Molecular imprinted nanoelectrodes for ultra sensitive detection of ovarian cancer marker

The relentless discovery of cancer biomarkers demands improved methods for their detection. In this work, we developed protein imprinted polymer on three-dimensional gold nanoelectrode ensemble (GNEE) to detect epithelial ovarian cancer antigen-125 (CA 125), a protein biomarker associated with ovarian cancer. CA 125 is the standard tumor marker used to follow women during or after treatment for epithelial ovarian cancer. The template protein CA 125 was initially incorporated into the thin-film coating and, upon extraction of protein from the accessible surfaces on the thin film, imprints for CA 125 were formed. The fabrication and analysis of the CA 125 imprinted GNEE was done by using cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) techniques. The surfaces of the very thin, protein imprinted sites on GNEE are utilized for immunospecific capture of CA 125 molecules, and the mass of bound on the electrode surface can be detected as a reduction in the faradic current from the redox marker. Under optimal conditions, the developed sensor showed good increments at the studied concentration range of 0.5–400 U mL−1. The lowest detection limit was found to be 0.5 U mL−1. Spiked human blood serum and unknown real serum samples were analyzed. The presence of non-specific proteins in the serum did not significantly affect the sensitivity of our assay. Molecular imprinting using synthetic polymers and nanomaterials provides an alternative approach to the trace detection of biomarker proteins.

Oriented Tailoring of Plastic Antibodies for Prostate Specific Antigen and Application of the Imprinted Material as Ionophore in Potentiometric Detection

Prostate Specific Antigen (PSA) is the biomarker of choice for screening prostate cancer throughout the population, with PSA values above 10 ng/mL pointing out a high probability of associated cancer1. According to the most recent World Health Organization (WHO) data, prostate cancer is the commonest form of cancer in men in Europe2. Early detection of prostate cancer is thus very important and is currently made by screening PSA in men over 45 years old, combined with other alterations in serum and urine parameters. PSA is a glycoprotein with a molecular mass of approximately 32 kDa consisting of one polypeptide chain, which is produced by the secretory epithelium of human prostate. Currently, the standard methods available for PSA screening are immunoassays like Enzyme-Linked Immunoabsorbent Assay (ELISA). These methods are highly sensitive and specific for the detection of PSA, but they require expensive laboratory facilities and high qualify personal resources. Other highly sensitive and specific methods for the detection of PSA have also become available and are in its majority immunobiosensors1,3-5, relying on antibodies. Less expensive methods producing quicker responses are thus needed, which may be achieved by synthesizing artificial antibodies by means of molecular imprinting techniques. These should also be coupled to simple and low cost devices, such as those of the potentiometric kind, one approach that has been proven successful6. Potentiometric sensors offer the advantage of selectivity and portability for use in point-of-care and have been widely recognized as potential analytical tools in this field. The inherent method is simple, precise, accurate and inexpensive regarding reagent consumption and equipment involved. Thus, this work proposes a new plastic antibody for PSA, designed over the surface of graphene layers extracted from graphite. Charged monomers were used to enable an oriented tailoring of the PSA rebinding sites. Uncharged monomers were used as control. These materials were used as ionophores in conventional solid-contact graphite electrodes. The obtained results showed that the imprinted materials displayed a selective response to PSA. The electrodes with charged monomers showed a more stable and sensitive response, with an average slope of -44.2 mV/decade and a detection limit of 5.8X10-11 mol/L (2 ng/mL). The corresponding non-imprinted sensors showed smaller sensitivity, with average slopes of -24.8 mV/decade. The best sensors were successfully applied to the analysis of serum samples, with percentage recoveries of 106.5% and relatives errors of 6.5%.

QuEChERS: a sample preparation for extraction of carbaryl from rat feces

Carbamate compounds are an important group of cholinesterase inhibitors. There is a need for creating awareness regarding the risks of the inadequate carbamate use in the residential areas due to potential adverse human effects. Carbaryl is a commonly used pesticide worldwide. A simple, fast, and high throughput method was developed employing liquid chromatography with fluorescence detector to determine carbaryl residues in rat feces. The extraction was performed by using a rapid, easy, cheap, effective, reliable, and safe (QuEChERS) method, using acetonitrile as the extracting solvent. The parameters for the performance of the extraction method were optimized, such as ratio of mass of sample per volume of extraction solvent, QuEChERS content, and cleanup columns. Linear response was obtained for all calibration curves (solven and matrix-matched) over the established concentration range (5 500 mg/L) with a correlation coefficients higher than 0.999. The achieved recovery was 97.9% with relative standard deviation values of 1.1% (n D 4) at 167 mg/kg fortified concentration level and the limits of detection and quantification were 27.7 and 92.3 mg/kg respectively.

Being ahead of its time the consumer resistance phenomenon: A comparative case study research

Double Degree.

Calculation of the optical conductivity for the Anderson impurity model using the slave boson technique

The optical conductivity of the Anderson impurity mode l has been calculated by emp l oying the slave boson technique and an expansion in powers of l i N, where N is the d egeneracy o f the f electron level . This method has been used to find the effective mass of the conduction electrons for temperatures above and below the Kondo tempera ture. For low temperatures, the mass enhancement is f ound to be large while a t high t emperatures, the mass enhancement is sma ll. The conductivity i s f ound to be Drude like with frequency dependent effective mass and scattering time for low independent effective mass and temperatures and scattering time f requency for high t emperatures. The behavior of both the effective mass and the conductivity is in qualitative agreement with experimental r esul t s .

The influence of recreational ball hockey play on cardiovascular risk factors in sedentary males

Introduction: The prevalence of coronary artery disease (CAD) is ever increasing in western industrialized societies. An individuals overall risk for CAD may be quantified by integrating a number of factors including, but not limited to, cardiorespiratory fitness, body composition, blood lipid profile and blood pressure. It might be expected that interventions aimed at improving any or all of these independent factors might improve an individual 's overall risk. To this end, the influence of standard endurance type exercise on cardiorespiratory fitness, body composition, blood lipids and blood pressure, and by extension the reduction of coronary risk factors, is well documented. On the other hand, interval training (IT) has been shown to provide an extremely powerful stimulus for improving indices of cardiorespiratory function but the influence of this training type on coronary risk factors is unknown. Moreover, the vast majority of studies investigating the effects of IT on fitness have used laboratory type training protocols. As a result of this, the influence of participation in interval-type recreational sports on cardiorespiratory fitness and coronary risk factors is unknown. Aims: The aim of the present study was to evaluate the effectiveness of recreational ball hockey, a sport associated with interval-type activity patterns, on indices of aerobic function and coronary risk factors in sedentary men in the approximate age range of 30 - 60 years. Individual risk factors were compiled into an overall coronary risk factor score using the Framingham Point Scale (FPS). Methods: Twenty-four sedentary males (age range 30 - 60) participated in the study. Subject activity level was assessed apriori using questionnaire responses. All subjects (experimental and control) were assessed to have been inactive and sedentary prior to participation in the study. The experimental group (43 ± 3 years; 90 ± 3 kg) (n = 11) participated in one season of recreational ball hockey (our surrogate for IT). Member of this group played a total of 16 games during an 11 week span. During this time, the control group (43 ± 2 years; 89 ± 2 kg) (n = 11) performed no training and continued with their sedentary lifestyle. Prior to and following the ball hockey season, experimental and control subjects were tested for the following variables: 1) cardiorespiratory fitness (as V02 Max) 2) blood lipid profile 3) body composition 5) waist to hip ratio 6) blood glucose levels and 7) blood pressure. Subject V02 Max was assessed using the Rockport submaximal walking test on an indoor track. To assess body composition we determined body mass ratio (BMI), % body fat, % lean body mass and waist to hip ratio. The blood lipid profile included high density lipoprotein, low density lipoprotein and total cholesterol levels; in addition, the ratio of total cholesterol to high density was calculated. Blood triglycerides were also assessed. All data were analyzed using independent t - tests and all data are expressed as mean ± standard error. Statistical significance was accepted at p :S 0.05. Results: Pre-test values for all variables were similar between the experimental and control group. Moreover, although the intervention used in this study was associated with changes in some variables for subjects in the experimental group, subjects in the control group did not exhibit any changes over the same time period. BODY COMPOSITION: The % body fat of experimental subjects decreased by 4.6 ± 0.5%, from 28.1 ± 2.6 to 26.9 ± 2.5 % while that of the control group was unchanged at 22.7 ± 1.4 and 22.2 ± 1.3 %. However, lean body mass of experimental and control subjects did not change at 64.3 ± 1.3 versus 66.1 ± 1.3 kg and 65.5 ± 0.8 versus 64.7 ± 0.8 kg, respectively. In terms of body mass index and waist to hip ratio, neither the experimental nor the control group showed any significant change. Respective values for the waist to hip ratio and body mass index (pre and post) were as follows: 1 ± 0.1 vs 0.9 ± 0.1 (experimental) and 0.9 ± 0.1 versus 0.9 ± 0.1 (controls) while for BMI they were 29 ± 1.4 versus 29 ± 1.2 (experimental) and 26 ± 0.7 vs. 26 ± 0.7 (controls). CARDIORESPIRATORY FITNESS: In the experimental group, predicted values for absolute V02 Max increased by 10 ± 3% (i.e. 3.3 ± 0.1 to 3.6 ± 0.1 liters min -1 while that of control subjects did not change (3.4 ± 0.2 and 3.4 ± 0.2 liters min-I). In terms of relative values for V02 Max, the experimental group increased by 11 ± 2% (37 ± 1.4 to 41 ± 1.4 ml kg-l min-I) while that of control subjects did not change (41 ± 1.4 and 40 ± 1.4 ml kg-l min-I). BLOOD LIPIDS: Compared to pre-test values, post-test values for HDL were decreased by 14 ± 5 % in the experiment group (from 52.4 ± 4.4 to 45.2 ± 4.3 mg dl-l) while HDL data for the control group was unchanged (49.7 ± 3.6 and 48.3 ± 4.1 mg dl-l, respectively. On the other hand, LDL levels did not change for either the experimental or control group (110.2 ± 10.4 versus 112.3 ± 7.1 mg dl-1 and 106.1 ± 11.3 versus 127 ± 15.1 mg dl-1, respectively). Further, total cholesterol did not change in either the experimental or control group (181.3 ± 8.7 mg dl-1 versus 178.7± 4.9 mg dl-l) and 190.7 ± 12.2 versus 197.1 ± 16.1 mg dl-1, respectively). Similarly, the ratio of TC/HDL did not change for either the experimental or control group (3.8 ± 0.4 versus 4.5 ± 0.5 and 4 ± 0.4 versus 4.2 ± 0.4, respectively). Blood triglyceride levels were also not altered in either the experimental or control group (100.3 ± 19.6 versus 114.8 ± 15.3 mg dl-1 and 140 ± 23.5 versus 137.3 ± 17.9 mg dl-l, respectively). BLOOD GLUCOSE: Fasted blood glucose levels did not change in either the experimental or control group. Pre- and post-values for experimental and control groups were 92.5 ± 4.8 versus 93.3 ± 4.3 mg dl-l and 92.3 ± 11.3 versus 93.2 ± 2.6 mg dl-1 , respectively. BLOOD PRESSURE: No aspect of blood pressure was altered in either the experimental or control group. For example, pre- and post-test systolic blood pressures were 131 ± 2 versus 129 ± 2 mmHg (experimental) and 123 ± 2 and 125 ± 2 mmHg (controls), respectively. Pre- and post-test diastolic blood pressures were 84 ± 2 and 83 ± 2 mmHg (experimental) and 81 ± 1 versus 82 ± 1 mmHg, respectively. Similarly, calculated pulse pressure was not altered in the experimental or control as pre- and post-test values were 47 ± 1 versus 47 ± 2 mmlHg and 42 ± 2 versus 43 ± 2 mmHg, respectively. FRAMINGHAM POINT SCORE: The concerted changes reported above produced an increased risk in the Framingham Point Score for the subjects in the experimental group. For example, the pre- and post-test FPS increased from 1.4 ± 0.9 to 2.7 ± 0.7. On the other hand, pre- and post-test scores for the control group were 1.8 ± 1 versus 1.8 ± 0.9. Conclusions: Our data confirms previous studies showing that interval-type exercise is a useful intervention for increasing aerobic fitness. Moreover, the increase in V02 Max we found in response to limited participation in ball hockey (i.e. 16 games) suggests that recreational sport may help reduce this aspect of coronary risk in previously sedentary individual. On the other hand, our results showing little or no positive change in body composition, blood lipids or blood pressures suggest that one season of recreational sport in not in of itself a powerful enough stimulus to reduce the overall risk of coronary artery disease. In light of this, it is recommended that, in addition to participation in recreational sport, the performance of regular physical activity is used as an adjunct to provide a more powerful overall stimulus for decreasing coronary risk factors. LIMITATIONS: The increase in the FPS we found for the experimental group, indicative of an increased risk for coronary disease, was largely due to the large decrease in HDL we observed after compared to above one season of ball hockey. In light of the fact that cardiorespiratory fitness was increased and % body fat was decreased, as well as the fact that other parameters such as blood pressure showed positive (but non statistically significant) trends, the possibility that the decrease in HDL showed by our data was anomalous should be considered. FUTURE DIRECTIONS: The results of this study suggesting that recreational sport may be a potentially useful intervention in the reduction of CAD require to be corroborated by future studies specifically employing 1) more rigorous assessment of fitness and fitness change and 2) more prolonged or frequent participants.

View of Falls from Steel Bridge, Niagara, U.S.A.

The description of the image reads "(4)-8972-General view of Falls from new steel bridge - Maid of the Mist at landing - Niagara, U.S.A." The reverse of the image includes the description, "We are standing on the new steel bridge over Niagara River, 190 feet above the water and looking a little west of south, up the river towards Lake Erie. The high cliff at the extreme left, on the American side, is Prospect Point, where a crowd is gathered at this moment to view the Falls that we see just beyond Prospect Point. That dark, tree-covered mass of rock beyond is Goat Island; and just this side of Goat Island we see a bit of its precipice has been cut off separate from the rest by the powerful current of the waters - the smaller portion is Luna Island, and the Luna Falls go pouring down between the two islands. The face of the precipice curves inward beneath the Luna Falls leaving behind the 160 foot sheet of water the unearthly hollow known as the Cave of the Winds. Beyond Goat Island we see the gigantic curve of the Horseshoe Falls, 3,010 feet long and 158 feet high, reaching around through the clouds of spray to the farther Canadian shore. (The boundary line between British and American territory is in mid-stream.) It has been estimated that every minute 375,000 tons of water pour over these Horseshoe Falls, and they are wearing away the cliffs, moving back up the stream at the rate of 2.4 feet per year. It was probably only about a thousand years ago that they took their plunge just about where we stand now. Down there below us, at the wharf is the Maid of the Mist at the American landing taking on passengers who have come down the steep bank by the inclined railway. Its course takes it through those clouds of spray almost to the very foot of both Falls, - waters falling from 167 feet overhead, and water surging at least as many feet deep under the staunch little vessel. See special 'keyed' maps of Niagara pub. by Underwood and Underwood, also the Niagara Book by Mark Twain, W.D. Howells and others."

Role of Phosphorylation of the Oxysterol Binding Protein (OSBP) in (PI(4)P) and Sterol-Containing Membrane Recognition and Binding

Studies have demonstrated that the oxysterol binding protein (OSBP) acts as a phosphatidylinositol phosphate (PIP)-sterol exchanger at membrane contact sites (MCS) of the endoplasmic reticulum (ER) and Golgi. OSBP is known to pick up phosphatidylinositol-4-phosphate (PI(4)P) from the ER, transfer it to the trans-Golgi in exchange for a cholesterol molecule that is then transferred from the trans-Golgi to the ER. Upon further examination of this pathway by Ridgway et al. (1), it appeared that phosphorylation of OSBP played a role in the localization of OSBP. The dephosphorylation state of OSBP was linked to Golgi localization and the depletion of cholesterol at the ER. To mimic the phosphorylated state of OSBP, the mutant OSBP-S5E was designed by Ridgway et al. (1). The lipid and sterol recognition by wt-OSBP and its phosphomimic mutant OSBP-S5E were investigated using immobilized lipid bilayers and dual polarization interferometry (DPI). DPI is a technique in which the protein binding affinity to immobilized lipid bilayers is measured and the binding behavior is examined through real time. Lipid bilayers containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and varying concentrations of PI(4)Ps or sterols (cholesterol or 25-hydroxycholesterol) were immobilized on a silicon nitride chip. It was determined that wt-OSBP binds differently to PI(4)P-containing bilayers compared to OSBP-S5E. The binding behavior suggested that wt-OSBP extracts PI(4)P and the change in the binding behavior, in the case of OSBP-S5E, suggested that the phosphorylation of OSBP may prevent the recognition and/or extraction of PI(4)P. In the presence of sterols, the overall binding behavior of OSBP, regardless of phosphorylation state, was fairly similar. The maximum specific bound mass of OSBP to sterols did not differ as the concentration of sterols increased. However, comparing the maximum specific bound mass of OSBP to cholesterol with oxysterol (25-hydroxycholesterol), OSBP displayed nearly a 2-fold increase in bound mass. With the absence of the wt-OSBP-PI(4)P binding behavior, it can be speculated that the sterols were not extracted. In addition, the binding behavior of OSBP was further tested using a fluorescence based binding assay. Using 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol (22-NBD cholesterol), wt-OSBP a one site binding dissociation constant Kd, of 15 ± 1.4 nM was determined. OSBP-S5E did not bind to 22-NBD cholesterol and Kd value was not obtained.

Characterization of glutaraldehyde-immobilized chymotrypsin and an in-situ immobilized enzyme reactor using capillary electrophoresis-based peptide mapping

La digestion enzymatique des protéines est une méthode de base pour les études protéomiques ainsi que pour le séquençage en mode « bottom-up ». Les enzymes sont ajoutées soit en solution (phase homogène), soit directement sur le gel polyacrylamide selon la méthode déjà utilisée pour l’isolation de la protéine. Les enzymes protéolytiques immobilisées, c’est-à-dire insolubles, offrent plusieurs avantages tels que la réutilisation de l’enzyme, un rapport élevé d’enzyme-sur-substrat, et une intégration facile avec les systèmes fluidiques. Dans cette étude, la chymotrypsine (CT) a été immobilisée par réticulation avec le glutaraldehyde (GA), ce qui crée des particules insolubles. L’efficacité d’immobilisation, déterminée par spectrophotométrie d’absorbance, était de 96% de la masse totale de la CT ajouté. Plusieurs différentes conditions d’immobilisation (i.e., réticulation) tels que la composition/pH du tampon et la masse de CT durant la réticulation ainsi que les différentes conditions d’entreposage tels que la température, durée et humidité pour les particules GA-CT ont été évaluées par comparaison des cartes peptidiques en électrophorèse capillaire (CE) des protéines standards digérées par les particules. Les particules de GA-CT ont été utilisés pour digérer la BSA comme exemple d’une protéine repliée large qui requit une dénaturation préalable à la digestion, et pour digérer la caséine marquée avec de l’isothiocyanate de fluorescéine (FITC) comme exemple d’un substrat dérivé afin de vérifier l’activité enzymatique du GA-CT dans la présence des groupements fluorescents liés au substrat. La cartographie peptidique des digestions par les particules GA-CT a été réalisée par CE avec la détection par absorbance ultraviolet (UV) ou fluorescence induite par laser. La caséine-FITC a été, en effet, digérée par GA-CT au même degré que par la CT libre (i.e., soluble). Un microréacteur enzymatique (IMER) a été fabriqué par immobilisation de la CT dans un capillaire de silice fondu du diamètre interne de 250 µm prétraité avec du 3-aminopropyltriéthoxysilane afin de fonctionnaliser la paroi interne avec les groupements amines. Le GA a été réagit avec les groupements amine puis la CT a été immobilisée par réticulation avec le GA. Les IMERs à base de GA-CT étaient préparé à l’aide d’un système CE automatisé puis utilisé pour digérer la BSA, la myoglobine, un peptide ayant 9 résidus et un dipeptide comme exemples des substrats ayant taille large, moyenne et petite, respectivement. La comparaison des cartes peptidiques des digestats obtenues par CE-UV ou CE-spectrométrie de masse nous permettent d’étudier les conditions d’immobilisation en fonction de la composition et le pH du tampon et le temps de réaction de la réticulation. Une étude par microscopie de fluorescence, un outil utilisé pour examiner l’étendue et les endroits d’immobilisation GA-CT dans l’IMER, ont montré que l’immobilisation a eu lieu majoritairement sur la paroi et que la réticulation ne s’est étendue pas si loin au centre du capillaire qu’anticipée.

Molecular cloning and homology modelling of a subtilisin-like serine protease from the marine fungus, Engyodontium album BTMFS10

An alkaline protease gene (Eap) was isolated for the first time from a marine fungus, Engyodontium album. Eap consists of an open reading frame of 1,161 bp encoding a prepropeptide consisting of 387 amino acids with a calculated molecular mass of 40.923 kDa. Homology comparison of the deduced amino acid sequence of Eap with other known proteins indicated that Eap encode an extracellular protease that belongs to the subtilase family of serine protease (Family S8). A comparative homology model of the Engyodontium album protease (EAP) was developed using the crystal structure of proteinase K. The model revealed that EAP has broad substrate specificity similar to Proteinase K with preference for bulky hydrophobic residues at P1 and P4. Also, EAP is suggested to have two disulfide bonds and more than two Ca2? binding sites in its 3D structure; both of which are assumed to contribute to the thermostable nature of the protein.

Two isoforms of anti-lipopolysaccharide factors identified and characterized from the hemocytes of portunid crabs, Portunus pelagicus and Scylla tranquebarica

Anti-lipopolysaccharide factors (ALFs), a type of cationic antimicrobial peptides (AMPs), and their derivatives are becoming predominant candidates for potential drugs in viral and bacterial diseases. This study reports the first ALF from the mud crab Scylla tranquebarica (StALF, JQ899453) and the second ALF isoform from the blue swimmer crab Portunus pelagicus (PpALF2, JQ899452). Both sequences encoded for precursor molecules, starting with a signal peptide containing 26 amino acid residues, followed by a highly cationic mature peptide, containing two conserved cysteine residues flanking a putative lipopolysaccharide (LPS)-binding domain. BLAST analysis revealed that both PpALF2 and StALF exhibited significant similarity with crustacean ALF sequences. The predicted molecular mass of the mature ALFs was 11.2 kDa with an estimated pI of 10.0. PpALF2 and StALF also showed the typical pattern of alternating hydrophobic and hydrophilic residues in their putative disulphide loop, suggesting that they comprise the same functional domain. Phylogenetic analysis showed that PpALF2 and StALF have similar evolutionary status and they were phylogenetically ancient immune effector molecules which may play an essential role in the host defense mechanism. The spatial structures of PpALF2 and StALF possessed four beta-strands and two alpha-helices. The results indicated that there were more than one ALF involved in crab immunity against various pathogens. ALFs would provide candidate promising therapeutic or prophylactic agents in health management and diseases control in crustacean aquaculture

Alkaline protease from a non-toxigenic mangrove isolate of Vibrio sp. V26 with potential application in animal cell culture

Vibrio sp. V26 isolated from mangrove sediment showed 98 % similarity to 16S rRNA gene of Vibrio cholerae, V. mimicus, V. albensis and uncultured clones of Vibrio. Phenotypically also it resembled both V. cholerae and V. mimicus.Serogrouping, virulence associated gene profiling, hydrophobicity, and adherence pattern clearly pointed towards the non—toxigenic nature of Vibrio sp. V26. Purification and characterization of the enzyme revealed that it was moderately thermoactive, nonhemagglutinating alkaline metalloprotease with a molecular mass of 32 kDa. The application of alkaline protease from Vibrio sp. V26 (APV26) in sub culturing cell lines (HEp-2, HeLa and RTG-2) and dissociation of animal tissue (chick embryo) for primary cell culture were investigated. The time required for dissociation of cells as well as the viable cell yield obtained by while administeringAPV26 and trypsin were compared. Investigations revealed that the alkaline protease of Vibrio sp. V26 has the potential to be used in animal cell culture for subculturing cell lines and dissociation of animal tissue for the development of primary cell cultures, which has not been reported earlier among metalloproteases of Vibrios.

Potential application of β-1, 3 glucanase from an environmental isolate of Pseudomonas aeruginosa MCCB 123 in fungal DNA extraction

Pseudomonas aeruginosa MCCB 123 was grown in a synthetic medium for β-1,3 glucanase production. From the culture filtrate, β-1,3 glucanase was purified with a molecular mass of 45 kDa. The enzyme was a metallozyme as its β-1,3 glucanase activity got inhibited by the metal chelator EDTA. Optimum pH and temperature for β-1,3 glucanase activity on laminarin was found to be 7 and 50 °C respectively. The MCCB 123 β-1,3 glucanase was found to have good lytic action on a wide range of fungal isolates, and hence its application in fungal DNA extraction was evaluated. β-1,3 glucanase purified from the culture supernatant of P. aeruginosa MCCB 123 could be used for the extraction of fungal DNA without the addition of any other reagents generally used. Optimum pH and temperature of enzyme for fungal DNA extraction was found to be 7 and 65 °C respectively. This is the first report on β-1,3 glucanase employed in fungal DNA extraction

Molecular cloning and homology modelling of a subtilisin-like serine protease from the marine fungus, Engyodontium album BTMFS10

An alkaline protease gene (Eap) was isolated for the first time from a marine fungus, Engyodontium album. Eap consists of an open reading frame of 1,161 bp encoding a prepropeptide consisting of 387 amino acids with a calculated molecular mass of 40.923 kDa. Homology comparison of the deduced amino acid sequence of Eap with other known proteins indicated that Eap encode an extracellular protease that belongs to the subtilase family of serine protease (Family S8). A comparative homology model of the Engyodontium album protease (EAP) was developed using the crystal structure of proteinase K. The model revealed that EAP has broad substrate specificity similar to Proteinase K with preference for bulky hydrophobic residues at P1 and P4. Also, EAP is suggested to have two disulfide bonds and more than two Ca2? binding sites in its 3D structure; both of which are assumed to contribute to the thermostable nature of the protein.

Characterization of an extracellular alkaline serine protease from marine Engyodontium album BTMFS10

An alkaline protease from marine Engyodontium album was characterized for its physicochemical properties towards evaluation of its suitability for potential industrial applications. Molecular mass of the enzyme by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) analysis was calculated as 28.6 kDa. Isoelectric focusing yielded pI of 3–4. Enzyme inhibition by phenylmethylsulfonyl fluoride (PMSF) and aprotinin confirmed the serine protease nature of the enzyme.Km, Vmax, and Kcat of the enzyme were 4.727 9 10-2 mg/ml, 394.68 U, and 4.2175 9 10-2 s-1, respectively. Enzyme was noted to be active over a broad range of pH (6–12) and temperature (15–65 C), withmaximumactivity at pH 11 and 60 C. CaCl2 (1 mM), starch (1%), and sucrose (1%) imparted thermal stability at 65 C. Hg2?, Cu2?, Fe3?, Zn2?, Cd?, and Al3? inhibited enzyme activity, while 1 mMCo2? enhanced enzyme activity. Reducing agents enhanced enzyme activity at lower concentrations. The enzyme showed considerable storage stability, and retained its activity in the presence of hydrocarbons, natural oils, surfactants, and most of the organic solvents tested. Results indicate that the marine protease holds potential for use in the detergent industry and for varied applications.