Proteases: Introduction, activation, catalytic mechanism, different sources (Plants,animals and microbial), physiological functions,industrial applications and clinical applications. References Institute of Industrial Biotechnology, Government College University, Lahore (Pakistan)

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.