BIO 304 Lab Worksheet 4 Spring 2006

NAME______________________________________________

Coverage: Ex 15, 16: Protein Catabolism

Exercise 17: Catalase and oxidase tests

Draw the structure of a generic amino acid: label the amino group and the carboxyl group.

/R

H3N-CH-COOH

amino carboxyl

What would be the name of an enzyme that removes the carboxyl group?

decarboxylase

What would be the name of an enzyme that removes the amino group?

deaminase

Gelatin is a protein polymer: what are the basic building blocks of gelatin?

amino acids

Describe the test done in lab that looks for gelatinase.

A gelatin deep is stabbed with a culture. After growth occurs, if the deep has turned to a liquid, the culture is deemed positive for the enzyme gelatinase

Is gelatinase an exoenzyme or an endoenzyme?

exoenzyme

What is the test we did in lab for carbohydrate exoenzymes that would be the counterpart to the gelatin test?

The starch hydrolysis test would be the counterpart. It looked for the exoenzyme amylase, which is an exoenzyme that breaks down the complex carbohydrate starch.

The litmus milk tubes tests for activities by bacteria on the major sugar and the major protein in milk.

The major sugar in milk is: lactose

The major protein in milk is: casein

What is litmus? a pH indicator that is blue under alkaline conditions and pink under acid conditions

If the litmus milk turns blue, what happened? an alkaline reaction occurred

If the litmus milk becomes clear, what happened? the proteins were partially hydrolyzed, also known as peptonization

If the litmus milk turns pink, what happened? the major sugar, lactose, was fermented to acid products

If the litmus milk turns white, what happened? the bacterial culture was able to reduce the litmus, so that is no longer an effective red/blue pH indicator

Urea is a product of protein catabolism in the human body. Is is excreted in the urine. What is the name of the enzyme we looked for using the urea slant? UREASE

Write the chemical equation for the action of the enzyme named above on urea.

see lab manual for strucures: urea is broken down to ammonia and CO₂

How do you know if the urea test is positive?

the ammonia lowers the pH of the medium, turning the phenol red indicator to bright pink, or fuschia

What do the letters MIO stand for? Fully describe these three tests that are in the MIO deep below:

Motility: determined by growth observed throughout the tube, away from the stab

Indole: the product of tryptophanase, and enzyme which converts tryptophan to indole and pyruvic acid. Indole is detected by adding kovac’s reagent to the tube. A red color indicates the presence of indole, while yellow is negative for indole

If a culture is describes as Hydrogen sulfide (H₂S) positive, what test was used to determine that result? Peptone Iron Deep

Describe how that test works:

The peptone iron deep contains peptone, a rich source of all amino acids. If cysteine, a sulfur-containing amino acid, is acted upon by the enzyme cysteine desulfhydrase, the sulfhydryl group will be released in the form of H₂S gas. The gas will react immediately with the iron in the medium to make a black precipitate.

Broad groups of organisms are differentiated with tests that look for key enzymes that are used to process oxygen. What are the two enzymes we tested for in lab? Explain their function in the cell AND how we tested for them.

Enzyme. 1:

name catalase

function converts hydrogen peroxide to water and oxygen

test: mix a sample of the culture with hydrogen peroxide. If bubbles are observed, the culture is positive for the presence of catalase.

Enzyme 2:

name cytochrome oxidase C

function terminal enzyme in electron transport: puts electrons onto oxygen

test: oxidase dryslide is smeared with some of the culture to be tested. If the indicator in the paper turns dark blue almost immediately, the culture is positive for oxidase. If there is no color change, the culture is negative for oxidase.