Carrier-Free Enzyme
Immobilization for Stabilization of
Biocatalysts.
Presented by Curtis Walton
Supervisor Dr. Roberto Chica
November 24, 2011
Introduction
Enzymes are the most powerful catalysts:
 High catalytic activity
• Rate enhancements of 107-1017
• High turnover number
 High selectivity
• Stereo-, chemo-, and regioselectivity
 Mild reaction conditions
• Near-ambient temperature and pH
 Environmentally Friendly
• Biodegradable
• Easy to produce
Richard Wolfenden, Acc. Chem. Res. 2001, 34, 938-94
2
Introduction
Richard Wolfenden, Acc. Chem. Res. 2001, 34, 938-94
3
Industrial Applications of Enzymes
Production
Scale [tpy]
Product
Enzyme
>1 000 000
High-fructose
corn syrup
Glucose
isomerase
>100 000
Lactose-free milk
lactase
> 10 000
Acrylamide
nitrilase
> 1000
6-aminopenillanic
acid
Penicillin amidase
> 100
Ampicillin
Penicillin amidase
The
catalytic power of enzymes allows for the production of many products on
an industrial scale. However, general enzyme stability has limited enzymatic
catalyst in industrial preparations.
Bommarius, Biocatalysis, 2004, Wiley
4
Enzyme Stability
Denaturant Type
Target
End Product
Temperature
Hydrogen bonds
Highly disordered structure
Aggregates
Acids
Buried uncharged group
(histidine, peptide bonds)
Random coil
Alkali
Buried uncharged groups
(tyrosine, cysteine)
Random coil
Salts
Polar and non-polar
groups
Highly disordered structure
Solvents
Non-polar groups
Highly disordered peptide with large
helical regions
Surfactants
Hydrophobic domains
Disordered with large helical regions
Denaturants disrupt the secondary structure of the enzymes resulting in
catalytically inactive enzymes
P. V. Iyer et al., Process Biochemistry 43 (2008) 10191032
5
Overcoming Enzyme Stability
Issues
Several approaches have been used to overcome these
deficiencies including:
 Enzymes from extremophiles
 pH extremes or high temperatures
 Taq & Vent Polymerase
 The enzymes may not exist in extremophiles
Ksenia Egorova, Current Opinion in Microbiology Volume
8, Issue 6, December 2005, Pages 649-655
6
Overcoming Enzyme Stability
Issues
 Genetic manipulation of enzymes by rational design
and directed evolution
Genetic manipulation to stabilize enzymes can be a time consuming process
requiring multiple rounds of screening mutants, especially to gain large in enzyme
stability beyond the native enzyme
Bertus Van den Burg et al., PNAS March 3, 1998 vol. 95
no. 5 2056-2060
Christopher K. Savile, et al. Science 329, 305 (2010);
7
Enzyme Immobilization
 Immobilization is the most widely used technique. This
is due to:
 Ease of separation of the enzyme from the reaction
products
 Enzyme reuse is economically advantageous
(reduction in waste and catalyst production)
 Stabilization of the enzyme
8
Overcoming Enzyme Stability
Issues
 Immobilization
Immobilization provides a fast method for stabilizing enzymes by restricting the
enzymes structural mobility
R. A. Sheldon, Adv. Synth. Catal. 2007, 349, 1289-1307
P. Lopez-Serrano et al., Biotechnology Letters 24: 13791383, 2002
9
Enzyme Immobilization
 There are two approaches for immobilizing enzymes:
 Carrier-bound immobilization
 Carrier-free immobilization
P. Lopez-Serrano et al., Biotechnology Letters 24: 13791383, 2002
10
Carrier-free immobilization
Extensive research over the past 20 years has been
performed for the immobilization of enzymes on carriers.
However, carrier-free immobilization has several
advantages:
 Elimination of solid support carrier and reagents
 Ease of preparation
 Reduced activity loss
 Increased space-time yield
Carrier-free immobilization can be performed by cross-linking enzymes crystals
and enzyme aggregates
11
Cross-Linked Enzyme Crystals
(CLECs)
In the 1960’s cross-linking reagents were first used to
stabilize enzyme crystals for crystallography.
Richards et al., J. Mol. Biol. (1968) 37, 231-233
Guli, M., et al., Angewandte Chemie
Volume 122, Issue 3, pages 530-533, 10 DEC 2009
12
CLECs - Glutaraldehyde
 Glutaraldehyde’s first application as a cross-linking
reagent was described by Quiocho and Richards in
1960’s.
 It was determined to exist as monohydrates,
dihydrates, cyclic hemi-acetals and oligomers.
 The ratio of these forms of glutaraldehyde is
dependent on pH, temperature and concentration.
 The mechanism of protein reticulation by
glutaraldehyde is poorly understood.
Richards et al., J. Mol. Biol. (1968) 37, 231-233
A. H. Korn et al., J. Mol. Biol. (1972) 65, 525-529
B. M. Salem, et al., Acta Cryst. (2010). F66, 225-228
13
CLECs - Glutaraldehyde
Glutaraldehyde can exist in several forms that could react with lysine residues.
Richards et al., J. Mol. Biol. (1968) 37, 231-233
A. H. Korn et al., J. Mol. Biol. (1972) 65, 525-529
14
CLECs - Glutaraldehyde
 A proposed mechanism for the reaction between
glutaraldehyde and proteins
Cross-linking by glutaraldehyde could occur through schiff base formation but the mechanism is still not
fully elucidated. Thus, the optimal concentration of glutaraldehyde for cross-linking is determined
empirically.
Richards et al., J. Mol. Biol. (1968) 37, 231-233
A. H. Korn et al., J. Mol. Biol. (1972) 65, 525-529
15
Determination of Optimal
Glutaraldehyde Concentration
Low concentrations of glutaraldehyde can result in the reconstitution of the soluble
enzyme. Excess glutaraldehyde can restrict enzyme flexibility abolishing catalytic activity
and disrupt the lattice structure.
Sobolov et al., Tetrahedron Letters, Vol. 35, No. 42, pp.
7751-7754, 1994
16
CLECs are Diffusion Controlled
The crystallization procedure can be modified to form small enzyme crystals which is
important to prevent mass-transfer limitations. Once the enzyme crystals are cross-linked
determination of the catalytic and mechanical properties is necessary.
A. L. Margolin, Angew. Chem. Int. Ed. 2001, 40, 22042222
17
Analysis of CLECs
pH
dependence
Temperature
dependence
Catalytic
properties
Stability in
solvents
Stability
against
proteolysis
New CLEC
Preparation
Solubility
Enzyme assay
Enzyme
kinetics
Size and shape
Physical/
Mechanical
properties
Shear stability
To determine if a cross-linked enzyme crystal can be used as a biocatalyst several criteria
must be evaluated. Can CLECs retain stability under hash conditions and for long periods
of time?
J. D. Vaghjiani et at., Biocatalysis and Biotransformation,
Vol. 18, pp. 151-175, 1999
18
Thermolysin CLECs
 Thermolysin is a protease found in various Bacillus
species.
 Used in manufacturing of the artificial sweetener
aspartame.
3N21
N. L. St. Clair et al., J. Am. Chem. Soc. 1992, 114,
7314-7316
19
Preparation of Thermolysin CLECs
 The purified enzyme was crystallized in 1M Calcium acetate,
30% dimethyl sulfoxide, 50 mM Tris buffer, pH 7.0
 Cross-linked with 12.5% glutaraldehyde in 50 mM Tris buffer
for 1 hour and and subsequently lyophilized.
 Rehydration of thermolysin CLECs allows for retention of
80% activity compared to soluble non-lyophilized enzyme.
The prepared CLECs can be assayed for activity in the presence of organic solvents.
N. L. St. Clair et al., J. Am. Chem. Soc. 1992, 114,
7314-7316
20
Thermolysin CLECs on Organic
Solvents
 An equal volume of organic solvent was added to free and
cross-linked enzyme preparations for 1 hour at 40oC
 The enzyme activity was determined following the
incubation period
% maximum activity
Solvents
Free
thermolysin
CLECs of
Thermolysin
acetonitrile
42
102
dioxane
66
97
acetone
75
99
tetrahydrofuran
36
96
CLEC retain >95% activity in the presence of organic solvents. Can they retain activity at
elevated temperatures?
N. L. St. Clair et al., J. Am. Chem. Soc. 1992, 114,
7314-7316
21
Thermolysin CLECs Stability
 Thermolysin was
incubated at 65oC
for 5 days
CLEC retain >90% activity during incubation at 65oC . CLECs retained activity at pH
extremes.
N. L. St. Clair et al., J. Am. Chem. Soc. 1992, 114,
7314-7316
22
Thermolysin CLECs
 Thermolysin was
incubated with a
mixture of proteases
called Pronase
CLECs are resistant to degradation by proteases. Can other enzymes be stabilized under
these conditions?
N. L. St. Clair et al., J. Am. Chem. Soc. 1992, 114,
7314-7316
23
Aldolase CLECs
 Rabbit Muscle Fructose diphosphate aldolase (RAMA) catalyzes
a key carbon-carbon bond forming step in the synthesis of
eukaryotic RNA polymerase inhibitor.
 Due to its broad substrate
specificity, RAMA has been
exploited to synthesize
other compounds.
Dihydroxyacetone phosphate
D-glyceraldehyde 3 -phosphate
 However, many desired
substrates are insoluble in
aqueous medium.
Can we apply CLEC technology to stabilize RAMA in organic solvents?
Sobolov et al., Tetrahedron Letters, Vol. 35, No. 42, pp.
7751-7754, 1994
24
Aldolase CLECs in Organic Solvents
 An equal volume of organic solvent was added to free and
cross-linked enzyme preparations for 1 hour at 40oC
 The enzyme activity was determined following the
incubation period
Solvent
% Starting activity
Lyophilized RAMA
CLEC of
RAMA
DMF
25
94
acetonitrile
5
90
acetone
37
92
DMSO
3
89
THF
3
99
dioxane
14
99
CLECs remain active in the presence of organic solvents. Are CLECs of RAMA stable for
long periods of time?
Sobolov et al., Tetrahedron Letters, Vol. 35, No. 42, pp.
7751-7754, 1994
25
Aldolase CLECs
CLEC RAMA
65% activity
after 6 months
High [10mg/mL]
soluble RAMA and
FDP
Dilute solutions
(0.2mg/mL)
of soluble RAMA and
FDP
CLECs of RAMA remain active for over 6 months.
Sobolov et al., Tetrahedron Letters, Vol. 35, No. 42, pp.
7751-7754, 1994
26
CLECs Conclusions
 CLECs retain enzymatic activity for significantly
longer periods of time than the soluble enzyme
 Thermolysin (>4 days)
 Aldolase (>6 months)
 CLECs retain enzymatic activity at higher
temperatures (>40oC) than the soluble enzyme
 CLECs retain enzymatic activity in the presence of
organic solvents
However, the high cost of using purified enzymes crystals on industrial scale has limited
their use
27
Cross-Linked Enzyme Aggregates
(CLEAs)
 Proteins aggregate in
the presence of high
concentrations of salts.
 Do aggregates retain
activity?
 Can we immobilize them
in a similar fashion to
CLECs?
How do we prepare enzyme aggregates?
P. Lopez-Serrano et al., Biotechnology Letters 24: 13791383, 2002
28
Preparation of CLEAs
Purification and aggregation can be performed in a single step. However, are the
aggregates active?
Richard R. Burgess, Methods in Enzymology, Volume 463
29
Activity of Penicillin Acylase CLEAs
• Penicillin Acylase was
the first enzyme to be
tested for activity
following aggregation
CLEA retain activity albeit lower than cross-linked crystals. What do these aggregates
look like?
Cao et al., Organic letters, 2000, Vol. 2, No. 10, 13621364
30
Electron Micrograph of CLEAs
Unlike enzyme crystals, aggregates do not form a lattice but they do form a superstructure. An
individual aggregate can millions of enzyme molecules. Since enzyme aggregates can be prepared very
quickly can prepare a high throughput method to optimize the aggregates activity?
R. Schoevaart, et al., Biotechnology and
Bioengineering, Vol. 87, NO. 6, September 20, 2004
31
High Throughput Development of
CLEAs
R. Schoevaart, et al., Biotechnology and
Bioengineering, Vol. 87, NO. 6, September 20, 2004
32
High Throughput Development of
CLEAs
Not all precipitants are appropriate for use with each enzyme. It is necessary to determine the best
precipitant for each enzyme to retain maximal activity. Some enzymes experience hyperactivation by
precipitation.
R. Schoevaart, et al., Biotechnology and
Bioengineering, Vol. 87, NO. 6, September 20, 2004
33
Hyperactivation of Lipase B CLEAs
Lipase B experiences a conformation change from a closed to open state resulting in
hyperactivation of the active site that does not occur in the soluble enzyme. Do CLEA
exhibit similar characteristics to CLEC in terms of stability.
R. Schoevaart, et al., Biotechnology and
Bioengineering, Vol. 87, NO. 6, September 20, 2004
U. Hanefeld et al., Chem. Soc. Rev., 2009, 38, 453–468
34
Nitrile Hydratase CLEAs
 >100,000 tons of acrylamide produced annually
 Enzymatic process replaced heterogeneous copper
catalyst at 120oC
 The enzyme is unstable in cell free formulation and used
in whole cell formulations
 Product can be destroyed by cells
(presence of amidases and protease
make a cell-free methods
preferable)
S. Van Pelt et al, Green Chem. 2008, 10, 395-400
Brant J Bassam & Peter M Gresshoff
Nature Protocols 2, 2649 - 2654 (2007)
35
Preparation of Nitrile Hydratase
CLEA with Glutaraldehyde
Glutaraldehyde [wt%]
Protein
[mg/mL]
Activity in
supernatant after
1st wash (%)
Activity in
supernatant after
3rd wash (%)
Remaini activity in
CLEA (%)
0.3
3.8
4.5
0.7
15
0.6
3.8
2
0.3
23
1.2
3.7
0.02
0
21
2.3
3.5
0.02
0
16
3.3
3.4
0
0
10
4.2
3.2
0
0
4
The optimal concentration of glutaraldehyde was determined by measuring the amount of
remaining soluble enzyme. Does cross-linking increase stability of Nitrile Hydratases?
S. Van Pelt et al, Green Chem. 2008, 10, 395-400
36
Nitrile Hydratases CLEA stability
The nitrile hydratase is stable at 21oC for >21 days. Do CLEA of nitrile hydratase allow for
stable production of acrylamide?
S. Van Pelt et al, Green Chem. 2008, 10, 395-400
37
Nitrile Hydratases CLEA Activity
The CLEA is capable of producing acrylamide and out performs whole cell and crude cellfree extracts. Can the CLEA be reused?
S. Van Pelt et al, Green Chem. 2008, 10, 395-400
38
Fed-batch production of Acylamide
with CLEAs of Nitrile Hydratases
The CLEA is capable of producing acrylamide and out performs whole cell and crude cellfree extracts and were catalytically activity through multiple cycles.
S. Van Pelt et al, Green Chem. 2008, 10, 395-400
39
Antifouling CLEAs
 To prevent fouling of vessels by microorganisms
tributyltin was previously as an antifouling agent
 However, tributyltin has been banned by European
countries.
 Protease have been shown to prevent fouling by
degrading biofilms
Each year fouling cost the
shipping industry millions
of dollars.
Are CLEAs capable of preventing fouling in salt water after being mixed and dried with
marine paint?
Messugeur et al., Progress in Organic Coatings Volume
50, Issue 2, July 2004, Pages 75-104
Skovgaard et al., J. Mater. Chem., 2010, 20, 7626-7629
J. Kim et al., Biotechnology Progress
Volume 18, Issue 3, pages 551–555, 2002
40
Subtilisin CLEAs in Artificial Salt
Water
ASW – 547.6mM NaCl, 56.8 mM MgSO4x7H2O, 2.4 mM NaHCO3
Incubated in Xylene for 24 hours
The CLEA remain activity in ASW and Xylene had no irreversible effect on the structure of the aggregates. Overall, the
CLEA have a better stability in ASW although the unmodified subtilisin absolute rate of catalytic activity was highest.
Skovgaard et al., J. Mater. Chem., 2010, 20, 7626-7629
41
Stability of CLEAs in dried marine
paint
The free enzyme activity decrease over a 28 day period. CLEAs remain active over 28 days. Why does
the free enzyme activity decrease significantly?
Skovgaard et al., J. Mater. Chem., 2010, 20, 7626-7629
42
Hydration Layer of CLEAs in Marine
Paint
Unmodified particles are extremely small (8 nm3) and will leach quickly from the paint resulting in a lost
of activity, having aggregated enzymes is advantageous in reducing leaching of the enzymes.
As the hydration layer increase additional CLEA are exposed to ASW
Skovgaard et al., J. Mater. Chem., 2010, 20, 7626-7629
43
CLEA Conclusions
 Simple method of generating immobilized enzymes.
 Purification and aggregation in one step.
 Increased storage lifetime compared to soluble
enzyme.
 Retain stability over multiple batch reactions
 Retain stability in the presence of organic solvents
44
Future Prospects
 Renewable and selective catalytic technology
 Increase in the number of enzymes used in industrial
processes with CLEC/CLEAA technology such as
biosensors
 Potential for catalytic cascades (combination of
multiple catalytically relevant enzymes aggregated
together)
45
Acknowledgements
Thanks to Jamie Davey and Samuel Oteng-Pabi.
Thank you for listening!
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