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The use ofThe use of αααααααα--amylase in starch processingamylase in starch processing
Soetijoso Soemitro Muhammad FadhlillahSoetijoso Soemitro Muhammad Fadhlillah
Sadiah Djajasoepena Ceci Hani Handayani
Safri Ishmayana Teni Mardiah
Biochemistry Laboratory
Department of Chemistry, FMIPA
Padjadjaran University
Background
- More production and processing of starch
- Green process: Degradation of raw starch by αααα-amylases
Structure and properties of Saccharomycopsis fibuligera
Outline
Structure and properties of Saccharomycopsis fibuligera
α-amylase
Raw starch processing by Saccharomycopsis fibuligera
α-amylase
Ongoing studies
Background
- More production and processing of starch
- Green process: Degradation of raw starch by αααα-amylases
Structure and properties of Saccharomycopsis fibuligera
Outline
Structure and properties of Saccharomycopsis fibuligera
α-amylase
Raw starch processing by Saccharomycopsis fibuligera
α-amylase
Ongoing studies
Modified starch processing technologies and products
(Jin Shuren, 2001)
Sport drinks: DP 3-6,moderate osmolality and highly absorbed
(Marchal et al., 1999)
Raw starch
Gelatinization process
(high-temperature) by Enzyme with SBD
(starch binding domain)
Advantages of raw-starch digesting enzyme process
(high-temperature) by thermostable enzyme
(starch binding domain)in the room temperature
Low-cost production High-cost production
Conventional ethanol production from starch No cook starch-ethanol processing
Reduction of energy consumption
Conventional ethanol production from starch No cook starch-ethanol processing
(van der Maarel, 2006)
~ 10% of total process energy
Performance of the raw starch digesting α-amylase
Raw starch-digesting
α-amylase
(Giraud et al., 1994)
(Rodriguez-Sanoja et al., 2003)
Raw starch-digesting
α-amylase
10% of amylolytic enzymes are able to degrade the raw starches directly
High activities of the Saccharomycopsis fibuligera R-64 amylase were
produced by using raw sago starch as a carbon source (RUT I, 1996)
Background
- More production and processing of starch
- Green process: Degradation of raw starch by αααα-amylases
Structure and properties of Saccharomycopsis fibuligera
Outline
Structure and properties of Saccharomycopsis fibuligera
α-amylase
Raw starch processing by Saccharomycopsis fibuligera
α-amylase
Ongoing studies
Modular structure of carbohydrate-acting enzymes
Carbohydrate Binding ModulesCatalytic Module Carbohydrate Binding ModulesCarbohydrate Binding Modules
Specific for
- Cellulose
- Chitin
- Xylan
- Other polysaccharides
- Starch ����Starch Binding Domain
Primary structure classification of Glycoside Hydrolases
49 CBM families � 7 SBDs: CBM20
CBM21
CBM25
CBM26
CBM34
CBM41
CBM45
(Machovic & Janecek, 2006; Liu et al., 2007)
Arrangement of the starch binding domain in the structure of
the raw starch-digesting enzymes
(Rodriguez-Sanoja et al. 2005)
The structural features of SBD from the individual CBM families
Aspergillus nigerglucoamylase (CBM20)
Rhizopus oryzaeglucoamylase (CBM21)
Bacillus halodurans maltohexaose-forming amylase
(CBM25)
Model
(Machovic and Janecek, 2006)
Bacillus halodurans maltohexaose-forming amylase
(CBM26)
Thermoactinomyces vulgaris TVA1 α-amylase (CBM34)
Klebsiella pneumoniae pullulanase (CBM41)
The roles of the starch binding domain
• Enabling the enzyme molecule to interact with the insoluble substrate in solution
• Delivering the substrate to the active site in the catalytic domaincatalytic domain
• Disrupting the surface of the starch granule
(Machovic and Janecek, 2006)
Saccharomycopsis fibuligera α-amylase (ALP1)
• Biochemical characteristic
- Optimum pH: 5.0
- Optimum temperature: 50oC
- Molecular weight: 54 kDa
(Soemitro et al., 1996)
• alp1 gene � amino acid sequence (Ismaya et al., 2003)
-CHO
Calcium Glycosylation
Dom
ain
s A/B
• Domain organization
(Hasan et al., 2005)
• Kinetics
(Syahbana et al., 2005)
• Host: Pichia pastoris
(Shabarni et al., 2007)
Catalytic site
Dom
ain
s A/B
Dom
ain
C
001 MQISKAALLA SLAALVYAQP VTLFKRETNA DKWRSQSIYQ IVTDRFARTD 050
051 GDTSASCNTE DRLYCGGSFQ GIIKKLDYIK DMGFTAIWIS PVVENIPDNT 100
101 AYGYAYHGYW MKNIYKINEN FGTADDLKSL AQELHDRDML LMVDIVTNHY 150
151 GSDGSGDSID YSEYTPFNDQ KYFHNYCLIS NYDDQAQVQS CWEGDSSVAL 200
N V N
201 PDLRTEDSDV ASVFNSWVKD FVGNYSIDGL RIDSAKHVDQ GFFPDFVSAS 250
GVYSVGEVFQ GDPAYTCPYQ NYIPGVSNYP LYYPTTRFFK TTDSSSSELT
Sequence of the S. fibuligera α-amilase (ALP1) (Itoh et al., 1987)
251 GVYSVGEVFQ GDPAYTCPYQ NYIPGVSNYP LYYPTTRFFK TTDSSSSELT 300
301 QMISSVASSC SDPTLLTNFV ENHDNERFAS MTSDQSLISN AIAFVLLGDG 350
351 IPVIYYGQEQ GLSGKSDPNN REALWLSGYN KESDYYKLIA KANAARNAAV 400
401 YQDSSYATSQ LSVIFSNDHV IATKRGSVVS VFNNLGSSGS SDVTISNTGY 450
451 SSGEDLVEVL TCSTVSGSSD LQVSIQGGQP QIFVPAKYAS DICS
(Ismaya et al., 2003)Catalytic sites
Sequence homology of ALP1 with α-amylase of A. oryzae
AoALP1
(T-Coffee program; Notredame et al., 2003)
(Ismaya et al., 2003)
Saccharomycopsis fibuligera α-amylase (ALP1)
• Biochemical characteristic
- Optimum pH: 5.0
- Optimum temperature: 50oC
- Molecular weight: 54 kDa
(Soemitro et al., 1996)
• alp1 gene � amino acid sequence (Ismaya et al., 2003)
-CHO
Calcium Glycosylation
Dom
ain
s A/B
• Domain organization
(Hasan et al., 2005)
• Kinetics
(Syahbana et al., 2005)
• Host: Pichia pastoris
(Shabarni et al., 2007)
Catalytic site
Dom
ain
s A/B
Dom
ain
C
Performance of domains for raw starch adsorption
0
20
40
60
80
100
0 25 50 75 100 125 150 175 200
Raw starch (mg)
Adso
rption (
%)
0
20
40
60
80
100
0 25 50 75 100 125 150 175 200
Raw starch (mg)Adso
rptio
n (
%)
0
20
40
60
80
100
0 25 50 75 100 125 150 175 200
Raw starch (mg)
Adso
rptio
n (
%)ALP1 Domain A/B~39 kDa Domain C~10 kDa
C-domain lost its ability to adsorb raw starch!
0
20
40
60
80
100
0 25 50 75 100 125 150 175 200
Raw starch (mg)
Adso
rption (
%)
Glucoamylase (positive control)
Glm (S. fibuligera IFO0111 glucoamylase)(Hostinova et al., 2003)
Glm (S. fibuligera IFO0111 glucoamylase) (Host: A. awamori)(Hostinova et al., 2003)
(Kusumawidjaja & Andiyana, 2007)
S. fibuligera R64 a-amylase activity towards raw starches
Granule
COMPARISON OF STARCH PROPERTIES FROM DIFFERENT SOURCES
0.000
0.050
0.100
0.150
0.200
0.250
0.300(µ
mol m
alto
se/m
L)
Corn
Cassava
Sago
Potat o
Raw Starch
Granule
Diameter
(µm)
Area : Volume Composition
Corn 2 – 30 0,2-3,0 : 125% amylose
75% amylopectin
Cassava 5 – 35 0,17-1,2 : 115% amylose
85% amylopectin
Sago 5 – 65 - -
Potato 5 – 100 0,06-1,2 : 120% amylose
80% amylopectin
(Ansharullah, 1997; Mishra & Rai, 2005; Tester et al., 2006)
A. niger glucoamylase (CBM20)
R. oryzae glucoamylase (CBM21)
B. halodurans maltohexaose-forming amylase (CBM25)
Model
Comparison of the structural features of domain C of ALP1 with
other CBM families
(Machovic and Janecek, 2006)
B. halodurans maltohexaose-forming amylase (CBM26)
Thermoactinomyces vulgaris TVA1 α-amylase (CBM34)
Klebsiella pneumoniae pullulanase (CBM41)
SBD TVA1 (Machovic and Janecek, 2006)
High homology !
Homology level
Study on homology between domain C of S. fibuligera α-amylase (ALP1) and SBD of T. vulgaris α-amylase TVAI
ALP1
Domain C model of the ALP1
ALP1
ALP1
Study on homology between domain C of S. fibuligera α-amylase (ALP1) and SBD of T. vulgaris α-amylase TVAI
Gap
Gap
7 β-strands
Several gaps !
SBD TVA1 (Machovic and Janecek, 2006)
8 β-strands
7 β-strands
ALP1
GapGap
Gap
Domain C model of the ALP1
7 β-strands
ALP1
ALP1
Study on homology between domain C of S. fibuligera α-amylase (ALP1) and SBD of T. vulgaris α-amylase TVAI
Gap
Gap
Domain C has no Trp (stacking interactions with raw starch)
7 β-strands
SBD TVA1 (Machovic and Janecek, 2006)
8 β-strands
7 β-strands
ALP1
GapGap
Gap
Domain C model of the ALP1
7 β-strands
ALP1
ALP1
Background
- More production and processing of starch
- Green process: Degradation of raw starch by αααα-amylases
Structure and properties of Saccharomycopsis fibuligera
Outline
Structure and properties of Saccharomycopsis fibuligera
α-amylase
Raw starch processing by Saccharomycopsis fibuligera
α-amylase
Ongoing studies
- Starch source
- Granule size
- Amylose/amylopectin ratio
- Amylose-lipid complexes
- Degree of crystallinity
Differences in the digestibility ����Interplay of many factors � Physicochemical properties
- Degree of crystallinity
- Type of crystalline polymorphic form
- Physical insulation of starch by thick walled cells
- α-Amylases inhibitors
- Influence of drying and storage conditions
(Hoover & Zhou, 2003)
(van der Maarel et al., 2002)
(Tester et al., 2004)
EXPERIMENTAL DESIGNSaccharomycopsis
fibuligera R-64
• Enzyme production in a batch culture
(1% sago starch dan 1% yeast extract)
• separation of a-amylase and glucoamylase
on HIC using Butyl-Toyopearl
α-Amylase
Raw Starches (1,2,3,4,5,6)
Glucoamylase(7, 8) (8)
1 : Proximate Analysis
2 : Freeze-Thaw Stability
3 : Swelling Volume
4 : Viscosityα-Amylase
• Optimization ( time, temperature, starch
concentration, and unit activity of enzyme)
• Hydrolysis (at optimized condition)
Supernatant Partially hydrolyzed raw
starch
(1,2,3,4,5,6,)(9,10)
Glucoamylase(7, 8) (8)4 : Viscosity
5 : Clarity
6 : Scanning Electron
Micrograph (SEM)
7 : Enzyme Activity
8 : Enzyme Adsorbability
9 : Dextrose equivalent (DE)
10 : Thin Layer
Chromatography (TLC)
ADSORPTION OF AMYLASES
ON RAW STARCHES
60
70
80
90
100
% Adsorption
Raw cassava starch Raw corn starch
α-amylase glucoamylase α-amylase glucoamylase
0
10
20
30
40
50
% Adsorption
MORPHOLOGY (SEM ANALYSIS) OF NATIVE AND
PARTIALLY HYDROLYZED RAW STARCHES
Partially hydrolyzed Partially hydrolyzed
Raw Cassava Starch
Native Partially hydrolyzed
at room temperature
Partially hydrolyzed
at 50oC
Raw Corn Starch
NativePartially hydrolyzed
at room temperaturePartially hydrolyzed
at 50oC
35.00
40.00
NCa = Native raw cassava starch
PCaR = Partially hydrolyzed raw cassava starch
(room temperature)
PCaO = Partially hydrolyzed raw cassava starch
(50oC)
NCo = Native raw corn starch
PCoR = Partially hydrolyzed raw corn starch
(room temperature)
PCoO = Partially hydrolyzed raw corn starch
(50oC)
AMYLOSE CONTENT
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
Nca PCaR PCaO NCo PCoR PCoO
Type of Starch
% Amylose
V X
30
40
Swelling Volume (%)
NCa
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
Nca PCaR PCaO NCo PCoR PCoO
Type of Starch
% Amylose
AMYLOSE CONTENT� Intramolecular H-bonding
� crystallinity
(cassava < corn)SWELLING
VOLUME
0
10
20
30
55 65 75 85 95
Temperature (oC)
Swelling Volume (%)
NCa
PCaR
PCaO
NCo
PCoR
PCoO
VISCOSITYSample Gel Temp.
(oC)
Viscosity at
93oC (BU)
Viscosity
93oC for
20 min.*(BU)
Viscosity at
50oC# (BU)
Viscosity
50oC for
20 min.
(BU)
NCaNCa 67.567.5 350350 210210 330330 370370
PCaR 69.0 80 70 100 90
PCaO 69.0 90 60 80 80PCaO 69.0 90 60 80 80
NCoNCo 85.585.5 9090 9090 160160 200200
PCoR 90.0 40 60 160 160
PCoO 91.5 10 30 50 50
Native
50ºC: Cassava > corn
93ºC: Cassava > corn↔ Amylose content
(↔ lipid)
↔ Corn more compact
SAMPLE RETROGRADATION
RATE
NCa 1.57
PCaR 1.43
*
#RATE GRADATION RETRO =
Rate of recrystalization:
cassava < corn(Amylose content: cassava < corn,
cassava more stable at low temp)PCaR 1.43
PCaO 1.33
NCo 1.78
PCoR 2.67
PCoO 1.67
cassava more stable at low temp)
FREEZE THAW STABILITY
Sample Syneresis Degree (%)
4oC -20oC
NCa 0.25 ±0.12 0.36 ±0.11
PCaR 0.18 ±0.00 0.18 ±0.02
PCaO 0.19 ±0.01 0.16 ±0.02PCaO 0.19 ±0.01 0.16 ±0.02
NCo 21.34 ±0.71 2.13 ±0.52
PCaR 27.70 ±0.77 2.75 ±0.08
PCaO 23.99 ± 0.84 0.22 ± 0.21
Syneresis degree: water loss
Stability: Cassava > corn(Amylose content:: cassava < corn)
CLARITY
60
70
80
90
100
Clarity:
Cassava > corn(Amylose content: cassava < corn)
� Lipid complex
SS: Commercial starch
0
10
20
30
40
50
SS NCa PCaR PCaO NCo PCoR PCoO
Starch Type
% T
Background
- More production and processing of starch
- Green process: Degradation of raw starch by αααα-amylases
Structure and properties of Saccharomycopsis fibuligera
Outline
Structure and properties of Saccharomycopsis fibuligera
α-amylase
Raw starch processing by Saccharomycopsis fibuligera
α-amylase
Ongoing studies
-Starch processing (� Carmencita Tjachjadi et al., Unpad)
α
Ongoing studies on
S. fibuligera αααα-amylase
- Improving biosynthesis and secretory of α-amylase in Pichiapastoris (� Dessy Natalia et al., ITB)
- Improving raw starch binding
Proteolytic fragmentation of S. fibuligera α-amylase
-CHO
Calcium Glycosylation
Domains A/B ~39 kDa
Catalytic sitesite
Domains A/B ~39 kDa
Domain C ~ 10 kDa
Proteolytic fragments separation on Sephadex G-50
ALP1
39 kDa
66
43
30
ALP1Sample: ALP1
0
0,2
0,4
0,6
0,8
1
1,2
1,4
0 10 20 30 40 50
A280 n
m
0
0,2
0,4
0,6
0,8
1
1,2
0 10 20 30 40 50
Eluent volume (mL)
A280 n
m
Trypsin
10 kDa
20
14
0 1 5 24 48 72
Time of incubation (h)
39 kDa
10 kDa
Sample: 72 h-tryptic digestion
0 10 20 30 40 50
Eluent volume (mL)
(Tester et al., 2004)
(Tester et al., 2004)
Amylolytic activity of the Saccharomycopsis fibuligera α-amylase
on the raw starches
0,276
0,1310,150
0,200
0,250
0,300
(µm
ol m
altose
/mL)
ALP1 is able to digest raw starches
0,044
0,009
0,000
0,050
0,100
(µm
ol m
altose
/mL)
Maize Tapioca Sago Potato
Raw starch characteristics
Raw starches
Granular shape (µm)
Surface area (µm2)a
Volume (µm3)b SA:Vc Double
helixdCrystallinity
levele
Maize 2-30 12.6-2.877 4.2-14.137 0.2-3.0:1 0.38-0.43 0.39-0.43
Tapioca 5-35 78.5-3.849 65.4-22.449 0.17-1.2:1 0.44 0.38-0.44Tapioca 5-35 78.5-3.849 65.4-22.449 0.17-1.2:1 0.44 0.38-0.44
Sago 5-65 - - - - -
Potato 5-100 78.5-31.416 65.4-523.599 0.06-1.2:1 0.29-0.64 0.23-0.53
;Tester et al., 2006)a 4πr2, d NMR
b 4/3 πr3 e X-ray scattering
c Surface area : volume ratio (Ansharullah, 1997; Tester et al., 2006)