The protein says We are the basis of structure and function of lifeComposed of twenty amino acids the building blocks; Organized into primary, secondary, tertiary, quaternary structure and classified as simple, conjugated and derived proteins.
Proteolytic systems of Lactic Acid Bacteria P. K. Choudhury National Dairy Research Institute Karnal, Haryana, 132001
The Proteolytic System……… Three component system• The cell envelop-associated proteinases (CEPs)• Peptide transporters• Intracellular peptidases
1. The cell envelop associated proteinase (CEPs)
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.
Cell envelope associates proteinases of different LAB strainsCW -Cell wall, M -membrane , C cytoplasm, PP- pre prodomain, PR- Catalyticdomain, I- insert domain, A- A domain, B- B domain, H- helix domain, W- Cell wallspacer domain, black dot -sorting signal, and AN -anchor domain Siezen, R. J., 1999
• 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
• 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
• 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
• 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
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)
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
• Intracellular enzyme• Cause peptidolytic cleavage of accumulated peptides• 3 types 1. Endopeptidases 2. Dipeptidases and Tripeptidases 3. Aminopeptidases
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
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)
Amino acid and peptide transport systems Oligopeptides Di- and tri- (4 – 18 a.a.’s) peptides Amino acidsOUT Di-, tri- AA Opp transporter Permease transporter ATPIN H + ADPOligopeptide permease (Opp) Di- and tri-peptide A.A. transporters areis a member of the ATP- transporters: ABC specific to an A.A. or abinding cassette (ABC) transporters or group of A.A.stransporter class of symport (with proton)transporters
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
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.
Selection of strains having the ability to degrade the Bitter peptides and to produce flavor compounds…………
Proteolytic system summaryLAB. employ an elaborate proteolytic system to getthe amino acids they require for growth. Cell envelope-associated protease Peptide and amino acid transporters PeptidasesLAB proteolysis impacts foods and food processing… Fermentation time Flavor and aroma Texture