Hi, Swape!

I use the following protocol, wait that it could be useful.

Preparation of the enzymatic extract from a daily culture in rich way (extract of malte and/or Czapeck or YPD), for come of the culture collection, to make the counting in chamber of Neubauer and to inoculate suspension of spores in the concentration of 5 X 106 spores/mL in 50 mL of Solution of Manachini, with inductive substratum, keeping constant agitation in 140 rpm at 28ºC, during 72 hours.

Inductive substratum supplement 0.5% of carboximetilcelulose (for celulase).

Solution of Manachini (Half liquid used for the enzymatic induction):

KH2PO4..........................................….2,0g

(NH4) 2SO4.…………..…………………1,0g

MgSO4.7H2O.......................................0,1g

NaH2PO4H2O......................................0,9g

Yeast extract.....................................1,0g

Water.........................................1000mL

Preparation of the enzymatic extract

To filter under vacuum in Millipore filter of 0,45μm, to separate the mycelia mass of the enzymatic extract. The filtered ones can be conditioned in freezer, and defrosting in aliquots, one for determination of the dosage of proteins and another enzymatic one.

Protein dosage Enzymatic tests (Teixeira et al 2000 and Teixeira, 1994)

Preparation of the plates :the determination of the enzymatic activity in

half solid (agar 4%) , being for celulases drain plug Buffer Sodium Acetate 0,2M pH 5,0 supplemented with its respective substratum 0,5%:

Carboximetilcelulase agar: used for revelation of the enzymatic characterization of celulases.

agar.....................................................9,0g

Carboximetilcelulase.........................10,0g

Buffer Sodium Acetate.................1000mL

The half solid carboximetlcelulase are distributed in 15 plates of x 100 mm, forming a layer of 4 mm, and done 3 punctures (cup-plate) in the surface of agar, with hollow glass (8 mm of diameter). In the gotten sockets 150 μL of the filtered extract of the culture (extract enzymatic) and incubated in 37ºC for 24 hours.

Revelation of the Qualitative Enzymatic Activity

After the incubation period, the surface of the agar will be spreed with Solution of Red of Kongo 0.1%, for determination of the enzymatic activity of celulase.

Red of Kongo 0.1% - Solution used for revelation of celulases:

Red of the Kongo ................................. 0,1 g

Alcohol 96.8% ...................................... 100mL

Sorry for my fast English...