this journal is © the royal society of chemistry 2013 ... entrapment cross-linking enzyme...

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)


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


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


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


Cross-Linking with Glutaraldehyde

The monomeric structure of glutaraldehyde does not reflect the complexity of glutaraldehyde behavior in solution and its reactivity with proteins!


Glutaraldehyde in Practice

slide | 7

I. Migneault, BioTechniques, 37 (2004) 790


Glutaraldehyde Reactions with Proteins

slide | 8

I. Migneault, BioTechniques, 37 (2004) 790


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


The CLEA Technology


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


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


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


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


Combi CLEAs

slide | 15

Synthesis of CombiCLEAs

• Two or more enzymes in one CLEA

• Used for cascade reactions


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


Thermostability – Papain CLEA

• Papain (protease from C. papaya) incubated at pH 7 and 50 °C.

slide | 17


Recyclability – PaHNL CLEA

• Effect of recycling on the performance of (R)-oxynitrilase CLEA in the hydrocyanation of o-chlorobenzaldehyde

slide | 18


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


Scope of the Technology

slide | 20

Hydrolases


• Lipases (19)

• Esterases (3)

• Proteases (9)

• Nitrilases (5)

• Aminoacylase

• Phytase

• Galactosidase



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


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


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


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


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


Peptide Synthesis

slide | 26

Nuijens et al, Advan. Synth. Catal. 352 (2010) 2399 – 2404


Resolution of Amino Ester with Alcalase-CLEA

Ferraboschi, P., De Miere, F., Galimberti, F. Tetrahedron Asymm. (2010) 21, 2136.


Rhodococcus erythropolis amidase CLEA: Enantioselective Hydrolysis (Astra Zeneca)

A. Wells, presented at the SCI Meeting on Biocatalysis &

Biotransformations, London, October 14, 2010


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



Zhao, L. et al. J. Mol. Catal. B: Enzymatic 54 (2008) 7

Yu, H. W. et al. J. Mol. Catal. B: Enzymatic



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



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



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


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


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


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



slide | 37

Hydrolases


• Lipases (19)

• Esterases (3)

• Proteases (9)

• Nitrilases (5)

• Aminoacylase

• Phytase

• Galactosidase



• FDH

• Glucose oxidase



• Laccase (3)

• Catalase


• HRP


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%


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)



slide | 40

Hydrolases


• Lipases (19)

• Esterases (3)

• Proteases (9)

• Nitrilases (5)

• Aminoacylase

• Phytase

• Galactosidase



• FDH

• Glucose oxidase



• Laccase (3)

• Catalase


• HRP

Lyases


• PDC

• DERA



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.”*


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


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


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


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



slide | 47

Hydrolases


• Lipases (19)

• Esterases (3)

• Proteases (9)

• Nitrilases (5)

• Aminoacylase

• Phytase

• Galactosidase



• FDH

• Glucose oxidase



• Laccase (3)

• Catalase


• HRP

Lyases


• PDC

• DERA


Transferases

• Transaminases

•(R) selective (3)

•(S) selective (5)


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


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


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


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


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)


53

www.cleatechnologies.com

Thank you…….


slide | 54


slide | 55


this journal is © the royal society of chemistry 2013 ... entrapment cross-linking enzyme...

Documents