Resumo em Inglês:

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.

Resumo em Inglês:

Penicillium restrictum was identified as a promising strain for lipase production due to enzyme production yield and thermal stability of the enzyme. This work presents results of lipase purification and enzyme stability versus pH. Ultrafiltration and precipitation with ammonium sulphate were used as initial purification steps. The partially purified enzyme preparation showed an increase in stability as pH increased. The crude enzymatic preparation was assayed with different oils and tributirin and showed a major catalytic activity for triglycerides of medium/long-chain fatty acids. Further purification steps were conducted on an analytical scale. The initial attempt to use gel filtration was abandoned as lipase lost its stability after this chromatographic procedure. The fast ion-exchange chromatography was performed on a Mono Q column, and two peaks with lipolytic activity were isolated and analysed by electrophoresis.

Resumo em Inglês:

Two adsorption techniques were studied as potential methods for the recovery of aprotinin: a biospecific adsorption (using immobilized trypsin and chymotrypsin as ligants) and a pseudobiospecific adsorption of aprotinin-trypsin complex onto IMAC matrix. These studies indicated that ionic strength has an important effect on aprotinin adsorption on immobilized trypsin, and the pH was the most significant variable in aprotinin desorption from both immobilized enzymes. The aprotinin-trypsin complex adsorption onto the IMAC matrix was not as significantly affected by changes in pH as it was for changes in ionic strength. For the desorption step, both variables significantly affected the complex recovery. However, the effect of ionic strength was markedly stronger for this desorption step.

Resumo em Inglês:

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.

Resumo em Inglês:

Penicillin G acylase is the second most important enzyme used by industry in an immobilized form. Penicillin hydrolysis is its main application. This reaction is used to produce 6-aminopenicillanic acid (6-APA), an intermediate in the synthesis of semisynthetic antibiotics. This work aims to compare catalytic properties of different penicillin G acylase (PGA) derivatives obtained by multipoint immobilization of the enzyme on macroporous silica. Enzyme amino groups react with different aldehyde groups produced in the support using either glutaraldehyde or glyoxyl activation. In the former method, silica reacts with <FONT FACE="Symbol">g</FONT>-aminopropyltriethoxysilane (<FONT FACE="Symbol">g</FONT>-APTS) and glutaraldehyde; in the latter, a reaction with glycidoxypropyltrimethoxysilane (GPTMS) is followed by acid hydrolysis and oxidation using sodium periodate. This work determines the influence of degree of activation, using glutaraldehyde, on immobilization parameters. PGA was immobilized on these two different supports. Maximum enzyme load, immobilized enzyme activity (derivative activity), rate of immobilization and thermal stability were checked for both cases. For glutaraldehyde activation, the results showed that 0.5% of the <FONT FACE="Symbol">g</FONT>-APTS is sufficient for all the hydroxyl groups in the silica to react. They also showed that degree of activation only affects immobilization yield and reaction velocity and that reduction of the glutaraldehyde derivatives with sodium borohydride does not affect their thermal stability. In comparing the derivatives obtained using glyoxyl and glutaraldehyde activation, it was observed that the glyoxyl derivatives presented better immobilization parameters, with a maximum enzyme load of 264 IU/g silica and a half-life of 20 minutes at 60 °C.

Resumo em Inglês:

In this work a kinetic model for the enzymatic conversion of sucrose to glucose and fructose using the free form of the enzyme invertase was studied. The initial rates of conversion of sucrose into glucose and fructose were evaluated for different initial concentrations of sucrose, fructose and glucose. The results of the ?-square test showed that the partially non competitive inhibition model for glucose and for fructose can be used to describe the reaction kinetics with a degree of confidence higher than 99.9%. An analysis of the sum of squares of the residuals for several inhibition models cited in the literature showed that the partially noncompetitive inhibition model is the best one for quantifying the effect of the concentration of glucose and fructose on sucrose hydrolysis. A comparison of a simulation of this model with the experimental results showed that the model can describe well the reaction in a batch reactor using initial concentrations of 600 g/L.

Resumo em Inglês:

In many continuous fermentation processes, the control objective is to maximize productivity per unit time. The optimum operational point in the steady state can be obtained by maximizing the productivity rate using feed substrate concentration as the independent variable with the equations of the static model as constraints. In the present study, three model-based control schemes have been developed and implemented for a continuous fermenter. The first method modifies the well-known dynamic matrix control (DMC) algorithm by making it adaptive. The other two use nonlinear model predictive control algorithms (NMPC, nonlinear model predictive control) for calculation of control actions. The NMPC1 algorithm, which uses orthogonal collocation in finite elements, acted similar to NMPC2, which uses equidistant collocation. These algorithms are compared with DMC. The results obtained show the good performance of nonlinear algorithms.

Resumo em Inglês:

Lignin peroxidase (LiP) production has been extensively studied due to the potential use of this enzyme in environmental pollution control. Important aspects of the production of the enzyme by S. viridosporus T7A which have been studied include the improvement of yield and enzyme stabilization. In experiments performed in agitated flasks containing culture media composed of yeast extract as the source of nitrogen, mineral salts and different carbon sources, the use of glucose resulted in the highest values for LiP activity (350 U/L), specific LiP activity (450 U/g) and productivity (7 U/L/h). As the profile obtained with glucose-containing medium suggested enzyme instability, the effect of calcium carbonate was evaluated. The addition of CaCO3 in two different concentrations, 0.5% and 5.0%, resulted in higher values of maximum LiP activity, 600 and 900 U/L, respectively. The presence of this salt also anticipated enzyme activity peaks and allowed the detection of higher enzyme activities in the extracellular medium for longer periods of time. These results indicate a positive effect of calcium carbonate on LiP production, which is extremely relevant for industrial processes.

Resumo em Inglês:

The optimization problem of a fed-batch alcoholic fermentation by Zymomonas mobilis, solved using Pontryagin's maximum principle and the singular control theory, is used to discuss the main challenges that usually arise during the implementation of optimal strategies. For each problem a practical solution is proposed. In order to solve the optimization problem, a hybrid neural model is developed using experimental data from batch and fed-batch fermentations. The calculated optimal solution is experimentally implemented in open-loop scheme.

Resumo em Inglês:

This paper reports on the use of the gas balance and dynamic methods to obtain an estimate of the volumetric oxygen transfer coefficient (kLa) in a conventional reactor during the growth phase of the microorganism Cephalosporium acremonium. A new way of calculating kLa by the dynamic method employing an electrode with a slow response, is proposed. The calculated values of kLa were used in the training of a feedforward neural network, for which the inputs were the parameter measurements of the related variables. The neural network technique proved effective, predicting values of kLa accurately from input data not used during the training phase. In contrast, the gas balance method was shown to be less useful. This could be attributed to the poor data obtained with the apparatus used to measure the oxygen in the exhaust gas, explained by the low rate of oxygen consumption by the microorganism.

Resumo em Inglês:

A culture medium for a wild strain of Bacillus cereus was developed for chitosanase production by using an experimental design. The factors having the strongest influence on chitosanase production were ammonium sulfate concentration, aeration, pH and the interaction between the first two parameters. Optimal conditions for chitosan hydrolysis were pH 5.8 and 54 oC; however, hydrolysis activity drastically decreased at pH 7.0. The enzyme was purified (single-electrophoretic band) by partitioning in an aqueous two-phase system (ATPS), followed by cation-exchange chromatography with a 66% yield. Chitosanase was mainly collected in the top phase (K = 129) of a 22% PEG 1,500, 13% phosphate (pH = 5.8) and 12% NaCl (w/w) solution, and the main protein contaminants were evenly distributed between the phases (K = 1.07). The apparent molecular weight and the isoelectric point of the chitosanase, determined by SDS-PAGE electrophoresis and by isoelectric focalization, were 47 kDa and 8.8, respectively.

Resumo em Inglês:

In this study, a preparative size-exclusion chromatography of two different clarified media obtained from submerse and semisolid culture of the mold Aspergillus awamori was carried out. Characterization and comparison of the quantities of glucoamylase and contaminant proteins present in these media were possible. Glucoamylase is the protein with the higher molecular weight in both media analyzed, varying from 72 to 80kDa in the submerse culture and from 68 to 90kDa in the semisolid culture. Also, glucoamylase protein concentration is higher in the submerse culture than in the semisolid culture. The other proteins in the submerse culture presented molecular weights lower than 12kDa and in the semisolid culture their molecular weights varied from 21 to 37kDa and below 10kDa.

Resumo em Inglês:

In biochemical processes, the stoichiometry can be the result of macroscopic balances (where the microorganism is specified by the elementary composition and only the chemical reactions of the conversion process are considered) or the balances of the metabolic pathway, where biochemical knowledge available for metabolic reactions, in addition to the chemical characteristics of the system, are considered. It is possible to identify several linear relationships among the conversion rates in these processes. While several rates are measured, others can be calculated. If a calculated conversion rate is also measured, measurement errors or errors of the described model can be detected or even diagnosed by comparing these values, and accurate estimates can be obtained by combining them.

Resumo em Inglês:

This paper presents a cybernetic structured mathematical model developed for the fermentation step of the process of production of the copolymer of polyhydroxyalkanoates by the bacteria Alcaligenes eutrophus. This process is performed in two different fermentation stages. The first emphasizes the growth of the microorganism in a batch operation without substrates limitations, while in the second, the focus is on copolymer production by a fed-batch operation in the absence of the nitrogen source. This paper presents the results of the treatment of experimental data and of preliminary parameter estimation. The fitting of the proposed model to the experimental data of a standard experiment showed a good agreement.