RESUMO
In the present study, it was presented a strategy to maximize the cutinase production by solid-state fermentation from different microorganisms and substrates. The best results were observed using Fusarium verticillioides, rice bran being the main substrate. Maximum yield of cutinase obtained by the strain was 16.22 U/g. For concentration, ethanol precipitation was used, and the purification factor was 2.4. The optimum temperature and pH for enzyme activity were 35 °C and 6.5, respectively. The enzyme was stable at a wide range of temperature and at all pH values tested. The concentrated cutinase was used as an adjuvant in a formulation containing cutinase + bioherbicide. The use of enzyme increased the efficiency of bioherbicide, since cutinase was responsible to remove/degrade the cutin that recovery the weed leaves and difficult the bioherbicide absorption. Cutinase showed to be a promising product to be used in formulation of bioherbicides.
Assuntos
Hidrolases de Éster Carboxílico , Proteínas Fúngicas , Fusarium/enzimologia , Herbicidas/metabolismo , Controle Biológico de Vetores , Hidrolases de Éster Carboxílico/biossíntese , Hidrolases de Éster Carboxílico/química , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Herbicidas/química , Concentração de Íons de Hidrogênio , Oryza/químicaRESUMO
The effects of ethanol/broth proportions and the number of steps at varying pH in the presence or absence of sodium chloride (NaCl) were studied as precipitation strategies for the recovery and purification of high molar mass bio-hyaluronic acid (Bio-HA). Bio-HA was synthesized by Streptococcus zooepidemicus in a culture medium containing glucose and soy peptones. A single-step precipitation was more attractive than multistep precipitation in terms of recovery and purity as well as decreased use of ethanol. The best conditions in the absence and presence of salt were 2:1 ethanol/broth (v/v) at pH 4 (55.0 ± 0.2% purity and 85.0 ± 0.7% recovery) and 2:1 ethanol/broth (v/v) at pH 7 + 2 mol L-1 NaCl (59.0 ± 0.9% purity and 82.0 ± 4.3% recovery). Dynamic light scattering (DLS) spectra showed different particle sizes as a consequence of the changes in the molecular structure of HA, mainly with changes in pH. Although slight changes in distribution were observed, the average HA molar mass was not affected by the precipitation strategy, remaining on the order of 105 Da. Therefore, pH and NaCl modulated the precipitation performance of HA. These findings are relevant to further optimizing the precipitation step, thus minimizing costs in the later stages of HA purification.
Assuntos
Ácido Hialurônico/isolamento & purificação , Biotecnologia , Precipitação Química , Meios de Cultura , Etanol , Fermentação , Ácido Hialurônico/biossíntese , Ácido Hialurônico/química , Concentração de Íons de Hidrogênio , Estrutura Molecular , Peso Molecular , Tamanho da Partícula , Cloreto de Sódio , Streptococcus equi/metabolismoRESUMO
Pneumonia caused by Streptococcus pneumoniae is a major bacterial disease responsible for many deaths worldwide each year and is particularly dangerous in children under 5 years old and adults over 50. The capsular polysaccharide (CPS) constitutes the outermost layer of the bacterial cell and is the main virulence factor. Regardless of whether pharmaceutical agents are composed of CPS alone or protein-conjugated CPS, CPS purification is essential for the development of vaccines against S. pneumoniae. These vaccines are effective and safe but remain quite expensive. This review describes the methods currently available for CPS purification. Advances in CPS purification methods are aimed at improvements in quality and yield and, above all, process simplification.
RESUMO
Background Thermostable DNA polymerase (Taq Pol ?) from Thermus aquaticus has been widely used in PCR, which was usually extracted with Pluthero's method. The method used ammonium sulfate to precipitate the enzyme, and it saved effort and money but not time. Moreover, we found that 30-40% activity of Taq Pol I was lost at the ammonium sulfate precipitation step, and the product contained a small amount of DNA. Results We provided a novel, simplified and low-cost method to purify the Taq Pol ? after overproduction of the enzyme in Escherichia coli, which used ethanol instead of ammonium sulfate to precipitate the enzyme. The precipitate can be directly dissolved in the storage buffer without dialysis. In addition, DNA and RNA contamination was removed with DNase I and RNase A before precipitation, and the extraction procedure was optimized. Our improvements increase recovery rate and specific activity of the enzyme, and save labor, time, and cost. Conclusions Our method uses ethanol, DNase I, and RNase A to purify the Taq Pol ?, and simplifies the operation, and increases the enzyme recovery rate and quality.