Carrier-bound Entrapment Cross-Linking Enzyme Immobilisation Enzyme Immobilization: Why, What and How Roger A. Sheldon Electronic Supplementary Material (ESI) for Chemical Society Reviews This journal is © The Royal Society of Chemistry 2013
Carrier-bound
Entrapment
Cross-Linking
Enzyme Immobilisation
Enzyme Immobilization: Why, What and How
Roger A. Sheldon
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Why use enzymes?
slide | 2
• Renewable, biodegradable feedstock • Mild conditions (pH, T & P)
• High rates • Higher quality product • High chemo, regio & enantioselectivity
• No special equipment needed • Environmentally & economically attractive (GREEN)
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
The Challenge
slide | 3
• Disadvantages of enzymes
– Low operational stability & shelf life
– Cumbersome recovery & re-use – Product contamination – Allergic reactions to proteins
• Non viable biocatalytic applications
– Enzyme costs too high
– Not practical
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
The Solution: Immobilization
slide | 4
• Immobilization is Enabling Technology
3/20/2013
High waste / low profit
Low waste / high profit
• Advantages - stability, stability, stability ...
- repeated re-use of biocatalyst (batch)
- easier downstream processing
- continuous process technology
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Types of Immobilization • Binding to a carrier
– e.g. ion exchange resins
• Entrapment
– e.g. in silica sol-gel
• Cross-Linking
– e.g. Cross-Linked Enzyme Aggregate (CLEA)
slide | 5
Carrier-bound / entrapped enzymes have inherently low volumetric & catalyst productivities (90->99% non-catalytic mass) R.A. Sheldon, Adv. Synth. Catal., 349 (2007) 1289
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Cross-Linking with Glutaraldehyde
The monomeric structure of glutaraldehyde does not reflect the complexity of glutaraldehyde behavior in solution and its reactivity with proteins!
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Glutaraldehyde in Practice
slide | 7
I. Migneault, BioTechniques, 37 (2004) 790
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Glutaraldehyde Reactions with Proteins
slide | 8
I. Migneault, BioTechniques, 37 (2004) 790
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Cross-Linking with Glutaraldehyde
• Common, inexpensive and effective protein cross-linking agent
• Cross-linking chemistry still not fully understood
• Type of covalent bond formed depends heavily on glutaraldehyde
concentration, amine concentration, pH, and temperature
• Reduction of Schiff bases with NaBH4 or NaCNBH3 usually not
necessary
• Other aldehyde cross-linkers, such as dextranpolyaldehyde
generally do need a reduction step
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
The CLEA Technology
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Basic CLEA Properties Very high enzyme loading Particle size typically 5-50 µm Good filterability and centrifugability Packed bed possible Mechanically robust
Excellent operational stability
heat, organic solvents and proteolysis (autolysis)
Tuneable hydrophobicity/hydrophillicity
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Advantages of CLEAs 1. Improved properties
• Better storage and operational stability
• Hypoallergenic
• No leaching of enzyme in aqueous media
2. Cost-effective
• No need for pure enzyme (crude cell lysate sufficient)
• Easy recovery and recycle (easier DSP)
• High productivities (kg product/kg enzyme)
3. Broad scope & short time to market
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Magnetic CLEAs
• Synthesis of magnetic nanoparticles in silica
• Functionalisation of nanoparticles with aminopropyl groups
• CLEAtion: cross-linking the enzyme and the nanoparticles
• Magnetic decantation
• Magnetic strength can be adjusted
• No change in CLEA activity
• e.g. hydrolases, oxidoreductases, nitrile hydratases
Characteristics
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Additional Properties of mCLEAs Separation of the enzyme catalyst by magnetic decantation
Magnetic strength of the mCLEA can be adjusted for the particular application
No changes in the structure by the introduction of magnetic particles
No changes in enzyme activity of the immobilised enzyme by the introduction of magnetic particles
mCLEA of any enzyme can be manufactured – currently examples with hydrolases and oxidoreductases
Potential application in the pharmaceutical, food and feed industries, and diagnostics
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Combi CLEAs
slide | 15
Synthesis of CombiCLEAs
• Two or more enzymes in one CLEA
• Used for cascade reactions
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Storage Stability – NHase CLEA
slide | 16
7 months storage
no decrease in activity!
0
20
40
60
80
100
120
0.0 5.0 10.0 15.0 20.0
Exposure to 21 oC (Days)
Resid
ual
Acti
vit
y (
%)
Free enzyme, no additions (%) Whole cell (%) CLEA (%)
• Storage in 0.01M TRIS buffer pH 8, no additions at room temperature
• Instability of free enzyme due to dissociation of multimeric enzyme
van Pelt, Green Chem. 10 (2008) 395-400
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Thermostability – Papain CLEA
• Papain (protease from C. papaya) incubated at pH 7 and 50 °C.
slide | 17
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Recyclability – PaHNL CLEA
• Effect of recycling on the performance of (R)-oxynitrilase CLEA in the hydrocyanation of o-chlorobenzaldehyde
slide | 18
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Lyases
• R- & S- HNLases (5)
• PDC
• DERA
• Nitrile hydratase (9)
Transferases
• Transaminases
•(R) selective (3)
•(S) selective (5)
Scope of the CLEA Technology
slide | 19
Hydrolases
• Pen. Acylases (2)
• Lipases (19)
• Esterases (3)
• Proteases (9)
• Nitrilases (5)
• Aminoacylase
• Phytase
• Galactosidase
• Carbonic anhydrase
Oxidoreductases • KRED
• FDH
• Glucose oxidase
• Galactose oxidase
• Amino acid oxidase
• Laccase (3)
• Catalase
• Chloroperoxidase
• HRP
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Scope of the Technology
slide | 20
Hydrolases
• Pen. Acylases (2)
• Lipases (19)
• Esterases (3)
• Proteases (9)
• Nitrilases (5)
• Aminoacylase
• Phytase
• Galactosidase
• Carbonic anhydrase
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Hydrolases – Pen. Acylase
slide | 21
ampicillin
N
S
C O O H O
N H
N H 2
O
NH 3
pen acylase
N
S
C O O H
H 2 N
O
+
N H 2
O
N H 2
Biocatalyst Conv. (%) S/H ratio Rel. Productivity
Free enzyme 88 2.0 100
T-CLEA 85 1.58 151
PGA-450 86 1.56 0.8
L.Cao, L.M.van Langen, F. van Rantwijk, and R.A.Sheldon, J. Mol. Catal. B:Enzym. 11 (2001) 665
Conclusion – High productivity and S/H
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Hydrolases – Protease
slide | 22
• Alcalase CLEA : B. licheniformis protease • Savinase CLEA: B. clausii protease • Esperase CLEA: B. lentus protease
• BS CLEA : B. subtilis protease
• Papain CLEA : C. papaya protease
• Protease CLEA Discovery Platform
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Hydrolases - Proteases
slide | 23
• To replace toxic organotin compounds
(banned in the EU since 2008)
• Cross-linked enzyme aggregates (CLEAs) of
proteases were tested in artificial seawater (ASW)
both as it is and as a component of the paint.
• It is found that all CLEAs have tolerance to xylene and have
great stability in dried paint.
• The maximum increase in relative activity was found for CLEA B.licheniformis.
• CLEA B.licheniformis has shown 900% activation during storage in ASW.
• In the paint, non-modified subtilisin lost more that 90% of activity in 28 days.
Antifouling Agent in Paint
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Hydrolases – Proteases
slide | 24
N
O OR
O H
CbzHN
PhNH2
A l c a l a s e - C L E A N
O NHPh
O H
CbzHN
M T B E / 5 0 o C
Nuijens, Cusan, Kruijtzer, Rijkers, Liskamp, Quaedflieg, J. Org. Chem. 74 (2009) 5145
Amidation in Organic Media
R Yield (%)
CH3 93
PhCH2 94
H 93
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Regioselective Esterifications
slide | 25
OH
O
R1 H N
OH
O n
O R2
O
R1 H N
OH
O n
A l c a l a s e - C L E A
R2 O H
R1 = C b z , B o c , F m o c
R2 = a l l y l , M e3SiCH2CH2-
n = 1 o r 2
9 2 - 9 8 % Y i e l d
8 4 - 8 9 % I s o l a t e d y i e l d
Nuijens, Cusan, Kruijtzer, Rijkers, Liskamp, Quaedflieg, Synthesis (2009) 809
O H
OH
O
R1 H N
n
O R2
O
O R2
O
R1 H N
n
A l c a l a s e - C L E A
H2O
OH
O
OR2
O
R1 H N
n
R2 O H
n = 1 o r 2 R1 = CBz, BOC, FMOC
R2 = a l l y l o r ( CH3)3SiCH2CH2-
9 5 - 9 8 % Y i e l d
7 7 - 8 7 % I s o l a t e d Y i e l d
O
Nuijens, Kruijtzer, Cusan, Rijkers, Liskamp, Quaedflieg, Tetrahedron Lett. 50 (2009) 2719
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Peptide Synthesis
slide | 26
Nuijens et al, Advan. Synth. Catal. 352 (2010) 2399 – 2404
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Resolution of Amino Ester with Alcalase-CLEA
Ferraboschi, P., De Miere, F., Galimberti, F. Tetrahedron Asymm. (2010) 21, 2136.
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Rhodococcus erythropolis amidase CLEA: Enantioselective Hydrolysis (Astra Zeneca)
A. Wells, presented at the SCI Meeting on Biocatalysis &
Biotransformations, London, October 14, 2010
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Hydrolases - Lipases
slide | 29
Candida antarctica Lipase B CLEA The only commercially available immobilized form of CaL B completely stable to leaching in
water
• Candida antarctica lipase B (CaLB)
• Candida antarctica lipase A (CaLA)
• Thermomyces lanuginosus (Lipolase)
• Rhizomucor miehei
• Candida rugosa
• Alcaligenes sp.
• Pseudomonas stutzeri
Lipase CLEA discovery platform
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Hydrolases - Lipases
Zhao, L. et al. J. Mol. Catal. B: Enzymatic 54 (2008) 7
Yu, H. W. et al. J. Mol. Catal. B: Enzymatic
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Hydrolases - Lipases
P. Hara, U. Hanefeld, L. T. Kanerva, J. Mol. Catal. B: Enzymatic 50 (2008) 80
Free Enzyme E = 19 CLEA E = 74
Majumder, A. B., Mondal, K., Singh, T. P., Gupta, M. N. Biocat. Biotrans 26 (2008) 235
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Hydrolases - Lipases
slide | 32
Ar R
OH
OAc O
Ar R
OH
+ Ar R
OAc
1 A r
= Ph,
R
= M e
2 A r = 2 - f u r y l , R = M e 3 A r = P h , R = C H 2 N H C O
P r
B . c e p a c i a l i p a s e - C L E A
E = 3 5 - > 2 0 0
i n P h M e o r M T B E
4 7 - 5 6 % c o n v e r s i o n
R T , 2 4 - 4 8 h
n
OH C a L A - C L E A
OAc O
n
OH
+
n
OAc
n = 1 o r 2
P. Hara, U. Hanefeld, L. T. Kanerva, J. Mol. Catal. B: Enzymatic 50 (2008) 80
D. Özdemirhan, S. Sezer, Y. Sönmez, Tetrahedron Asymm. 19 (2008) 2717
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Hydrolases - Lipases
slide | 33
R
H
O
OEt
OEt
O
O
OEt
OH
O
C a L B C L E A
H 2 O R
H
K. Robins, Lonza
CaL B CLEA in a Fixed Bed Reactor 100% activity after >300 h on stream
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
CalB CLEA in Organic Media
slide | 34
HN O
(i-Pr)2O
NH2
O
O
OPr Lipase
+
Activity in H2O (U/g)
Activity in (i-Pr)2O (U/g) Ratio
CaL B CLEA–ST 38000 50 21
CaL B CLEA-OM 31000 1500 760
Novozym 435 7300 250 29
O
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
CalB CLEA in scCO2
slide | 35
OH OH OAc
AcO
CaLB CLEA
scCO2
O
+
H.R. Hobbs, M. Poliakoff, R.A. Sheldon, et al., Green Chem. 8 (2006) 816
Catalyst Conversion (%) E
Novozym 435 17 280
CaL B CLEA 48 640
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
CalB CLEA in Ionic Liquid
slide | 36
*dca = (CN)2N
+ + CaLB
4OoC
O
OEt OH
O
O EtOH
Solvent Lipase Time (h) Conv. (%)
t-BuOH Nov 435 6 83
[bmim][dca]* Nov 435 24 0
t-BuOH CaL B CLEA 3 83
[bmim][dca] CaL B CLEA 6 80
A. Ruiz Toral, F. van Rantwijk, R. A. Sheldon et al, Enz. Microb. Technol. 40 (2007) 1095-1099
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Scope of the Technology
slide | 37
Hydrolases
• Pen. Acylases (2)
• Lipases (19)
• Esterases (3)
• Proteases (9)
• Nitrilases (5)
• Aminoacylase
• Phytase
• Galactosidase
• Carbonic anhydrase
Oxidoreductases • KRED
• FDH
• Glucose oxidase
• Galactose oxidase
• Amino acid oxidase
• Laccase (3)
• Catalase
• Chloroperoxidase
• HRP
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Oxidoreductases – Combi CLEA
slide | 38
HO O
HO OH
OH
OH
OH
OH O HO HO O O2 / water / pH 7
O R
O
HO
HO OH
O
O R
O
HO
HO O H
OH
O2 / water / pH 7
combiCLEA 1
combiCLEA 2
combiCLEA 1 = Glucose oxidase / catalase
combiCLEA 2 = Galactose oxidase / catalase
Activity Free enzyme CLEA
1st use 100% 100%
2nd use - 100%
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Oxidoreductases – Laccase
slide | 39
• TEMPO/NaOCl environmentally
unfriendly
• Laccase / TEMPO / O2 :
— Green Alternative
— Enzyme costs too high
(owing to suicide inactivation)
• Increase operational stability with a
laccase CLEA
— Recycle
• Also with cellulose to
carboxycellulose (shampoo)
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Scope of the Technology
slide | 40
Hydrolases
• Pen. Acylases (2)
• Lipases (19)
• Esterases (3)
• Proteases (9)
• Nitrilases (5)
• Aminoacylase
• Phytase
• Galactosidase
• Carbonic anhydrase
Oxidoreductases • KRED
• FDH
• Glucose oxidase
• Galactose oxidase
• Amino acid oxidase
• Laccase (3)
• Catalase
• Chloroperoxidase
• HRP
Lyases
• R- & S- HNLases (5)
• PDC
• DERA
• Nitrile hydratase (9)
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Lyases – Hydroxynitrile Lyases
slide | 41
• Low reaction temp. (5oC)
• Microaqueous environment (0.18% H2O in DCM)
• Immobilization as a CLEA
N
O
+ HCN
M e H N L - C L E A
Pa H N L - C L E A
N
CN
OH
( 93% ee)
( S ) ( 94% ee )
N
CN
OH
( R )
*C. Roberge, F. Fleitz, D. Pollard, P. Devine, Tetrahedron Letters 48(8) (2007) 1473-1477
”The use of a dichloromethane reaction system with enzyme
aggregates and free hydrogen cyanide was crucial in improving
cyanohydrin stereoselectivity through minimizing background racemic
cyanide addition and enzyme-catalyzed racemization of the product.”*
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Lyases – Combi CLEA
slide | 42
Step Economy a Tri-enzymatic Cascade with a Triple-Decker Combi CLEA
• Buffer : DIPE (10:90)
• pH 5.5 / RT / < 5h
• HnL/ NLase / Pen.acylase Combi-CLEA
• Conv. 96% / ee >99%
O
OH
CN
HCN /(S)-HnL OH
COOH
OH
CONH2
NLase
Pen G amidase H2O
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
3/20/2013 slide | 43
(1) Aggregation/purification using ammonium sulfate
(2) Cross linking using glutaraldehyde
Remaining activity in CLEA: >50%
NHase CLEA Formation
1 2
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Lyases – Nitrile Hydratase
slide | 44
0
200
400
600
800
1000
1200
1400
1600
1800
2000
0 2 4 6
Time (h)
Ac
ryla
mid
e C
on
ce
ntr
ati
on
(m
mo
l/L
)
Cells 1600 mmol/L
Cells 2000 mmol/L
extract 1600 mmol/L
extract 2000 mmol/L
CLEA 1600 mmol/L
CLEA 2000 mmol/L
1
2
Run Yield
1 90 %
2 95 %
C N
NH2
O10 mM Tris-HCl pH8
21°C
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
3/20/2013 slide | 45
0
500
1000
1500
2000
2500
3000
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Time (h)
Co
ncen
trati
on
Acry
lam
ide (
mm
ol/
L) CLEA
Extract
Cells1
2
3
4
Run Residual Activity
1 95 %
2 85 %
3 65 %
4 50 %
21 oC
Fed-Batch Production Acrylamide Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
NHases - Storage and Recycle Stability
slide | 46
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 25 26 27 28 29 30 31 32 33 34 35 3624
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
Cycle #
Pro
du
ce
d H
ex
an
ea
mid
e (
mm
ol/
L)
21 C
29 C
35 C
Sto
rage: 3
days o
n ic
e
Exposure
to 2
phase s
yste
m fo
r 60 m
inute
s
Sto
rage: 2
weeks o
n ic
e
Sto
rage: 5
days o
n ic
e
Sto
rage: 2
0 h
ours
at rt.
Sto
rage: 1
day o
n ic
e
Sto
rage: 5
days a
t rt.
Sto
rage: 7
days a
t rt.
Sto
rage: 1
day a
t rt.
Sto
rage: 1
day a
t rt.
Sto
rage: 5
days a
t rt.
Sto
rage: 1
0 d
ays a
t rt.
•1000 µL 0.01 M Tris buffer pH 8
•80 mM hexanenitrile
•3.9 mg protein in CLEA
C N CNH2
ONHase
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Scope of the Technology
slide | 47
Hydrolases
• Pen. Acylases (2)
• Lipases (19)
• Esterases (3)
• Proteases (9)
• Nitrilases (5)
• Aminoacylase
• Phytase
• Galactosidase
• Carbonic anhydrase
Oxidoreductases • KRED
• FDH
• Glucose oxidase
• Galactose oxidase
• Amino acid oxidase
• Laccase (3)
• Catalase
• Chloroperoxidase
• HRP
Lyases
• R- & S- HNLases (5)
• PDC
• DERA
• Nitrile hydratase (9)
Transferases
• Transaminases
•(R) selective (3)
•(S) selective (5)
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
0
10
20
30
40
50
60
70
80
90
TA1 FE TA1 CC TA1 CLEA
TA1 CLEA (s)
TA1a CC TA1a CC 2
TA1a CC 3
TA1a CLEA
% 88 13 11 24 24 69 60 46
% C
on
vers
ion
to
MP
PA
Conversion to MPPA after 24 h
Transferases - Transaminases
slide | 48
NADHNAD+
Transaminase
L-Alanine pyruvate
Alanine Dehydrogenase
Glucose DehydrogenaseGlucose Gluconic acid
H O-NH4+
O
O NH2
• Combi CLEA – TAm and AlaDH
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Triple Combi CLEAs
Different TAm : LDH : GDH ratios in the triple combi CLEAs
0
10
20
30
40
50
60
70
80
TC:1:1 CC 1:1:1 CC 1:2:1 CC 2:1:1 CC 2:2:1
% c
om
par
iso
n t
o F
E o
f co
nve
rsio
n t
o M
PP
A
Comparison to FE (TA:LDH:GDH)
O NH2
TA/LDH/GDH
24 h
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
CLEAs in Reactors
slide | 50
Pen Acylase in Filter Slurry Reactor (FSR)
M. J. Sorgedrager, D. Verdoes, H. van der Meer, R. A. Sheldon, ChimicaOggi, 26 (2008) 23-25
Alcalase® CLEA in fluidized bed
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Microchannel Reactors
slide | 51
• Numbering up vs Scaling up
• 102 x larger surface/volume ratio
• Efficient mass & heat transfer
• Rapid screening of enzyme scope
• γ-Lactamase CLEAs in microchannel reactor
– 100% Activity retention
A. M. Hickey, B. Ngamsom, C. Wiles, G. M. Greenway, P. Watts and J. A. Littlechild,
Biotechnol. J., 4(4) (2009) 510-516
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
Conclusions
slide | 52
• Biocatalysis is Green & Sustainable
• Immobilization is a Key Enabling Technlogy
• The CLEA Technology has many Benefits
— Simple, broadly applicable & cost-effective
— Improved stability & operational performance
— High productivity & product quality
— Applicable to crude cell lysates
— No leaching of the enzyme in aqueous media
— Combi-CLEAs for biocatalytic cascades
— Smart CLEAs (e.g. magnetic CLEAs)
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
53
www.cleatechnologies.com
Thank you…….
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013
slide | 54
Electronic Supplementary Material (ESI) for Chemical Society ReviewsThis journal is © The Royal Society of Chemistry 2013