Selection of CalB immobilization method to be used in continuous oil transesterification: analysis of the economical impact

Enzymatic transesterification of triglycerides in a continuous way is always a great challenge with a large field of applications for biodiesel, bio-lubricant, bio-surfactant, etc. productions. The lipase B from Candida antarctica (CalB) is the most appreciated enzyme because of its high activity and its non-regio-selectivity toward positions of fatty acid residues on glycerol backbone of triglycerides. Nevertheless, in the field of heterogeneous catalysis, we demonstrated that the medium hydrophilic nature of the support used for its commercial form (Lewatit VPOC1600) is a limitation. Glycerol is adsorbed onto support inducing drastic decrease in enzyme activity. Glycerol would form a hydrophilic layer around the enzyme resulting in diffusional limitations during triglyceride transfer to the enzyme. Accurel MP, a very hydrophobic macroporous polymer of propylene, was found not to adsorb glycerol. Immobilization conditions using this support were optimized. The best support was Accurel MP1001 (particle size<1000 μm) and a pre-treatment of the support with acetone instead of ethanol enables the adsorption rate and the immobilized enzyme quantity to be maximized. An economical approach (maximization of the process net present value) was expanded in order to explore the impact of immobilization on development of an industrial packed bed reactor. The crucial ratio between the quantity of lipase and the quantity of support, taking into account enzyme, support and equipped packed bed reactor costs was optimized in this sense. The biocatalyst cost was found as largely the main cost centre (2-10 times higher than the investments for the reactor vessel). In consequence, optimal conditions for immobilization were a compromise between this immobilization yield (90% of lipase immobilized), biocatalyst activity, reactor volume and total investments.     

Praziquantel,Mefloquine-Praziquantel,and Mefloquine-Artesunate-Praziquantel against Schistosoma haematobium: A Randomized,Exploratory, Open-Label Trial

Background

Treatment and morbidity control of schistosomiasis relies on a single drug, praziquantel. Hence, there is a pressing need to develop additional therapeutics against schistosomiasis. The antimalarial drug mefloquine shows antischistosomal activity in animal models and clinical trials, which calls for further investigations.

Methodology

We comparatively assessed the efficacy and tolerability of the following treatments against Schistosoma haematobium in school-aged children in Côte d''Ivoire: (i) praziquantel (40 mg/kg; standard treatment); (ii) mefloquine (25 mg/kg) combined with praziquantel (40 mg/kg); and (iii) mefloquine-artesunate (3× (100 mg artesunate +250 mg mefloquine)) combined with praziquantel (40 mg/kg) (treatments administered on subsequent days). Two urine samples were collected before, and on days 21–22 and 78–79 after the first dosing.

Principal Findings

Sixty-one children were present on all examination time points and had complete datasets. No difference in efficacy was observed between the three treatment groups on either follow-up. On the 21–22 day posttreatment follow-up, based on available case analysis, cure rates of 33% (95% confidence interval (CI) 11–55%), 29% (95% CI 8–50%), and 26% (95% CI 5–48%) were observed for praziquantel, mefloquine-artesunate-praziquantel, and mefloquine-praziquantel, respectively. The corresponding egg reduction rates were 94% and above. On the second follow-up, observed cure rates ranged from 19% (praziquantel) to 33% (mefloquine-artesunate-praziquantel), and egg reduction rates were above 90%. Praziquantel monotherapy was the best tolerated treatment. In the mefloquine-artesunate-praziquantel group, adverse events were reported by 91% of the participants, and in the mefloquine-praziquantel group, 95% experienced adverse events. With the exception of abdominal pain at moderate severity, adverse events were mild.

Conclusions/Significance

The addition of mefloquine or mefloquine-artesunate does not increase the efficacy of praziquantel against chronic S. haematobium infection. Additional studies are necessary to elucidate the effect of the combinations against acute schistosomiasis.     

Degradation and fermentation of α-gluco-oligosaccharides by bacterial strains from human colon: in vitro and in vivo studies in gnotobiotic rats

The ability of several human gut bacteria to break down α-1,2 and α-1,6 glycosidic linkages in α-gluco-oligosaccharides (GOS) was investigated in vitro in substrate utilization tests. Bacteroides thetaiotaomicron, Bifidobacterium breve and Clostridium butyricum , which are usually found in the infant gut and have been associated with both beneficial and deleterious effects on health, were studied. α-Gluco-oligosaccharide degradation was compared in vitro and in vivo in gnotobiotic rats associated with these organisms, inoculated alone or in combination. Oligomer breakdown and short chain fatty acid and gas production indicated hydrolysis and fermentation of the substrate. In vitro and in vivo, Cl. butyricum was the least efficient in utilizing GOS, whereas Bact. thetaiotaomicron was the most efficient. Kinetic studies on GOS hydrolysis in pH-regulated fermenters showed that α-1,2 glucosidic bonds, which characterize the substrate, were more resistant than α-1,6 linkages. Adaptation of gnotobiotic rats to a diet containing 2% (w/w) GOS significantly increased the hydrolysis of α 1,2 glucosidic bonds. Combination of bacteria in trixenic rats improved GOS degradation and inhibited Cl. butyricum metabolism. This inhibition was confirmed in pH-regulated fermenters containing GOS as the principal carbon source. The association of beneficial bacteria and GOS may therefore have a potential health-promoting effect in human necnates.     

Growth and energetics of Leuconostoc mesenteroides NRRL B-1299 during metabolism of various sugars and their consequences for dextransucrase production.

The metabolic and energetic properties of Leuconostoc mesenteroides have been examined with the goal of better understanding the parameters which affect dextransucrase activity and hence allowing the development of strategies for improved dextransucrase production. Glucose and fructose support equivalent specific growth rates (0.6 h-1) under aerobic conditions, but glucose leads to a better biomass yield in anaerobiosis. Both sugars are phosphorylated by specific hexokinases and catabolized through the heterofermentative phosphoketolase pathway. During sucrose-grown cultures, a large fraction of sucrose is converted outside the cell by dextransucrase into dextran and fructose and does not support growth. The other fraction enters the cell, where it is phosphorylated by an inducible sucrose phosphorylase and converted to glucose-6-phosphate (G-6-P) by a constitutive phosphoglucomutase and to heterofermentative products (lactate, acetate, and ethanol). Sucrose supports a higher growth rate (0.98 h-1) than the monosaccharides. When fructose is not consumed simultaneously with G-1-P, the biomass yield relative to ATP is high (16.8 mol of ATP.mol of sucrose-1), and dextransucrase production is directly proportional to growth. However, when the fructose moiety is used, a sink of energy is observed, and dextransucrase production is no longer correlated with growth. As a consequence, fructose catabolism must be avoided to improve the amount of dextransucrase synthesized.     

Kinetic and thermal stability studies of rulactine,a proteolytic enzyme fromMicrococcus caseolyticus

Summary Kinetics and thermostability of Rulactine, a protease fromMicrococcus caseolyticus were investigated. Study of the enzyme activity as a function of the temperature showed an optimum peak of 45°C. The effeci of the substrate concentration on the initial velocity at various temperatures was examined, and V_max and K_M were determined using a Lineweaver-Burk reciprocal plot. The activation energy evaluation gave a value of 9500 cal/mole. Studies of additives such as polyhydric alcohols (glycerol, erythritol, xylitol and sorbitol) and disaccharides (sucrose and lactose) to Rulactine at 58°C proved that they have a stabilizing effect on Rulactine.     

Rheology of native dextrans in relation to their primary structure

High molecular weight dextrans were synthesized at five temperatures (3, 10, 20, 25 and 30°C) using an in-vitro enzymatic method. The rheological properties of these dextrans in aqueous solution were assessed through their flow behaviour and their viscoelastic characteristics. The results were interpreted in relation to their primary structure and particularly to their branching.

It was shown that the relatively expanded conformation of the dextrans synthesized at 3, 10 and 20°C gives to these dextrans comparable properties which are not too different from those described in literature for random-coil linear polysaccharides. Dextran synthesized at 30°C exhibited flow properties which are typical of particle suspensions in dilute and semi-dilute solution. In the concentrated domain, this dextran yielded structured systems with properties typical of weak gels. This unexpected behaviour could be related to the highly-ramified structure of this dextran in comparison with the dextrans synthesized between 3 and 20°C. On the other hand, the dextran synthesized at 25°C displayed rheological behaviour which could also be related to an intermediate primary structure between those of dextran synthesized at 20°C and dextran synthesized at 30°C.     

High-level production and purification of a fully active recombinant dextransucrase from Leuconostoc mesenteroides NRRL B-512F

Recombinant expression of the dextransucrase dsrS gene by Escherichia coli was optimized to produce 5850 U L(-1) (culture) of DSR-S, corresponding to a 30-fold increase compared with previous studies. Rational deletions of the signal peptide, the beginning of the variable region and the last four repeats of the C-terminal end caused no loss of activity. This new variant successfully purified was remarkably stable. With a k(cat) of 584 s(-1), it is the most efficient recombinant glucansucrase described to date. The synthesized polymer possesses more than 95% of alpha-1,6 links, like the dextran produced by the native enzyme, and innovative gel properties were obtained.