Enzyme : All living organisms from bacteria to man are built and maintained by biological catalysts called enzymes. These enzymes are made from proteins which have each been evolved over millions of years to perform very specific biochemical tasks.

Enzyme classification:

Hydrolases:In biochemistry, a hydrolase is an enzyme that catalyzes the hydrolysis of a chemical bond

SOME COMMERCIALLY IMPORTANT HYDROLASES:

1. LIPASE

2. PROTEASE

3. ASPERGINASE

4. TANNASE

SOURCES OF L-ASPERGINSE: This enzyme is widely distributed, being found in animal, microbial and plant sources. it's presence in guinea pig serum was first reported by Clementi (Clement,l922). The enzyme is present in liver and kidney of certain birds, rats and chicken(Krebs, 1950). Large number of microorganisms that include Erwinia caratovora, Pseudomonas stutzeri, Pseudomonas aerugenosa and E.coli. It has been observed that eukaryotic microorganisms like yeast and fungi have a potential for asparaginase production.

Mechanism of Action as Food Processing Aid:

Asparaginase is intended for use as a processing aid during food manufacture to reduce the level of L-asparagine by its hydrolysis to L-aspartic acid and ammonia. Free L-asparagine present in food is the main precursor of acrylamide, which is considered to be a probable human carcinogen. Acrylamide is formed from L-asparagine and reducing sugars primarily in starchy foods that are baked or fried at temperatures above l20 ͦ C.

Asparginase will be used during preparation of carbohydrate-rich foods that are major sources of dietary acrylamide, such as bread and other cereal-based products, baked and fried potato-based products, and reaction flavours (also known as “thermal process flavours"). The levels of L-asparagine would be reduced in these foods prior to heating, thereby reducing the availability of L-asparagine for acrylamide formation. The enzyme will be inactivated by denaturing during the heating step.

MATERIAL AND METHODs:-Media used:-1.Enrichment media for isolation of microorganism from soil

Nutrient Broth 13 g/LGlucose 20 g/L

Peptone 20 g/LKH₂PO₄ 1 g/LK₂HPO₄ 0.5 g/L

Distilled Water to make 1000 mLMaintain pH at 7.0 at 37 ͦ C

2.Nutrient Agar medium

Nutrient Agar 28 g/L

Agar Powder 4 g/L

3. Nutrient Broth Medium

Nutient Broth 13 g/L

Distilled Water to make 1000 ml

4.Screening Medium( for L- asparaginase)

Na₂HPO₄ 6 g/L

KH₂PO₄ 3 g/L

NaCl 0.75 g/L

L- asparagine 10 g/L

PROCEDURE:

Collection of soil sample

The soil sample was collected from the garden area of Delhi University South Campus.

Culture Enrichment

The soil samples collected were then added to the enriched media prepared earlier. Soil sample was added 4g/L of enriched media for microbes present in the soil to grow. For that they were incubated overnight in shaking incubator at 37°C.

Isolation of pure cultures

In natural habitats microorganisms usually grow in complex mixed populations containing several species. This presents a problem for the microbiologist because a single type of microorganism cannot be studied adequately in a mixed culture. One needs a pure culture, a population of cells arising from a single cell, to characterize an individual species.

Simpler methods for isolation of a pure culture include:

(i) Spread plating on solid agar medium with a glass spreader and

(ii) Streak plating with a loop. The purpose of spread plating and streak plating is to isolate.

SPREAD PLATE TECHNIQUE:

In this technique, the number of bacteria per unit volume of sample was reduced by serial dilution before the sample was spread on agar plate.

Serial Dilution

●12 test tubes were taken and 9 mL of distilled water in each test tube was added and autoclaved.

●These test tubes were then marked from 10-1 to 10-12 serially.

●1 mL of medium (containing the soil sample) was added in test tube marked as 10-1.

●Now take 1 mL of sample from the test tube marked 10-1 and add it to the tube marked 10-2 and repeat the sample process till 10-12.

Nutrient Agar plates were made and marked and these plates were then marked from 10-1 to 10-12 serially.

PLATINGA spreader was taken and heated to make it sterile. Now from each test tube 50 µL of sample was taken and added to the corresponding agar plate. With the help of spreader, spreading was done till the surface of agar becomes rough.

Then , colony were developed on the agar surface and we characterize the colony according to their morphology.

Slant Preparation

For slant preparation, nutrient agar was prepared in a flask (250 mL).

The flask was then placed in the heating mantle and was continuously shaken for even heating. The flask was heated till it boils

The flask was then removed from the heating mantle.

Test tubes were placed in the stand and media was poured in the test tube

Test tubes were sealed by the cotton plugs and then autoclaved.

After autoclaving they were kept at an angle to prepare the slants.

Fig: slant

Gram Staining

Gram positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50-90% of cell wall), which are stained purple by crystal violet, whereas Gram-negative bacteria have a thinner layer (10% of cell wall), which are stained pink by the counter-stain. Alcohol does not readily penetrate to decolorize the cell wall of the previously applied crystal violet stain. Gram-negative cells have a thinner cell wall through which the alcohol readily penetrates. The crystal violet is removed from these cell walls that are then stained with the safranin counterstain.

There are four basic steps of the Gram stain:-(1) Applying a primary stain (crystal violet) to a heat-fixed (death by heat) smear of a bacterial culture.

(2) The addition of a trapping agent (Gram's iodine)

(3) Rapid decolorization with alcohol or acetone, and

(4) Counterstaining with safranin.

(5) Observe the slide under the microscope

Negative StainingNegative staining is an established method for contrasting a thin specimen

with an optically opaque fluid. In this technique, the background is stained, leaving the actual specimen untouched, and thus visible. This contrasts with 'positive staining', in which the actual specimen is stained.

To conduct a proper negative stain the following procedure should be followed:

1. Place a very small drop (more than a loop full--less than a free falling drop from the dropper) of nigrosin near one end of a well-cleaned and flamed slide.

2. Remove a small amount of the culture from the slant with an inoculating

loop and disperse it in the drop of stain without spreading the drop.

3. Use another clean slide to spread the drop of stain containing the organism using the following technique.

4. Rest one end of the clean slide on the center of the slide with the stain. Tilt the clean slide toward the drop forming an acute angle and draw that slide toward the drop until it touches the drop and causes it to spread along the edge of the spreader slide. Maintaining a small acute angle between the slides, push the spreader slide toward the clean end of the slide being stained dragging the drop behind the spreader slide and producing a broad, even, thin smear.

5. Allow the smear to dry without heating.

6. Focus a thin area under oil immersion and observe the unstained cells surrounded by the gray stain

Screening MediaAfter preparing plates of screening media, point inoculation was performed.

The plate was divided into 8 parts and a point was marked in each of the eight sections.

The slant of the all the colonies were taken one by one.

And with the help of loop a small amount of colony was picked from slant.

Then the loop was touched at the point marked in the plate.

These steps were repeated until all the colonies were inoculated.

The plates were then incubated at 37 ͦC for 12-24 hours.

Growth CurveGrowth is an orderly increase in the quantity of cellular constituents. The growth of

microorganisms reproducing by binary fission can be prepared by plotting as the logarithm of the number of viable cells versus the incubation time.

Lag Phase:Bacteria are becoming "acclimated" to the new environmental conditions to which

they have been introduced (pH, temperature, nutrients, etc.). There is no significant increase in numbers with time.

Exponential Growth Phase:

The living bacteria population increases rapidly with time at an exponential growth in umbers, and the growth rate increasing with time. Conditions are optimal for growth.

Stationary Phase:

With the exhaustion of nutrients and build-up of waste and secondary metabolic products, the growth rate has slowed to the point where the growth rate equals the death rate. Effectively, there is no net growth in the bacterial population.

Death phase:

The depletion of nutrients and the subsequent accumulation of metabolic waste products and other toxic materials in the media will facilitates the bacterium to move on to the Death phase. During this, the bacterium completely loses its ability to reproduce. Individual bacteria begin to die due to the unfavourable conditions and the death is rapid and at uniform rate. The number of dead cells exceeds the number of live cells. Some organisms which can resist this condition can survive in the environment by producing endospores .

Enzyme ActivityEffect of temperature

The temperature has a vital role to play in activity of any enzyme. The readings of the enzyme activity were calculated for temperatures ranging from 20 ͦC to 70 ͦC. In which the highest activity was noticed at 60 ͦC. The following readings were observed and subsequent graphs were plotted.

Temperature( ͦC) Enzyme

Activity(IU/mL)

20 203.829

30 274.283

40 351.664

50 450.456

60 555.818

70 168.63

203.829

274.283

351.664

450.456

555.818

168.63

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Enzyme Activity

Enzyme Activity

Effect of pHThe pH has a vital role to play in activity of enzyme. The readings of the Enzyme

activity were measured from pH ranging from 2 to 12. In which the highest activity was noticed at pH 9. The following readings were observed and subsequent graphs were plotted.

pH Enzyme Activity(IU/mL)

4 6.715

5 24.31

6 70.073

7 91.7896

8 129.912

9 150.456

10 91.7896

11 62.389

12 50.6963

6.715

24.31

70.073

91.7896

129.912

150.456

91.7896

62.38950.6963

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Enzyme Activity