EXPERIMENTAL PROGEDURES IN LIFE ScIENCES

Prepare the beads by controlled drop wise addition of slurry to the 4% calcium
Keep the beads in calcium chloride solution about 30 minutes for chloride solution.
gelation.
Activation of immobilized beads
Perform activation of immobilized beads with the help of Growth mnedium.
Inoculate required volume of beads into the rich medium and keptit for 30 minutes
Note the movemnents of beads.
Fermentation of alcohol using immobilized beads
Add 25 mL of beads to 250 mL of fermentation medium and carryout the
room temperature.
fermentation under static condition at
Estimate alcohol content through chromic acid method (Refer experiment 160 - Page 408).
(Measure volume of beads using water displacement method).
Observation

Spherical shaped immobilized beads are prepared and activated in Growth medium.
Result

Immobilized Saccharomyces cerevisiae cells produces alcohol, which is estimated through chromic acid method.
Viva questions
What is immobilization?
What are the purposes of immobilization?
What are the types of immobilization?
What are the principles of cell immobilization?
What is gelation?
Name the chêmicals used in immobilization of cells?
How do you activate immobilized cells
apotters: Calcium chloride, sodium alginate., immobilized beads.
115. Amylase Production
Aim

To produce amylaoe fronm microorganisms.

Background Informations
Amylase is an enzyme that break down starch or glycogen. Amylase is produced b÷ avariety of living organisms,
ranging from bacteria to plants and humans. Bacteria and fungi secrete amylase to the outside of their cells to carrv
outextra-cellular digestion. When they have broken down the insoluble starch, the soluble end products such as
(glucose or maltose) are absorbed into their cells. Amylases are classified based on how they break down starch
molecules
i. a-amylase (alpha-amylase)- Reduces the viscosity of starch by breaking down the bonds at random, therefore
producing varied side chains of glucose.
iü. B-amylase (Beta-amylase)- Breaks the glucose-glucose bonds down by removing two glucose units at atime,
thereby producing maltose.
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ii, Amyloglucosidase (AMG) -Breaks successive bonds from the non-reducing end of the straight chain,
producing glucose.
Many microbial amylases usually contain a mixture of these amylases.
Humans exploit microbial amylases for the following purposes, they are High Fructose Corn syrup preparation,
Additives to detergents for removing stains, Saccharification of starch for alcohol production and Brewing, Most
commonly used for their industrial production are Bacilus subtilis, Bacillus licheniformis, Bacillus amyloliquifaciens and
Aspergillus niger.
Materials Required
Autoclave, Microwave oven, Nutrient Agar, Potato Dextrose Agar, Soluble starch, Balance, Shaker
Spectrophotometer, Water bath, disposable spoons, micro pipettes, sterile water, Petri dishes, inoculation, Dissecting
needle or Cork borer,Bunsen burner, matches, Glass spreader,95% ethanol.
Amylase production medium (g/1)
Bacteriological Peptone óg; MgS0,- 0.5g; KCL O.5g; Starch 1g. Sterilize by autoclaving at 121°C for 15 minutes
Procedure

Isolation of Amylase producers

Collect 100g of the top soil and transfer into a "Ziploc" bag.
Suspend about 10 grams of soilin 100 mL sterile distilled water,mix properly (10)
Pipette 10 mL of the above and transfer to another 90 mL of water (102)
Dilute further in two more 90 mL sterile water blanks (10 and 104).
Spread 0.1 mL of the diluted samples (10 and 10) on Nutrient Agar plates (4 plates) containing 1 %w/v
soluble starch and incubate at 30°C for 24 hours

Starch-hydrolysing colonies will have an area of clearing around them.

(Itis confirmed by flooding plates with Gram's iodine,)
Transfer distinguishable amylase-producing bacteria by streak on a fresh plate of Nutrient Agar containing
1% starch.

Transfer isolated amylase-producing bacteria to Nutrient Agar and allow to grow for 24 hours, then store in the
refrigerator until needed.
Amylase production
Seed culture

Prepare amylase production medium, sterilize properly.

Inoculate loopful of amylase producing Bacillus sp.,
Incubate for 24 hours at 37°C
Fermentation
Prepare 250mL of amylase production medium in 500mL conical flask.
Sterilize at 121C for 15 minutes.
Inoculate 25mL of seed culture.

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 ExPERIMENTAL PRocEDURES IN LIFE ScIENCES

Incubate at 37°C for 24 hours under shaking condition.

Withdrw culture and subject to extraction of enzyme.
Extraction of Enzyme from bacteria
Pour the bacterial culture into centrifuge tubes, and spin for 20 minutes at 5000 rpm.
Decant the supernatant in to a sterile beaker, which is the crude enzyme extract.
Enzyme Activity
i. Pipettel mL of culture extract"enzyme" into a test tube.
ii. Add 1mLof 1% soluble starch in citrate-phosphate buffer (pH6.5).
iü, Incubate in a water bath at 40°Cfor 30minutes
0v. Setup ablank consisting of 2mLof the enzyme extract that has been boiled for 20minutes (boiling inactivates
the enzyme), added to the starch solution and treated with the same reagent as the experimental tubes.
v. Stop the reaction by adding 2mL of DNS reagent and estimateredscing sugar (Refer experiment No 148).
Enzyme activity may be defined as the amount of glucose produced per mL in the reaction mixture per unit
time.

Result
Isolated soil bacteria have the capability to produce amylase. Quantity of amylase may vary on the basis of
environmental condition. Unit of amylase production is studied indirectly by analyzing reducing sugar by DNS
method.

Viva questions
What is amylase?
What are the uses of amylase enzyme?
Mention types of amylase enzyme
Mention about the sources of amylase
Name any two organisms responsible for amylase production
How do you check amylase production in laboratory?
Mention media used for amylase production
Spotters:Starch, Iodine
116. Protease Production
Aim

To Produce Protease from microorganisms

Background Informations
Proteases and metalloprotease constitute one of the most important groupsof industrial enzymes, accounting
for about 60% of the total enzyme market. Among the various proteases, bacterialproteases are the most significant,
compared with animal and fungal proteases and among bacteria, Bacillus sp are specific producers of extra-cellular
proteases. These enzymes have wide industrial application, including pharmaceutical industry, leather industry,
manufacture of protein hydrolizates, food industry and waste processing industry. Thermostable proteases are
advantageous in some applications because higher processing temperatures can be employed, resulting in faster
reaction rates, increase in the solubility of nongaseous reactants and products, and reduced incidence of microbial
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