Specificity ofthe collagenase enzyme from NW4327 for Rhopaloeides odorabile spongin fibres was assessed by measuring its ability to degrade tissue fibres from other marine sponges. Material from Ianthella flabelliformis, Cacospongia sp., Ircinia sp. and Luffariella sp. was prepared by removing the non-fiber components by thorough rinsing with running tap water and homogenizing the remaining air-dried tissue in a Waring blender. Specificity was further assessed using commercial casein and gelatin (Sigma- Aldrich) and bird feather keratin obtained by homogenizing the vanes cut off from the central shaft ofthe moulted tail feather ofthe bush turkey (Alectura lathami). Since large amount ofenzyme was required for these experiments and because of an observed loss of specific activity as the protein became more pure (see Results), these substrates were digested with collagenase obtained after semi-preparative molecular size exclusion chromatography. Assays were conducted by suspending 5 mg ofthe above- mentioned substrates in 5 ml of 50 mM Tris HCl (pH 7.5) buffer with 1 mM CaCl 2 , as used for the sensitive ninhydrin assay, see
L - Glutaminase, a therapeutically and industrially important enzyme, was produced from marine Vibrio costicola by a novel solid state fermentation process using polystyrene beads as inert support. The new fermentation system offered several advantages over the conventional systems, such as the yield of leachate with minimum viscosity and high specific activity for the target product besides facilitating the easy estimation of biomass. Theenzyme thus produced was purified and characterised. It was active at physiological pH, showed high substrate specificity towards L - glutamine and had a Km value of 7.4 x 10 -2 M. It also exhibited high salt and temperature tolerance indicating good scope for its industrial and therapeutic applications.
In the literature, ACE2 isoforms were described with molecular mass ranging between 70 and 130 kDa. Donoghue and collaborators were able to express a 90 kDa recombinant ACE2 in the extracellu- lar medium and cellular lysate using transfected CHO cells . The same molecular mass was described for the enzymes identified in rat and mouse tissues [1,14]. However, ACE2 expressed in mammal cells has a molecular mass of 120 kDa reduced to 85 kDa after enzy- matic deglycosylation . Other study also described ACE2 isoform with molecular masses ranging from 120 to 130 kDa in rat kidney . The authors suggest that these differences could be explained by post-transductional modifications. An ACE2 presenting a molec- ular mass similar to theenzyme described in this study, 70 kDa, was purified from astrocyte cell culture, and was able to convert Ang II into Ang 1–7, suggesting that this protein is a secreted form oftheenzyme . Therefore, the lower molecular mass of purified ACE2 described by this study may be explained by deglycosylation process or this enzyme could be a secreted ACE2 isoform.
Several studies suggest that membrane form of catechol-O-methyltransferase OMT (MB-COMT) is the main responsible for O-methylation at physiologically low concentrations of catecholamines. Despite this, until now no studies have been allowed the total isolation of MB-COMT. Then, a sustainable chromatographic step should be developed in order to obtain significant quantities of active and pure enzyme for posterior application on biochemical, kinetic and structural studies. For the first time, we intend to compare the performance of three hydrophobic adsorbents (Butyl-, Epoxy- and Octyl-sepharose) in thepurificationof human membrane-bound COMT (hMBCOMT) from crude Brevibacillus choshinensis cell lysates. The hydrophobic matrices were evaluated in terms of selectivity, binding and elution conditions. Results show that the isolation of MB-COMT was possible using 375 mM monosodium phosphate concentrations for its adsorption. The hMB-COMT, as the soluble form, was found to elute at four different fractions at 0.25; 0.7 and 1% Triton X-100. Preliminary chromatographic trials indicate that hMBCOMT may be isolated on octyl- with mild salt conditions but on epoxy- were required high salt concentrations to complete enzyme adsorption. Thereby and in conclusion, in this work and for the first time was possible the total isolation of significant amounts of MB-COMT on a single chromatography step with high selectivity. Although successful applications of Hydrophobic Interaction Chromatography (HIC) in thepurificationof membrane proteins are uncommon, in this work we prove that traditional hydrophobic matrices can open a promising unexplored field in order to fulfill specific requirements for kinetic and pharmacological trials.
by precipitation with 30-90% ammonium sulphate fractionation. The precipitates were dissolved in 5 ml of Tris-HCl buffer (pH 6.8) and dialyzed overnight against 2 L ofthe same buffer. Each fraction was checked for enzyme activity as well as protein content. 30% ammonium sulphate fraction showing maximum lipase activity was mixed with ammonium sulphate to a final concentration of 0.25 M in 50 mM Tris-HCL buffer, pH 6.8 and applied to 15 ml of preactivated phenyl Sepharose CL-4B (1.5 X24 cm). Theenzyme was eluted with linear gradient of 1% (w/ v) cholate in 50 mM Tris-HCl buffer, pH 6.8 with flow rate of 1ml/ min. All the fractions were checked for enzyme activity. The active fractions were pooled and applied on pre-equilibrated Mono Q HR5/5 Column (1X6cm). Theenzyme was eluted by gradient NaCl (0-1.0M) in the same buffer at a flow rate of 1 ml/ min. The active fractions that contained lipase enzyme were pooled, desalted and rechromatographed on Mono Q HR5/5 Column (1X6cm). Theenzyme was eluted with linear gradient of NaCl (0-0.5M) in the same buffer at a flow rate of 0.5 ml/min. The lipase containing fractions were pooled and assessed for protein content. The resulting enzyme was utilized for the characterization ofthe extracellular lipase.
The partial characterization and purificationof milk clotting enzyme obtained from the (root latex) of Jacaratia corumbensis O. kuntze was studied, by fractional precipitation with ammonium sulphate and ion exchange chromatography. The ammonium sulphate precipitate showed five fractions (AS1– 0-20%; AS2 – 20-40%; AS3 – 40- 60%; AS4 – 60-80%; AS5 – 80-100%) and among the fractions obtained, the 40-60% fraction (AS3) showed the highest milk clotting activity with a purification factor of 1.2 fold in relation to the crude extract. This fraction when applied on Mono Q column yielded two protein peaks (p1 and p2), but p1 pool showed the best milk-clotting activity. The optimal pH for the crude and partially purified extract was 6.5 and 7.0, respectively. The maximum milk-clotting activity was at 55ºC for the both crude and partially purified extracts. Theenzyme was inhibited by iodoacetic acid which suggested that this enzyme was a cysteine protease, with molecular weight of 33 kDa.
20 mM Tris HCl, pH 8.0, 100 mM NaCl. Peak fractions eluted from a linear NaCl gradient were pooled, diluted in a buffer containing 20 mM potassium phosphate, pH 7.0, 50 mM NaCl and loaded onto a ceramic hydroxyapatite column (7 ml; Bio- Rad). Peak fractions eluted from a linear potassium phosphate gradient were pooled and concentrated to ~1.5 ml (4 mg/ml) using a Centricon 70 plus concentrator (MWCO = 10 kDa; Millipore). To remove SAM potentially bound to and co-purified with RM.BpuSI, 5 mg (50 nmol) ofthe concentrated protein were incubated with 100 nmol of a oligonucleotide duplex containing a BpuSI site (sub01, Figure 1) in a 1-ml solution containing 20 mM Tris HCl, pH 8.0, 50 mM NaCl, 5 mM DTT, 1 mM EDTA at 37°C for 1 h. The reaction was then diluted in a buffer containing 20 mM Tris HCl, pH 8.0, 50 mM NaCl and loaded into a Source 15Q column (7 ml; GE Life Sciences). Peak fractions eluted from a linear NaCl gradient were pooled, concentrated and stored at -20°C in a buffer containing 25 mM Tris, pH 7.5, 300 mM NaCl, 40% glycerol. The purity ofthe protein preparations was evaluated using SDS-PAGE followed by Coomassie Blue staining (SimplyBlue SafeStain; Invitrogen). The bpuSIM1 ORF was PCR-amplified and cloned into pT7SL. The construct pT7SL-bpuSIM1 was used to transform ER3081. Expression and purification procedures were essentially the same as for RM.BpuSI.
After precipitation ofthe crude extract with ammonium sulphate up to 100% of saturation, the active fractions with fibrinolytic activity were loaded into a DEAE-Sephadex A50 ion-exchange column (25 x 12 x 2.0 cm) equilibrated with 150 mM Tris-HCl buffer, pH 8. The sample was then eluted with the same buffer containing 0.5 M potassium chloride. The protein-containing fraction was pooled, and theenzyme solution concentrated for further analysis. All the process was monitored at 280 nm absorbance. The main fractions with fibrinolytic activity were dialyzed in 15 mM Tris-HCl buffer, pH 8. The dialysate was concentrated by lyophilization and subsequently gel filtrated through Superdex 75 HR10/300, Äkta Avant 25 System (GE Healthcare, Uppsala, Sweden) that had previously been equilibrated with 100 mM Tris–HCl buffer, pH 8.0, at a flow rate of 0.5 mL/min. The fractions possessing protease activity were pooled and concentrated.
The inhibition rate of CBE hydrolysates towards ACE involved in the regulation of blood pressure (Li et al., 2015) were determine, All hydrolysates at 10 µg/mL showing different DH, the CBE hydrolyzed by Protease G6 (PG6H) had significantly higher ACE-O activity (94.22%) than other hydrolysates. On the other hand, CBE hydrolyzed by Alcalase (ALH) and Protamex (PTH) showed very low antihypertensive activity (P<0.05; Table 2). The PG6H and ALH have similar DH values, however, these are significant differences in ACE-O activity. This result is in agreement with other studies that reported different enzymes with almost the same DH did not show the same ACE inhibitory potency. According to Barzideh et al. (2014), collagen hydrolysates (ribbon jellyfish) hydrolyzed by Alcalase and Trypsin resulted in similar DH values (26 & 25%, respectively), but different ACE-O activity (59 & 89%, respectively). Thus, the protein sequence ofthe substrate or protease activity would be the most important factor of hydrolysates exhibiting ACE-O activity (Cheung & Li-Chan, 2010).
The kosmotropes are a class of anions that are “water-structure makers”  as they possess the ability to perform hydrogen bonding with water molecules and contribute for the enhancement of hydrophobic interactions and salting-out effects. The chaotropic salts act by disordering water molecules, increasing the solubility of non-polar groups and reducing the hydrophobic interactions . The urea, guanidine and detergents as sodium dodecyl sulphate (SDS) are also strong chaotropic agents [36, 258-259]. The greatest disadvantage of using chaotropic agents as eluents consists on the structural changes ofthe target protein, namely the loss of secondary and tertiary structure, leading to incorrect folding and denaturation, which require further removal after elution [34, 36, 247, 260]. The use of organic solvents also can affect the polarity ofthe affinity interaction, being the most common methanol, polyols as ethylene glycol (0-50%) and glycerol [247-248]. These solvents are polar reducing agents through the binding to hydrophobic sites ofthe target protein decreasing the hydrophobic interactions between the ligand and protein [261-262]. In general, ethylene glycol does not induce large conformational alterations in the protein, but high concentrations can cause ligand leaching . The temperature and chelating substances are agents that also can improve the elution efficiency [34, 36]. The chelating agents such as EDTA are particular efficient in those affinity systems that require metals to enhance binding .
purity values above 98%, after concentration by distillation, precipitation on ice-cold methanol and recrystallization (50). Besides chlorinated solvents, other classes of compounds were also studied to extract PHAs. Diols, like 1,2-propandiol, were also tested to extract PHAs from C. necator resulting in a recovery of 79% and a purity of 99.1% at 140ºC (49). Also with the same biomass, acetalized triols, as glicerol formal, resulted in 85% of recovery yield, and in a purity of 99.7% at 120ºC. A di-carboxilic acid ester, as diethyl succinate, led to 90 % of recovery yield and a purity of 100% at 110ºC (49), and butyrolactone allowed for a recovery yield of 90% and a purity of 99,5% at 110ºC (49). When using solvent extraction, the biopolymer solutions obtained, could present more than 5% (w/v) of P(3HB), with high viscosities. However, the removal of cell debris proves to be difficult (2). In order to overcome this problem, Vanlautem and Gilain et all (1982) (49) proposed a process were the P(3HB) was solubilized in a solvent immiscible with water, at temperatures above 120ºC (49). The recovery of PHA was then made by simply adding cool water, leading to the separation ofthe complex solvent and PHA. Rosengart et al. (51) also tested other non-chlorinated solvents, namely anisole and cyclohexanone (Figure 4) for PHA separation from Burkholderia sacchari. To a mass of lyophilized cells, 0.6g, were added to 40 mL of solvent (anisole and cyclohexanone), in a glass balloon, and the solutions were left to rest in an oil bath, at 120ºC, for 1h. A temperature of 60ºC for 2h was also tested, but the results were unsatisfactory. Cell debris
During Ho-FACS ofthe different mammalian species, it was notable that Ho-red and blue fluorescence alone could discriminate round and elongated spermatid populations in the dog sample, it was insufficient to further refine this population in the remaining species (Figure 5, Table 1). Given that round (rSpd) and elongating (eSpd) spermatids are molecularly very distinct in terms of transcription activity as well as the differentiation occurring in the latter during spermiogenesis, we sought to evaluate a different strategy to isolate different mouse spermatid subpopulations by FACS. It has been previously suggested that rSpd and eSpd could be gated based on high forward scatter (FSC high) and low forward scatter (FSC low), respectively . Interestingly, we observed that gating based on the FSC parameter alone introduced some contamination in the sorted populations. Microscopy quantification of purity of sorted populations based on cell morphology and Ho fluorescence revealed and enrichment of ~62% for eSpd and 84% for rSpd (Fig. S2). Gating for events with low FSC and high VS low side scatter (SSC), we increased purity levels to 92% of eSpd and 86% rSpd (Fig. S2). Finally, we observed that the lowest levels of contamination could be obtained by the combination of FSC and SSC gating followed by Hoechst red/blue fluorescence. It seems that eSpd can be isolated gating for low FSC&SSC with 83-92% enrichment range, whilst rSpd appear to have higher FSC&SSC values and can be separated with 86-95% accuracy (Fig. 6; Table 1). Importantly, this gating strategy is based on cell size, shape and complexity and thus potentially applicable to Ho-FACS of any species undergoing spermiogenesis during gamete development.
As previously mentioned, MRS broth is the most commonly used laboratory medium to grow LAB and promote the production of EPS. The composition of MRS broth listed in Table 1 reveals considerable amounts of polymeric extracts that are often found as contaminants of EPS (Kimmel & Roberts, 1998; Vaningelgem et al., 2004). As shown, YE is a major source of mannoproteins and other glycans, as well as of non-glycosidic material (Table 2 and Fig. 1). Furthermore, beef extract, which is a complex nutrient also present, has high glycogen content and has been reported as a source of contamination which may interfere with EPS determination, as well as peptones that may be responsible mainly for the introduction of proteinaceous material (Kimmel & Roberts, 1998).To evaluate the extent ofthe possible contamination of MRS components and the performance ofthepurification methods under study to purify the EPS from MRS medium, experiments were carried out using model solutions containing MRS broth medium and xanthan gum. The influence of lactose was also determined.
Most genes investigated are housekeeping genes necessarily conserved for the survival ofthe organism. The know pathogenicity islands are phages or phage-like mobile elements responsible for the lateral transfer of non essential genes coding cholera toxin (CT) and toxin co- regulated pilli (TCP) (Karaolis et al. 1999, Faruque et al. 1999) and genes coding somatic O antigens as in the Bengal O139. This latter strain is genetically an El Tor 7th pandemic zymovar 14 A which received from a non-O1 vibrio, genes coding a modified LPS (Johnson et al. 1994, Bik et al. 1995). It is therefore unlikely that strong correla- tion may be found between zymovars and antigenic or virulence factors carried by these mobile elements.
An example ofthe application of these custom zinc finger proteins involves several kinds of methyltransferases, which have been linked to zinc-finger motifs and analyzed in vitro and in vivo conditions. This targeted DNA methylation al- lows artificial gene silencing and can be used to prevent an overexpressed gene in cancer by adding a methyl group to the target gene. Targeted DNA methylation was demon- strated to be effective at repressing Herpes Simples Virus type I (HSV-1) infection in cell culture . Several groups have tested zinc fingers to inhibit the replication of inte- grated HIV [30,31] and high-risk HPV type 18, which is in- volved in cervical cancer . Thus, custom zinc finger proteins can be engineered to control the expressions of target genes and have therapeutic potential for the study and treatment of diseases. Moreover, with further advances in zinc finger protein research, it may become possible to construct synthetic cell networks and control high-level vital functions .
In this paper, we consider incomplete information games with a finite number of players in which each player’s payoff depends only on his type- action character and on the average distribution of type-action characters ofthe others. Our purification result shows that, if players’ types are in- dependent and their payoff functions are selected from an equicontinuous family, then all (Bayesian) Nash equilibria of sufficiently large games can be approximately purified. More precisely, our result shows that for every Nash equilibrium of such games there exists a pure strategy approximate equilibrium that yields approximately the same payoff to all players.
Two experiments were carried out. The first one consisted ofthe use ofthe herbicide imazapic [2-((RS)-4-isopropyl- 4-methyl-5-oxo-2-imidazolin-2-yl)-5- methylnicotinic acid] and the second ofthe use of metsulfuron-methyl [methyl 2-(4-methoxy-6-methyl-1,3,5-triazin-2- ylcarbamoylsulfamoyl) benzoate] herbicide. The sulfonylurea herbicide was used to confirm that imidazolinone resistant canola does not have cross-resistance to this chemical group and to be a positive control in the enzymatic assay, inhibiting ALS enzyme activity. The herbicides used were those suitable for use in laboratory analysis, with purity of approximately 99% and without inert ingredients.
purification and preservation steps. ILs, commonly described as molten salts at temperatures below 100 °C, and composed of large and unsymmetrical organic cations and organic or inorganic anions, have been reported as appropriate stabilizing media for proteins and enzymes 20-22 . During the last years, ILs have also been applied to improve some properties of nucleic acids, mainly of DNA, such as solubility, stability and bioavailability 23-29 . Despite its high importance, few reports dealing with ILs and RNA are available 14,23 . In these works, cholinium dihydrogen phosphate and cholinium chloride were investigated. However, it is known that the stability, structure and functionality of RNA depend on the concentration and chemical identity ofthe ions in solution 7 , justifying a more intensive search on adequate IL ions, which is even of higher relevance given the ILs designer ability. Recently, we proposed a novel class of self- buffering imidazolium- and tetraalkylammonium-based ILs containing anions derived from biological buffers (Good’s buffers) – GB-ILs 30 . These ILs display high stabilizing effects over proteins and allow their complete extraction from aqueous media when used as phase-forming components of liquid-liquid systems 31 . Cholinium chloride is a water-soluble nutrient, and appropriate cholinium-based ILs usually present high biodegradability, low toxicity, and are able to maintain the structure and function of proteins and/or other labile biomolecules 31 . Based on the exposed and considering that nucleic acids are not stable in extreme ranges of pH values 12 , aqueous solutions of GB-ILs comprising the cholinium cation seem thus to be an attractive and biocompatible milieu to maintain the integrity and improve the stability of RNA. Furthermore, the use of self-buffering ILs can avoid the need of adding an additional buffer. Accordingly, in this work, we synthesized several cholinium-based GB-ILs and evaluated their performance to stabilize a recombinant Escherichia coli small RNAs fraction (sRNAs) containing a microRNA precursor (pre-miR-29). The integrity, structural stability, cytotoxicity, and superficial charge of RNA in aqueous solutions comprising 20 and 50 wt% of each IL were evaluated, as well as the medium-term storage stability of RNA in these aqueous solutions. Zeta potential measurements and molecular dynamics simulations were used to appraise the ILs effects on the RNA stability. Finally, it was demonstrated that the dissolved RNA can be successfully recovered from the aqueous solutions of ILs, further allowing the IL recycling.
The sea anemone Heteractis magnifica [identified by Dr. Daphne Fautin (17)] was collected from Andaman Islands, India, at a depth of 5 m by scuba diving. Samples were deposited in the National Marine Repository at the National Institute of Oceanography, Goa, India. In the laboratory, the live animal was induced by osmotic thermal stress to eject the epithelial mucus that contains the toxins (6). Cnidarian toxins are stored in the cnidocytes, which are intracellular organelles that fire under pressure or osmotic variations and deeply injects the venom. Approximately 2 kg of anemone was placed in 1 L of warm distilled water (40 to 45°C) for 15 minutes with intermittent stirring to minimize tissue damage. After 15 minutes, the anemone was removed and the solution was filtered. The filtrate was stored as 100 mL aliquots in liquid nitrogen during transportation. The extract was removed from liquid nitrogen after transportation and stored at –70°C until analysis. When required, the aliquots were thawed and concentrated by lyophilization and reconstituted in phosphate buffered saline at pH 7.4.