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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 10 7 -10 17 High turnover number

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introduction
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

introduction1
Introduction

Richard Wolfenden, Acc. Chem. Res. 2001, 34, 938-94

3

industrial applications of enzymes
Industrial Applications of Enzymes

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
Enzyme Stability

Denaturants disrupt the secondary structure of the enzymes resulting in catalytically inactive enzymes

P. V. Iyer et al., ProcessBiochemistry 43 (2008) 1019-1032

5

overcoming enzyme stability issues
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

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overcoming enzyme stability issues1
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);

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enzyme immobilization
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 issues2
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., BiotechnologyLetters 24: 1379-1383, 2002

9

enzyme immobilization1
Enzyme Immobilization
  • There are two approaches for immobilizing enzymes:
    • Carrier-bound immobilization
    • Carrier-free immobilization

P. Lopez-Serrano et al., BiotechnologyLetters 24: 1379-1383, 2002

10

carrier free immobilization
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
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., AngewandteChemieVolume 122, Issue 3, pages 530-533, 10 DEC 2009

12

clecs glutaraldehyde
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

H. Korn et al., J. Mol. Biol. (1972) 65, 525-529

M. Salem, et al., Acta Cryst. (2010). F66, 225-228

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clecs glutaraldehyde1
CLECs - Glutaraldehyde

Glutaraldehyde can exist in several forms that could react with lysine residues.

Richards et al., J. Mol. Biol. (1968) 37, 231-233

H. Korn et al., J. Mol. Biol. (1972) 65, 525-529

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clecs glutaraldehyde2
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

H. Korn et al., J. Mol. Biol. (1972) 65, 525-529

15

determination of optimal glutaraldehyde concentration
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

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clecs are diffusion controlled
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, 2204-2222

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analysis of clecs
Analysis of CLECs

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

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thermolysin clecs
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
Preparation of Thermolysin CLECs
  • The purified enzyme was crystallized in 1M Calcium acetate, 30% dimethyl sulfoxide, 50 mMTris buffer, pH 7.0
  • Cross-linked with 12.5% glutaraldehyde in 50 mMTris 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
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

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 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 clecs1
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
Aldolase CLECs
  • Rabbit Muscle Fructose diphosphatealdolase (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.
  • However, many desired substrates are insoluble in aqueous medium.

D-glyceraldehyde 3 -phosphate

Dihydroxyacetone phosphate

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
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

CLECs remain active in thepresence 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 clecs1
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 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
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., BiotechnologyLetters 24: 1379-1383, 2002

28

preparation of cleas
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
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, 1362-1364

30

electron micrograph of cleas
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
High Throughput Development of CLEAs

R. Schoevaart, et al., Biotechnology and Bioengineering, Vol. 87, NO. 6, September 20, 2004

32

high throughput development of cleas1
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

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hyperactivation of lipase b cleas
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
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
Preparation of Nitrile Hydratase CLEA with Glutaraldehyde
  • 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
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
Nitrile Hydratases CLEA Activity
  • The CLEA is capable of producing acrylamide and out performs whole cell and crude cell-free 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
Fed-batch production of Acylamide with CLEAs of Nitrile Hydratases
  • The CLEA is capable of producing acrylamide and out performs whole cell and crude cell-free extracts and were catalytically activity through multiple cycles.

S. Van Pelt et al, Green Chem. 2008, 10, 395-400

39

antifouling cleas
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 OrganicCoatings 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
Subtilisin CLEAs in Artificial Salt Water
  • ASW – 547.6mM NaCl, 56.8 mM MgSO4x7H2O, 2.4 mMNaHCO3
  • 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
Stability of CLEAsin 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
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
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
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
Acknowledgements

Thanks to Jamie Daveyand Samuel Oteng-Pabi.

Thank you for listening!

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