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