Trypsin including sources, extraction, purification and applications

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   μ L ) of a solution containing TRP 1500   μ M and ChTRP 1500   μ M ( 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 ( K pTRP and K pChTRP ) 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 K p 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 K p 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