a
Enzyme
detergen
•
•
liquid
10/30/2014
2
NOVOZYMES PRESENTATION
CONTENT
•
Denaturation and degradation of
enzymes
•
Factors influencing the enzyme
stability in liquid and solutions,
and formulation guideline for
enzymatic liquid detergents
3
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NOVOZYMES PRES£TAT70N
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ENZYME
DEGRADATION
AND DENATURATION
•
10/30/2014
Novo2Yes PRESENTATION
When the enzyme looses its structure it
looses its function
Denaturation:
• High temperature (frying an egg)
• Chemical attack
• (pH, bleach)
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Protein inactivation
Autoproteolysis
or proteolysis
Active enzyme
in solution
Denaturation /
unfolding
over time
Eh
d
r---'
Chemical
modification
j
5%%
Precipitation
Inhibition
6
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NOVOZYMES PRESENTATION
Factors influencing enzyme stability in a
liquid detergent
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• Water content
• pH
• Type and amount of enzyme stabilizers
• Time
• Temperature
• Enzyme dose
• Major components such as surfactants, builders
• Other components in the reaction mix (e.g.
preservatives, bleach)
These factors interact
Each enzyme has a specific range of operating
parameters for its optimum performance
'
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Activity - effect of temperature
Effect of temperature on the
activity of Alcalase
100
:•
•
80
60
l
40
2&
20
%
0
20
30
40
50
Temperature (C)
60
70
80
SUBSTRATE
DENATURED HAEMOGLOBIN
METHOD> ANSON
PH
8.5
IO MIN TIME
•
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NOVOZYMES SENTA710
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Activity - effect of pH
Activity of Alcalase at
different pH-values
100
tli
t
%
2
80
60
40
20
�
0
4
5
6
7
8
9
10
11
12
pH
SUBSTRATE
HAEMOGLOBIN
TEMPERATURE
25 (
10 MIN REACTION TIME
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9
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Stability depends on conditions
Enzyme stability over time
100
z«
-
1 year
1 hour
60
16 weeks
3
3 40
2
Storage
In detergent
During Wash
20
0
Time - log scale
SUBSTRATE
HAEMOGLOBIN
TEMPERATURE 25C
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Enzyme stability
Inactive enzyme does NOT contribute to
performa nee
I
100
--►
80
2
60
3
6
«<t
•
•
%
0r
R
Active enzyme
contributes to
performance
40
20
.
0
0
1
.
.
2
3
4
Storage period (weeks)
5
6
7
8
11
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Enzyme denaturation
During storage in liquid detergent, the 3-dimensional
structure of the enzyme molecule may be distorted
(denatured) through interaction with (mainly):
• Surfactants
• Builders
• Buffers
•
•
•
The denaturation may result in loss of enzyme
activity as well as increased risk of enzyme
degradation.
••
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Enzyme degradation
During storage in liquid detergent, the amino-acid chain which
forms the "backbone" of the enzyme molecule may be degradated
by hydrolysis ( reaction with water).
The hydrolysis is accelerated (mainly) by:
• Protease
• High pH
• High water activity
-•
•
•
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•
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•
•
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FACTORS INFLUENCING THE
ENZYME STABILITY IN LIQUID
SOLUTIONS, AND FORMULA1oN
GUIDELINE FOR ENZYMATc
LIQUID DETERGENTS
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Formulation guidelines 1
Water content
Preferably
: < 50 %
Possible
: 50-60 %
Not Advisable : > 60 %
The risk of hydrolytic degradation of the
enzyme molecule may be reduced by
lowering the water activity of the detergent
formulation
1$
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Formulation guidelines 2
pH
Preferably
: 7-9
Possible
: 9-9.5
Not Advisable :9.5-11
High pH increases the risk of enzyme
molecule degradation
16
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Formulation guidelines 3
Anionic surfactants
Charged surfactants:
Negative influence on enzyme stability
AES = None > MES > AOS = soap > LAS
alkyl ethoxy sulfate
methyl ether sulfate
Alpha Olefin Sulfonate
linear alkyl.benzene sulphooarte
CH,-(CH,)»-CH-(CH,,-CH,
Recommendation: Use a combination of
non-ionic and anionic surfactants
1
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Formulation guidelines 4
Nonionic surfactants
AEO, > NP, > NP, > None
Amides and alkyl-polyglucosides are good
Recommendation: Preferably use non-ionic
surfactants or combine non-ionic and anionic
surfactants
18
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Formulation guidelines 5
Solvent
OH
A
CH,
Alcohols/hydroxy groups stabilize proteases
and facilitate solubilization of surfactants in
water phase
Propylene-glycol > Ethanol > >
Sorbitol/Glycerol > None
Other coupling agents like Na-Xylene
sulfonate, Na-cumene sulfonate, phosphate
esters, other hydrotropes are also used
19
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Formulation guidelines 6
Buffer system
TEA (Triethanolamine) > DEA > MEA = Urea
H
H
\I
I
(
(
H
H
/
H
Keep ethanol amines (MEA, TEA) levels low
(below 5%) in case 4-FPBA is used
H
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Formulation guidelines 7
Builder system
R.P°
'
[-CH-C-]
2,n
Preferably:
Citrate, polyacrylates (mild builders)
Possible:
Zeolite, phosphate
Questionable:
MGDA (methylglycinediacetic acid)
Not Possible:
EDTA
H
Most enzymes contain Calcium ions for stabilization of the threedimensional structure of the enzyme molecule
During storage in liquid detergent, strong builder components tend to
extract Ca-ions from the enzyme molecules, increasing the risk of
enzyme denaturation
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Formulation guidelines 8
Bleaching system
Preferably: Peroxide, but not very active over
a long period of time
HYDROGEN PEROXIDE
• May require oxidative
stable enzymes like
Everlase
Not Possible: Chlorine
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Formulation guidelines 9
Preservative
Almost all the preservatives compatible with enzymes:
0
CI
S
O
'S
0
• Kathon 886 MW,
• Bronopol,
• Proxel CRL or GXL
• Nipasol M,
• Irgasan DP300,
• Dowicide A
Aldehydes are generally not advisable
Not possible: formaldehyde
0
II
C
4"
23
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Formulation guidelines 10
Enzyme stabilization system
Calcium (CaCl,) and formate (HCOONa)
important for stability of proteases and
amylases
Dosage: 0.02 - 0.1 % (w/w) CaCl,
(100 ppm free Calcium)
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Calcium binding sites
Lipex with Ca-binding site
+
'"LI
r
44
�-
25
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Formulation guidelines 11
Enzyme stability
Acetamide, glycine, maltodextrin may be
added to improve enzymatic stability
Dosage: 0-5 % (w/w)
,Jl
0
NH
foH
NH
Not compatible: Glutamic acid (Phase sep.)
OH
26
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Formulation guidelines 12
Enzyme stability
• Boric acid and Borax (1-3 wt%), use at pH>8
• Sodium formate (1-3 wt%), use at pH<8
• 4-FPBA (0.005-0.02 wt%), included in "Ultra"
proteases
• Propylene glycol (5-10%)
Since enzymes are proteins, protease molecules in
a liquid detergent will tend to decompose
(hydrolyse) other enzyme molecules present in the
formulation
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Example 1
Relative Protease Stability in a Liquid Detergent (30 °C)
as a function of mono-propylene glycol & borax content at pH 9.3
4,5
Enzyme T, life at 110 F
isostability lines
4
3,5
-••
3
•
•
R
525
"0
•
10 days
2
c
1,5
1
3 days
0,5
0
0
2
4
6
8
10
Propylene Glycol (wt. %)
12
14
16
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Example 2
·c)
Protease Stability in a Liquid Detergent (30
as a Function of pH
25 ,--------------------------,
No Stabilizer
- 5% Propylene Glycol + 3% borax
20
-•
'
T 45
•E
3
w
>
10
5
6
6.5
7
7,5
8
8,5
Formulation pH
9
"°
% '
35
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Example 3
Protease Stability in a Liquid Detergent (30 °C)
as a Function of pH
45
No Stabilizer
40
t%Sodium Formate
35
-•
r•
z2
-± 30
• :I
%• •
••
�
4
f 2o
0
-••
>
..
t%
15
0r
10
5
0
6
6.5
7
7.5
8
8,5
9
95
10
""be'·"
10,5
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4-FPBA - highly effective protease inhibitor
• Highly efficient protease
inhibitor compared to
borax/propylene glycol
combination.
• Results in good stability of
protease and other enzymes in
the presence of protease
Molecular structure
Ho
B-
H-O
• 4-FPBA can replace borax and
propylene glycol with minor
formulation adjustments
including lowering of pH.
I
H
I
O)do
4-formylphenyl
boronic acid
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201«4
3l
NOVOZYMES PRESENTATION
4-FPBA is a reversible protease inhibitor
that stabilizes proteases in liquid detergents
Boric acid
4-formyl phenyl boronic acid
(4-FPBA)
H-O
B-<
I
H-0
Borax = di-Sodiumtetraborate, 10.H,0
K,= 10 mM
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H
I
0 C=O
4-FPBA requires less or no
propylene glycol for
stabilization
K,= 0.03- 0.1 mM
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201«4
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Benefits of stabilizer
- the Savinase protease product containing a stabilizer
Formulation
optimization
Cost savings
Due to removal
or reduction in
level of traditional
stabilizers
Due to removal
or reduction in
level of traditional
stabilizers
Reduced level
of boric
compounds
4-FPBA
represents a boric
acid substitution
option: 0.1% of
4-FPBA can
replace 1-3%
boric acid
normally used
EU have reclassified boric
acid as reprotoxic
substance above
5.5%
Simpler
manufacturing
processes
Decrease in
number of
ingredients
Avoid dissolving
step for Borax
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How do borate stabilizers work?
Protease Enzyme
_I ave site
f
0¢ gy
.°
I I
7
0g°
1:1 Borate-Diol Complex
1.2 Borate-Diol Complex
Acidification of borax in a
liquid detergent yields
boric acid and borates.
The borate species react
with propane-diol to form
1: 1 and 1: 2 borate-diol
ester complexes. The 1: 1
complex weakly
associates with the
protease at the active
site and provides
competitive inhibition.
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Mechanism of boric acid
• Boric acid is a subtilisin inhibitor
(alkaline serine protease
inhibitor).
• Dissolution of Borax in a liquid
detergent yields boric acid and
borates. Boric acid associates
with the protease at the active
site and provides competitive
inhibition.
• The borate species react with
propane-dial to form 1: 1 and 1: 2
borate-dial ester complexes. The
1: 1 complex may also provide
inhibition.
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This is what 4-FPBA does?
Protease Enzyme
4-FPBA has the same
effect as borate, but in
much smaller amounts.
y Active site
,&gs,
I I
00
o's
¢
4-FPBA weakly
associates with the
protease at the active
site and provides
competitive inhibition.
36
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Mechanism of 4-FPBA
• 4-FPBA is a subtilisin inhibitor as
boric acid.
• 4-FPBA fits well in the binding
pocket of the enzyme and is
effective in much smaller
amounts than boric acid.
4-FPBA requires less or no
propylene glycol for stabilisation.
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4-FPBA in US type model detergent
with MPG, without Borax, pH 7, 30°C
Savinase Stability
100 ■
75
--
I
■
I
Lipolase Stability
•
100
50
50
25
25
0
"' "'-
75
0
0
7
14
21
-Savinase w/o inhibitor - Savinasew. 4-FPBA
0
7
-No protease
-Savinase w. 4.Fps
14
21
--Savinase w/o inhibitor
• Improved protease stability
• Lipolase stable in basis formulation without protease
• 4 FPBA significantly reduces proteolysis of Lipolase
38
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4-FPBA effect in EU structured, 40C
Savinase Stability
100
Lipolase Stability
75
75
so
so
25
25
0
0
0
7
14
=
100
'
21
-Savinase w/o inhibitor -t Savinase w. 4-FPBA
----0
7
No protease
-Savinasew, 4.Fp8A
-
~
14
21
--Savinase w/o inhibitor
• Savinase stability improved
• Lipolase activity from poor to very good
• 4-FPBA is 1000 X more efficient than Boric Acid based
on weight
39
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Formulation guidelines 13
Physical stability
Sorbitol or glycerol (glycerine) may be added to
improve physical stability.
Dosage: 0-5 % (w/w)
ooH
OH
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Formulation guidelines
- Improve the physical stability in the detergent
I
I
I
• Add glycerol 2%
• Lower CaCl,2H,0 to 0.06 %
• Lower amount of surfactant
• Add hydrotrope, e.g. SXS 2%
• Increase amount of ethanol or MPG
• Lower amount of carboxylic acids:
citrate, borate or formate
• Change ratio between anionic and
non-ionic surfactants
• Increase pH
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Formulation guidelines
- Improve the enzymatic stability in the detergent
Add Cacl,2H,0 to 0.06 %
Add borate + MPG
Add formate
Lower the water content to 40 %
Lower the anionic surfactant
content, especially LAS (AS)
• Examine "strange" detergent
components
• Lower pH
•
•
•
•
•
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Formulation guidelines - detergent influence
Stability
depends largely
on the
formulation of
the liquid
detergent
And only to a very
limited extend on
the formulation of
the enzyme
product
0VO7vOS
HAN' Io1