Estudo da produção de lipase por Burkholderia cepacia / Study of lipase production by Burkholderia cepacia

RESUMO O uso de enzimas pelas indústrias possibilita o desenvolvimento de processos tecnológicos com eficiência similar aos realizados pela natureza, o que faz dessa tecnologia um dos campos mais promissores na síntese de compostos de alto valor agregado. O presente trabalho teve por objetivo estudar a produção de lipase por Burkholderia cepacia utilizando a metodologia de superfície de resposta. Foram utilizadas as variáveis concentrações de fonte de potássio, magnésio, óleo de soja, água de maceração de milho e pH. Foi observado que, dentro das concentrações utilizadas, o potássio, a água de maceração de milho e o óleo de soja influenciaram positivamente na produção de lipase. O Bioflo III se destacou dentre os biorreatores empregados para a produção da enzima, possivelmente devido a melhor distribuição dos fenômenos de transferência de massa e movimento, alcançando valores de até 2,43 U mL-1 em 120 horas de fermentação.

ABSTRACT The use of enzymes on an industrial scale has enabled the development of technological processes with an efficiency similar to those made by nature, which makes this technology one of the most promising fields in new technologies for synthesis of high-added value compounds. This paper aimed to study the production of lipase by Burkholderia cepacia using the response surface methodology. We used the following variables: concentrations of potassium, magnesium, soybean oil, corn steep liquor and pH. We observed that, among the used concentrations, potassium, corn steep liquor and soybean oil positively influenced the lipase production. Bioflo III showed the best performance among the bioreactors used for the enzyme production, possibly due to a better distribution of mass and movement transfer phenomena, reaching values of up to 2.43 U mL-1 at 120 hours of fermentation.

A cost effective fermentative production of glutathione by Saccharomyces cerevisiae with cane molasses and glycerol

This work aimed to evaluate the effect of sugar cane molasses and glycerol on glutathione (GSH) fermentation by Saccharomyces cerevisiae ATCC 7754 in flask culture using response surface methodology. Under optimized conditions (80 g/L of molasses and 60 g/L of glycerol), the highest GSH and biomass concentration achieved were 119.6 mg/L and 25.3 g/L, respectively. Further studies done in 5 L bioreactor resulted 241.3 mg/L GSH after 96 h in batch fermentation without amino acids addition and the concentration of biomass was 12.1 g/L. In batch fermentation with the addition of the three amino acids (4 mM cysteine, glycine and glutamic acid at 32 h), biomass reached to 25 g/L and GSH, 236.1 mg/L at 96 h of fermentation. The strategy of precursor amino acids addition is a key aspect in increasing the synthesis of GSH.

Glutathione production using magnetic fields generated by magnets

The objective of this work was to study the production of GSH by Saccharomyces cerevisiae ATCC 7754 in a fermentor (5 L) using a cell recycle system with magnets. The fermentation conditions were 20°C, 500 rpm, 5% (v/v) of inoculum, pHinitial 5, 1.1 vvm aeration and total fermentation time of 72 h. The time of application of MF ranged from 24, 48 or 72 h. In comparison to the control experiment, the best results were obtained with 72 h of application of MF. The cell concentration reached 19.5 g/L and GSH concentration was 271.9 mg/L that corresponded to an increase of 2.63 and 32.1% compared to the control experiment, respectively.

Extraction of lipase from Burkholderia cepacia by PEG/Phosphate ATPS and its biochemical characterization

This work aimed to study the partitioning of a lipase produced by Burkholderia cepacia in PEG/Phosphate aqueous two phase system (ATPS) and its characterization. Lipase was produced by B. cepacia strains in a fermenter. Enzyme partitioning occurred at pH 6.0 and 8.0, using PEG 1500 and 6000 on two tie lines. Metal ions, pH and temperature effects on enzyme activity were evaluated. Five milliliter of 7.5% olive oil emulsion with 2.5% gumarabic in 0.1M sodium phosphate buffer at pH 8.0 and 37ºC were used for the activity determinations. Results showed that crude stratum from B. cepacia was partitioned by PEG1500/phosphate ATPS at pH 6.0 or 8.0 for, which the partitioning coefficients were 108-and 209-folds. Lipase presented optimal activity conditions at 37ºC and pH 8.0; it showed pH-stability for 4 h of incubation at different pH values at 37ºC. Metal ions such as Mn2+ , Co2+, I-and Ca2+ sustained enzymatic activities; however, it was inhibited by the presence of Fe2+, Hg2+ and Al3+ . Km and Vmax values were 0.258 U/mg and 43.90 g/L, respectively. A molecular weight of 33 kDa and an isoelectric point at pH 5.0 were determined by SDS-PAGE and IFS electrophoresis, respectively.

Catechol biodegradation kinetics using Candida parapsilopsis

This study evaluated the biodegradation of catechol by a yeast strain of Candida parapsilopsis in standard medium in Erlenmeyer flasks. Results shown that the highest concentration of catechol caused the longer lag period, demonstrating that acclimatized cultures could completely degrade an initial catechol concentration of 910 mg/L within 48 h. Haldane's model validated the experimental data adequately for growth kinetics over the studied catechol concentration ranges of 36 to 910 mg/L. The constants obtained for this model were µmax = 0.246 h-1, Ks = 16.95 mg/L and Ki = 604.85 mg/L.

Neste trabalho foi estudada a biodegradação de catecol em frascos de Erlenmeyers em água residuária sintética pela levedura Candida parapsilopsis. As respostas dos ensaios cinéticos mostraram que altas concentrações de catecol ocasionaram uma fase lag longa para a levedura. Portanto, a aclimatização da cultura de levedura empregada para biodegradação de catecol é de fundamental importância, sendo possível reduzir toda a concentração inicial de catecol da água residuária sintética de 910 mg/L em 48 horas. Os dados experimentais da cinética de biodegradação do catecol foram ajustados pelo modelo de Haldane adequadamente, sobre a faixa de concentração de catecol investigada de 36 a 910 mg/L. Os parâmetros cinéticos obtidos do modelo de Haldane foram: µmax = 0,246 h-1, Ks = 16,95 mg/L e Ki = 604,85 mg/L.

Evaluation of emulsifier stability of biosurfactant produced by Saccharomyces lipolytica CCT-0913

Surface-active compounds of biological origin are widely used for many industries (cosmetic, food, petrochemical). The Saccharomyces lipolytica CCT-0913 was able to grow and produce a biosurfactant on 5 percent (v/v) diesel-oil at pH 5.0 and 32ºC. The cell-free broth emulsified and stabilized the oil-in-water emulsion through a first order kinetics. The results showed that the initial pH value and temperature influenced the emulsifier stability (ES), which was the time when oil was separated. The biosurfactant presented different stabilization properties for vegetable and mineral oil in water solution, despite the highest values of the ES occurring with vegetable oil. The biosurfactant presented smallest ES when compared to commercial surfactants; however, this biosurfactant was not purified.

Os tensoativos de origem biológica são amplamente utilizados em diversas aplicações. O microrganismo Saccharomyces lipolytica CCT-0913 possui a habilidade de crescer em 5 por cento (v/v) óleo diesel a pH 5,0 e 32ºC e produzir biosurfactante. O caldo fermentado livre de células e produzido por S. lipolytica emulsiona e estabiliza emulsões óleo em água de acordo com uma cinética de primeira ordem. Os resultados mostram que o valor do pH inicial e a temperatura influenciam a estabilidade emulsificante (ES), que é medido pelo tempo que a quantidade de óleo. O biosurfactante apresenta diferentes valores de estabilidade emulsificante para óleos vegetais e minerais em emulsões óleo-água, os maiores valores de ES ocorrem nas emulsões utilizando óleo vegetal. O biosurfactante apresenta valores baixos de ES quando comparado com emulsificantes comerciais, entretanto sem sofrer nenhum processo de purificação.

Expanded bed adsorption of an alkaline lipase from Pseudomona cepacia.

An extracellular lipase was isolated from Pseudomona cepacia by expanded bed adsorption on an Amberlite 410 ion-exchange resin. Enzyme characterization and hydrodynamic study of a chromatography column were done. Enzyme purification was done at three condition of expanded bed height (H): at one and half (6cm), at two (8cm) and at three (12cm) times the fixed bed height (H(0)=4cm). The results showed that the experimental data was fitted to the Richardson and Zaki equation, and the comparison between the experimental and calculated terminal velocities showed low relative error. In enzyme purification for better condition, a purification factor of about 80 times was found at 6cm of expanded bed height, or 1.5 times of expansion degree. Purified lipase had an optimal pH and a temperature of 8 and 37 degrees C, respectively.

Screening of Metarhizium spp. strains for anticancer indolizidine alkaloid production and its rapid detection by MS analysis

Six fungi strains (M. anisopliae 3935, 4516, 4819, PL57, PL43 and M. flavoviride CG291) were studied regarding their ability to produce an anticancer indolizidine alkaloid. The culture process was carried out in Shaken flask at 26ºC and 200 rpm using three different culture medium containing oat meal extract supplemented with glucose and DL-lysine or Czapek culture medium. The mycelial extracts produced by Metarhizium spp. cultures were directly submitted to electrospray ionization mass spectrometry (ESI-MS) analysis and the highest alkaloid concentration (approximately, 6 mg.L-1) was reached when M. anisopliae 3935 was tested.

O presente trabalho teve como objetivo avaliar diferentes cepas dos fungos M. anisopliae e M. flavoviride ao respeito da sua capacidade de produzir um alcalóide anticancerígeno, por fermentação em frascos erlenmeyers usando três meios de cultura distintos. De seis cepas testadas, quatro foram capazes de produzir o composto de interesse, M. anisopliae 3935, PL57 e PL43 e M. flavoviride CG291, sendo que a maior concentração de alcalóide (aproximadamente, 6 mg.L-1) foi produzida pelo M. anisopliae 3935, contendo um meio constituído de extrato de farinha de aveia, glicose e DL-lisina a 26ºC e 200 rpm.

Growth of Escherichia coli under extremely low-frequency electromagnetic fields.

The influence of extremely low-frequency (ELF) electromagnetic fields on Escherichia coli cultures in submerse fermentation was studied. The fermentation processes were carried out recycling the culture medium externally through a stainless steel tube inserted in a magnetic field generator (solenoid). The exposure time and electromagnetic induction were varied in a range of 1 to 12 h and 0.010 to 0.10 T, respectively, according to a Box-Wilson Central Composite Designs of face centered with five central points. Growth of E. coli could be altered (stimulated or inhibited) under magnetic fieldinduced effects. E. coli cultures exposed at 0.1 T during 6.5 h exhibited changes in its viability compared to unexposed cells, which was 100 times higher than the control. The magnetic field generator associated with the cellular suspension recycle is a new way of magnetic treatment in fermentation processes and could be appropriate to industrial scale up.

Produçäo de proteína microbiana a partir de Saccharomyces lipolytica cultivada em emulsäo de óleo solúvel de corte / Production of single cell protein from Saccharomyces lipolytica growing on emulsion of cutting oil

Culture media containing used emulsion of cutting oil (ECO) as carbon source were fermented bu Saccharomyces lipolytica CCT 0913 for single cell protein (SCP) production and chemical oxygen demand (COD) reduction. Two media were used, the first containing (NH4)so4 as nitrogen source with concentration in the range 2.5 to 15.0g/l, and the second containing NH4Cl with concentration range 0.1 to 1.0g/l. Fermentations were carried out in Erlenmeyer flasks and fermentor of 6 liters, showing that cellular dried mass (CDM) increased with the scale up as a consequence of better oxygen transfer. A yeld of 1.12 g/l o CDM was achieved utilizing 15g/l of (NH4)2 so4 and 5 per cent (v/v) of ECO in the Erlenmeyer flasks fermentation. A yeld of 3.12 g/l was obtained utilizing 1.0 g/l of NH4Cl and 20 per cent (v/v) of ECO in the six liters fermentor. In this last case the COD was reduced from 6,188 to 4,773 mg/l

Producao e propriedades de beta-galactosidade de Kluyveromyces fragilis NRRL Y-2415 / Production and properties of the Kluyveromyces fragilis NRRL Y-2415 beta-galactosidase

Permeado de soro de queijo e soro de proteinado por acidificacao podem ser utilizados como meio de crescimento de Kluyveromyces fragilis NRRL Y-2415 para obtencao de beta-galactosidase. A suplementacao destes meios com 0,1//de (NH4)2SO4 e 0,05//de KH2PO4 aumenta as producoes de massa celular seca e beta-galactosidase. A extracao da enzima das celulas de levedura feita por autolise em tampao fosfato e 2//de cloroformio foi mais rapida do que quando se substituiu o cloroformio por 2//de tolueno, sendo a atividade obtida funcao da concentracao celular na suspensao. Esta enzima tem pH otimo entre 6,6 e 6,8; a sua estabilidade com respeito a pH de pre-incubacao por uma hora e baixa, o que indica que durante o processo de sua obtencao deve-se trabalhar na faixa otima de pH e temperaturas baixas. Quanto a temperatura de atividade maxima, nao ha diferenca em trabalhar a 30 graus centigrados e a 37 graus centigrados; a 50 graus centigrados a enzima e totalmente inativada. Uma alternativa para pre-purificar esta enzima e utilizar membranas. Usando membrana de 0,08 micro, foi possivel obter o extrato enzimatico livre de fragmentos celulares; com outra de 15.000 Daltons, foi possivel aumentar sua concentracao, obtendo-se no final extrato com 2.500 unidade internacionais de ortonitrofenil beta-D-galactopiranosideo por mL