Amylase development for starch liquefaction

Carsten Andersen

Novozymes R&D

Starch From Starch to Sugar

Glucose/Fructose

-Amylases – one of many enzyme types used in the Starch Industry

Liquefaction

Saccharification

Speciality syrups

and dextrins

Liquefying amylases

Saccharifying amylases

Glucoamylase

Pullulanase

Acidic -amylase

-amylase

CGTase

Maltogenic -amylase

High fructose corn syrup process

Steeping

pH 4.0-4.5

50°C

48H

pH 6

105°C, 0.1H

95°C, 1-2H

pH 4.0-4.5

60°C

30-60H

Liquefaction

Saccharification

Isomerization

Na2CO3

Mg2+ pH 8.2

60°C

0.3-3H

NaOH

Ca2+

HCl

High temp.

NaOH

Liquefying -amylase

Glucoamylase

Pullulanase Acidic -amylase

Glucose Isomerase

}

}

}

pH and Temperature changes during the starch conversion process

34

567

89

Ste

eping

Liqu

efac

tion-

1

Liqu

efac

tion-

2

Inac

tivatio

n

Sac

char

ifica

tion

Ion

exch

ange

Isom

erizat

ion

pH

020

406080

100120

Tem

pera

ture

(°C

)

•

High Fructose Corn Syrup Production

Steeping Steeping

Liquefaction

Saccharification

Isomerization

pH 4.0-4.5

60°C

pH 5.8-6.2

105°C 0.1 h

95°C 1-2 h

pH 4.0-4.5

HCl High temp. NaOH

Process by 1997

NaOH

Ca2+

pH 4.5

60°C

pH 4.5

105°C 0.1 h

95°C 1-2 h

pH 4.5

50°C

Optimal process

Liquefaction

Saccharification

Isomerization

} Na2CO3 Mg2+ }

}

pH 8.2

60°C

pH 8.2

60°C

No inactivation

•

The development of a new liquefying -amylase

was divided into three steps

Calcium independency

Low pH stability/activity

Product specificity

B.Licheniformis -amylase with substitutions and hybrid part

H156

A181 A209

Q264

N190

BAN (1-35)

•

Calcium dependent stability 95°C, pH 6,2, No free Calcium, 5% Starch, 0,1M Acetate

0 5 10 15 20 25 30 0

20

40

60

80

100

120

H156Y,A181T,A209V Hybrid,H156Y,A181T,A209V

Q264S N190F Termamyl

Hybrid: AA.1-35 from B.amyloliquefaciens, 35-483 from B.licheniformis

Minutes

% activity

N190F: 11100

Specific activities in Units/mg:

H156Y,A181T,A209V: 7000

Hybrid,H156Y,A181T,A209V: 8500

Q264S: 10000

•

DE development - pilot plant trials

time (mins) @ 95°C

DE

0

2

4

6

8

10

12

14

0 20 40 60 80 100

Termamyl pH 6.0 5 ppm Ca ++

Termamyl pH 6.0 40 ppm Ca ++

Next step towards the ideal liquefying -amylase

Addressing • stability and activity at pH 4,5

• Reducing pH from the current level of 5,6 to pH 4,5

• degradation specificity • A more robust liquefaction process – no inactivation • Allowing the industry to liquefy to a higher DE

Resulting in • reduce operating cost • improved glucose yield and reduction of the

saccharification enzymes needed

•

A random protein engineering approach to obtain higher stability

• 10 amino acid regions chosen from 3D structure

• Interfaces between the three domains A/B, A/C

included

• Calcium coordinating regions included

• B-domain regions included

Primary Filter Assay

• Every single colony of

positive variants were

picked up and incubated

in medium for 22h at

37°C

Colonies on cellulose

acetate, nitrocellulose

filters and TY agar

Detection on 0.2%

starch + 1% agarose in

citrate buffer, pH 6.0

stained with Lugol

Incubation in citrate buffer, pH 4.5

for 10-20 min. at 80°C

Nitrocellulose filter

with bound protein

Screening Assays

Residual activity at pH 4.5 after

0 5 10 20 30 40 50 60 70 80 min

80°C

87°C

Wt

Ter. LC

Amy 1

Amy 2

Amy 3

Wt

Ter. LC

Amy 1

Amy 2

Amy 3

•The bacillus culture

was incubated in citrate

buffer, pH 4,5 at 80°C or

87°C and samples were

taken at time intervals

from 0 to 100 min.

A 3 ml sample was

spotted on an assay

plate (0,2% starch,1%

agarose)

The assay plate was

stained with 10% Lugol

solution

Variants from the random PE program Stability at 95°C, pH 5.0, No calcium, 5% Starch, 0,1M Acetate

0 5 10 15 20 25 30 0

20

40

60

80

100

Termamyl pH 5,5 Termamyl LC plus I201F plus E211Q plus D207Y

% activity

Minutes

Designing the product specificity of Bacillus -amylases

Problem of existing liquefying amylases

Idea generating studies

Rational Design of product specificity

Liquozyme X – the new amylase from Novozymes

High fructose corn syrup process

Steeping

pH 4.0-4.5

50°C

48H

pH 5-5.5

105°C 0.1H

95°C 1-2H

pH 4.0-4.5

60°C

30-60H

Liquefaction

Saccharification

Isomerization

Na2CO3

Mg2+ pH 8.2

60°C

0.3-3H

NaOH

- HCl

- High temp.

- NaOH

}

} Inactivation

of -amylase

Active liquefaction enzyme present during saccharification causes panose formation

(AMG E, initial DS 30%)

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

20 30 40 50 60 70 80 90 100

Hours

To

tal D

P3

Termamyl LC

Inactivated amylase

•

Panose is formed because bacterial -amylases hydrolyse amylopectin close to the branching point

Amylopectin

panose precursors

B. Licheniformis amylase

Panose

A protein engineering concept aiming at a BLA based amylase with the desired specificity:

B.licheniformis:

Asp.niger -amylase:

Active Site

Important and Strong

in Termamyl

Less Important

Space for

branched

dextrins

-5-4

-3 -2 -1 + 1+ 2

+ 3

Active Site

Important and Strong

in Acidic -amylaseLess Important

No Space for

Branching

Points Near

Cleavage Site -2 -1 + 1+ 2

+ 3

V54W, a bulky amino acid introduced near the predicted branching point in the substrate binding crevice

V54W substitution resulted in much lower panose

formation during saccharification

V54W is assumed to have the desired degradation pattern

Unfortunately the specific activity (activity/mg enzyme)

was severely reduced to 25% of wild type

The stability at 95°C was significantly reduced

A larger amino residue in position T49, A52, V54 and G107 results in lower panose formation.

Mutations in Termamyl LC DP1 DP2 DP3

T49L 96,3 1,77 1,11

A52S 95,9 1,80 1,11

V54N 96,1 1,75 1,18

G107A 94,4 1,89 1,04

T49L+G107A 96,4 1,87 0,72

Reference 95,9 1,85 1,26

Mutations in Termamyl LC DP1 DP2 DP3DP1 DP2 DP3

T49L 96,3 1,77 1,11

A52S 95,9 1,80 1,11

V54N 96,1 1,75 1,18V54N 96,1 1,75 1,18

G107A 94,4 1,89 1,04G107A 94,4 1,89 1,04

T49L+G107A 96,4 1,87 0,72T49L+G107A 96,4 1,87 0,72

Reference 95,9 1,85 1,26Reference 95,9 1,85 1,26

The new enzyme, Liquozyme X, does not give rise to panose formation, when active during saccharification (AMG E, initial DS 30%)

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

20 30 40 50 60 70 80 90 100

Hours

To

tal

DP

3

Termamyl LC

Liquozyme X

Inactivated amylase

Liquozyme X has further increased stability – pH interval 5.2-5.6

Identified and modified key residues for the product profile

Less panose

No inactivation necessary

A more robust liquefaction process

New liquefaction amylase :

-amylase products launched by Novozymes

Laundry and Detergent Termamyl Termamyl Ultra Duramyl Stainzyme Stainzyme Plus

Starch Liquefaction Termamyl L Termamyl LC Liquozyme X

Biofuel (1. generation) Termamyl SC Termamyl Supra Novozym BPX