RESUMO
AIMS: Different cultural conditions for forming and reverting protoplasts were systematically studied to establish a rapid and efficient protocol for Sclerotium rolfsii ATCC 201126. METHODS AND RESULTS: Osmotic stabilizer, lytic enzymes and mycelial age were the main factors influencing protoplast yields. An optimized protocol involving 1-h hydrolysis of 45-h-old mycelium with Trichoderma harzianum enzymes in a 1 : 1 (w/w) biomass : enzyme ratio and 0.6 mol l-1 MgSO4 as osmotic stabilizer was designed to produce approx. 2 x 109 protoplasts per gram biomass dry weight, with 99% viability. Differences on the lytic activity between batches of commercial enzymes were clearly evidenced. Protoplast release was highly efficient showing no remaining cell wall material as witnessed by fluorescent brightener 28. Up to 26% of purified protoplasts developed into the typical filamentous form after 50 h of incubation on 0.6 mol l-1 sucrose agar media. CONCLUSIONS: The methodology herein proposed allowed a rapid, inexpensive and efficient protoplast production. Optimum yields were higher or in the order of that elsewhere reported for other S. rolfsii strains and the required lytic time was significantly shorter. Purified protoplasts successfully reverted to the filamentous morphology. SIGNIFICANCE AND IMPACT OF THE STUDY: The present research reports the former protocol for the isolation and reversion of protoplasts in S. rolfsii ATCC 201126 providing key factors to ensure optimum results. In addition, the described procedure constitutes a starting point for downstream genetic manipulation.
Assuntos
Basidiomycota/fisiologia , Protoplastos/fisiologia , Biomassa , Parede Celular/fisiologia , Meios de Cultura , Hidrólise , Micélio/fisiologia , Osmose/fisiologia , Fatores de Tempo , Trichoderma/enzimologiaRESUMO
The measurement of the colony radial growth rate (Kr) on solid medium of colonies of Sclerotium rolfsii Proimi F-6656 for the evaluation of scleroglucan production medium and other different media, incubation temperature and tolerance to diverse concentrations of sucrose and NaCl were studied. The optimum growth temperature observed was 30 degrees C. The Kr value reached on the Production Medium used (0.66 mm.h-1) showed no differences compared with those of the other media tested, indicating that all the requirements for growth were provided. Poor growth was only observed on Soil Extract Agar. The fungus tolerated concentrations of sucrose from 0.15 to 1.17 M, on both Czapek and production medium. Growth was limited by the highest concentrations of sucrose tested (0.88 and 1.17 M), as indicated by a slower increase in colony size. Addition of 0.86 M NaCl to the production medium and YM agar did not inhibit growth completely, but decreased the radial growth rate considerably (80 and 70% respectively).
Assuntos
Ascomicetos/crescimento & desenvolvimento , Ascomicetos/fisiologia , Meios de Cultura , Concentração Osmolar , Cloreto de Sódio , TemperaturaRESUMO
Para la produccion de proteina unicelular a partir de medula de bagazo de cana de azucar, se uso un cultivo mixto de bacterias identificadas como Cellulomonas sp. y Bacillus subtilis. Los maximos valores de rendimiento (0,17 g proteina/g medula) y de produccion (3,43 g proteina/I) fueron obtenidos a 34 graus C. (pH 7,0) con nitrato de sodio como fuente de nitrogeno.El cultivo mixto mostro un crescimiento satisfactorio con 20% de oxigeno disuelto con respecto a saturacion, condicion esta alcanzada con 400 rpm y 0,15 vvm de aire.El contenido proteico (N x 6,25) del cultivo misto es de 50%, respondiendo la composicion aminoacidica con alto contenido en lisina (6,4%) a los requerimientos de la FAO para aminoacidos esenciales excepto em metionina