Question

1. exoenzymes. know how to test for the production of specific enzyme in each of the following test, know the substrate, the enzyme, and the reaction that occurs in the media. also know the name of the media, reagents and/or indicator that are used, and how to interpret the results. it would help to complete the following chart summarizing the information. (note: dnase will be used later in the semester, and will not be on the first practical)

exoenzyme tested	amylase	caseinase	gelatinase	lipase	dnase
type of macro - molecule catabolyzed			protein
reaction catalyzed by the enzyme				fats->glycerol + 3 fatty acids
additional reagent needed	iodine
expected (+) result, (enzyme is present)			the gelatin liquefies
expected (-) result (enzyme is not present)				no clear zone around the colony

1. name of organisms:

• be able to write the name of the organisms using binomial nomenclature
• spelling of the name is important. incorrect spelling will have point deducted
• important: when asked to provide the name of an organism or the species of organisms, this means both the genus and species. in general, unless you are asked only the name of the genus, then you need to write a complete name, with genus and species.
• know both the genus and species names of bacteria that have been covered in lab, where they are normally found, disease they cause, and any special characteristics

Explanation:

Step1: Recall enzyme - related knowledge

Amylase catalyzes the hydrolysis of starch. The media for testing amylase is starch plate. The substrate is starch (a polysaccharide). Iodine is used as an additional reagent. A positive result (+) for amylase is a clear zone around the colony after adding iodine because amylase breaks down starch and iodine no longer gives a blue - black color with the broken - down products. A negative result (-) is a blue - black color around the colony due to the presence of unhydrolyzed starch.

Step2: Caseinase information

The media for testing caseinase is milk agar (casein is the main protein in milk). Caseinase catalyzes the breakdown of casein (a protein). A positive result is a clear zone around the colony as casein is broken down. A negative result is no clear zone.

Step3: Gelatinase details

The media for testing gelatinase is nutrient gelatin. Gelatin is a protein. Gelatinase catalyzes the hydrolysis of gelatin. As mentioned, a positive result is the liquefaction of gelatin. A negative result is no liquefaction (gelatin remains solid).

Step4: Lipase facts

The media for testing lipase is spirit - blue agar or tributyrin agar. Lipase catalyzes the breakdown of fats into glycerol and fatty acids. A positive result is a clear or colored zone (depending on the indicator) around the colony due to the breakdown of fats. A negative result is no clear zone.

Step5: DNAse information

The media for testing DNAse is DNA - agar. DNAse catalyzes the breakdown of DNA. A positive result is a clear zone around the colony after adding toluidine blue or methyl green as the DNA is hydrolyzed. A negative result is no clear zone.

Answer:

Exoenzyme tested	Amylase	Caseinase	Gelatinase	Lipase	DNAse
Type of macro - molecule catabolyzed	Polysaccharide (starch)	Protein (casein)	Protein (gelatin)	Lipid (fats)	Nucleic acid (DNA)
Reaction catalyzed by the enzyme	Starch→Maltose + Dextrins	Casein→Amino acids	Gelatin→Peptides + Amino acids	Fats→Glycerol + 3 fatty acids	DNA→Oligonucleotides + Nucleotides
Additional reagent needed	Iodine	None (but milk has natural properties for detection)	None (natural liquefaction is observed)	Spirit - blue dye (in spirit - blue agar) or none (in tributyrin agar, just observe clear zone)	Toluidine blue or methyl green
Expected (+) result, (enzyme is present)	Clear zone around colony after adding iodine	Clear zone around colony	Gelatin liquefies	Clear or colored zone around colony	Clear zone around colony after adding indicator
Expected (-) result (enzyme is not present)	Blue - black color around colony (due to unhydrolyzed starch)	No clear zone	No liquefaction (gelatin remains solid)	No clear zone	No clear zone