RESUMO
Penicillin G acylase (PGA) is a strategic enzyme in the production processes of beta-lactam antibiotics. High demand for ß-lactam semisynthetic antibiotics explain the genetic and biochemical engineering strategies devoted towards novel ways for PGA production and application. This work presents a fermentation process for the heterologous production of PGA from Alcaligenes faecalis in Bacillus megaterium with optimization. The thermal stability from A. faecalis PGA is considerably higher than other described PGA and the recombinant enzyme is secreted to the culture medium by B. megaterium, which facilitates the separation and purification steps. Media optimization using fractional factorial design experiments was used to identify factors related to PGA activity detection in supernatant and cell lysates. The optimized medium resulted in almost 6-fold increased activity in the supernatant samples when compared with the basal medium. Maximum enzyme activity in optimized medium composition achieves values between 135 and 140 IU/ml. The results suggest a promising model for recombinant production of PGA in B. megaterium with possible extracellular expression of the active enzyme.
Assuntos
Alcaligenes faecalis , Bacillus megaterium , Penicilina Amidase , Alcaligenes faecalis/genética , Alcaligenes faecalis/metabolismo , Penicilina Amidase/genética , Penicilina Amidase/metabolismo , Antibacterianos , beta-LactamasRESUMO
Under specific environmental conditions, Pseudomonas aeruginosa produces a biodegradable surfactant rhamnolipid. Evidences suggest that this biosurfactant is involved in protecting cells against oxidative stress; however, the effects of oxidative stress on its production and other virulence factors are still unclear. Here we show that rhamnolipid production is dependent on the aeration surface when P. aeruginosa is cultured in shaken flasks, as well as in production of elastases and alkaline proteases. The production of alginate, lipase, and pyocyanin was not detected in our shaken-flask experiments. P. aeruginosa was treated with hydrogen peroxide to trigger its oxidative stress response, and the proteome profile was analyzed. We identified 14 proteins that were expressed differently between samples that were treated and not treated with peroxide; these proteins are potentially involved in the rhamnolipid production/secretion pathway and oxidative stress.
Assuntos
Proteínas de Bactérias/metabolismo , Glicolipídeos/biossíntese , Estresse Oxidativo , Proteoma/metabolismo , Pseudomonas aeruginosa/metabolismo , Fatores de Virulência/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Eletroforese em Gel Bidimensional , Endopeptidases/química , Endopeptidases/genética , Endopeptidases/metabolismo , Regulação Bacteriana da Expressão Gênica , Elastase Pancreática/química , Elastase Pancreática/genética , Elastase Pancreática/metabolismo , Proteoma/química , Proteoma/genética , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Fatores de Virulência/química , Fatores de Virulência/genéticaRESUMO
The influence of different nutrients on biosurfactant production by Rhodococcus erythropolis was investigated. Increasing the concentration of phosphate buffer from 30 up through 150 mmol/L stimulated an increase in biosurfactant production, which reached a maximum concentration of 285 mg/L in shaken flasks. Statistical analysis showed that glycerol, NaNO3,MgSO4 and yeast extract had significant effects on production. The results were confirmed in a batchwise bioreactor, and semi-growth-associated production was detected. Reduction in the surface tension, which indicates the presence of biosurfactant, reached a value of 38 mN/m at the end of 35 hours. Use of the produced biosurfactant for washing crude oil-contaminated soil showed that 2 and 4 times the critical micellar concentration (CMC) were able to remove 97 and 99 percent of the oil, respectively, after 1 month of impregnation.
Assuntos
Arquivos , Biodegradação Ambiental , Indústria Química , Remoção de Contaminantes , Hidrocarbonetos , Petróleo/classificação , Petróleo/efeitos adversos , Rhodococcus/química , Interpretação Estatística de Dados , Métodos , ToxicidadeRESUMO
The influence of different nutrients on biosurfactant production by Rhodococcus erythropolis was investigated. Increasing the concentration of phosphate buffer from 30 up through 150 mmol/L stimulated an increase in biosurfactant production, which reached a maximum concentration of 285 mg/L in shaken flasks. Statistical analysis showed that glycerol, NaNO3, MgSO4 and yeast extract had significant effects on production. The results were confirmed in a batchwise bioreactor, and semi-growth-associated production was detected. Reduction in the surface tension, which indicates the presence of biosurfactant, reached a value of 38 mN/m at the end of 35 hours. Use of the produced biosurfactant for washing crude oil-contaminated soil showed that 2 and 4 times the critical micellar concentration (CMC) were able to remove 97 and 99% of the oil, respectively, after 1 month of impregnation.