RESUMO
The complexity of biological processes often makes impractical the development of detailed, structured phenomenological models of the cultivation of microorganisms in bioreactors. In this context, data pre-treatment techniques are useful for bioprocess control and fault detection. Among them, principal component analysis (PCA) plays an important role. This work presents a case study of the application of this technique during real experiments, where the enzyme penicillin G acylase (PGA) was produced by Bacillus megaterium ATCC 14945. PGA hydrolyzes penicillin G to yield 6-aminopenicilanic acid (6-APA) and phenyl acetic acid. 6-APA is used to produce semi-synthetic beta-lactam antibiotics. A static PCA algorithm was implemented for on-line detection of deviations from the desired process behavior. The experiments were carried out in a 2-L bioreactor. Hotteling's T(2) was the discrimination criterion employed in this multivariable problem and the method showed a high sensibility for fault detection in all real cases that were studied.
Assuntos
Bacillus megaterium , Proteínas de Bactérias/biossíntese , Reatores Biológicos , Penicilina Amidase/biossíntese , Bacillus megaterium/enzimologia , Bacillus megaterium/crescimento & desenvolvimento , Proteínas de Bactérias/química , Ácido Penicilânico/análogos & derivados , Ácido Penicilânico/química , Penicilina Amidase/química , Penicilina G/química , Fenilacetatos/químicaRESUMO
The production of extracellular and mycelia-associated penicillin G acylase (maPGA) with Mucor griseocyanus H/55.1.1 by surface-adhesion fermentation using Opuntia imbricata, a cactus, as a natural immobilization support was studied. Enzyme activity to form 6-aminopencillanic acid (6-APA) from penicillin G was assayed spectrophotometrically. The penicillin G hydrolysis to 6-APA was evaluated at six different times using PGA samples recovered from the skim milk medium at five different incubation times. Additionally, the effect of varying the penicillin G substrate concentration level on the PGA enzyme activity was also studied. The maximum reaction rate, V (max), and the Michaelis constant, K (M), were determined using the Michaelis-Menten model. The maximum levels for maPGA and extracellular activity were found to be 2,126.50 international unit per liter (IU/l; equal to 997.83 IU/g of support) at 48 h and 755.33 IU/l at 60 h, respectively. Kinetics of biomass production for total biomass showed a maximum growth at 60 h of 3.36 and 2.55 g/l (equal to 0.012 g of biomass per gram of support) for the immobilized M. griseocyanus biomass. The maPGA was employed for the hydrolysis of penicillin G to obtain 6-APA in a batch reactor. The highest quantity of 6-APA obtained was 226.16 mg/l after 40-min reaction. The effect of substrate concentration on maPGA activity was evaluated at different concentrations of penicillin G (0-10 mM). K(M) and V(max) were determined to be 3.0 x 10(-3) M and 4.4 x 10(-3) mM/min, respectively.
Assuntos
Enzimas Imobilizadas/biossíntese , Fermentação , Ácido Penicilânico/análogos & derivados , Penicilina Amidase/biossíntese , Penicilina G/química , Biofilmes , Adesão Celular , Ativação Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Hidrólise , Opuntia/enzimologia , Ácido Penicilânico/síntese química , Ácido Penicilânico/química , Ácido Penicilânico/metabolismo , Penicilina Amidase/química , Penicilina Amidase/metabolismo , Penicilina G/metabolismo , Propriedades de SuperfícieRESUMO
Penicillin G acylase (PGA) is one of the most important enzymes for the pharmaceutical industry. Bacillus megaterium has the advantage of producing extra-cellular PGA. This work compares two neural networks (NNs) architectures for on-line inference of B. megaterium cell mass in an aerated stirred tank bioreactor, during the production of PGA. Nowadays, intelligent computing tools such as artificial NNs and fuzzy logic are commonly applied for state inference and modeling of bioreactors. Combining these two approaches in hybrid, neuro-fuzzy systems, may be advantageous. Our results indicate that a neuro-fuzzy inference system showed a better performance to infer cell concentrations, when compared to multilayer perceptrons networks.
Assuntos
Bacillus megaterium/citologia , Bacillus megaterium/enzimologia , Redes Neurais de Computação , Penicilina Amidase/biossíntese , Reatores Biológicos , Contagem de Colônia Microbiana/estatística & dados numéricos , Lógica Fuzzy , Modelos Biológicos , Sistemas On-Line , Biologia de SistemasRESUMO
Aiming at to enhance the production of penicillin G acylase (PGA) by Bacillus megaterium, we have performed flasks experiments using different medium composition. Using 51 g/L of casein hydrolyzed with Alcalase and 2.7 g/L of phenylacetic acid (PhAc), the following carbon substrates were tested, individually and combined: glucose, glycerol, and lactose (present in cheese whey). Glycerol and glucose showed to be effective nutrients for the microorganism growth but delayed the PGA production. Cheese whey always increased enzyme production and cell mass. However, lactose (present in cheese whey) was not a significant carbon source for B. megaterium. PhAc, amino acids, and small peptides present in the hydrolyzed casein were the actual carbon sources for enzyme production. Replacement of hydrolyzed casein by free amino acids, 10.0 g/L, led to a significant increase in enzyme production (app. 150%), with a preferential consumption of alanine, aspartic acid, glycine, serine, arginine, threonine, lysine, and glutamic acid. A decrease of the enzyme production was observed when 20.0 g/L of amino acids were used. Using the single omission technique, it was shown that none of the 18 tested amino acids was essential for enzyme production. The use of a medium containing eight of the preferentially consumed amino acids lead to similar enzyme production level obtained when using 18 amino acids. PhAc, up to 2.7 g/L, did not inhibit enzyme production, even if added at the beginning of the cultivation.
Assuntos
Aminoácidos/metabolismo , Bacillus megaterium/enzimologia , Penicilina Amidase/biossíntese , Aminoácidos/biossíntese , Bacillus megaterium/metabolismo , Técnicas Bacteriológicas , Caseínas/química , Meios de Cultura , HidróliseRESUMO
Cheese whey (CW) is the major subproduct from cheese manufacturing and it is considered as a waste pollutant since its high content of lactose. In this work a fermentation process for the production of penicillin acylase (PA) by a recombinant Escherichia coli and using CW as unique carbon source and inducer was developed. A design factorial 3(2) was used to evaluate the influence of independent variables (dissolved oxygen and CW concentration) on the ability of E. coli W3110/pPA102 to produce PA. Maximum specific PA activity of 781 U g(-1) was attained at 5 g L(-1) of CW and 3% dissolved oxygen. The results showed that CW can be used successfully as unique carbon source and inducer for the production of recombinant proteins using constructions driven by the lac promoter and this way reducing the discharges of that pollutant to the environment.
Assuntos
Escherichia coli/crescimento & desenvolvimento , Penicilina Amidase/biossíntese , Proteínas Recombinantes/biossíntese , Queijo , Meios de Cultura , Escherichia coli/enzimologia , Escherichia coli/genética , Óperon Lac , Lactose/metabolismo , Penicilina Amidase/genética , Proteínas Recombinantes/genéticaRESUMO
In this study, several fermentation media were tested for the production of penicillin G acylase (PGA) using Bacillus megaterium. The carbon sources studied were glucose and lactose. The nitrogen sources studied were enzymatic casein hydrolysates produced with proteases of different specificities. The replacement of glucose with cheese whey and the addition of free amino acids in the PGA production were also tested. The results showed a strong correlation between the nitrogen source and enzyme yield and the presence of glucose repression. The highest enzyme concentration achieved was 138 IU/L using casein hydrolyzed with 0.6 L of Alcalase and cheese whey.
Assuntos
Bacillus megaterium/crescimento & desenvolvimento , Penicilina Amidase/biossíntese , Bacillus megaterium/enzimologia , Técnicas Bacteriológicas , Caseínas/química , Meios de Cultura , Endopeptidases/metabolismo , Fermentação , Hidrólise , CinéticaRESUMO
Penicillin acylase is a key enzyme for the production of semisynthetic beta-lactam antibiotics. The intracellular enzyme from Escherichia coli has been thoroughly studied and characterized. The extracellular enzyme from Bacillus megaterium, despite its potential advantages, has received less attention in the recent scientific literature. A comparative study is presented for the production of penicillin acylase with two strains of Bacillus megaterium in batch fermentation in previously optimized complex and defined media. The enzyme produced by the selected strain has been recovered, partially purified and its kinetic behaviour determined.
Assuntos
Bacillus megaterium/enzimologia , Penicilina Amidase/biossíntese , Bacillus megaterium/crescimento & desenvolvimento , Bacillus megaterium/isolamento & purificação , Escherichia coli/enzimologia , Escherichia coli/isolamento & purificação , Fermentação , Concentração de Íons de Hidrogênio , Cinética , Penicilina Amidase/análise , Temperatura , Fatores de TempoRESUMO
Penicillin acylase is a key enzyme for the production of semisynthetic beta-lactam antibiotics. The intracellular enzyme from Escherichia coli has been thoroughly studied and characterized. The extracellular enzyme from Bacillus megaterium, despite its potential advantages, has received less attention in the recent scientific literature. A comparative study is presented for the production of penicillin acylase with two strains of Bacillus megaterium in batch fermentation in previously optimized complex and defined media. The enzyme produced by the selected strain has been recovered, partially purified and its kinetic behaviour determined