F. Maugeri

Optimization of extracellular lipase production by Geotrichum sp. using factorial design

Response surface methodology was employed to study the effects of carbon source (soy oil, olive oil and glucose) and nitrogen source concentrations (corn steep liquor and NH(4)NO(3)) on the lipase production by Geotrichum sp. The experiment included a 2(4) central composite rotatable design (CCRD) and four others 2(3) CCRD. According to the responses from the experimental designs, the effects of each variable were calculated and the interactions between them were determined. The response surface methodology was applied for the optimization of the nutrient concentrations in the culture medium for the enzyme production, at 30 degrees C. The optimum medium composition for lipase production by Geotrichum sp. was ammonium nitrate 2.1-2.5%, corn steep liquor 13-15% and soy oil 0.6% as carbon source, which lead to a lipase activity of about 20 U/ml. Using olive oil as carbon source, the optimum composition was ammonium nitrate 0.8-1%, corn steep liquor 13-15% and olive oil 0.6%, leading to an activity of 17 U/ml.

Production of lipase from Geotrichum sp and adsorption studies on affinity resin

There is a growing interest in microbial lipase production due to its great potential for industrial applications such as food additives, industrial reagents and stain removers, as well as for medical applications. Specially for medical applications a high degree of purity is required, which is accomplished with high resolution chromatographic techniques. Affinity chromatography is considered a very high resolution chromatographic technique. In this work the adsorption isotherms and kinetics of the adsorption of lipase from Geotrichum sp on biospecific resin were determined. The resin was prepared using EAH sepharose 4B gel (Pharmacia), made to react with oleic acid as the specific ligand.The lipase was produced in a five-liter fermenter, with both complex and synthetic media. Fermentation conditions were a temperature of 30°C, an aeration of 1VVM and an agitation of 400 rpm. Maximum lipase activity was around 28 U/ml after 10 hours of fermentation for the complex medium. The kinetic model and parameters were determined by dynamic fitting to experimental results using the fourth-order Runge-Kutta method.

In vitro synthesis of oligosaccharides by acceptor reaction of dextransucrase from Leuconostoc mesenteroides

Glucose was used as acceptor to obtain small chain oligossaccharides from sucrose using dextransucrase from Leuconostoc mesenteroides NRRL B-512F. Better conditions for the synthesis of the oligosaccharides were obtained using experimental design and response surface methodology. Yield of oligosaccharides was increased from 5% to 45% following an increase in both sucrose and glucose/sucrose concentrations, from 58 g/l to 142 g/l and from 0.02 to 0.18, respectively. Molecular weight increased from 2800 to 4500 daltons with a temperature shifting from 10°C to 30°C.

Thermal stability of the immobilized fructosyltransferase from Rhodotorula sp

The thermal stability of the extracellular fructosyltransferase (FTase) from Rhodotorula sp., recovered from cultivation medium by ethanol precipitation and immobilized onto niobium ore, was studied by Arrhenius plot, half - life profile, half - inactivation temperature (T50) and thermodynamic parameters. The Arrhenius plot showed two different behaviors with different deactivation energies (Ead) only after immobilization, the transition occurring in the temperature interval between 51 and 52oC. T50 for the free enzyme was estimated to be around 62oC and, after immobilization, 66oC. After 15 minutes at 52oC, it was also possible to observe enzymatic activation for both the free and immobilized forms, but greater activation was achieved at pH 4.5 with the immobilized enzyme. Between 47 - 51oC the immobilized enzyme was more stable than the free enzyme, with pH 6.0 being the more stable condition for the immobilized enzyme. However, above 52oC the free form was more stable.

Comparative studies of the stability of free and immobilized inulinase from Kluyveromyces marxianus NRRL Y-7571 in aqueous-organic solutions

Abstract - Enzymes have been extensively used in organic solvents to catalyze a variety of reactions of biological and industrial significance. In this work, the characteristics of free and immobilized inulinase were investigated in buffered solutions of butyl acetate. The influences of the organic solvent content on the optimal temperature and pH, the stabilities to temperature and pH and the kinetic parameters were systematically evaluated. The results showed that the organic solvent content had no effect on the optimal pH, either in the free or immobilized inulinase. For the immobilized enzyme, the optimal temperatures ranged from 55°C to 60°C, depending on the content of butyl acetate. At higher butyl acetate content, the stability of the immobilized enzyme increased for both pH and temperature. The organic solvent showed the tendency to increase the values of the kinetic parameters K m and v max for both free and immobilized inulinase. Keywords : Organic solvent; Inulinase; Stability; Kinetic parameters.

Optimization of dextran syntesis and acidic hydrolisis by surface response analysis

The influence of some variables in the in vitro synthesis of dextran by dextransucrase from Leusconostoc mesenteroides NRRL B512F, as well as in the acidic hydrolysis of the dextran produced, were studied in order to maximize the production of clinical dextran (dextran 70 and dextran 40). The experiments were conducted using a factorial design and surface response analysis.

The Control Problem for Protein Separation by the Continuous Affinity Recycling Extraction Process

Process modelling and simulation are important tools for better knowledge of systems, narrowing the ranges of experiments and leading to lower costs of process implementation. Both recovery yield and productivity in biochemical processes have been improved by genetic manipulation of microorganisms and by new techniques leading to better process control. It is important for the process economics to maximize yields, while maintaining high recovery efficiency and productivity. In this work, the dynamic modelling of the Continuous Affinity Recycling Extraction (CARE) process was carried out and its performance was studied against different disturbances. The parametric analysis and parameters sensibility studies were performed considering the steady state behavior. The appropriate manipulated variable was determined from the dynamic responses of the system, so that the control structure could be defined. The classical PI and PID feed back controls strategies were studied and both of them showed good performance when a time-delay of 8 minutes in the on-line analysis of the control variable was considered The non-conventional feedback feedforward control strategy was also studied and the results showed that the performance was good with time delays up to 24 minutes. In particular, this work showed that the CARE process under control can be a very attractive way to achieve continuous protein separation in industrial environments.

DYNAMIC MODELLING AND ADVANCED PREDICTIVE CONTROL OF A CONTINUOUS PROCESS OF ENZYME PURIFICATION

A dynamic mathematical model, simulation and computer control of a Continuous Affinity Recycle Extraction (CARE) process, a protein purification technique based on protein adsorption on solid-phase adsorbents is described in this work. This process, consisting of three reactors, is a multivariable process with considerable time delay in the on-line analyses of the controlled variable. An advanced predictive control configuration, specifically the Dynamic Matrix Control (DMC), was applied. The DMC algorithm was applied in process schemes where the aim was to maintain constant the enzyme concentration in the outlet of the third reactor. The performance of the DMC controller was analyzed in the feed-flow disturbances and the results are presented.