Enzyme assays
Degradative enzymes can be assayed by growing up mycelium, grinding in buffer, and testing extracts for enzymatic activities. Problems for survey level assay – enzymes might not survive extraction, might be in low concentration; buffer characteristics must be optimized for individual enzymes; cells will be homogenized which may lead to antagonistic interactions; some enzymes are induced by their substrate and mightn’t be present
Alternative – grow fungal isolates on solid media containing specific complex substrates; compare colony growth and assay substrate degradation. Note that the media contain a minimal level of nutrients to support initial growth in case the enzyme is substrate-induced.
Most of these are two part assays : grow the isolates on specialized media, and then (for some assays) develop the plate to reveal substrate degradation. For tests with developer, wait about 10 min after applying the reagent for full colour to show. Note that lack of substrate utilization will also reduce growth rate and/or hyphal density due to relative nutrient lack.
Amylolytic activity
Medium: Difco Nutrient agar plus 0.2% soluble starch, pH 6.
Assay: Flood the plate with dilute iodine, about the colour of light maple syrup. Amylolytic activity indicated by a yellow, starch-depleted zone
Cellulolytic activity
Medium: two-layered plates.
Bottom layer (16 ml): 50 ml 20x nitrate salts, 1 ml trace elements, 1 g yeast extract, 15 g agar,
1000 ml ddHOH.
Top layer (8 ml): 12.5 g carboxy methyl cellulose, 15 g agar, 500 ml ddHOH
Assay: flood plates with 10% copper acetate, which will reveal clear zones where soluble cellulose has been degraded. Fibrous cellulose gives blue precipitate where degraded
Lipolytic acticity
Medium: Autoclave the two solutions separately, and then combine:
1) 10 g Difco peptone, 5 g NaCl, 0.1 g CaCl
2
.2H
2
O, 15 g agar, 900 ml ddHOH, pH 6.
2) 10 ml Tween 20 + 90 ml ddHOH. After autoclaving, add the detergent solution (Tween
20 is polyoxyethylene sorbitan monolaurate) to the sterilized and cooled medium. Mix very gently, avoiding froth. Pour.
Assay: formation of lipases will cause a visible precipitate of Ca-laurate crystals, followed by a complete clearing of the crystals due to continued degradation.
Pectolytic activity
Medium: 50 ml 20x nitrate salts, 1 ml trace elements, 1 g yeast extract, 15 g agar, 5 g pectin,
1000 ml ddHOH. Buffer to pH 7 to detect pectate lyase (pectate transeliminase) activity.
Buffer to pH 5 to detect pectinase (pectin depolymerase) activity.
Assay: flood with 1% aqueous hexadecyl trimethyl ammonium bromide (may need to warm slightly to dissolve). HTAB is also called CTAB, and is a common part of plant DNA isolation protocols. It precipitates intact pectin, leaving a clear zone around a colony with an otherwise opaque medium.
Proteolytic activity
Medium: 1% glucose; 0.5% peptone; 3 % gelatin. Autoclave. Cool. Pour.
Secretion of proteases will result in a liquefied zone around the culture after 2-3 d