-hydroxybutyrate (PHB) is a biodegradable biopolymer, synthesized by some bacterial genera. The objective of the current study is to explore the ability of some fungi to biodegrade PHB. The degradation of (PHB) was detected in Petri dish by the formation of a clear zone around the fungal colonies due to the production of depolymerase enzyme which has an interesting role in the PHB degradation process. Among 10 tested fungi, the most active PHB biodegraded fungi were identified as Trichoderma asperellum using morphological and molecular characters. The highest PHB degradation was at 25°C, pH 7.5 after 7 days of incubation for the tested fungi. Finally, the depolymerase enzyme was isolated, purified using column chromatography and characterized. In conclusion, PHB can be biodegraded in solid and liquid medium using depolymerase enzyme from T. asperellum.

Keywords: degradation, depolymerase enzyme, PHB, Trichoderma asperellum

Procedia PDF Downloads 72
6444 Identification and Characterization of Inhibitors of Epoxide Hydrolase from Trichoderma reesei

Authors: Gabriel S. De Oliveira, Patricia P. Adriani, Christophe Moriseau, Bruce D. Hammock, Felipe S. Chambergo

Abstract:

Epoxide hydrolases (EHs) are enzymes that are present in all living organisms and catalyze the hydrolysis of epoxides to the corresponding vicinal diols. EHs have high biotechnological interest for the drug design and chemistry transformation for industries. In this study, we describe the identification of substrates and inhibitors of epoxide hydrolase enzyme from the filamentous fungus Trichoderma reesei (TrEH), and these inhibitors showed the fungal growth inhibitory activity. We have used the cloned enzyme and expressed in E. coli to develop the screening in the library of fluorescent substrates with the objective of finding the best substrate to be used in the identification of good inhibitors for the enzyme TrEH. The substrate (3-phenyloxiranyl)-acetic acid cyano-(6-methoxy-naphthalen-2-yl)-methyl ester showed the highest specific activity and was chosen for the next steps of the study. The inhibitors screening was performed in the library with more than three thousand molecules and we could identify the 6 best inhibitors. The IC50 of these molecules were determined in nM and all the best inhibitors have urea or amide in their structure, because It has been recognized that these groups fit well in the hydrolase catalytic pocket of the epoxide hydrolases. Then the growth of T. reesei in PDA medium containing these TrEH inhibitors was tested, and fungal growth inhibition activity was demonstrated with more than 60% of inhibition of fungus growth in the assay with the TrEH inhibitor with the lowest IC50. Understanding how this EH enzyme from T. reesei responds to inhibitors may contribute for the study of fungal metabolism and drug design against pathogenic fungi.

Keywords: epoxide hydrolases, fungal growth inhibition, inhibitor, Trichoderma reesei

Procedia PDF Downloads 110
6443 High Catalytic Activity and Stability of Ginger Peroxidase Immobilized on Amino Functionalized Silica Coated Titanium Dioxide Nanocomposite: A Promising Tool for Bioremediation

Authors: Misha Ali, Qayyum Husain, Nida Alam, Masood Ahmad

Abstract:

Improving the activity and stability of the enzyme is an important aspect in bioremediation processes. Immobilization of enzyme is an efficient approach to amend the properties of biocatalyst required during wastewater treatment. The present study was done to immobilize partially purified ginger peroxidase on amino functionalized silica coated titanium dioxide nanocomposite. Interestingly there was an enhancement in enzyme activity after immobilization on nanosupport which was evident from effectiveness factor (η) value of 1.76. Immobilized enzyme was characterized by transmission electron microscopy, scanning electron microscopy and Fourier transform infrared spectroscopy. Immobilized peroxidase exhibited higher activity in a broad range of pH and temperature as compared to free enzyme. Also, the thermostability of peroxidase was strikingly improved upon immobilization. After six repeated uses, the immobilized peroxidase retained around 62% of its dye decolorization activity. There was a 4 fold increase in Vmax of immobilized peroxidase as compared to free enzyme. Circular dichroism spectroscopy demonstrated conformational changes in the secondary structure of enzyme, a possible reason for the enhanced enzyme activity after immobilization. Immobilized peroxidase was highly efficient in the removal of acid yellow 42 dye in a stirred batch process. Our study shows that this bio-remediating system has remarkable potential for treatment of aromatic pollutants present in wastewater.

Keywords: acid yellow 42, decolorization, ginger peroxidase, immobilization

Procedia PDF Downloads 167
6442 Efficiency for Enzyme Production of Fungi Isolated from the Stomach of Buffalo

Authors: Suphalucksana, Wichai, Sangsoponjit Settasit, Soytong Kasem

Abstract:

A study on the efficiency for enzyme production of fungi isolated from stomach of buffalo was conducted. The fungi were collected from 4 parts of stomach as rumen, reticulum, omasum and abomasums. The objective to study the efficiency of fungi from stomach of buffalo had effected to produced enzyme and to selected fungi for their ability to produced enzyme cellulase, hemicellulase and ligninase. Results shown that the fungi isolated from rumen were: Eupenicillium sp. (B-RU-01-1), Eupenicillium sp. (B-RU-02-3G), Rhyzopus stolonifer (B-RU-01-4) and Trichoderma sp. (B-RU-01-2). From the reticulum, Aspergillus glaucus (B-RET-02-3), Aspergillus orchraceus (B-RET-02-2) and Penicillium sp. (B-RET-02-4) were found. In the omasum Aspergillus fumigatus (B-OMA-01-1G), Eurotium sp. (B-OMA-01-4) and Rhizopus stolonifer (B-OMA-02-3) were isolated and in the abomasums Aspergillus flavas (B-ABO-02-3), Aspergillus fumigatus (B-ABO-02-1), Aspergillus niger (B-ABO-01-3G), Aspergillius terreus (B-ABO-02-4) and Mucor sp. (B-ABO-02-4G). Results of enzyme analysis revealed that cellulase was produced by isolated: Eupenicillium sp. (B-RU-02-3G), Eupenicillium sp. (B-RU-01-1), Penicillium sp. (B-RET-02-4), Aspergillius glaucus (B-RET-02-3), Aspergillus ochraceus (B-RET-02-2), Aspergillius fumigatus (B-OMA-01-1G), Eurotium sp. (B-OMA-01-4), Aspergillius flavus (B-ABO-02-3), Aspergillius fumigatus (B-ABO-02-1), Aspergillius niger (B-ABO-01-3G), Aspergillius terreus (B-ABO-02-4). Hemicellulase was produced Eupenicillium sp. (B-RU-02-3G), Eupenicillium sp. (B-RU-01-1), Rhizopus stolonifer (B-RU-01-4), Trichoderma sp. (B-RU-01-2), Aspergillius glaucus (B-RET-02-3), Aspergillus ochraceus (B-RET-02-2), Penicillium sp. (B-RET-02-4), Aspergillius fumigatus (B-OMA-01-1G), Eurotium sp. (B-OMA -01-4), Aspergillius flavus (B-ABO-02-3), Aspergillius fumigatus (B-ABO-02-1) Aspergillius niger (B-ABO-01-3G), Aspergillius terreus (B-ABO-02-4), Mucor sp. (B-ABO-02-4G). For the enzyme ligninase, two isolates were found to produced this enzyme namely : Trichoderma sp. (B-RU-01-2) and Mucor sp. (B-ABO-02-4G).

Keywords: enzyme production from fungi, enzyme production, fungi, agricultural technology

Procedia PDF Downloads 265
6441 Improved Production, Purification and Characterization of Invertase from Penicillium lilacinum by Shaken Flask Technique of Submerged Fermentation

Authors: Kashif Ahmed

Abstract:

Recent years researchers have been motivated towards extensive exploring of living organism, which could be utilized effectively in intense industrial conditions. The present study shows enhanced production, purification and characterization of industrial enzyme, invertase (Beta-D-fructofuranosidase) from Penicillium lilacinum. Various agricultural based by-products (cotton stalk, sunflower waste, rice husk, molasses and date syrup) were used as energy source. The highest amount of enzyme (13.05 Units/mL) was produced when the strain was cultured on growth medium containing date syrup as energy source. Yeast extract was used as nitrogen source after 96 h of incubation at incubation temperature of 40º C. Initial pH of medium was 8.0, inoculum size 6x10⁶ conidia and 200 rev/min agitation rate. The enzyme was also purified (7 folds than crude) and characterized. Molecular mass of purified enzyme (65 kDa) was determined by 10 % SDS-PAGE. Lineweaver-Burk Plot was used to determine Kinetic constants (Vmax 178.6 U/mL/min and Km 2.76 mM). Temperature and pH optima were 55º C and 5.5 respectively. MnCl₂ (52.9 %), MgSO₄ (48.9 %), BaCl₂ (24.6 %), MgCl₂ (9.6 %), CoCl₂ (5.7 %) and NaCl (4.2 %) enhanced the relative activity of enzyme and HgCl₂ (-92.8 %), CuSO₄ (-80.2 %) and CuCl₂ (-76.6 %) were proved inhibitors. The strain was showing enzyme activity even at extreme conditions of temperature (up to 60º C) and pH (up to 9), so it can be used in industries.

Keywords: invertase, Penicillium lilacinum, submerged fermentation, industrial enzyme

Procedia PDF Downloads 66
6440 Implication of Fractal Kinetics and Diffusion Limited Reaction on Biomass Hydrolysis

Authors: Sibashish Baksi, Ujjaini Sarkar, Sudeshna Saha

Abstract:

In the present study, hydrolysis of Pinus roxburghi wood powder was carried out with Viscozyme, and kinetics of the hydrolysis has been investigated. Finely ground sawdust is submerged into 2% aqueous peroxide solution (pH=11.5) and pretreated through autoclaving, probe sonication, and alkaline peroxide pretreatment. Afterward, the pretreated material is subjected to hydrolysis. A chain of experiments was executed with delignified biomass (50 g/l) and varying enzyme concentrations (24.2–60.5 g/l). In the present study, 14.32 g/l of glucose, along with 7.35 g/l of xylose, have been recovered with a viscozyme concentration of 48.8 g/l and the same condition was treated as optimum condition. Additionally, thermal deactivation of viscozyme has been investigated and found to be gradually decreasing with escalated enzyme loading from 48.4 g/l (dissociation constant= 0.05 h⁻¹) to 60.5 g/l (dissociation constant= 0.02 h⁻¹). The hydrolysis reaction is a pseudo first-order reaction, and therefore, the rate of the hydrolysis can be expressed as a fractal-like kinetic equation that communicates between the product concentration and hydrolytic time t. It is seen that the value of rate constant (K) increases from 0.008 to 0.017 with augmented enzyme concentration from 24.2 g/l to 60.5 g/l. Greater value of K is associated with stronger enzyme binding capacity of the substrate mass. However, escalated concentration of supplied enzyme ensures improved interaction with more substrate molecules resulting in an enhanced de-polymerization of the polymeric sugar chains per unit time which eventually modifies the physiochemical structure of biomass. All fractal dimensions are in between 0 and 1. Lower the value of fractal dimension, more easily the biomass get hydrolyzed. It can be seen that with increased enzyme concentration from 24.2 g/l to 48.4 g/l, the values of fractal dimension go down from 0.1 to 0.044. This indicates that the presence of more enzyme molecules can more easily hydrolyze the substrate. However, an increased value has been observed with a further increment of enzyme concentration to 60.5g/l because of diffusional limitation. It is evident that the hydrolysis reaction system is a heterogeneous organization, and the product formation rate depends strongly on the enzyme diffusion resistances caused by the rate-limiting structures of the substrate-enzyme complex. Value of the rate constant increases from 1.061 to 2.610 with escalated enzyme concentration from 24.2 to 48.4 g/l. As the rate constant is proportional to Fick’s diffusion coefficient, it can be assumed that with a higher concentration of enzyme, a larger amount of enzyme mass dM diffuses into the substrate through the surface dF per unit time dt. Therefore, a higher rate constant value is associated with a faster diffusion of enzyme into the substrate. Regression analysis of time curves with various enzyme concentrations shows that diffusion resistant constant increases from 0.3 to 0.51 for the first two enzyme concentrations and again decreases with enzyme concentration of 60.5 g/l. During diffusion in a differential scale, the enzyme also experiences a greater resistance during diffusion of larger dM through dF in dt.

Keywords: viscozyme, glucose, fractal kinetics, thermal deactivation

Procedia PDF Downloads 39
6439 Synthesis and Anti-Inflammatory Activity of Pyrazol-3-yl Thiazole 4-Carboxylic Acid Derivatives Targeting Enzyme in the Leukotriene Pathway

Authors: Shweta Sinha, Mukesh Doble, Manju S. L.

Abstract:

Pyrazole scaffold is an important group of compound in heterocyclic chemistry and is found to possess numerous uses in chemistry. Pyrazole derivatives are also known to possess important biological activities including antitumor, antimicrobial, antiviral, antifungal, anticancer and anti-inflammatory. Inflammation is associated with pain, allergy and asthma. Leukotrienes are mediators of various inflammatory and allergic disorders. 5-Lipoxygenase (5-LOX) is an important enzyme involved in the biosynthesis of leukotrienes and metabolism of arachidonic acid (AA) and thus targeted for anti-inflammation. In vitro inhibitory activity of pyrazol-3-yl thiazole 4-carboxylic acid derivatives is tested against enzyme 5-LOX. Most of these compounds exhibit good inhibitory activity against this enzyme. Binding mode study of these compounds is determined by computational tool. Further experiments are being done to understand the mechanism of action of these compounds in inhibiting this enzyme. To conclude, these compounds appear to be a promising target in drug design against 5-LOX.

Keywords: inflammation, inhibition, 5-lipoxygenase, pyrazole

Procedia PDF Downloads 168
6438 Isolation and Screening of Fungal Strains for β-Galactosidase Production

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

Abstract:

Enzymes are the biocatalysts which catalyze the biochemical processes and thus have a wide variety of applications in the industrial sector. β-Galactosidase (E.C. 3.2.1.23) also known as lactase, is one of the prime enzymes, which has significant potential in the dairy and food processing industries. It has the capability to catalyze both the hydrolytic reaction for the production of lactose hydrolyzed milk and transgalactosylation reaction for the synthesis of prebiotics such as lactulose and galactooligosaccharides. These prebiotics have various nutritional and technological benefits. Although, the enzyme is naturally present in almonds, peaches, apricots and other variety of fruits and animals, the extraction of enzyme from these sources increases the cost of enzyme. Therefore, focus has been shifted towards the production of low cost enzyme from the microorganisms such as bacteria, yeast and fungi. As compared to yeast and bacteria, fungal β-galactosidase is generally preferred as being extracellular and thermostable in nature. Keeping the above in view, the present study was carried out for the isolation of the β-galactosidase producing fungal strain from the food as well as the agricultural wastes. A total of more than 100 fungal cultures were examined for their potential in enzyme production. All the fungal strains were screened using X-gal and IPTG as inducers in the modified Czapek Dox Agar medium. Among the various isolated fungal strains, the strain exhibiting the highest enzyme activity was chosen for further phenotypic and genotypic characterization. The strain was identified as Rhizomucor pusillus on the basis of 5.8s RNA gene sequencing data.

Keywords: beta-galactosidase, enzyme, fungal, isolation

Procedia PDF Downloads 175
6437 Magnetite Nanoparticles Immobilized Pectinase: Preparation, Characterization and Application for the Fruit Juices Clarification

Authors: Leila Mosafa, Majid Moghadam, Mohammad Shahedi

Abstract:

In this work, pectinase was immobilized on the surface of silica-coated magnetite nanoparticles via covalent attachment. The magnetite-immobilized enzyme was characterized by Fourier transform infrared spectroscopy, X-ray powder diffraction, scanning electron microscopy and vibrating sample magnetometry techniques. Response surface methodology using Minitab Software was applied for statistical designing of operating conditions in order to immobilize pectinase on magnetic nanoparticles. The optimal conditions were obtained at 30°C and pH 5.5 with 42.97 µl pectinase for 2 h. The immobilization yield was 50.6% at optimized conditions. Compared to the free pectinase, the immobilized pectinase was found to exhibit enhanced enzyme activity, better tolerance to the variation of pH and temperature, and improved storage stability. Both free and immobilized samples reduced the viscosity of apple juice from 1.12 to 0.88 and 0.92 mm2s-1, respectively, after 30 min at their optimum temperature. Furthermore, the immobilized enzyme could be reused six consecutive cycles and the efficiency loss in viscosity reduction was found to be only 8.16%.

Keywords: magnetite nanoparticles, pectinase enzyme, immobilization, juice clarification, enzyme activity

Procedia PDF Downloads 323