INTRODUCTION PROPERTIES FUNCTION MECHANISM SOURCES EXTRACTION AND PURIFICATION PHARMACEUTICAL , THERAPEUTIC AND CLINICAL APPLICATIONS REFERENCES

1. trypsin
By
ZAHID YASEEN
BRANCH : PHARMACEUTICAL BIOTECHNOLOGY
ROLL NO. : 143/MPB/SPS/2020
Govt. of NCT of Delhi
DELHI PHARMACEUTICAL SCIENCES AND
RESEARCH UNIVERSITY
Pushp Vihar, Sect-III, New Delhi-110017

2. CONTENTS
INTRODUCTION
PROPERTIES
FUNCTION
MECHANISM
SOURCES
EXTRACTION AND PURIFICATION
PHARMACEUTICAL , THERAPEUTIC AND CLINICAL
APPLICATIONS
REFERENCES

3. INTRODUCTION
 Trypsin is a serine protease , found in the digestive
system of many vertebrates, where
it hydrolyzes proteins.
 Trypsin is formed in the small intestine when
its proenzyme form, the trypsinogen produced by
the pancreas, is activated.
 The process is commonly referred to as
trypsin proteolysis or trypsinisation, and proteins that
have been digested/treated with trypsin are said to
have been trypsinized.
 Trypsin was discovered in 1876 by Wilhelm Kühne and
was named from the Ancient Greek word for rubbing
since it was first isolated by rubbing the pancreas
with glycerin.

4. Trypsin cuts peptide chains mainly at
the carboxyl side of the amino
acids lysine or arginine.

5. PROPERTIES
 Human trypsin has an optimal operating temperature of about
37 °C. In contrast, the Atlantic cod has several types of trypsins for
the poikilothermic fish to survive at different body temperatures.
 As a protein, trypsin has various molecular weights depending on the
source. For example, a molecular weight of 23.3 kDa is reported for
trypsin from bovine and porcine sources.
 Trypsin should be stored at very cold temperatures (between −20
and −80 °C) to prevent autolysis, which may also be impeded by
storage of trypsin at pH 3 or by using trypsin modified by reductive
methylation. When the pH is adjusted back to pH 8, activity returns.

6. FUNCTION
 In the duodenum , trypsin catalyzes the hydrolysis of peptide bonds,
breaking down proteins into smaller peptides.
 Trypsin is produced as the inactive zymogen trypsinogen in the
pancreas. When the pancreas is stimulated by cholecystokinin, it is then
secreted into the first part of the small intestine (the duodenum) via
the pancreatic duct.

7. MECHANISM
X-ray crystallographic structure of
Trypsin
The function of Trypsin is to break down peptides using a
hydrolysis reaction into amino acid building blocks. This
mechanism is a general catalytic mechanism that all Serine
proteases use. The active site where this mechanism occurs
in Trypsin is composed of three amino acids and called a
catalytic triad. The three catalytic residues are Serine 195,
Histidine 57, and Aspartate 102 . In the mechanism, serine is
bonded to the imidazole ring of the histidine. When histidine
accepts a proton from serine an alkoxide nucleophile is
formed. This nucleophile attacks the substrate when the
substrate is present. The role of the aspartate residue is to
hold histidine in the proper position to make it a good
proton acceptor. What makes this mechanism works is that a
pocket if formed from the three residues and the three
residues function to hold each other in proper position for
nucleophilic attack. The steps of the mechanism involve two
tetrahedral intermediates and an Acyl-enzyme intermediate .

8. SOURCES
BOVINE PANCREAS
PORCINE PANCREAS
DOG PANCREASE
MICROBES LIKE Streptomyces griseus

9. EXTRACTION AND PURIFICATION
 Extraction of trypsin from bovine pancreas by applying
polyethyleneglycol/sodium citrate aqueous two-phase systems
 aqueous two-phase systems (ATPSs) have become an attractive
separation technology due to the mild conditions and the easy
scaling up to an industrial level.
 Due to the low cost of the chemicals forming phases, polymer–salt
systems have been preferred as a first step for practical reasons.
 polyethyleneglycol/ sodium citrate (PEG/NaCit) ATPSs could be
employed as a viable and potentially useful tool for separating
proteins

10. Chemicals
Trypsin, alpha-chymotrypsin from bovine pancreas,
polyethyleneglycols of average molecular masses: 600,
1000, 1450, 3350 and 8000 (PEG600, PEG1000, PEG1450,
PEG3350, PEG8000), -N-benzoyl-dl-arginine-p-nitroanilide
(BAPNA) and N-benzoyl-L- tyrosine ethyl ester (BTEE) were
purchased from Sigma Chem. Co. and used without further
purification. All the other reagents were of analytical quality.

11. Preparation of pancreatic homogenate
Pancreas was removed from freshly killed bovine and stored
at −80 ◦C. When using, pancreatic tissue was thawed, cut in
small pieces and washed exhaustively with buffer Tris–HCl
100mM pH 6.80, containing 100mM NaCl. Then, it was
settled with three volumes of washing solution in a
homogenizer for 5 min. The resultant homogenate was
centrifuged for 5 min at low rate (2500 rpm), and then, the
supernatant was filtered through a cheese cloth, thus
obtaining the working extract.

12. Preparation of the aqueous biphasic
system
To prepare the biphasic aqueous systems, stock solutions of
the phase components: PEG of different molecular weight
40% (w/w) and sodium citrate 20% (w/w) of a given pH were
mixed accordingly . The desired pH (5.20 and 8.20) of the
sodium citrate solution was adjusted by the addition of
sodium hydroxide . Low-speed centrifugation was used after
a thorough gentle mixing of the system components to
speed up phase separation, then 1 g of each phase Was
mixed to reconstitute several two-phase systems in which
the protein partition was assayed.

13. Extraction process
 TRP extraction with ATPS was applied on both an artificial mixture (standard) and a pancreatic
homogenate. A given amount (10–14 μL) of a solution containing TRP 1500 μM and ChTRP
1500 μM (artificial mixture) was partitioned in the selected two-phase systems containing 1 g of
each equilibrated phase. The partitioning behavior of both enzymes when formed the mixture
was evaluated by measuring their partitioning coefficients (KpTRP and KpChTRP)
 where [P]T and [P]B are equilibrium concentrations of the partitioned protein in the PEG- and
citrate-rich phases, respectively

14. RESULTS AND DISCUSSION
We had observed that both proteins showed a
decrease in their Kp value as the PEG molecular
weight increased, due to an increase in the polymer
hydrophobic character
On the other hand, for most of the assayed systems
an increase in the Kp values was observed when pH
increased from5.20 to 8.20

15. Pharmaceutical ,therapeutic and clinical
applications
 Trypsin is given to people who lack enzymes needed for
digestion.
 Trypsin can also be used to dissolve blood clots in its
microbial form and treat inflammation in its pancreatic
form . It is also given in combination with bromelain and
rutin for treatment of osteoarthritis.
 Some people apply trypsin directly to wounds and ulcers
to remove dead tissue and improve healing.
 In a tissue culture lab, trypsin is used to re-suspend cells
adherent to the cell culture dish wall during the process
of harvesting cell.
 Trypsin is commonly used in biological research
during proteomics experiments to digest proteins into
peptides for mass spectrometry analysis, e.g. in-gel
digestion.

16. Other applications
 Commercial protease preparations usually consist of a mixture of various
protease enzymes that often includes trypsin. These preparations are widely
used in food processing.
 as a baking enzyme to improve the workability of dough
 in the extraction of seasonings and flavorings from vegetable or animal
proteins and in the manufacture of sauces
 to control aroma formation in cheese and milk products
 to improve the texture of fish products
 to tenderize meat
 during cold stabilization of beer
 in the production of hypoallergenic food where proteases break down
specific allergenic proteins into non allergenic peptides, for example, proteases
are used to produce hypoallergenic baby food from cow's milk, thereby
diminishing the risk of babies developing milk allergies.

17. REFERENCES
Extraction of trypsin from bovine pancreas by applying
polyethyleneglycol/sodium citrate aqueous two-phase
systems by Gisela Tubio, Guillermo A. Picó, Bibiana B. Nerli
Journal of Chromatography B, 877 (2009) 115–120
https://en.wikipedia.org/wiki/Trypsin
https://proteopedia.org/wiki/index.php/Trypsin

18. Thank you