An industrial scale process for the enzymatic removal of steryl glucosides from biodiesel

Background: Biodiesels produced from transesterification of vegetable oils have a major quality problem due to thepresence of precipitates, which need to be removed to avoid clogging of filters and engine failures. These precipitateshave been reported to be mostly composed of steryl glucosides (SGs), but so far industrial cost-effective methods toremove these compounds are not available. Here we describe a novel method for the efficient removal of SGs frombiodiesel, based on the hydrolytic activity of a thermostable β-glycosidase obtained from Thermococcus litoralis.Results: A steryl glucosidase (SGase) enzyme from T. litoralis was produced and purified from Escherichia coli culturesexpressing a synthetic gene, and used to treat soybean-derived biodiesel. Several optimization steps allowed for theselection of optimal reaction conditions to finally provide a simple and efficient process for the removal of SGs fromcrude biodiesel. The resulting biodiesel displayed filterability properties similar to distilled biodiesel according to thetotal contamination (TC), the cold soak filtration test (CSFT), filter blocking tendency (FBT), and cold soak filter blockingtendency (CSFBT) tests. The process was successfully scaled up to a 20 ton reactor, confirming its adaptability toindustrial settings.Conclusions: The results presented in this work provide a novel path for the removal of steryl glucosides from biodieselusing a cost-effective, environmentally friendly and scalable enzymatic process, contributing to the adoption ofthis renewable fuel.Fil: Peirú, Salvador. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Keclon; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Aguirre, Andres. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Keclon; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Eberhardt, Maria Florencia. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Braia, Mauricio Javier. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cabrera, Rodolfo Ariel. Unitec Bio; ArgentinaFil: Menzella, Hugo Gabriel. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Keclon; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Enzymatic hydrolysis of fish frames using pilot plant scale systems

Papain was used to hydrolyse fish frames under controlled conditions at a batch-pilot plant scale-process, for the pro-duction of fish protein hydrolysates (FPH). Mass balance calculations were carried out so that the rate of hydrolysis, rate of protein solubilisation and yields could be estimated. Almost complete hydrolysis could be achieved in 1 hour, at 40°C, with no pH adjustment, at 0.5% (5 g·kg−1) enzyme to substrate ratio (E/S, were S is Kjeldahl protein) using whole fish frames (including heads and flaps). This was achieved both with the addition of water (1/1 to 2/1 frames/water) but more importantly from commercial considerations without the initial addition of water (after mincing of the fish mate-rial). The degree of protein solubilisation ranged between 71% - 86% w/w. Four different processes are described, namely: 1) a soluble spray-dried FPH powder; 2) a liquid FPH; 3) a partly soluble, spray dried FPH powder and; 4) a crude, drum-dried protein for animal consumption. The amino acid profile of the FPH was identical to that of the par-ent substrate (fish frames)

Filtration characteristics of hollow fiber microfiltration membranes used in a specific double membrane bioreactor

The performance of the microfiltration in a specifically designed membrane bioreactor operating under various transmembrane pressures with periodic backwashing was investigated for model media. These media were representative of some usual components of a fermentation medium: BSA solution (2 g L−1), yeast suspension (8 g L−1, dry mass) and a mixture of BSA/yeast (2 g L−1/8 g L−1). In this system, the separation was provided by a 0.1 μm polysulfone hollow fiber membrane. The net permeate fluxes observed for yeast/BSA mixture were proportional to the transmembrane pressure applied (ΔP) but were less than those obtained with water osmosis, showing that, in spite of the periodic backwash, a small amount of irreversible fouling remained. This fouling can be assumed to be due to internal fouling by protein and/or external fouling by a residual yeast cake. Moreover, the net permeate flux obtained with the yeast/BSA mixture was higher than that obtained with the BSA alone, showing that a thin yeast cake probably acted as a primary filtration layer that could protect the polysulfone membrane against protein fouling. These experiments enable operating recommendations to be made for the use of this specific bioreactor concerning the transmembrane pressure value and the possible addition of inert particles

Gel filtration chromatography

Gel-filtration chromatography is a popular and versatile technique that permits the effective separation of proteins and other biological molecules in high yield. Here, the basis of the method is described and typical matrix types are contrasted. The selection of suitable operating conditions and applications of the method are also discussed

1,2,6-thiadiazinones as novel narrow spectrum calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) inhibitors

We demonstrate for the first time that 4H-1,2,6-thiadiazin-4-one (TDZ) can function as a chemotype for the design of ATP-competitive kinase inhibitors. Using insights from a co-crystal structure of a 3,5-bis(arylamino)-4H-1,2,6-thiadiazin-4-one bound to calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), several analogues were identified with micromolar activity through targeted displacement of bound water molecules in the active site. Since the TDZ analogues showed reduced promiscuity compared to their 2,4-dianilinopyrimidine counter parts, they represent starting points for development of highly selective kinase inhibitors

A saposin-lipoprotein nanoparticle system for membrane proteins.

A limiting factor in membrane protein research is the ability to solubilize and stabilize such proteins. Detergents are used most often for solubilizing membrane proteins, but they are associated with protein instability and poor compatibility with structural and biophysical studies. Here we present a saposin-lipoprotein nanoparticle system, Salipro, which allows for the reconstitution of membrane proteins in a lipid environment that is stabilized by a scaffold of saposin proteins. We demonstrate the applicability of the method on two purified membrane protein complexes as well as by the direct solubilization and nanoparticle incorporation of a viral membrane protein complex from the virus membrane. Our approach facilitated high-resolution structural studies of the bacterial peptide transporter PeptTSo2 by single-particle cryo-electron microscopy (cryo-EM) and allowed us to stabilize the HIV envelope glycoprotein in a functional state

Antitumor studies. Part 4: Design, synthesis, antitumor activity, and molecular docking study of novel 2-substituted 2-deoxoflavin-5-oxides, 2-deoxoalloxazine-5-oxides, and their 5-deaza analogs

Various novel 10-alkyl-2-deoxo-2-methylthioflavin-5-oxides and their 2-alkylamino derivatives were prepared by facile nitrosative cyclization of 6-(N-alkylanilino)-2-methylthiopyrimidin-4(3H)-ones followed by nucleophilic replacement of the 2-methylthio moiety by different amines, and acidic hydrolysis of the 2-methylthio moiety afforded the corresponding flavin derivatives. 2-Deoxo-2-methylthio-5-deazaalloxazines and 2-deoxo-2-methylthioalloxazine-5-oxides were also prepared by Vilsmeier reaction and by nitrosation of 6-anilino-2-methylthiopyrimidin-4(3H)-ones, respectively. Then, they were subjected to nucleophilic replacement with appropriate amines to produce the corresponding 2-alkylamino derivatives. Regiospecific N-3-alkylation of 2-deoxo-2-methylthioalloxazine-5-oxides was carried out with various alkylating agents in the usual way, The antitumor activities against CCRF-HSB-2 and KB tumor cells have been investigated in vitro, and many compounds showed promising antitumor activities. Furthermore, AutoDock molecular docking into PTK (PDB: 1t46) has been done for lead optimization of the aforementioned compounds as potential PTK inhibitors

Surface Mutation Thr34His Facilitates Purification of Haemophilus influenza Carbonic Anhydrase via Metal Affinity Chromatography

In order to pursue Haemophilus influenza carbonic anhydrase (HICA) as a potential drug target, easy and efficient purification methods must be developed. While immobilized metal affinity chromatography (IMAC) may be used, complications with polyhistidine tags is a concern. Inspired by the endogenous metal affinity of Escherichia coli β-carbonic anhydrase (ECCA), we suggest that the generation of histidine clusters on HICA’s surface will facilitate its purification by metal affinity chromatography without the potential interference of His-tags. Here we investigate the Thr34His mutation as a method to generate metal affinity in HICA. Since Thr34His is located only 5.3 Å away from His32, the two residues make a vicinal histidine pair that can interact with nickel resin. We report successful generation of Thr34His HICA mutant plasmid via site-directed mutagenesis. To obtain mutant protein for metal affinity chromatography, Thr34His HICA was overexpressed in E. coli cells and isolated as a cell lysate with a concentration of 20.2 ± 0.6 mg/mL. Metal affinity chromatography was performed on the sample, and the chromatography fractions were analyzed by SDS-PAGE in order to assess the metal affinity of the mutant. SDS-PAGE revealed that while Thr34His HICA eluted at low 10 mM and 25 mM concentrations of imidazole, 150 mM imidazole was required to fully elute the mutant. These results suggest that through the generation of surface histidine pairs, HICA can be engineered to have metal affinity and thus be easily purified via IMAC

Properties of Endoglucanase of Penicillium chrysogemum PCL501

Crude extracellular enzyme from a 3-day culture of Penicillium chrysogenum (PCL 501), in basal medium containing cellulose as the sole carbon source, yielded 0.67 ± 0.03, 19.94 ± 1.30 and 8.50 ± 0.50 units mg protein-1 of 1, 4- â-endoglucanase, â-glucosidase and xylanase activity respectively. The crude enzyme was subjected to ammonium sulphate precipitation (80% saturation) and gel filtration. A purification-fold of 7.5 was achieved. Two active fractions of 1, 4 âendoglucanase (EC 3. 2. 1. 4), which exhibited about the same activity towards carboxymethylcellulose (CMC), were obtained and pooled for the subsequent analyses. The endoglucanase gave a Vmax of 10.0 ± 0.4 μmol min-1 mg protein-1 and Km of 11.8 ± 0.4 gL-1 with CMC. The enzyme was most active at pH of 4.5 – 5.0 and temperature range of 40 – 50 OC. The optimum pH was 4.9 while the Optimum temperature was 48 OC. Divalent metal ions and EDTA affected the enzyme activity at 2.0 mM concentrations. Mn2+ and Fe2+ had stimulatory effects on the enzyme whereas Mg2+, Cu2+, Zn2+, Hg2+ and EDTA inhibited the enzyme activity. The effect of Ca2+ was not significant. Over 3- fold increase in the enzyme activity was recorded with Mn2+. Percentage inhibition of 65.9 and 79.7 respectively was obtained with Hg2+ and EDTA. The organism appears to produce two types of endoglucanase which differed in their molecular weight but not significantly in their activity. The enzyme activity was highly stimulated by manganese ion and inhibited by the metal-chelating agent, EDTA