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Starch degradation
Starch: amylose (linear)
amylopectin (branched)
A-chain
B-chain
reducing end
C-chain
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A
OH
OH
CHO
OH
OH
OH
OH
H O
OH
OH
OH
CH2OH
5(
CH2OH
5(
OH
OH
O
H
OH
CH2OH
5(
Amylolytic enzymes
Glucoamylase
langzaam
slow
CH2O H
O
CH2O H
O
CH2O H
CH2O H
O
OH
OH
O
OH
OH
OH
OH
OH
-amylase
CH2
OH
O
OH
OH
OH
O
OH
CH2O H
OH
O
OH
CH2O H
CH2O H
OH
O
OH
CH2O H
OH
O
OH
CH2O H
O
O
OH
O
OH
O
CH2O H
OH
O
OH
OH
O
OH
OH
CH2O H
OH
CH2O H
OH
O
OH
-amylase
OH
CH2O H
OH
OH
HO
CH2O H
OH
OH
OH
O
OH
CH2O H
OH
CH2O H
OH
CH2O H
OH
OH
OH
OH
CH2O H
CH2O H
OH
CH2O H
OH
OH
OH
Isoamylase
Iso-amylase
of pullulanase
pullulanase
or
CH2O H
OH
OH
OH
OH
OH
OH
CH2O H
OH
OH
OH
CH2O
O
OH
OH
CH2O H
OH
OH
OH
CH2O H
CH2O H
O
CH2O H
O
CH2O H
CH2O H
CH2O H
Glucoamylase
Glucoamylase
etc.
CH2O H
O
OH
O
OH
OH
OH
Pullulanase
CH 2
CH2O H
OH
OH
HO
CH2O H
OH
O
OH
O
OH
Pullulanase
OH
CH 2
CH2O H
O
OH
OH
HO
CH2O H
OH
O
OH
O
OH
O
OH
CH 2
CH2O H
OH
OH
HO
CH2O H
OH
O
OH
O
OH
O
OH
CH 2
CH2O H
OH
OH
HO
CH2O H
OH
O
OH
O
OH
O
OH
CH 2
CH2O H
OH
OH
HO
CH2O H
OH
O
OH
O
OH
OH
OH
+
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Glu 230
(catalyse)
Maltohexaose
(substraat)
7 7
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2
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Cellulose
Cellulases
Cellobiohydrolase (CBH)
activity towards crystalline cellulose
older names: avicellase, C1-cellulase
acts in synergy with endoglucanase
possible disruptive function
Endoglucanase
acts in synergy with CBH
mainly active towards CM-cellulose (decrease in
viscosity)
Cellobiase
-glucosidase
cleaves cellobiose (inhibitor of CBH)
Active cleft
Catalytic domain
Cellulose
binding
domain
(CBD)
crystalline
Kristallijn
gebied
Enzymic degradation
of cellulose
amorphous
Amorf gebied
Endoglucanase
CBH
Endoglucanase
CBH
CBH
CBH
CBH
=Cellobiose
Cellobiase
Endoglucanase
CBH
Glucose
Endoglucanase
by CBD
Intact
Core
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Smooth Region
(Homogalacturonan)
Hairy Region
SR
Homogalacturonan
II
III
methyl ester
galacturonic acid
rhamnose
galactose
xylose
arabinose
OH
OH
OH
O
O
OH
OH
OH
OH
CO O CH 3
CO O H
O
O
CO O CH 3
CO O CH 3
CO O CH 3
OH
OH
OH
Pectine lyase
Pectine esterase
OH
OH
OH
O
OH
OH
O
O
OH
CO O H
CO O H
O
O
CO O H
CO O H
CO O H
OH
OH
OH
Pectaat lyase
Polygalacturonase
OH
Polygalacturonase
COOH
COOH
O
O
OH
OH
COOH
OH
O
OH
+ H2 O
Polygalacturonase
OH
COOH
O OH
+
OH
HO
OH
O
OH
start
Right handed parallel -helix
-65x34x36 angstrom
-42 -strands
-4 twisted parallel -sheets (PB1 PB1a PB2 PB3)
end
Octagalacturonate
Pectate lyase
COOH
COOH
O
O
OH
OH
COOH
OH
O
OH
O
Pectaat lyase
OH
COOH
O OH
+
OH
OH
H
OH
Pectin lyase
COOCH3
COOCH3
O
O
OH
OH
COOCH3
OH
O
O
OH
COOCH3
O
Pectine lyase
OH
OH
OH
OH
H
OH
Pectine esterase
Pectine esterase
OH
OH
OH
O
O
OH
OH
OH
OH
COOCH3
COOH
O
O
COOCH3
COOCH3
COOCH3
OH
OH
OH
Pectine esterase
OH
OH
OH
O
OH
OH
O
O
OH
COOH
COOH
O
O
COOH
COOH
COOH
OH
O
OH
OH
OH
Step 1
Step 2b
Step 2a
Step 3
Arabino
oligosaccharides
,
R1 O
X
R2 O
R1 O
R2 O
C
HO
protease
OH
peptide bond
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Aminopeptidase (exo)
H2N
COOH
Endo-protease
Carboxypeptidase (exo)
Trypsin
Chymotrypsin, subtilisin
Staphylococcus V8
protease
Papain
Thermolysin
Pepsin
Protease
Type
Important features
in active site
Example(s)
Serine
protease
Endo
Subtilisine
Trypsine
Aspartate
protease
Endo
Cysteine
protease
Endo
Papaine
Metalloprotease
Exo
Sulfhydryl group of
cysteine
Imidazole group of
histidine
2+
Zn
2+
Mn
Pepsine
Chymosine
Collagenase
7
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7 !
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CH(CH 3)2
O
Enzyme-Ser-OH
O
CH(CH 3 )2
DFP
CH(CH 3)2
O
Enzyme-Ser-O
O
CH(CH 3 )2
Inhibited enzyme
,2
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,
@ 0
CH3
CH
H 3C
H 3C
CH3
CH CH2 CO
(Leu)2
CH2 OH
NH CH CH CH2
CO
OH
Ala
NH CH CH CH2
CH2
Statine
CH
CH3
CH3
Statine
CO2H
,
Catalytic residues
Metallo-proteinases
Contain divalent metal ion (usually Zn 2+)
Inhibition by gelating agents (EDTA)
Can be divided in:
Exopeptidases
carboxypeptidases
aminopeptidases
Endopeptidases
collagenases
thermolysin
Metal ion binding motif: His-Glu-X-X-His serves as
Zn-ligand
Thermolysin
His 146
His 142
Substraat
Glu 166
Zn2+
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Supersubstrates
Molecular solution
Micelles/Emulsion
Closed
Open
H2 C O C
R2
C O CH
O
Sn-1,2,3-Triglyceride
H2 C O C
R3
Step 1
O
O C
R1
O C
R1
H2 C OH
R2
C O CH
O
H2 C O C
R2
H2 C O C
R3
O CH
H2 C
R1
1,2-Diglyceride
2,3-Diglyceride
Examples:
Pancreatic lipase
Several microbial lipases
Mucor miehei
Rhizopus javanicus
Rhizopus niveus
Rhizopus delemar
Rhizopus rhizopodiformis
Penecillium roquefortii
R3
Step 2
O
H2C OH
R2
C O CH
O
H2C OH
O C
R1
respectivelijk
O
O C
2-Monoglyceride
R3
Step 3
H2C OH
HO C H
H2C OH
Glycerol
O
O C
R2
Specificity of lipases
1: Position-specificity
1,3-specific lipases
Mode of action: see slide degradation of triglycerides
Pancreatic lipase
Several microbial lipases
Mucor miehei
Rhizopus javanicus
Rhizopus niveus
Rhizopus delemar
Rhizopus rhizopodiformis
Penecillium roquefortii
Pseudomonas fluorescence (heat stable lipase in milk)
Non-specific lipases
Mode of action: does not distinguish between 1-, 2- or 3-position
Candida cilindracea
Humicola lanuginosa
Specificity of lipases
2: Fatty-acid specificity
Lipase makes a distinction between types of fatty acids
Saturated/unsaturated
Geotrichum candidum prefers unsaturated fatty acids with
cis-double band at position 9 (oleic acid;linoleic acid; -linolenic acid)
Glycerol esters of saturated fatty acids are only slowly hydrolysed
Length
Pancreatic lipase prefers long-chain fatty acids (C16)
Lingual or gastric lipase prefer short-chain fatty acids (C8)
Aspergillus niger lipase I prefers long-chain fatty acids
Aspergillus niger lipase II prefers short chain fatty acids
R=CH2CH2N(CH3)3
Phosphatidyl ethanolamine
R=CH2CH2NH3
N-Acylphosphatidyl ethanolamine
R=CH2CH2NHCO.R
Phosphatidyl serine
R=CH2CHNH2
CO2H
Phosphatidyl inositol
P-Lipase-A1
(1)
O
R2
(2)
C O
P-Lipase-A2
P-Lipase-C
(3)
OH
P-Lipase-D
R1
O C
(4)
R=C6H6(OH)6
Lipoxygenase
cis, cis-1,4-pentadiene group
16
CH
15
HH
13
12
10
CH 2
CH 2
CH 2
H H
O2
Lipoxygenase
H
H
CH
16
C
CH 2
15
CH2
13
CH
H
10
C
R
C
H
O
O
H
Instable hydroxyperoxide
Follow-up reactions