Information on EC 3.4.22.33 - Fruit bromelain

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The expected taxonomic range for this enzyme is: Bromeliaceae

EC NUMBER
COMMENTARY hide
3.4.22.33
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RECOMMENDED NAME
GeneOntology No.
Fruit bromelain
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Hydrolysis of proteins with broad specificity for peptide bonds. Bz-Phe-Val-Arg-/-NHMec is a good synthetic substrate, but there is no action on Z-Arg-Arg-NHMec (c.f. stem bromelain)
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of peptide bond
CAS REGISTRY NUMBER
COMMENTARY hide
9001-00-7
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
fragment
UniProt
Manually annotated by BRENDA team
another cysteine endopeptidase, with similar action on small molecule substrates, pinguinain (formerly EC 3.4.99.18), is obtained from the related plant, Bromelia pinguin, but pinguinain differs from fruit bromelain in being inhibited by chicken cystatin
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Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
Angiotensin II + H2O
Hydrolyzed angiotensin II
show the reaction diagram
Azoalbumin + H2O
?
show the reaction diagram
-
-
-
-
?
azocasein + H2O
?
show the reaction diagram
-
-
-
-
?
Benzoyl-Gly-Gly-Lys + H2O
?
show the reaction diagram
-
enzyme form FBA is much more active than FBB
-
-
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benzoyl-L-arginine ethyl ester + H2O
?
show the reaction diagram
-
artificial substrate
-
-
?
benzoyl-Phe-Val-Arg-4-methylcoumarin 7-amide + H2O
benzoyl-Phe-Val-Arg + 7-amino-4-methylcoumarin
show the reaction diagram
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convenient substrate for fruit bromelain, scarcely affected by stem bromelain
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-
?
benzoyl-Phe-Val-Arg-p-nitroanilide + H2O
benzoyl-Phe-Val-Arg + p-nitroaniline
show the reaction diagram
-
-
-
-
?
Benzyloxycarbonyl-Ala-Leu + H2O
Benzyloxycarbonyl-Ala + Leu
show the reaction diagram
-
enzyme form FBA is much more active than FBB
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benzyloxycarbonyl-Arg-Arg-p-nitroanilide + H2O
benzyloxycarbonyl-Arg-Arg + p-nitroaniline
show the reaction diagram
-
-
-
-
?
Benzyloxycarbonyl-Gly-Leu + H2O
Benzyloxycarbonyl-Gly + Leu
show the reaction diagram
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enzyme form FBA is much more active than FBB
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Benzyloxycarbonyl-Gly-Phe + H2O
Benzyloxycarbonyl-Gly + Phe
show the reaction diagram
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-
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Benzyloxycarbonyl-Gly-Tyr + H2O
Benzyloxycarbonyl-Gly + Tyr
show the reaction diagram
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Benzyloxycarbonyl-Gly-Val + H2O
Benzyloxycarbonyl-Gly + Val
show the reaction diagram
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enzyme form FBA is much more active than FBB
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benzyloxyxarbonyl-Phe-Arg-7-amido-4-methylcoumarin + H2O
?
show the reaction diagram
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-
-
?
Bradykinin + H2O
Hydrolyzed bradykinin
show the reaction diagram
bromelain + H2O
?
show the reaction diagram
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rapid self-digestion in aqueous solution, can be prevented by addition of alpha2-macroglobulin
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?
Carbobenzoxy-Gly-Ala + H2O
Carbobenzoxy-Gly + Ala
show the reaction diagram
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enzyme form FBA is much more active than FBB
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casein + H2O
?
show the reaction diagram
casein + H2O
hydrolyzed casein
show the reaction diagram
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epsilon-NH2-Cap-Ala-Cys(S-Bzl)-7-amido-4-methylcoumarin + H2O
?
show the reaction diagram
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?
epsilon-NH2-Cap-Cys(S-Bzl)-Leu-7-amido-4-methylcoumarin + H2O
?
show the reaction diagram
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-
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?
epsilon-NH2-Cap-Leu-Cys(S-Bzl)-7-amido-4-methylcoumarin + H2O
?
show the reaction diagram
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-
-
?
epsilon-NH2-Cap-Leu-Gly-7-amido-4-methylcoumarin + H2O
?
show the reaction diagram
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?
epsilon-NH2-Cap-Phe-Arg-7-amido-4-methylcoumarin + H2O
?
show the reaction diagram
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?
epsilon-NH2-Cap-Pro-Cys(S-Bzl)-7-amido-4-methylcoumarin + H2O
?
show the reaction diagram
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?
Gelatin + H2O
?
show the reaction diagram
Hemoglobin + H2O
?
show the reaction diagram
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-
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?
Hemoglobin + H2O
Hydrolyzed hemoglobin
show the reaction diagram
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Isolated soybean protein + H2O
Hydrolyzed soybean protein
show the reaction diagram
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N-alpha-benzoyl-DL-arginine-2-naphthylamide + H2O
N-alpha-benzoyl-DL-arginine + 2-naphthylamine
show the reaction diagram
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?
N-Benzoyl-L-Arg ethyl ester + H2O
N-Benzoyl-L-Arg + ethanol
show the reaction diagram
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N-benzoyl-L-tyrosine ethyl ester + H2O
?
show the reaction diagram
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artificial substrate
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?
Nalpha-Benzoyl-Arg amide + H2O
Nalpha-Benzoyl-Arg + NH3
show the reaction diagram
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Nalpha-Benzoyl-DL-Arg 4-nitroanilide + H2O
Nalpha-Benzoyl-DL-Arg + 4-nitroaniline
show the reaction diagram
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Nalpha-benzoyl-L-arginine-4-nitroanilide + H2O
?
show the reaction diagram
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artificial substrate
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?
protein + H2O
peptides
show the reaction diagram
-
broad substrate specificity
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?
sodium caseinate + H2O
?
show the reaction diagram
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?
Wheat gluten + H2O
Hydrolyzed wheat gluten
show the reaction diagram
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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
protein + H2O
peptides
show the reaction diagram
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broad substrate specificity
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?
additional information
?
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bromelain has a profound immunmodulatory effect, overview, bromelain affects blood coagulation, fibrinolysis, and platelet functions, mechanism, overview
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?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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poor or no inhibition by ions K+, Ca2+, Mg2+, Mn2+, Co2+, Bo3+, Al3+, Cr2+, Ni2+, Sn2+, Ag+, Fe2+, Cd2+, Zn2+, W3+, Se2+, As3+, and Pb2+, overview
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Cystatin
Fe3+
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reduces activity
iodoacetic acid
5% residual activity at 2 mM
Mercurials
p-chloromercuribenzoate
15% residual activity at 2 mM
PCMB
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activity restored by cysteine
Phenylmethylsulfonylfluoride
84% residual activity at 2 mM
Potato cysteine proteinase inhibitor
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sulfhydryl reagents
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Urea
at 2.4 M urea the activity decreases continuously to 50% when using N-alpha-benzoyl-DL-arginine-2-naphthylamide as a substrate
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
highest activity at 96 mM, at 37C and pH 7.0
dithiothreitol
highest activity at 2.5 mM, at pH 7.0 and 37C
EDTA
28% increase of activity at 2 mM
Urea
activity decreases in the presence of 4 M urea and reaches the activity of the control again at 8 M when using casein as a substrate
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
43
N-benzoyl-L-Arg ethyl ester
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2.1 - 4
Nalpha-Benzoyl-Arg amide
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0094 - 0.033
Nalpha-Benzoyl-Arg amide
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3.5 - 4
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benzoyl-Glu-Tyr, benzoyl-Glu-Phe
3.5
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benzoyl-Ala-Leu, benzoyl-Ala-Phe, enzyme form FBA
4
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benzoyl-Ala-Leu, enzyme form FBA
5.5 - 8
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6
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assay at
8.3
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casein
additional information
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3.5 - 8.5
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
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assay at
45
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assay at
65
using N-alpha-benzoyl-DL-arginine-2-naphthylamide as a substrate
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50 - 60
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most suitable hydrolysate temperature of casein acids for bromelain is 50-60C
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.6 - 4.7
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isoelectric focusing
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
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bromelain is a crude extract containing a complex mixture of several components, such as different thiol-endopeptidases, phosphatases, glucosidases, peroxidases, cellulases, glycoproteins and carbohydrates, as well as several proteinase inhibitors
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
18000
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Ananas comosus, fruit bromelain A, gel filtration
19000
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Ananas comosus, fruit bromelain C, gel filtration
30000
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SDS-PAGE
31000
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Ananas comosus, gel electrophoresis
32500
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Ananas comosus, gel filtration
34000
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SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
no glycoprotein
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2
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at a concentration of 250 mg/ml the enzyme is resistant to inactivation
665208
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
-4
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bromelain retains 75% of its activity when stored without preservative at -4C
60
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inactivation increases if temperature exceeds 60C
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
incubation with trypsin (1 mg/ml, 1 h, 37C) results in 12% loss of activity (with benzyloxycarbonyl-Arg-Arg-p-nitroanilide as substrate) at bromelain concentration of 10 mg/ml , but has no effect on activity at bromelain concentrations of 100 mg/ml
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proteolytic activity of solutions containing either 10 mg/ml or 100 mg/ml bromelain in water or 100 mg/ml NaHCO3 is not changed against benzyloxycarbonyl-Arg-Arg-p-nitroanilide and benzoyl-Phe-Val-Arg-p-nitroanilide substrate by four freeze/thaw cycles
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
2 forms: FBA and FBB; fractionation
-
bromelain from pineapple stem and skin are recovered by a PEG 4000/phosphate aqueous two-phase systems (ATPs) liquid-liquid extraction
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CM-Sepharose column chromatography, SP-Sepharose column chromatography, and Sephadex G-50 gel filtration
fractionation; fruit bromelain A, B and C
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high-speed counter-current chromatography coupled with the reverse micelle solvent system consisting of 0.10 g/ml cetyltrimethylammonium bromide (CTAB)/isooctane-hexylalcohol and 0.05 M sodium phosphate buffer is successfully applied to separate bromelain from pineapple fruit
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reverse micellar extraction system with cetyltrimethylammonium bromide
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using centrifugation, salt precipitation technique, dialysis, ion-exchange chromatography. Ion exchange chromatography using diethylaminoethyl cellulose (DEAE) anion exchangers maintain the structural integrity of purified bromelain and thereby the product exhibits better proteolytic activity than crude extract
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli Top10 cells
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
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bromelain can be used as a phytotherapy agent in several aspects, overview
pharmacology
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use as additive to prevent lymphedema by reducing lymphocongestion, detritus, viscosity of the exsudate and stimulation of phagocytosis of associated leukocytes, use as additive for radiotherapy and surgery due to wound healing effect and reductive effect on inflammation and edema, use as additives for chemotherapy due to inhibitory effects on malignant growth of tumor cells