Pretreatment and enzymic saccharification of water hyacinth cellulose

Water hyacinth was pretreated, under variable conditions, with NaOH, alkaline H₂O₂, peracetic acid and sodium chlorite. Combined pretreatments included sodium chlorite with each of NaOH, alkaline H₂O₂ and peracetic acid. Combined pretreatment with 0.1% NaClO₂ for 1 h at 100 °C and peracetic acid at 100 °C for 15 min afforded the most promising sample. The recovered lignin, cellulose and hemicellulose of this sample was 2.56%, 96.69%, and 81.38%, respectively. The same sample, by cellulase hydrolysis showed the highest cellulose conversion (80.8%) and 90% saccharification using 200 FPU/g substrate. Some ambient factors affecting saccharification of pretreated water hyacinth were investigated. Enzymic saccharification after 6 h was about 50% of that at 48 h, indicating a slow hydrolysis rate by time. Addition of 8% glucose at the beginning of the enzymic hydrolysis decreased the saccharification to about its half while addition of 8% ethanol brought about complete inhibition of the enzyme. Addition of cellobiase to the reaction mixture increased cellulose conversion and saccharification by 10%.     

Lipase-catalyzed alcoholysis of triglycerides for the preparation of 2-monoglycerides

Rhizopus arrhizus lipase immobilized on celite was used to prepare isomerically pure 2-monoglycerides by alcoholysis of triglycerides in organic media. Reaction parameters such as choice of solvent, choice of alcohol, and alcohol concentration were studied. When 12.5 mM tripalmitin was used as substrate, methyl-tert-butyl ether was the best solvent for alcoholysis at water activity 0.11. Ethanol gave the highest yield (97%) at an optimal ethanol concentration of 200–300 mM. At higher alcohol concentrations, the enzyme activity was substantially lowered. The enzyme preparation showed high stability in repeated-batch reactions.     

Enantioselective hydrolysis of racemic methyl jasmonate using microorganisms and isolated enzymes

A variety of microorganisms were used to hydrolyze racemic methyl jasmonate [I] with varying degrees of enantioselectivity. The fungi tested included species from the genera Aspergillus, Penicillium, and Talaromyces. All fungi tested showed a preference for the [1S,2S(Z)]-(+)-isomer. The yeasts Saccharomyces cerevisiae and Candida albicans showed no activity. A number of bacterial genera were also tested. No activity could be shown for members of the genera Bacillus, Pseudomonas, Escherichia, Nocardia, and Thermoactinomyces. Hydrolytic activity was found in the genera Streptomyces and Mycobacterium. S. henetus showed the same enantioselectivity as the fungi, while M. phlei hydrolyzed the [1R,2R(Z)]-(−)-isomer preferentially. A number of isolated enzymes were also screened for activity. Varying degrees of hydrolytic activity and enantioselectivity were found.     

The Effect of Substrate Hydrophobicity on the Kinetic Behaviour of Immobilized Candida rugosa Lipase

Candida rugosa lipase (EC 3.1.1.3.) was immobilized in a hydrophilic polyurethane foam and used in the hydrolysis of olive oil, in H-hexane. The results obtained were compared with those from a previous study, in which the same lipase preparation was used in the esterification of ethanol with butyric acid.

The initial rate of hydrolysis increased exponentially with increasing olive oil concentration. In contrast, for the esterification reaction, Michaelis-Menten kinetics with inhibition by both substrates, had been observed.

The effect of medium viscosity, stirring conditions and size of immobilization particles could not explain the observed kinetics of the hydrolytic reaction. However, a direct relationship was observed between the log P values of the reaction medium and the initial rate of hydrolysis, i.e., activation of the immobilized Candida rugosa lipase appears to be promoted by a high hydrophobicity of the reaction medium.

In the case of the esterification reaction, no similar correlation was found.     

Studies toward a conjugate vaccine for anthrax. Synthesis and characterization of anthrose [4,6-dideoxy-4-(3-hydroxy-3-methylbutanamido)-2-O-methyl-D-glucopyranose] and its methyl glycosides

The key step in the first chemical synthesis of anthrose (16) and its methyl alpha- (6) and beta-glycoside (22) was inversion of configuration at C-2 in triflates 10, 2, and 18, respectively, obtained from the common intermediate, methyl 4-azido-3-O-benzyl-4,6-dideoxy-alpha-D-mannopyranoside (1). To prepare methyl alpha-anthroside (6), methylation at O-2 of the gluco product 3, obtained from 2, was followed by hydrogenation/hydrogenolysis of the formed 2-methyl ether 4, to simultaneously remove the protecting benzyl group and reduce the azido function. Subsequent N-acylation of the formed amine 5 with 3-hydroxy-3-methylbutyric acid gave the target methyl alpha-glycoside 6. Synthesis of methyl beta-anthroside (22) comprised the same sequence of reactions, starting from the known methyl 4-azido-3-O-benzyl-4,6-dideoxy-beta-D-mannopyranoside (17), which was prepared from 1. In the synthesis of anthrose (16), 1-thio-beta-glucoside 11, obtained from 1 through 10, was methylated at O-2, and the azido function in the resulting benzylated 1-thioglycoside 12 was selectively reduced to give amine 13. After N-acylation with 3-hydroxy-3-methylbutyric acid, 1-thioglycoside 14 was hydrolyzed to give the corresponding reducing sugar, aldol 15, which was debenzylated to afford anthrose.     

Development and validation of an experimental and theoretical method for the chiral discrimination of dinotefuran

Dinotefuran is a low-cost agrochemical considered a highly toxic product. In this sense, there is a need for its constant environmental, biological, and food control, aiming to ensure its use to humans as well as to preserve biodiversity and ecosystems. In the present work, we developed an experimental and theoretical method for dinotefuran chiral discrimination. According to the main results, the dinotefuran enantioselective separation was efficiently optimized by high-performance liquid chromatography evaluating the influence of different percentage compositions in the mobile phase to improve the resolution of the peaks in the chromatogram. The novelty of this work was the proposition of a reduced molecular model for the chiral selector amylose-Tris-(3,5-dimethylphenylcarbamate) polysaccharide that was able to adequately describe at the molecular level its interaction with the dinotefuran enantiomers. Besides, the thermodynamic and structural parameters obtained via density functional theory calculations pointed out the chiral discrimination as well as the enantiomeric elution order of the analyte studied, confirming the experimental data, thus validating our proposed method. Finally, hydrogen bonds and repulsive interactions played a key role in the discrimination between the diastereomeric complexes, and consequently, for the dinotefuran enantioselective separation.     

Acid-catalyzed steam pretreatment of lodgepole pine and subsequent enzymatic hydrolysis and fermentation to ethanol

Utilization of ethanol produced from biomass has the potential to offset the use of gasoline and reduce CO(2) emissions. This could reduce the effects of global warming, one of which is the current outbreak of epidemic proportions of the mountain pine beetle (MPB) in British Columbia (BC), Canada. The result of this is increasing volumes of dead lodgepole pine with increasingly limited commercial uses. Bioconversion of lodgepole pine to ethanol using SO(2)-catalyzed steam explosion was investigated. The optimum pretreatment condition for this feedstock was determined to be 200 degrees C, 5 min, and 4% SO(2) (w/w). Simultaneous saccharification and fermentation (SSF) of this material provided an overall ethanol yield of 77% of the theoretical yield from raw material based on starting glucan, mannan, and galactan, which corresponds to 244 g ethanol/kg raw material within 30 h. Three conditions representing low (L), medium (M), and high (H) severity were also applied to healthy lodgepole pine. Although the M severity conditions of 200 degrees C, 5 min, and 4% SO(2) were sufficiently robust to pretreat healthy wood, the substrate produced from beetle-killed (BK) wood provided consistently higher ethanol yields after SSF than the other substrates tested. BK lodgepole pine appears to be an excellent candidate for efficient and productive bioconversion to ethanol.     

Purification and partial characterization of an acid β-fructosidase from sweet-pepper (Capsicum annuum L.) fruit

The present study describes the biochemical characteristics of an acid -fructosidase (EC 3.2.1.26) purified from the fruit of sweet pepper (Capsicum annuum L.). The soluble form, which constitutes more than 95% of the total activity at pH 4.5, hydrolyzes sucrose, raffinose, and stachyose. Its pH and temperature optima are 4.5 and 55 °C, respectively. Metal cations such as Ag⁺ and Hg²⁺ strongly inhibit its activity, suggesting the presence of at least one sulfhydryl group at the catalytic site. After purification of the enzyme by means of ammonium sulfate fractionation, gel chromatography (diethyl-aminoethyl-Sephacel, hydroxylapatite, concanavalin A-Sepharose), and preparative gel electrophoresis, the purified enzyme was shown to be a 42 kDa glycoprotein interacting specifically with concanavalin A. After complete chemical deglycosylation with trifluoromethanesulfonic acid, the molecular weight of the constitutive polypeptide was estimated to be 39 kDa. The enzyme glycans were characterized using both affino- and immunodetection. The enzyme has at least two N-linked oligosaccharide sidechains, one of the high-mannose type, and the other of the complex type. The high-mannose glycan has a low molecular weight (1 kDa), and is responsible for the interaction between the enzyme and concanavalin A. The complex-type glycan has an estimated molecular weight of 2 kDa. It contains one 1 2-linked xylose residue, probably one fucose residue 1 3-linked to the chitobiose unit, and no terminal galactose residue. The two glycans, associated to the 39 kDa polypeptide, constitute the acid -fructosidase of the sweet-pepper fruit.Abbreviations F -fructosidase - ConA concanavalin A - DEAE diethylaminoethyl - DTNB dithionitrobenzoic acid - endo F endo--N-acetylglucosamidase F - endo H endo--N-acetylglucosamidase H - NEM N-ethylmaleimide - PCMB parachloromercurobenzoate - PNGase glycopeptide-N-glycosidase - TFMS trifluoromethane sulfonic acid This work was partly supported by a grant from the Commission Permanente de Coopération Franco-Québécoise to L. Faye, and S. Yelle. D. Michaud was a recipient of a graduate scholarship from the Natural Science and Engineering Research Council of Canada.     

Energy metabolism and ATP free-energy change of the intertidal wormSipunculus nudus below a critical temperature

The intertidal wormSipunculus nudus was exposed to various temperatures for an analysis of the integrated changes in energy and acid-base status. Animals were incubated in sea water or maintained for up to 8 days at 4 and 0°C while dwelling in the sediment. Cannulation of the animals prior to experimentation allowed the analysis of blood gas parameters ( , and pH). fell to 0 torr within 8 days at 0°C. A simultaneous reduction of ventilatory activity was derived from measurements of the pattern of coelomic fluid pressure changes associated with ventilatory movements. The increase in and an onset of anaerobic metabolism, indicated by the accumulation of end products like acetate and propionate both in the coelomic fluid and the body wall musculature, led to the development of a progressive acidosis and a deviation from the alphastat regulation of intracellular pH seen in unburied animals. The drop in intracellular pH together with the depletion of the adenylates and the phosphagen, phospho-l-arginine, reflect a significant decrease in the Gibb's free-energy change of ATP hydrolysis. These changes are interpreted to indicate lethal cold injuries, because recovery was not possible when the animals were returned to 12°C after more than 2 days of exposure to 0°C. A low critical temperature indicating the onset of cold-induced anaerobiosis is concluded to exist below 4°C owing to the insufficient response of the ventilatory system to the developing hypoxia.