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

Liquefying

Liquefaction

Saccharification

amylases

Saccharifying amylases
Glucoamylase
Pullulanase
Acidic -amylase

Speciality syrups
and dextrins

-amylase
CGTase
Maltogenic -amylase

High fructose corn syrup process

Steeping

NaOH
Ca2+

pH 4.0-4.5
50C
48H

Liquefaction

HCl
High temp.
NaOH

pH 6
Liquefying
105C, 0.1H
95C, 1-2H

-amylase

Glucoamylase
pH 4.0-4.5Pullulanase

Saccharification 60 C
Acidic -amylase
30-60H

Na2CO3
Mg2+

Isomerization

pH 8.2 Glucose
Isomerase
60C
0.3-3H

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pH
9
8
7
6
5
4
3
120
100
80
60
40
20
0

Temperature (C)

pH and Temperature changes during the

starch conversion process

High Fructose Corn Syrup Production

Process by 1997

Optimal process

pH 4.0-4.5

pH 4.5

Steeping

Steeping

50C

NaOH
Ca2+

pH 5.8-6.2

Liquefaction

pH 4.5

Liquefaction

105C 0.1 h
95C 1-2 h

HCl
High temp.
NaOH

Na2CO3
Mg2+

105C 0.1 h
95C 1-2 h

No inactivation
pH 4.0-4.5

Saccharification

Saccharification

60C

}
Isomerization

pH 4.5

pH 8.2
60C

60C

Isomerization

pH 8.2
60C

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

A209
A181
H156

Q264

N190

BAN (1-35)

Calcium dependent stability

% activity

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

120

Specific activities in Units/mg:

H156Y,A181T,A209V:
7000
Hybrid,H156Y,A181T,A209V: 8500
Q264S:
10000
N190F:
11100

100
80
60
40
20
0
0

10

15

H156Y,A181T,A209V
N190F

20

25

30

Minutes

Hybrid,H156Y,A181T,A209V
Q264S

Termamyl

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

DE development - pilot plant trials

14
12

DE

10

++
Termamyl pH 6.0 40 ppm Ca

8
6
4
Termamyl pH 6.0 5 ppm ++
Ca

0
0

20

40

60

80

100

time (mins)@ 95C

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

Colonies on cellulose
acetate, nitrocellulose
filters and TY agar

Nitrocellulose filter
with bound protein
Incubation in citrate buffer, pH 4.5
for 10-20 min. at 80C

Detection on 0.2%
starch + 1% agarose in
citrate buffer, pH 6.0
stained with Lugol

Every single colony of

positive variants were
picked up and incubated
in medium for 22h at
37C

Screening Assays
Residual activity at pH 4.5 after
0 5 10 20 30 40 50 60 70 80 min

The bacillus culture

was incubated in citrate
buffer, pH 4,5 at 80C or
87C 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

80C

Wt
Ter. LC
Amy 1
Amy 2
Amy 3

87C

Wt
Ter. LC
Amy 1
Amy 2
Amy 3

Variants from the random PE program

Stability at 95C, pH 5.0, No calcium, 5% Starch, 0,1M Acetate
% activity

100
80
60
40

20
0

10

Termamyl pH 5,5
plus E211Q

15

20

25

Termamyl LC
plus D207Y

30

Minutes

plus I201F

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

pH 4.0-4.5
50C
48H

Steeping

NaOH

Liquefaction

Inactivation - HCl
of - - High temp.
amylase - NaOH

pH 5-5.5
105C 0.1H
95C 1-2H

Saccharification

Na2CO3
Mg2+

pH 4.0-4.5
60C
30-60H

Isomerization

pH 8.2
60C
0.3-3H

Active liquefaction enzyme present during

saccharification causes panose formation
(AMG E, initial DS 30%)
1,6

Termamyl LC
1,4

Inactivated amylase

Total DP3

1,2
1
0,8
0,6
0,4
0,2
0
20

30

40

50

60

Hours

70

80

90

100

Panose is formed because bacterial -amylases

hydrolyse amylopectin close to the branching
point
Amylopectin
B. Licheniformis

amylase

panose precursors

Panose

A protein engineering concept aiming at a

BLA based amylase with the desired specificity:
B.licheniformis:

Active Site

Space for
branched
dext rins

-5

-4

-3

-2

-1

+1 +2
+3

Less Important
Import ant and St rong
in Termamyl

Asp.niger -amylase:

No Space for
Branching
Points Near
Cleavage Site

Active Site

-2

Less Important

-1

+1 +2
+3

Important and Strong

in Acidic -amylase

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 95C 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

Reference

95,9

1,85

1,26

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

The new enzyme, Liquozyme X, does not give rise

to panose formation, when active during
saccharification (AMG E, initial DS 30%)
1,6

Termamyl LC

1,4

Liquozyme X
1,2

Total DP3

Inactivated amylase
1
0,8
0,6
0,4
0,2
0
20

30

40

50

60

Hours

70

80

90

100

New liquefaction 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

-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