lab 3

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Microbial Enzymes I

Physiology of microbiology Bio 336

KING ABDULAZIZ UNIVERSITY

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Microbial Enzymes I

Enzymes: A substance produced by some microorganisms that has the potential to accelerate metabolic reactions by analysis many complex carbohydrate, protein and fatty

substances into small molecules that can be absorbed by the microbes.

Enzymes are divided into:

1- Constitutive Enzymes

Consists in cells on a continuous basis regardless of whether or not there is an interaction.

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• 1- Starch hydrolysis (Amylase enzyme)

• 2- Gelatin liquefaction (Gelatinase enzyme )

• 3- Casein hydrolysis ( caseinase enzyme)

• 4- Lipid hydrolysis (lipase enzyme)

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Microbial Enzymes I

Starch composite complex carbohydrates are two types of units:

1- Amylose (straight chains polymer of D-glucose units) and 2- Amylopectin (branched chains polymer of D-glucose units)

Some microbes can analyze starch BY secretion some enzymes

1- Starch hydrolysis (Amylase enzyme) :

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Amylose:

*It

constitutes About 20%

of the starch.

*Its stains blue with iodine.

*More soluble in water.

Amylopectin:

* It

constitutes About 80%

of the starch.

*Its stains reddish

brown with iodine.

*less soluble in water

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Experiment # 1

qMaterial and Experimental Protocols:

qObservation and results:

qConclusion:

Test the ability of bacteria to analyze starch Microbial Enzymes I

Purpose: To determine if a bacterium produces the amylase enzyme, which breaks down starch to glucose and

Distinguish microorganisms by their ability to analyze starch

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Bio 336

Microbial Enzymes I

Detects the ability of bacteria to decompose starch:

by using the (iodine detector) where the surface of the media is immersed in an appropriate amount

of iodine.

*The presence of a transparent halo on bacterial growth indicates that the bacteria are able to analyze starch, that is, it secrete the enzyme amylase to the outer medium, which breaks the starch into simple sugars (glucose), thus showing the transparent region free of starch and darkening the rest of the environment

in dark blue. If there is no colorless area around the

growth means the inability of bacteria to analyze starch (starch + iodine = blue color)

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Microbial Enzymes I

Hydrolysis of starch

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Bio 336

Microbial Enzymes I

2- Gelatin liquefaction (Gelatinase enzyme ):

Gelatin is a protein substance recorded from the

hydrolysis of collagen turns into the glacial state when dissolved in hot water and then refrigeration and some types of microbes can be analyzed this gelatin, which is lost by the nature of the transformation of the

state of gluten due to the ability of the microbe to

produce an external enzyme Gelatinase, which analyzes gelatin

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Microbial Enzymes I Experiment # 2

qConclusion:

test the ability of bacteria to analyze gelatin

Purpose: To differentiate between organisms that

produce the exoenzymes gelatinase and those that do not.

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• Detects the ability of the bacteria to analyze the gelatin by placing the tubes in a container containing ice and leave for 10 minutes, and then hold the tube and is tilted, if found that the media is still coherent means that the gelatin did not

decompose by bacteria . And If you notice the liquefaction of gelatin, that’s mean the microbes has the Gelatinase enzyme that analyzes gelatin

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Microbial Enzymes I

Gelatin liquefaction

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Bio 336

Microbial Enzymes I

Gelatin liquefaction

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Microbial Enzymes I

3- Casein hydrolysis ( caseinase enzyme):

The enzyme caseinase is secreted out of the cells (an exoenzyme) into the surrounding media, Casein is the predominant protein in milk

(gives white color) Some microbes can be analyzed it into small soluble units small

peptides and individual amino acids which are then

taken up by the organism for energy use or as building material. Producing a clear yellow

solution. This process is known as peptonization by secretion of the external enzyme Caseinase,

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Bio 336

qConclusion:

test the ability of bacteria to analyze casein

Purpose: To determine if an organism can produce the exoenzyme Caseinase .

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The result is recorded immediately after incubation.

If a transparent halo of bacterial growth is observed, the bacteria analyzed casein with the secretion of

casinase, and if there is no clear growth zone

evidence of the inability of the bacteria to dissolve Casein.

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Bio 336

Microbial Enzymes I

Hydrolysis of casein

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4- Lipid hydrolysis (lipase enzyme):

The ability of bacteria to analyze fat is related to the secretion of the lipase enzyme, which

analyzes the fat molecule into a glycerol molecule and 3 fatty acid molecules.

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Bio 336

qConclusion:

test the ability of bacteria to analyze lipase

Purpose: To determine whether a bacterium produces a lipase that will hydrolyze a neutral fat to fatty acid and glycerol.

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After incubation, dip the dishes in sufficient quantity of( copper sulfate solution detector)20-10% for

10 minutes, then get rid of the solution and leave the plates for 3-5 minutes and then examined.

The appearance of a blue-green color on the line

of the colony is evidence of the ability of bacteria to analyze fat by secretion of the lipase enzyme and the

color is formed by the union of copper sulfate with the fatty acids resulting from decomposition.

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Lipid hydrolysis

Bio 336

Microbial Enzymes I

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1-starch hydrolysis test

https://www.youtube.com/watch?v=zFhMbXSgve8 https://www.youtube.com/watch?v=FEf_ecmD_Uk

2- gelatin hydrolysis test

https://www.youtube.com/watch?v=ew0W0vFaUhU https://www.youtube.com/watch?v=XTh4FcW32Ck

3- Casein Hydrolysis

https://www.youtube.com/watch?v=c3tx4oY1ncQ