Information on EC 3.4.21.96 - Lactocepin

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The expected taxonomic range for this enzyme is: Bacteria

EC NUMBER
COMMENTARY hide
3.4.21.96
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RECOMMENDED NAME
GeneOntology No.
Lactocepin
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Endopeptidase activity with very broad specificity, although some subsite preferences have been noted, e.g. large hydrophobic residues in the P1 and P4 positions, and Pro in the P2 position. Best known for its action on caseins, although it has been shown to hydrolyse hemoglobin and oxidized insulin B chain
show the reaction diagram
endopeptidase
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of peptide bond
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CAS REGISTRY NUMBER
COMMENTARY hide
205510-58-3
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
subsp. lactis CRL 581
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Manually annotated by BRENDA team
strain Zuc2, from natural whey culture for Grana Padano cheese
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Manually annotated by BRENDA team
subsp. paracasei NCDO 151
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Manually annotated by BRENDA team
strains BGT43, BGT51-K, BGT54-K, BGT110, BGT266, BGT389, BGT390, BGT391, BGT392, BGT393, BGT394, BGT805, BGRI7, BGZL7, BGZL18, BGZL19, BGZL20, ATCC7469, CRL1036, CRL536 and EN1
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Manually annotated by BRENDA team
strain AM2
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Manually annotated by BRENDA team
strain C13
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Manually annotated by BRENDA team
strain E8
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Manually annotated by BRENDA team
strain FD21
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Manually annotated by BRENDA team
strain H61
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Manually annotated by BRENDA team
strain HP
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Manually annotated by BRENDA team
strain K14
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Manually annotated by BRENDA team
strain S4, commercial cheese starter culture, group h lactocepin
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Manually annotated by BRENDA team
strain UC317
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Manually annotated by BRENDA team
strain US3
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Manually annotated by BRENDA team
strain Wg2
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Manually annotated by BRENDA team
strain Z8
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
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compared to wild-type, protease PrtP knockout mutants show higher bacteriocin activity that has lost its growth-medium dependence. Lack of PrtP stimulates lactococcin LcnB activity and interdependence of LcnB activity and casitone concentration is lost
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
alpha(S1)-casein + H2O
?
show the reaction diagram
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?
alpha1-casein + H2O
Fragments of alpha1-casein
show the reaction diagram
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-
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-
?
alpha1-casein + H2O
fragments of alphas1-casein
show the reaction diagram
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-
-
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?
alphas1-Casein + H2O
Fragments of alphas1-casein
show the reaction diagram
alphas1-Casein fragment 1-23 + H2O
Fragments of alphas1-casein fragment 1-23
show the reaction diagram
beta-Casein + H2O
Fragments of beta-casein
show the reaction diagram
casein + H2O
?
show the reaction diagram
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nitrogen nutrition mechanism of lactic streptococci, process in cheese ripening
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casein + H2O
hydrolyzed casein
show the reaction diagram
Insulin B-chain + H2O
Fragments of insulin B-chain
show the reaction diagram
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-
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kappa-Casein + H2O
Fragemnts of kappa-casein
show the reaction diagram
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-
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kappa-casein + H2O
fragments of kappa-casein
show the reaction diagram
Methoxysuccinyl-Arg-Pro-Tyr 4-nitroanilide + H2O
?
show the reaction diagram
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide + H2O
N-succinyl-Ala-Ala-Pro-Phe + 4-nitroaniline
show the reaction diagram
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synthetic substrate
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?
Suc-Ala-Glu-Pro-Phe-p-nitroanilide + H2O
?
show the reaction diagram
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-
-
?
succinyl-Ala-Ala-Pro-Phe 4-nitroanilide + H2O
succinyl-Ala-Ala-Pro-Phe + 4-nitroaniline
show the reaction diagram
succinyl-Ala-Ala-Pro-Phe-p-nitroanilide + H2O
?
show the reaction diagram
Succinyl-Ala-Glu-Pro-Phe 4-nitroanilide + H2O
?
show the reaction diagram
Succinyl-Ala-Gly-Pro-Phe 4-nitroanilide + H2O
?
show the reaction diagram
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-
-
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Succinyl-Ala-His-Pro-Phe 4-nitroanilide + H2O
?
show the reaction diagram
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-
-
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Succinyl-Ala-Ile-Pro-Phe 4-nitroanilide + H2O
?
show the reaction diagram
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-
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Succinyl-Ala-Leu-Pro-Phe 4-nitroanilide + H2O
?
show the reaction diagram
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-
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Succinyl-Ala-Lys-Pro-Phe 4-nitroanilide + H2O
?
show the reaction diagram
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-
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Succinyl-Ala-Phe-Pro-Phe 4-nitroanilide + H2O
?
show the reaction diagram
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-
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Succinyl-Ala-Trp-Pro-Phe 4-nitroanilide + H2O
?
show the reaction diagram
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Usp 45 + H2O
?
show the reaction diagram
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
casein + H2O
?
show the reaction diagram
-
nitrogen nutrition mechanism of lactic streptococci, process in cheese ripening
-
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casein + H2O
hydrolyzed casein
show the reaction diagram
-
-
-
?
Usp 45 + H2O
?
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
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triggers stabilization, removal of weakly bound Ca2+ from the native cell-bound enzyme is coupled with a significant reversible decrease in specific activity and a dramatic reversible reduction in thermal stability
Cd2+
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can replace Ca2+
additional information
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Mn2+ is much less efficient, Zn2+, Mg2+, Ba2+, Ni2+, Co2+, Cu2+, and Sn2+ have no significant effect
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3,4-dichloroisocoumarin
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complete and irreversible inhibition with 0.1 mM
alpha-casein
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maximum activity is observed in a basal minimal defined medium, whereas in the presence of alpha-casein CEP activity is inhibited 1fold
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beta-casein
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maximum activity is observed in a basal minimal defined medium, whereas in the presence of beta-casein CEP activity is inhibited 1.2fold
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branched-chain amino acids
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maximum activity is observed in a basal minimal defined medium, whereas in the presence of branched-chain amino acids CEP activity is inhibited 1.7fold
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casamino acid
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maximum activity is observed in a basal minimal defined medium, whereas in the presence of casamino acid 1% (w,v) or 0.2% (w,v) CEP activity is inhibited 70fold or 20fold, respectively
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casitone
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maximum activity is observed in a basal minimal defined medium, whereas in the presence of casitone 1% (w,v) or 2% (w,v) CEP activity is inhibited 99fold or 51fold, respectively
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glycerol
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maximum activity is observed in a basal minimal defined medium, whereas in the presence of glycerol CEP activity is inhibited 1.3fold
leucylglycylglycine
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maximum activity is observed in a basal minimal defined medium, whereas in the presence of leucylglycylglycine 1 mM or 5 mM, CEP activity is inhibited 6.5fold or 12.9fold, respectively
leucylleucine
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maximum activity is observed in a basal minimal defined medium, whereas in the presence of leucylleucine 1 mM or 5 mM, CEP activity is inhibited 7.2fold or 15.1fold, respectively
leucylproline
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maximum activity is observed in a basal minimal defined medium, whereas in the presence of leucylproline 1 mM or 5 mM, CEP activity is inhibited 4fold or 8.8fold, respectively
low-molecular-mass peptides
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maximum activity is observed in a basal minimal defined medium, whereas in the presence of low-molecular-mass peptides 1% (w, u) CEP activity is inhibited 99fold
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NaCl
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maximum activity is observed in a basal minimal defined medium, whereas in the presence of NaCl 0.25%-1% (w, u) CEP activity is inhibited 1.4fold
Pepstatin
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1 mM inhibits approximately 10%, enzyme activity is almost completely restored after inhibitor removal
PMSF
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complete and irreversible inhibition with 1 mM
prolylleucine
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maximum activity is observed in a basal minimal defined medium, whereas in the presence of prolylleucine 1 mM or 5 mM, CEP activity is inhibited 3.5fold or 9.9fold, respectively
yeast extract
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maximum activity is observed in a basal minimal defined medium, whereas in the presence of yeast extract CEP activity is inhibited 68fold
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additional information
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no inhibition with the thiol-proteinase inhibitor E-64
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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the CEP activity levels are controlled by the peptide content of the growth medium. The maximum activity is observed in a basal minimal defined medium
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.8 - 6
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Ca-free enzyme
6.4
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Ca-loaded enzyme
6.5
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efficient degradation of beta-casein
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.8 - 7
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Ca-free enzyme
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
additional information
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in comparison to other CEPs, the PrtP proteinase does not have the same mechanism of release from the cell surface
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Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
72000
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SDS-PAGE
145000
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Ca-free enzyme, gel filtration
150000
180000
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mature active enzyme
250000
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gel filtration
300000
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gel filtration, enzyme pro135
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
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1 * 110000, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
construction of a model structure based on the crystal structure of Streptococcus C5a cell envelope proteinase, which is used to investigate the amino acid positions in the substrate-binding region. Amino acid positions are suggested, which can be relevant for the cleavage specificity
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
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Ca-free cell-bound enzyme is extremely unstable, lose 99% of its potential activity within 20 min
37 - 60
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retains 40% of activity when incubated for 2 h at 60°C
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
0°C, Ca-free enzyme, storage on ice results in a gradual loss of potential activity, 20% reduction in activity after 4 h
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by gel filtration and ion-exchange chromatography, 527.42fold
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant MG 1363 strains containg plasmid-located hybrid proteinase genes constructed from SK11 and Wg2
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stain SK11, cloned in Lactococcus lactis strains MG1363 and SK1128
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subsp. lactis MG611, carrying the genes prtP and prtM of subsp. cremoris Wg2 on its chromosome
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the enzyme of subsp. cremoris WG2 and strain SK11 are plasmid-encoded, the enzyme of subsp. cremoris BC101 is chromosomally -encoded
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
conversion of lactocepin substrate binding regions by allele exchange can effectively alter lactocepin specificity in industrial strains of Lactococcus lactis without significantly affecting other important strain properties. The methodology can be used to alter lactocepin specificity in commercial starter cultures with a propensity for bitter flavour defect; conversion of lactocepin substrate binding regions by allele exchange can effectively alter lactocepin specificity in industrial strains of Lactococcus lactis without significantly affecting other important strain properties. The methodology can be used to alter lactocepin specificity in commercial starter cultures with a propensity for bitter flavour defect
industry
nutrition
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cheese making, cheese starter organism, dairy industry
additional information
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exposure of PrtP, and not its proteolytic activity, is responsible for greater cell hydrophobicity and adhesion. The increased bacterial affinity to polar and apolar solvents indicates that exposure of PrtP on lactococcal cell surface can enhance the capacity to exchange attractive van der Waals interactions, and consequently increase their adhesion to different types of solid surfaces and solvents. PrtP or its derivatives may be used as a tool to construct strains with increased adhesion that form protective biofilms
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