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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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Aspergillus oryzae -amylase


Ca 2+
Asp 203
(catalyse)

Glu 230
(catalyse)

Maltohexaose
(substraat)

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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)

Domain structure of CBH


Linker, or hinge

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

CBH, niet hydrolytische


CBH, non-hydrolytic
action
activiteit
door 'binding domain'

=Cellobiose
Cellobiase

Endoglucanase
CBH

Glucose
Endoglucanase

by CBD

Cellulose degradation by core and


intact cellulases
Intact
Core

Intact
Core

Cellulases can degrade


barley -1,3/4-glucan

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Schematic structure of pectin

Smooth Region
(Homogalacturonan)

Hairy Region

pectin: hypothetical structure


Pectin
HR

SR
Homogalacturonan

II

III

methyl ester
galacturonic acid
rhamnose
galactose
xylose
arabinose

Points of attack of pectinases


Pectine esterase

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

Activity towards low-methoxyl pectin


cleaves next to a free carboxyl group
Two types
endopolygalacturonase
exopolygalacturonase

Source: fungi, bacteria, plants


pH optimum 4-5.5

HO
OH

O
OH

3D structure of polygalacturonase from


Aspergillus aculeatus (look from the side)
Glycosylation
sites

start
Right handed parallel -helix
-65x34x36 angstrom
-42 -strands
-4 twisted parallel -sheets (PB1 PB1a PB2 PB3)

end

Modelled structure of A. aculeatus


polygalacturonase-substrate complex
Space-filling model of
polygalacturonase

Octagalacturonate

Pectate lyase
COOH

COOH

O
O

OH
OH

COOH

OH

O
OH

O
Pectaat lyase

OH

COOH

O OH

+
OH

no hydrolase, but a lyase (-elimination)


activity towards low-methoxyl pectin
endo- and exo-pectate lyases
Ca2+ dependent
pH optimum 8-9.5
source: mainly bacteria, also in fungi and plants

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

Active towards high methoxyl pectin (-eliminatie)


cleaves next to a methoxylated carboxyl group
Not Ca2+-dependent
only endo-acting enzymes known
source: fungi
pH optimum 5-6

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

Starts next to a free carboxyl group


fungal PME: pH optimum 4.5; at random de-esterification
plant PME: pH optimum > 7; blockwise de-esterification

Arabinan is a side chain of rhamno


galacturonan
Main chain
-1,5-linked

Single unit side chains


-1,2 or -1,3 -linked
Two unit side chain

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

-Lys (or Arg) -----

Chymotrypsin, subtilisin

-Trp (or Tyr, Phe, Leu)------

Staphylococcus V8
protease
Papain

-Asp (or Glu)------

Thermolysin

---Leu (or Phe) ------

Pepsin

-Phe (or Tyr, Leu)- Trp (or Phe, Tyr)

-Phe (or Val, Leu)-Xaa-----

Arrow indicates the site of action of the protease.


Xaa, any amino acid residue.

Protease

Type

Important features
in active site

Example(s)

Serine
protease

Endo

Subtilisine
Trypsine

Aspartate
protease

Endo

hydroxyl group of serine


imidazole group of
histidine
Carboxyl group of 2
aspartic acids

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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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

3-methyl butanoic acid

(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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Lipases nest in oil/water interfaces

Supersubstrates

Molecular solution

Micelles/Emulsion

Rhizomucor miehei lipase


Ser 144

Closed

Open

Hydrolysis of triglycerides by lipase

H2 C O C
R2

C O CH
O

Lipase with 1,3-specificity:


Step 1: fast
Step 2: slower
Step 3: very slow

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

Free fatty acids influence taste and smell

Free fatty acids influence taste and smell

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

Phospholipids and their enzymatic degradation


Phosphatidyl choline

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

Some technological aspect of phospholipases

First step in the formation of rancid compounds by the release of


unsaturated fatty acids, which are substrate for lipoxygenase
(for instance in milk, soy protein, fish, meat)
Aroma biosynthesis by the formation of free unsaturated fatty acids
(for instance in cheese like roquefort, camembert)
Shelve live of cereals (contain lipase and phospholipase)
Foam stability of beer (lysophosphatidyl choline reduces foam stability)

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