To produce protease enzyme by free cells and by immobilized cells

Aim: To produce protease enzyme by free cells and by immobilized cells.

Requrements:
Slant culture if streptomycin
Sterile water
Sterile slantstarch casein agar
4% sodium alginate (sterile)
0.5M calcium chloride(sterile)
Inoculam and productin medium:
Potassium nitrate 0.1%
K2HPO4 0.05%
MgSO4 0.05%
NaCl 0.05%
FeSO4 0.001%
CaSO4 0.01%
Glucose 2%
Water Q.S
pH 6.5
Sterile pipette(5ml)
Sterile tubes

Procedure:
The stock culture was inoculated in sterile slant of starch calcium agar, and incubated at room temp. , for 1 week. The spores in this slant is gently scrapped in to sterile H2O and inoculated in inoculums medium and incubated at 280c for 48 hr on a rotary shaker.

Production by free cells:
Production medium sterilized and inoculated at 10% level with inoculums stage culture. Production is run on a rotary shaker at 280c for 5-7 days. Samples are withdrawn on 5th and 7th days using sterile pipette into sterile tubes.
Protease activity is determined by incubating the enzymatic broth with 3% casein solution for 30min. the material of tyrosine in present in the mixture was estimated by colorimetriy a 400nm by extracting it using trichloroacetic acid and approximately dilute it . the O.D reading is converted it to proteasecunits (caseinase unit) from standard graph. The value are tabulated.

Production by immobilized cells:
A 4% sodium alginate the carrier solution in prepared and sterilized. Similarely 0.5% M calcium chloride solution was prepared and sterilized. A slant of protease producing organism is taken and spores scrapped gently in to sterilized water. The spores suspension of the slants is transferred in to 50ml of sodium alginate solution and mix uniform on a rotary shaker for 30 min, care should take to avoid clumps. This spore containing alginate solution is taken in sterile glass syringe fitted with 21 gauge and drops of alginate is transfer slowly in to CaCl2 solution to give beads of the culture.
The beads were carefully prepared adjusting the distance between the needle tip and surface of calcium chloride solution. The beads were slightly agitated in calcium chloride solution.
About 100-150 beads is transfer into 100ml of production medium and incubated at 280c for 5-7 days on rotary shaker. Samples were withdrawn on 5th and 7th days.
Protease activity was determined as mentioned before in protease units. Results were tabulated. Beads size is determined by measuring diameter of 50 beads and calculating the mean diameter.
Beads stability:Disperse a known no. of bead in various concentrations of CaCl2 solution and shake than on rotary shaker. Note the No. of disintegrated beads at regular interval of time.


Result: Difference in tyrosine concentration of immobilized cell is 37 and free cell concentration is 10.
Interpretation: From the obtained data we interpret that difference in tyrosine concentration and casein activity of immobilized cell is higher than free cell.