The document discusses the function and classification of proteases, highlighting their roles in hydrolyzing peptide bonds and their various types including serine, cysteine, aspartic, and metalloproteases. It details the activation mechanisms for trypsin and chymotrypsin, as well as their applications in industries such as food, pharmaceuticals, and leather. Additionally, it outlines the environmental benefits of using enzymes over traditional methods in industry.

1. Muhammad Tahir Hayat
03-BH-BT-2017
GC University, Lahore

2. Definition:-
• catalyze the hydrolytic cleavage of peptide bonds
• Hydrolase
• trypsin(EC 3.4. 21.4)
• EC Tree
• 3 Hydrolases
• 3.4 Acting on peptide bonds (peptidases)
• 3.4.21 Serine endopeptidases
• 3.4.21.4 trypsin

3. Activation of Proteases:
• Zymogens or proenzymes
• no energy (ATP) is required.
• irreversible.
Chymotrypsin:
* hydrolyzes in the small intestine.
*chymotrypsinogen in pancreas (acinar cells).
*converted into a fully active enzyme when the
peptide bond joining Arg15 and Ile 16 is
cleaved by trypsin to form π - Chymotrypsin
• π - Chymotrypsin subsequently undergoes
autolysis to specifically excise two dipeptides,
Ser 14–Arg 15 and Thr147–Asn 148, yielding
active enzyme α- chymotrypsin.

4. Trypsin:
2 stages
• Enteropeptidase in the duodenal
mucosa, hydrolyzes trypsinogen’s
Lys 15-Ile 16 peptide bond at its N
terminal end, forming an
hexapeptide.
• Formed small amount of trypsin
cleaves trypsinogen to form its
active form. I.e. tripsinogen
activation is autocatalytic

5. Trypsin activates other proteases:

6. Papain
*active at pH 5-9
*stable to 80℃ to 90℃
Bromelain
*stem and juice of pineapples
(Trypsin, Chymotrypsin, Pepsin, Rennins)
Keratinases
* hair and wool degradation
* (Neutral and alkalaine)
* (Acid,neutral and alkaline)
* (Serine, Aspartic, and Cysteine )

7. Classification :
*
• acts at free N-Terminus of polypeptide
chain
• a single AA residue, a dipeptide, or a
tripeptide
• Examples
– Aminopeptidase I &
– Aminopeptidase II
*
– act at C terminals
– single AA or a dipeptide
Endopeptidases
 Serine protease
 Cysteine protease
 Aspartic protease
 Metalloprotease

8. Classification:
.
1. Serine Proteases
*serine group in active site,
* by using serine alcohol
Serine alkaline proteases '
(pH 10,Iso electric point 9)
e.g.Subtilisins
Bacillus,
pH 10

9. cysteine thiol, catalytic dyad (Cys-His or His-Cys), active in reducing agents e.g., HCN or
cysteine, neutral pH optima,susceptible to sulfhydrylagents e.g,
3. Aspartyl proteases:aspartate carboxylic acid, acidic proteases, three families,
namely, pepsin(A1),retropepsin(A2),andenzymes from pararetroviruses (A3) (13),pH 3 to
4, Ip 3 to 4.5, bilobal structure of pepsin, pepstatin,
4. Metalloproteases:divalent metal ion e.g, Zn2+,collagenases,

10. Mechanism of Proteases:
• One step Catalysis:
• activates H2O to act as
nucleophile.
• Two steps Catalysis:
• a residue within the
enzyme is activated to act
as a nucleophile (Nu) and
attack substrate.

11. Mechanism:

12. • Protein Turnover:balance between protein synthesis and protein degradation. ATP dependent protease La
(Lon gene product) for hydrolysis of abnormal proteins in E.coli.
• Germination:dormant spores lack aa, serine endoproteinsases make available,
• Nutrition: Pepsin, trypsin, chymotrypsin → Absorption by cell

13. • Detergents:
pI for best performance of protease, pI concides with pH. eg., Esperase and Savinase T (Novo Industry) from alkalophilic Bacillus sp.
high pI,
*25% of total sale of enzymes.
*1913,Burnus consisted of Na2CO3 +Crude pancreatic extract.
*1956, BIO-40 first detergent containing bacterial enzyme.
*13 billion tons per year production
*Currently used in market are Serine Proteases by Bacillus strains
• Leather Industry:
*Soaking (mic.alkaline proteases)→dehairing (alkaline proteases+hydrated lime+NaCl)→bating (trypsin)→tanning
*Skin+hair are proteinaceous
*conventional methods: sodium sulfide→pollution
*use of enzymes→improve leather quality + reduce pollution+save energy
• Food Industry→
1) Dairy Industry→Rannet from 4th stomach of calves contains chymosin acts on casein → cheese making
2) Baking Industry→gluten, dough quality, A.oryzae.
3) Soy Products→soy sause+soy products akaline and neutral protases from fungi,
Soy proteins + Alcalase at pH 8→ Soluble hydrolysate ( as feeds+drinks)
Aspartame→dipeptide(L-aspartic acid+L-phenylalanine) B.thermoprotyolyticus

14. Pharmaceutical Industry:
• Burns and wounds treatment:
*Subtilisin+ broad spectrum antibiotics
• Treatment of tumors:
1)L-asparaginase administration →extracellular asparagine to aspartic acid and
ammonia.(lymphocytic leukemia) Asparginase from E.coli
2)L-glutaminase: hydrolyses L-glutamine to L-glutamic acid and ammonia
• Digestive Aid: (to correct lytic enyme deficiency syndrome)→ A.oryzae (Luizym and Nortase)
• Augmenting surgery:
*injection of trypsin in 1980 By Dr. J.Spina to remove clouded lens (cataract) by liquefying it.
*cut 2.5cm → 0.3 or 0.025

15. References:
• Kulkarni N, Shendye A, Rao M. Molecular and biotechnological aspects of
xylanases. FEMS Microbiol Rev. 1999;23(4):411–56.
• Chanalia P, Gandhi D, Jodha D, Singh J. Applications of microbial proteases
in pharmaceutical industry: An overview. Rev Med Microbiol.
2011;22(4):96–101.
• Pratush A, Gupta A, Bhalla TC. Microbial proteases: prospects and
challenges. Microbiol Appl. 2013;(June 2016):30–48.