Starch and Casein hydrolysis, SIM, Decarboxylase

Define hydrolysis

Reactions that use water to split complex molecules

What enzymes are needed for a hydrolysis reaction?

Hydrolytic enzymes

What is meant by “intracellular” and “extracellullar” enzymes?

Intra: When the enzyme catalyzes its reaction INSIDE the cell

Extra: When the enzyme catalyzes its reaction OUTSIDE the cell

What is an exoenzyme?

Enzymes are secreted to catalyze reactions outside the cell

What does the nutrient gelatin test detect?

The exoenzyme gelatinase

Which test detects intracellular enzymes?

Urease and bile esculin tests

What does a PYR test identify?

A specific aminopeptidase that removes the amino acid on the end of a polypeptide

What do extracellular enzymes usually cause in the medium?

A clear zone or halo around bacteria

Describe starch

Made of complex polysacharide compound made of a-glucose polymers in either amylose or amylopectin

Describe the forms of amylose and amylopectin

Amylose is linear and amylopectin is branched

How are the a-glucose molecules joined?

By one 4-a-glycosidic (acetal) bonds

How do you amylopectin and amylose differ?

Amylopectin contains side chains covalently bonded to every 30th glucose by a one 6-a-glycosidic linkage

How is starch considered a metabolic value?

It must first be digested extracellularly into smaller molecules. If it is a gram negative bacteria it must be digested in the periplasmic space

What are the 3 enzymes involved in starch digestion?

a-amylase: breaks down starch polymer into monosaccharide a-glucose and disaccharide a-maltose

b-amylase: produces b-maltose

a-1,6 glucosidase: breaks limit dextrans down into short,linear oligosaccharides

Describe starch agar

Simple plated medium of beef extract, soluble starch and agar

How is starch hydrolysis detected in the agar?

Iodine is added to the plate, it reacts with the starch and turns blue or dark brown.

What are limit dextran?

Small branched molecules that remain after amylase activity

What indicates a positive result for starch hydrolysis?

A clear zone surrounding the bacterial growth where starch has been hydrolyzed.

Starxh agar was originally designed for cultivating what organism?

Neiisseria, not no more but with pH indicators it is used to isolate and identify Gardnella vaginalis

Starch agar helps differentiate what type of organisms?

Corymebacterium, Clostridium, Bacillus, Bacteroides and Enterococcus

Why do many bacteria require proteins?

As a source of amino acids for synthesis of their own proteins and other compounds.

What is Casease?

A proteolytic enzyme that some bacteria produce to hydrolyze the milk protein casein

What is casein?

Molecule that gives milk its color

Describe a milk agar

Undefined medium containing pancreatic digest of casein , yeast extract, dextrose and powdered milk

How is milk agar inoculated?

With a casease positive organism, the secreated cease will diffuse into medium around colonies and create a clear zone when casein is hydrolyzed

When is casein hydrolysis test used?

For the cultivation and differentiation of bacteria that produce the enzyme casease

What are decarboxylases?

Enzymes that catalyze the removal of an amino acid carboxyl group

What are deaminases?

Enzymes that catalyze the removal of an amino acid amine group

Describe the Mollers decarboxylase base medium

Contains peptone, glucose, pH indicators bromocresol purple and coenzyme pyridoxal phosphate

Describe bromocresol purple

Purple at 6.8 and above and yellow at 5.2

How is the decarboxylation medium sealed from external o2 after inoculation?

With mineral oil

Describe a glucose fermentation in anaerobic medium

Turns yellow due to the accumulation of acid end products

Describe the decarboxilation of amino acids results

Accumulation of amines, which are alkaline and turn medium purple

What will happen if the organism is a glucose fermenter but doesn’t produce appropriate decarboxylase?

Medium will turn yellow

What color indicates a positive decarboxylase result?

Only purple

What are the 3 amino acids tested most frequently for decarboxylase?

Arginine, lysine , ornithine

What does SIM medium test for?

Sulfur reduction, indole production, and motility (these are important characteristics of Enterobacteriaceae)

Function of enzyme tryptophanase

Hydrolyze tryptophan to pyruvate, ammonia and indole

What enzyme catalyzes sulfur reduction from cysteine?

Cysteine desulfurase which hydrolyzes cysteine to produce puruvates,H2S and ammonia

What enzyme reduces thiosulfate to H2S in anaerobic respiration?

Thiosulfate reductase

What indicates a positive sulfur reduction test in SIM medium?

Blackening of medium