Kinetics and model development for enzymatic synthesis of fructo-oligosaccharides using fructosyltransferase.

Experimental investigations were made to synthesize fructo-oligosaccharides (FOS) from sucrose using fructosyltransferase. The influence of various parameters such as temperature (45-55 °C), pH (4-5), initial sucrose concentration (ISC: 300-500 g/L) and enzyme concentration (4-32 U/mL) were varied. A maximum FOS yield of 60% was observed at ISC 500 g/L, pH 4.5 with enzyme activity 32 U/mL and at 55 °C. It was confirmed that 1-kestose (tri-) was the major product of FOS as compared to nystose (tetra-) and fructosylnystose (penta-saccharides). Further, the reaction rate increases with increase in temperature. From separate sets of experiments, it was observed that FOS formation was affected by glucose inhibition. Apart from the increase in the rate of FOS formation with increasing enzyme activity, the final values of FOS yield increase though till 16 U/mL and thereafter attain plateau. A kinetic model was also developed, based on Michaelis-Menten kinetics, and a five-step ten-parameter model, including glucose inhibition, was obtained. Model was solved using COPASI(®) (version 4.8) solver for kinetic parameter estimations followed by time course simulations.

Kinetics of sucrose conversion to fructo-oligosaccharides using enzyme (invertase) under free condition.

The study reports the synthesis of fructo-oligosaccharide (FOS) from sucrose using invertase derived from Saccharomyces cerevisiae. The reaction was conducted in a batch mode under free enzyme condition. Fructo-oligosaccharide formation was detected at a high sucrose concentration of over 200 g/L. The investigation was extended to study the effect of different parameters such as initial sucrose concentration (ISC), pH, and enzyme concentration. A maximum FOS yield of 10 % (dry basis) was observed using 525 g/L of ISC, with 6 U/mL of the enzyme, and pH 5.5 at 40 °C. 1-Kestose was the major product of among different forms of FOS. The FOS yield increased with an increase in sucrose concentration up to 525 g/L, beyond which it started to decrease. However, the maximum FOS yield was not affected by the increasing concentration of the enzyme beyond a certain level (2 U/mL). Furthermore, the activity of enzyme slightly increased with an increase in the pH up to 6, and thereafter it declined. Addition of glucose decreased the FOS yield because of enzyme inhibition. A five-step, ten-parameter model was developed, for which the simulation was performed in COPASI. The results predicted by the model were consistent with the experimental data.

Simulation studies of ammonia removal from water in a membrane contactor under liquid-liquid extraction mode.

Simulation studies were carried out, in an unsteady state, for the removal of ammonia from water via a membrane contactor. The contactor had an aqueous solution of NH(3) in the lumen and sulphuric acid in the shell side. The model equations were developed considering radial and axial diffusion and convection in the lumen. The partial differential equations were converted by the finite difference technique into a series of stiff ordinary differential equations w.r.t. time and solved using MATLAB. Excellent agreement was observed between the simulation results and experimental data (from the literature) for a contactor of 75 fibres. Excellent agreement was also observed between the simulation results and laboratory-generated data from a contactor containing 10,200 fibres. Our model is more suitable than the plug-flow model for designing the operation of the membrane contactor. The plug-flow model over-predicts the fractional removal of ammonia and was observed to be limited when designing longer contactors.

Kinetic studies and model development for the formation of galacto-oligosaccharides from lactose using synthesized thermo-responsive bioconjugate.

In an earlier study by us [47], thermo-responsive bioconjugate (poly-N-isopropylacrylamide-ß-galactosidase) was synthesized and characterized. This study utilizes the prowess of such smart bioconjugate for the enzymatic synthesis of galacto-oligosaccharides (GOS) from lactose at various initial lactose concentrations (ILC), enzyme concentrations, and temperatures, while maintaining a constant pH of 6. A maximum GOS yield of 35% (on dry basis) was observed at 100g/L ILC and 0.275mg/mL (0.055U/mL) conjugated protein. The GOS yield remained approximately the same for 50 and 100g/L ILC, beyond which, it decreased. As the enzyme concentration increased, the equilibrium formation of GOS increased and eventually attained a plateau when the concentration of conjugated protein exceeded 0.275mg/mL (0.055U/mL). GOS yield increased on raising the temperature from 30 to 40°C, and declined thereafter. The apparent kinetic parameters were estimated from a five-step, nine-parameter kinetic model, which was then simulated using the COPASI package. The simulated results demonstrated an excellent match with the experimental data.

Synthesis and characterization of thermo-responsive poly-N-isopropylacrylamide bioconjugates for application in the formation of galacto-oligosaccharides.

The study demonstrates the properties of conjugation of ß-galactosidase with a thermo-responsive polymer, poly-N-isopropylacrylamide (PNIPAAm) in comparison to a non-responsive polymer, poly-acrylamide (PAAm). The maximum formation of bioconjugate (PNIPAAm-ß-galactosidase) was 75% (yield) with 50% chemically modified enzyme (using itaconic anhydride). The process of bioconjugation (bioconjugate concentration: 7.4%) decreases lower critical solution temperature from 32.5 °C (with pure PNIPAAm) to 26.5 °C. The effect of temperature on the activities of PNIPAAm-ß-galactosidase, PAAm-ß-galactosidase and native enzyme was also compared. At 70 °C, the maximum activity was observed for PNIPAAm-ß-galactosidase while for others it was at 60 °C. However, the effect of pH was insignificant on activities of both the bioconjugates than the native enzyme. The addition of ethylene glycol (20%, v/v) enhances the activity (by 45%) of PNIPAAm-ß-galactosidase with no loss in stability; however; the trend is reversed with the addition of ethanol. Further, employing bioconjugates even up to 24 cycles of precipitation (at 40 °C) followed by re-dissolution (4 °C) around 90% of activity could be retained by PNIPAAm-ß-galactosidase. The PNIPAAm-ß-galactosidase also showed much-improved thermal and storage stabilities. A lower Michaelis-Menten constant (Km) was estimated with the PNIPAAm-ß-galactosidase than the native enzyme as well as PAAm-ß-galactosidase. Finally, PNIPAAm-ß-galactosidase was tested to synthesize galacto-oligosaccharides from lactose solution.

Kinetics of lactose conversion to galacto-oligosaccharides by ß-galactosidase immobilized on PVDF membrane.

Experimental studies were made for immobilization of enzymes on microporous polyvinylidene fluoride (PVDF) membrane in order to carry out enzymatic reaction of lactose into galacto-oligosaccharides using ß-galactosidase. The present work, however, is the first part in the direction of enzymatic membrane reactor studies for carrying out reaction followed by membrane based separation to purify galacto-oligosaccharides out of reaction mixture. The middle of the three compartment cell, separated by two immobilized (enzyme) membranes, was utilized to feed lactose solution; whereas, adjacent compartments were filled with distilled water. The reacted mixture solution was analyzed for tri-, tetra- and penta-forms of GOS. The formation of product GOS strongly depended on varying amounts of initial lactose concentration (ILC). Total GOS formation increased from 7% to 28% for ILC from 50 to 200 g/L. However, tri-saccharide was the major (67%) in comparison to tetra (27%) and penta (6%) forms of GOS. Further, based on Michaelis-Menten kinetics, a six-step-eleven-parameter model was developed. The model incorporated enzyme inhibition and formation of glucose and galactose separately. Simulated results from developed model matched exceeding well with experimental results.

Kinetics and design relation for enzymatic conversion of lactose into galacto-oligosaccharides using commercial grade ß-galactosidase.

The enzymatic synthesis of galacto-oligosaccharides (GOS) from lactose was studied using commercial grade ß-galactosidase (Biolacta FN5) from Bacillus circulans. The reaction was carried out under free enzyme condition varying initial lactose concentration (ILC: 55-525 g/L), enzyme concentration (0.05-1.575 g/L), temperature (30-50°C) and pH (5.0-6.0). Reaction mixture compositions were analyzed utilizing high performance liquid chromatography (HPLC). A maximum GOS formation of 39% (dry basis) was achieved at an ILC of 525 g/L converting 60% of the lactose fed. Tri-saccharides were the major types of GOS formed, accounting approximately 24%; whereas, tetra-saccharides and penta-saccharides account approximately 12% and 3%, respectively. Design correlation was developed in order to observe the quantitative effect of operating parameters on GOS yield. Further, based on Michaelis-Menten model, four-step reaction pathways were considered for simplistic understanding of the kinetics. Apart from predicting the reaction mixture composition, the approach also provided kinetic parameters though simulation using COPASI 4.7®. Excellent agreements were observed between simulated and experimental results.

Pervaporation from a dense membrane: roles of permeant-membrane interactions, Kelvin effect, and membrane swelling.

Dense polymeric membranes with extremely small pores in the form of free volume are used widely in the pervaporative separation of liquid mixtures. The membrane permeation of a component followed by its vaporization on the opposite face is governed by the solubility and downstream pressure. We measured the evaporative flux of pure methanol and 2-propanol using dense membranes with different free volumes and different affinities (wettabilities and solubilities) for the permeant. Interestingly, the evaporative flux for different membranes vanished substantially (10-75%) below the equilibrium vapor pressure in the bulk. The discrepancy was larger for a smaller pore size and for more wettable membranes (higher positive spreading coefficients). This observation, which cannot be explained by the existing (mostly solution-diffusion type) models ofpervaporation, suggests an important role for the membrane-permeant interactions in nanopores that can lower the equilibrium vapor pressure. The pore sizes, as estimated from the positron annihilation, ranged from 0.2 to 0.6 nm for the dry membranes. Solubilities of methanol in different composite membranes were estimated from the Flory-Huggins theory. The interaction parameter was obtained from the surface properties measured by the contact angle goniometry in conjunction with the acid-base theory of polar surface interactions. For the membranes examined, the increase in the "wet" pore volume due to membrane swelling correlates almost linearly with the solubility of methanol in these membranes. Indeed, the observations are found to be consistent with the lowering of the equilibrium vapor pressure on the basis of the Kelvin equation. Thus, a higher solubility or selectivity of a membrane also implies stronger permeant-membrane interactions and a greater retention of the permeant by the membrane, thus decreasing its evaporative flux. This observation has important implications for the interpretation of existing experiments and in the separation of liquid mixtures by pervaporation.