1. The protein says
We are the basis of structure
and function of life
Composed of twenty amino acids
the building blocks;
Organized into primary,
secondary, tertiary, quaternary
structure and classified as
simple, conjugated and derived
proteins.

2. Proteolytic systems of
Lactic Acid Bacteria
P. K. Choudhury
National Dairy Research Institute
Karnal, Haryana, 132001

3. The Proteolytic System………
Three component system
• The cell envelop-associated proteinases
(CEPs)
• Peptide transporters
• Intracellular peptidases

4. 1. The cell envelop associated
proteinase (CEPs)

5. From the N terminus the CEPs include:
a) Pre prodomain (PP) : corresponding to a signal sequence (∼40 residues)
required for secretion & pro sequence (∼150 residues) that is removed
by autocatalytic processing
b) Catalytic serine protease domain (PR) :(∼500 residues)
c) Insert domain (I) : (∼150 residues) modulates the substrate specificity of
CEPs
d) A domain :(∼400 residues) of unknown function;
e) B domain (∼500 residues) involved in stabilizing the CEP
activity/specificity;
f) Helix domain (H) :(∼200 residues) involved in positioning the A and B
domains outside the bacterial cell
g) Hydrophilic W domain :(∼100 residues) functioning as a cell wall spacer.

6. Cell envelope associates proteinases of different
LAB strains
CW -Cell wall, M -membrane , C cytoplasm, PP- pre prodomain, PR- Catalytic
domain, I- insert domain, A- A domain, B- B domain, H- helix domain, W- Cell wall
spacer domain, black dot -sorting signal, and AN -anchor domain
Siezen, R. J., 1999

7. • Degrades the protein into oligopeptides that are subsequently
taken up by the cells via specific peptide transport systems.
• Five different types characterized from LAB, including
Prt P - Lc. lactis and Lb. paracasei,
PrtH - Lb. helveticus,
PrtR - Lb. rhamnosus,
PrtS - S. thermophilus,
PrtB - Lb. delbrueckii subspp. bulgarius.
• Chromosomally encoded (Lactococci, prtP genes can either
be plasmid or genome encoded)
• Synthesized as pre-pro proteins of approximately 2,000
residues and are composed of several distinct functional
domains

8. • prtP gene is preceded by a divergently transcribed gene
encoding a membrane-bound lipoprotein (PrtM)
• PrtM has shown to be essentialRemoval of pro
for autocatalytic maturation of
sequence
Prt P
Preproteinase (PrtP) Mature PrtP
Prt M
• Both PrtP and PrtM are inducible.
• PrtP acts on C-terminal of β-casein, produce oligopeptides
mostly 4-30 amino acids residues

9. • Strong preference for hydrophobic caseins.
• Lactococcus PrtPs are divided into PI- and PIII-type enzymes,
distinguished by their substrate specificity for αS1-, β-, and κ-
caseins
• PI-type primarily degrades β-casein that is cleaved into more
than 100 different oligo- peptides ranging from 4 to 30 amino
acid residues, κ-casein is cleaved to a lesser extent by the PI-
type enzyme
• PIII-type is able to cleave αS1-, β−, and κ-caseins equally well

10. 2. Peptide transport system

11. • Transport substrates of varying size, polarity and structure.
• Some highly specific, some have broad specificity.
• Vary in use of energy as fuel for active transport.
Peptide
transporter
Oligo peptide
Transporter DtpP DtpT
system

12. a) Oligo-peptide Transport System
• Belong to a super family of ATP-binding cassette transporters
that mediate the uptake of casein derived peptides
• Necessary for growth in milk.
• Operon of OPP oppDFBCA (5 subunits)
2 Transmembrane proteins : (Opp B and Opp C )
2 ATP binding protein : ( Opp D and Opp F )
A membrane-linked substrate binding protein(Opp A)

13. b) Dipeptide tripeptide transport system
DtpP: DtpT
• Transport di and tri peptides • Transport di and tri-peptides
• ATP dependent transporter • PMF dependent transporter
• Have high affinity peptides • Have high affinity for
having hydrophobic hydrophilic charged di and
branched chain amino tripeptides
highest affinity for tri-
peptides.
• Milk is necessary
• Not necessary for growth in
milk

14. 3. Intracellular Peptidases

15. • Intracellular enzyme
• Cause peptidolytic cleavage of accumulated
peptides
• 3 types
1. Endopeptidases
2. Dipeptidases and Tripeptidases
3. Aminopeptidases

16. Endopeptidases
• Metalloenzymes that contain sequence typical of zinc-binding
domains.
• Hydrolyze oligopeptides of varying length as substrates.
• Have pH optima in alkaline range (7.5 – 9.0) (very low
activity at pH <6)
Dipeptidases and Tripeptidases
• Hydrolyze Dipeptides & Tripeptides formed from oligopeptides
• Substrate specific

17. Aminopeptidases
• Hydrolyze N-terminal peptides bonds and releases N-terminal
amino acids
• Most widespread peptidases in LAB
• Some specifically hydrolyze proline-containing peptides (e.g.
PepA, PepX, PepP, PepR, PepI)

21. Overall……

22. 1. Cell envelope-associated proteinase
= amino acid
Oligopeptides Di- and tri- Free amino
(4 – 18 a.a.’s) peptides acids (f.a.a.)
OUT
IN

23. Amino acid and peptide transport systems
Oligopeptides Di- and tri-
(4 – 18 a.a.’s) peptides Amino acids
OUT
Di-, tri- AA
Opp transporter Permease transporter
ATP
IN H
+
ADP
Oligopeptide permease (Opp) Di- and tri-peptide A.A. transporters are
is a member of the ATP- transporters: ABC specific to an A.A. or a
binding cassette (ABC) transporters or group of A.A.s
transporter class of symport (with proton)
transporters

24. 3. Intracellular peptidases
Permease
ATP
H
+
IN ADP
Aminopeptidases
Endopeptidases Dipeptidases
Tripeptidases
Dipeptidases/ Aminopeptidases
Tripeptidases
Used for making cellular proteins or converted to other compounds

25. Regulation of the proteolytic
system

26. Regulation of the proteolytic systems

27. Technological aspects of proteolysis
• Products of Proteinases and peptidases impart cheese
flavor and texture.
• PrtP produce bitter peptides
• Free amino-acids and small peptides contribute
“nutty and sweet flavor” ( Swiss, Parmesan Cheese)
• Products of free amino acid catabolism contribute
flavor Cheddar cheese
• Degradation products from amino acids e.g.
methanethiol and sulfur containing compounds
essential for flavoring surface ripened cheeses.

28. Selection of strains having the ability to
degrade the
Bitter peptides
and to produce flavor
compounds…………

29. Proteolytic system summary
LAB. employ an elaborate proteolytic system to get
the amino acids they require for growth.
Cell envelope-associated protease
Peptide and amino acid transporters
Peptidases
LAB proteolysis impacts foods and food processing…
Fermentation time
Flavor and aroma
Texture