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)