Preparation and characterization of pancreatic lipase immobilized in Eudragit-matrix

Pancreatic lipase (EC 3.1.1.3) was immobilized by entrapping in a commercial preparation of acrylic/methacrylic acid ester-based copolymer (Eudragit E 30 D). The activity of the immobilized lipase beads with a diameter of 1.5-2.0 mm was found to be lower than that of the free lipase. The optimum pH was shifted to the alkaline region and the thermal stability increased, whereas the optimum temperature level remained unchanged. The most important reason for the decreased activity was diffusion limitations. The diffusion of the substrate and products became more pronounced, and lipolytic activity increased upon addition of n-hexane into the reaction medium. The storage and operational stabilities of the immobilized lipase were investigated, and both characteristics were found to be increased when compared to the free enzyme. Furthermore, mechanical or magnetic stirring during the operation were found to have no influence on the carrier-matrix as determined by nephelometric measurements.     

Production of citronellyl acetate in a fed-batch system using immobilized lipase

Several reports exist in the literature citing the decrease in conversion rates of organic-phase catalytic synthesis reactions when acetic acid is present as a reaction component. This inhibition is thought to result from damage to either the hydration layer-protein interaction or the overall enzyme structure. In this work, the inhibitory effect of acetic acid on lipase enzyme activity was ameliorated by conducting syntheses under acetic acid-limiting conditions in a fed-batch system, resulting in higher product yields. Periodic additions of acetic acid at levels of 40 mM or less gave maximum yields of 65% conversion for the reaction of citronellol and acetic acid to form citronellyl acetate. The enzyme used was a fungal lipase fromMucor miehei, and was immobilized on macroporous synthetic resin (a Novo lipozyme Novo Nordisk, Denmark). These results represent a fourfold improvement over batch runs reported in the literature for direct esterification of terpene alcohol with acetic acid using lipozyme as a catalytic agent.     

Studies on the immobilization of glucuronidase (part 1)

beta-Glucuronidase (EC 3.2.1.31) was immobilized on various organic and inorganic carriers by different methods. Optimum coupling conditions have been worked out. The immobilization were characterized and compared to each other. Parameters resulting in most stable preparations with high activities are discussed.     

Simultaneous saccharification and fermentation of lignocellulose

Simultaneous saccharification and fermentation (SSF) processes for producing ethanol from lignocellulose are capable of improved hydrolysis rates, yields, and product concentrations compared to separate hydrolysis and fermentation (SHF) systems, because the continuous removal of the sugars by the yeasts reduces the end-product inhibition of the enzyme complex. Recent experiments using Genencor 150L cellulase and mixed yeast cultures have produced yields and concentrations of ethanol from cellulose of 80% and 4.5%, respectively. The mixed culture was employed because B.clausenii has the ability to ferment cellobiose (further reducing end-product inhibition), while the brewing yeastS. cerevisiae provides a robust ability to ferment the monomeric sugars. These experimental results are combined with a process model to evaluate the economics of the process and to investigate the effect of alternative processes, conditions, and organisms.     

Some engineering parameters for Propionic acid fermentation coupled with Ultrafiltration

The effect of circulation rate on permeate flux, the energy requirements for heating or cooling, the reactor homogeneity, and cell activity are discussed for a continuous culture system with cell recycle. The fermentation system was a continuous stirred-tank reactor with an ultrafiltration membrane unit for cell recycle. The membranes have a tubular configuration and are composed of a carbon support coated with zirconium oxide. The permeate flux obtained for a long-run fermentation with Propionibacterium acidi-propionici was higher when the circulation rate was increased: 5.13 L/m²-h for a circulation rate of 0.810 m³/h and 7.09 L/m²-h for 1.104 m³/h. The temperature rise inside the fermenter for different circulation rates was studied, allowing determination of the need of input or output of energy for temperature control. Residence time distribution studies showed that, with a circulation rate of 0.606 m³/h, the dead volume was 9.2%, whereas at 1.104 m³/h the reactor behavior was almost ideal. The influence of the circulation rate on loss of cell activity is also discussed, and rheological studies are suggested as an indirect indicator of cell viability. We hereby express our recognition for the ongoing collaborations with James Gaddy (University of Arkansas) and Gerard Goma (INSA, Toulouse, France).     

Purification and partial characterization of two lectin isoforms fromCratylia mollis mart. (camaratu bean)

Two additional electrophoretically distinct molecular forms, isoforms (iso) 2 and 3, with lectin properties were isolated fromCratylia mollis Mart, seeds (FABACEAE), by extraction with 0.15M NaCl and ammonium sulfate fractionation, followed by chromatography on Sephadex G-75 and Bio-Gel P-200 (iso 2), as well as CM-Cellulose and Sephadex G-75 (iso 3). Both isoforms were human group nonspecific and showed distinct specificity. Polyacrylamide gel electrophoresis resolved iso 2 and 3 in polypeptides of apparent mol wts 60 and 31 kDa, respectively; a distinct isoelectric focusing pattern was obtained for iso 2 and 3, under denaturing and reducing conditions.     

Thermal stability and energy of deactivation of free and immobilized amyloglucosidase in the saccharification of liquefied cassava starch

Amyloglucosidase from Novo (Copenhagen, Denmark) was immobilized in controlled pore silica particles with the silane-glutaraldehyde covalent method. Thermal stability of the free and immobilized enzyme (IE) was determined with 30% (w/v) α-amylase liquefied cassava starch, pH 4.5, temperatures from 35 to 75°C. Free amyloglucosidase maintained its activity practically constant for 240 min and temperatures up to 50°C. The IE has shown higher stability retaining its activity for the same period up to 60°C. Half-life for free enzyme was 20.6, 6.44, 2.07, 0.69, and 0.24 h for 55, 60, 65, 70, and 75°C, respectively, whereas the IE at the same temperatures had half-lives of 116.4, 30.88, 8.52, 2.44, and 0.73 h. The energy of thermal deactivation was thus 50.6 and 57.6 kcal/mol, respectively for the free and IE, confirming stabilization by immobilization.     

Evaluation of mass-transfer and kinetic parameters forRhodospirillum rubrum in a continuous stirred tank reactor

The photosynthetic bacteriumRhodospirillum rubrum has been evaluated for its ability to produce hydrogen from carbon monoxide and water in a continuous stirred tank reactor according to the watergas shift reaction. An assessment of mass-transfer parameters and reaction kinetics was made for this sparingly soluble substrate system. Experiments were conducted in a nonsteady-state fashion with continuous liquid and gas flow, which allowed for separation of the mass-transfer and kinetic-limited regions. Based on the data obtained, mass-transfer coefficients for the system were determined, and a mathematical expression for the reaction kinetics was formulated. The results showed that the hydrogen production was inhibited by elevated levels of dissolved carbon monoxide in the liquid.     

Tributyl phosphate degradation by immobilized cells of aCitrobacter sp.

Tributyl phosphate (TBP), a plasticizer and solvent, is used in nuclear fuel reprocessing, generating TBP wastes laden with residual uranium. ACitrobacter sp. accumulated heavy metals via a phosphohydrolase(s) that precipitated metals with inorganic phosphate liberated from an organic phosphate “donor” molecule (TBP). Mutant analysis suggested that TBP hydrolysis was not attributable to a previously documented acid phosphatase (monoesterase). Purified monoesterase had little activity against phospho di- and triesters, had no requirement for Mg²⁺ or Mn²⁺, and was EDTA-resistant. Conversely, TBP cleavage by immobilized cells was enhanced by Mg²⁺, and ininhibited by Mn²⁺ and EDTA. A separate phosphotri/diesterase was implicated.     

L-malic acid production using immobilized saccharomyces cerevisiae

L-Malate was produced from fumarate by using immobilized Saccharomyces cerevisiae cells entrapped in polyacrylamide. This preparation performed better when pretreated with malonate. Under the experimental conditions described here, succinate was not detected as a by-product of the reaction, as had been reported for other microorganisms.     

Characterization of F VIII concentrates produced by two methods incorporating double virus inactivation

The trend toward the production of high purity factor VIII concentrates for clinical use is still in progress. Although all plasma derivatives must undergo viral inactivation procedures, the possibility of transmission of viral diseases is not completely eliminated. In order to reduce such risk, we have included double virus inactivation in the procedure of factor VIII concentrate production. In a scale-up procedure for isolation of factor VIII from cryoprecipitate, two methods were used. The first is based on the chromatographic purification of factor VIII after pasteurization of cryoprecipitate solution and solvent/detergent (S/D) inactivation of viruses. The second is based on multistep precipitation of factor VIII by sodium chloride and glycine. Viral inactivation was performed by combination of S/D treatment and heating of final freeze-dried product 30 min at 100°C. The typical yield of factor VIII activity in the freeze-dried product was about 20% for the first method, and 25–30% for the second. Electrophoretic analyses of both factor VIII preparations by SDS-PAGE and IEF show very low content of contaminant proteins, in accordance with observed 400–650-fold increase of their specific activity over plasma. Both factor VIII products were stable in the liquid state for more than 24 h at room temperature. The final products, after double viral inactivation, are considered to be suitable for clinical evaluations.     

Mode of action and synergism of cellulases fromPenicillium funiculosum

A 1,4-β-d-glucan cellobiohydrolase (EC 3.2.1.91) and l,4-β-d-glucan glucanohydrolase (EC 3.2.1.4) were purified from the culture filtrates ofPenicillium funiculosum by using preparative isoelectric focusing. Both the enzymes were homogeneous on polyacrylamide gel with and without sodium dodecyl sulphate. The mol wt of the cellobiohydrolase and endoglucanase were 14,400 and 25,000 respectively. The purified enzymes were free of β-glucosidase activity. Acting in isolation, the cellobiohydrolase had little capacity for solubilizing Avicel or Walseth cellulose, but showed increased rates of hydrolysis when combined with endoglucanase. Cellobiose inhibition (50%) was observed in the initial rate of the hydrolysis of Walseth cellulose. It was also observed that cellobiohydrolase initiates the attack on crystalline cellulose. † NCL communication no. 3898.     

A kinetic study on the bioremediation of sodium cyanide and acetonitrile by free and immobilized cells ofPseudomonas putida

Pseudomonas putida capable of utilizing organic nitrile (acetonitrile) and inorganic cyanide (sodium cyanide) as the sole source of carbon and nitrogen was isolated from contaminated industrial sites and waste water. The bacterium possesses nitrile aminohydrolase (EC 3.5.5.1) and amidase (EC 3.5.1.4), which are involved in the transformation of cyanides and nitriles into ammonia and CO₂ through the formation of amide as an intermediate. Both of the enzymes have a high selectivity and affinity toward the⁻Cn group. The rate of degradation of aceotnitrile and sodium cyanide to ammonia and CO₂ by the calcium-alginate immobilized cells ofP. putida was studied. The rate of reaction during the biodegradation of acetonitrile and sodium cyanide, and the substrate- and product-dependent kinetics of these toxic compounds were studied using free and immobilized cells ofP. putida and modeled using a simple Michaelis-Menten equation.     

Kinetic Investigation and Mathematical Modeling of Methanogenesis of Glucose

The kinetic regularities of anaerobic conversion of glucose, and intermediates of its decomposition (ethanol, butyrate, and acetate) by a microbial methanogenic association from anaerobic digester were investigated. Kinetic scheme for conversion of glucose is suggested, and the mathematical model based on the scheme is evolved. The model includes growth and metabolism of three kinds of microorganisms—acid producents, and acetate- and hydrogen-utilizing methane producents; of cell lysis with consequent fermentation of “died biomass” to acetate, hydrogen, and carbon dioxide; of induction and repression of the enzyme responsible for decomposition of butyrate, and for a number of regulations depending on the concentrations of intermediates in glucose metabolism. The values of parameters of the model have been calculated, sufficiently describing the experimental regularities. The numerical experiments have enabled us to reveal and describe the principal regulating factors of glucose methanogenesis.     

Comparison between experimental and theoretical values of effectiveness factor in cephalosporin C production process with immobilized cells

Cells ofCephalosporium acretnonium ATCC 48272 immobilized in calcium alginate beads were utilized for cephalosporin C production and the results were compared with those obtained with free cells. The experiments were performed with synthetic medium containing glucose and sucrose as carbon and energy sources. Experimental effectiveness factor values were obtained at various cell and dissolved-oxygen concentrations, considering Monod kinetics for the respiration rate, and were compared with the values calculated with zero-order kinetics in spherical bioparticle. The results showed that the assumption of oxygen limitation by diffusion in the bioparticle was correct, and that cephalosporin C production with immobilized cells is perfectly viable, although a slightly lower rate than that obtained in the free cell process was observed.     

Purification and characterization of lamb pregastric lipase

Lamb pregastric lipase was purified from a commercial source using delipidation, solubilization with KSCN, acid-precipitation, pepsin-digestion, affinity chromatography with agarose-Cibacron Blue F3GA, gel filtration, and elution from a native 10% (w/v) polyacrylamide gel. The enzyme had a single subunit of 68,000 Da with maximum esterase activity when measured at pH 6.0 and 30 degrees C. The enzyme preferentially hydrolyzed short- and medium-chain (C4, C6, and C8) synthetic esters and short-chain (C4 and C6) monoacid triglycerides. The NH2-terminal sequence demonstrated high homology with gastric and lingual lipases.     

Synthesis of Cyclodextrin Glucosyl Transferase byBacillus cereus for the production of cyclodextrins

A potent indigenous bacillus isolate identified asBacillus cereus (RJ-30) was found to produce Cyclodextrin Glucosyl Transferase (CGTase) extracellularly. Process optimization of various fermentation parameters has been established for optimal growth of bacillus and the maximum enzyme synthesis. The organism had the highest specific growth rate (0.7μ) with a generation time of 1 h in glucose containing medium at the conditions of pH 7.0, 37°C at 300 rpm, 1.5 vvm of agitation, and aeration. At these conditions, it exhibited the maximum activity of 54 U/mL at the synthesis rate of 2.7 U/L/h. CGTase was produced from the early exponential growth and peaked during the midsporulating stage of about 16 h thereafter maintained at the same level of 50 U/mL. Saccharides containing media were better inducers than starch, and the influence of carbohydrate substrates has shown that enzyme synthesis is promoted by xylose (65 U/mL) and, more remarkably, by the supplementation of wheat bran extract in glucose medium (106 U/mL). This organism produced CGTase stably in a chemostat culturing over a period of 400 h with a maximum productivity of 5.4 kU/L/h (threefold higher than obtained in batch culturing [1.75 kU/L/h]). Comparatively, CGTase was produced by immobilized cells in a continuous fluidized bed reactor for over approx 360 h, at a relatively high dilution rate of 0.88 h⁻¹ resulting in the productivity of 23.0 kU/L/h.     

Production of fumaric acid with immobilized biocatalysts

Immobilization ofRhizopus arrhizus mycelium improved fumaric acid production. The optimum conditions for fumaric acid production with immobilized cells were investigated using a statistical experimental design. Substrate concentration, carbon:nitrogen ratio, and residence time were chosen as independent variables. In the repeated batch shake flask fermentation, the fumaric acid yield from xylose was as much as 3.5 times higher with immobilized mycelium than with free mycelium. Polyurethane foam cubes, in this case, gave better results than nylon net cubes as a carrier.     

A new kinetic approach to the fermentation of multisubstrate complex media

Ethanol production from natural complex media has been studied in this work. A new kinetic approach is presented for two-substrate media, such as hardwood hemicellulose hydrolysate, which predominantly consists of a mixture of xylose and glucose. It has been founded on the supposition that the whole ethanol production and biomass growth can be subdivided into two separated components imputable to glucose and xylose consumptions, respectively. A model describing the continuous fermentation in CSTR byPachysolen tannophilus has been also presented, and experimentally verified; it takes into account the different substrate consumption rates of these sugars contained in both natural and synthetic complex media.