1.

CENTRAL POWER RESEARCH INSTITUTE: From June 2010 - Till Date

Graduate Apprentice Trainee in UNIDO Project for Testing Polychlorinated Bipenyl

(PCB’s in Transformer oil using GC-MS/ECD, GC-MS/MS, Polychlorinated Bipenyl
(PCB’s) is Persistent Organic Pollutant (POPs) which causes

2. Sartorious Stedim Biotech: From Jun 2010 -€“ Till Date

Internship for a period of 6 months as Design Engineer:

A. Vessel and Heat Exchanger Design of of the bio- equipment like Bioreactor, Cross
flow filtration system, Harvest filtration system and Inactivation vessel.

B. Design of Piping and instrumentation (P& ID) of the bio- equipment like Bioreactor,
Cross flow filtration system, Harvest filtration system and Inactivation vessel.

Production, Purification and Characterization of Tannase from Serratia ficaria

1. Isolation of Pure culture of Serratia ficaria for The Preparation of inoculum.

Contamination of cell culture by Fungi .

2. Preparation of inoculum of Serratia ficariaI and Production

In MPSM medium having Tannic acid and hydrosylate as Carbon Source, Serratia
ficaria have retarded growth

3. Extraction of Extra-cellar and Cell- Associated Tannase for Characterization of

Total Tannase Production.

Release of cell Associated Tannase By

A. Sonication: cell wall associated enzymes not released
B. Cell Permeabilization using surfactants and solvents which Deactivated tannase
producted
 C. Cell Lysis using lysozyme could not able lyse the cell.

2. Background to the problem (300 words)

A. Isolation of Pure culture of Serratia ficaria for The Preparation of inoculum.

Contamination of cell culture by Fungi Particular by Aspergillus Sp because fungi
have less generation time when compared to Bacteria. Also Aspergillus Niger is able
to utilize Tannic acid as sole Carbon source. Since Aspergillus to Produce Tannase
which hydrolysis Tannic acid to Gallic acid and glucose.

B. Preparation of inoculum of Serratia ficaria and Production.

Retarded Growth of Serratia ficaria in MPSM medium having Tannic acid and
hydrosylate as Sole Carbon Source is due that Tannic acid can be utilized the Serratia
ficaria only when Tannase hydrolysis Tannic acid to Gallic acid and glucose. But
Tannase Tannase Tannase is an adaptive enzyme or inducible enzyme. Inducible
enzymes those which are expressed only under conditions in which it is clear of
adaptive value. Since Tannase is an adaptive enzyme, Tannase well be produced only
when glucose is not available. Although a small quantity of glucose has to be added
for initial growth for inoculum preparation. As Microbes required some time adjust to
new environment. So Media have both Glucose (0.1%) for Growth and Tannic Acid
(2%) to reduce the batch time the production.
3. Extraction of Extra-cellar and Cell- Associated Tannase for Characterization of
Total Tannase Production.

Release of cell Associated Tannase By

A. Sonication:
Sonication of cell is carried out to release tannase out of cell; sonication is done at
different time intervals from. Disruption of crude cells by sonication showed no
release of tannase from cell and tannase activity decreased with increase in sonication
time, finally became zero at 60 min of sonication as cell associated tannase may be
disrupted in its protein structure by sonication.

B. Cell Permeabilization :

Cell Permeabilization using surfactants and solvents 1Tween 60, Tween 80, , Sodium
cholate and Sodium tauro cholate , SDS, Toluene and Ethanol. The enzyme activity
was decreased with increase in increasing concentration of Solvent or surfactants
which due Destabilizing the protein structure tannase produced or not able to release
the cell as the tannase is a cell membrane bound enzyme. These Solvent or
surfactants was forming a viscous substance when mixed with the crude enzyme due
to the presence of tannic acid.

C. Cell Lysis using lysozyme could not able lyse the cell as the cell wall of Genus
Serratia contains glycoproteins From Literature survey (Josephc. Tsang . et.al) So
lysozyme cant able lyse cell wall of the Serratia ficaria . Thus the Cell- Associated
Tannase was not released into the media

3. Experiment to address the problem (300 words)

1. Isolation of Pure culture of Serratia ficaria for The Preparation of inoculum.

 Contamination of cell culture by Fungi Particular by Aspergillus Can be Prevent By
adding Antifungal agent like Imidazole, and triazole. Imidazole, and triazole belongs
to Azole antifungal drugs inhibit the enzyme lanosterol 14 α-demethylase; the
enzyme necessary to convert lanosterol to ergosterol. Depletion of ergosterol in
fungal membrane disrupts the structure and many functions of fungal membrane
leading to inhibition of fungal growth.

It is important note that most commonly used antifungal agent are the azole
antifungals such as ketoconazole or itraconazole can be both substrates and inhibitors
of the P-glycoprotein which the structural component of the cell wall of fungus . But
the azole antifungals cannot be used the structural component of the cell wall of
Serratia ficaria is also glycoprotein.

2. Preparation of inoculum of Serratia ficaria and Production.

Retarded Growth of Serratia ficaria in MPSM medium during inoculum and

production can be prevented by adding the tannic used to the media which is used for
the pure culture of Serratia ficaria. Thus inoculum development and Production time
of tannase enzyme can reduce. As the time required by the microbe to adjust to the
new environment can be reduced. Thus the production time is reduced and the
maintenance cost reduces by about 60%.

3. Extraction of Extra-cellar and Cell- Associated Tannase for Characterization of

Total Tannase Production.

As the cell associated tannase can’t be release by Sonication, Cell

Permeabilization and Cell Lysis using lysozyme as mentioned above, which can done by
following methed

A. Triton X-100 as surfactants: From Literature survey (Harrison et.el and Kopecny, J
et.el ), it was found the cell membranes bound can be released using Triton X-100 as
 surfactants without cell lysis the cell-associated tannase without lysing the cell using
Triton X-100 suggests that the tannase enzyme might be bound to the cell
membranes.

B. CELL LYSIS BY NaOH-SDS SOLUABLIZATION

Cell Lysis using lysozyme could not able lyse the cell as the cell wall of Genus Serratia
contains glycoproteins. So lysozyme can’t able lyses cell wall of the Serratia ficaria. Cell
lysis and release of the cell membranes bound tannase can released using
NaOH-SDS soluablization. The principle is that sodium dodecyl sulfate (SDS) is will
dissolve the Glycoprtein in the cell wall of the Serratia ficaria. Thus destabilizes the cell
wall of the Serratia ficaria. This destabilizes the cell wall went treated with alkaline soln
like NaOH, It lysis the Cell Membrane. Thus release of the cell membranes bound
tannase into the media.

The known dry weight pure cell pellets were taken and added with 1 ml of NaOH-SDS
(0.2M, 2.5%); these were incubated in water bath at 1000C for 5 min. The clear
supernatant is collected after centrifugation at 8000 x g for 20 min at 40C. 0.1 ml of
supernatant is used for protein determination using 3.1 assay protocol of Bradford
method. The protein content is expressed in terms of mg per gram dry weight.

Josephc. Tsang, Sandy Tattrie and Dennis kallvy, Outer Cell envelope Glycoprotien
From Two Stains of Serratia marcescens, American Society of Microbiology, Vol 21, p
27-31.

Harrison (1991) had reviewed the action of various surfactants on gram negative bacteria
and concluded that Triton X-100 can solubilize both inner and outer membranes in the
absence of Mg2+ ions. Kopecny, J and John Wallace, R (1982) reported the release of
membrane bound proteolytic enzymes from rumen bacteria using 0.05 % (w/v) Triton X-
100. Release of substantial cell-associated tannase with out lysing the cell using Triton X-
100 suggests that the tannase enzyme might be bound to the cell membranes.

C. Cell Lysis using lysozyme could not able lyse the cell as the cell wall of Genus
Serratia contains glycoproteins. So lysozyme cant able lyse cell wall of the Serratia
ficaria . Thus the Cell- Associated Tannase was not released into the media

4. Expected outcomes and alternative strategy if necessary (100 words)

5. References

(Scientific Referee 1)
Name, Affiliation, Email and Telephone contact

(Scientific Referee 2)
Name, Affiliation, Email and Telephone contact