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Enzyme Engineering

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(1)

Enzyme Engineering

5. Immobilized Enzymes

(2)

Why Enzyme?

Selectivity and specificity

Reaction under mild conditions

Environmental aspects

Shortcut steps

Ref.) C&EN, 80, 7, pp86-87, 2002

(3)

Why immobilized enzymes?

Definition : Immobilization means that the biocatalysts are limited in moving due to chemically or physically treatment

Reasons

- Reuse of enzyme(reducing cost) - Easy product separation

- Continuous processing

- Stabilization by immobilization

Limitations

-Cost of carriers and immobilization -Changes in properties(selectivity) -Mass transfer limitations

-Activity loss during immobilization

(4)

Immobilization

Cross-linking

Adsorption

Covalent binding

Entrapment

Encapsulation

Conventional Immobilization Methods

(5)

Enzyme Immobilization ; Adsorption

l DEAE-cellulose, activated carbon, etc.

l reversible electrostatic surface interaction -Van der Waals, Hydrogen interaction

Advantage

l Little damage to enzyme l Simple / cheap / quick

l No chemical change to support / enzyme l Reversible to allow regeneration

Disadvantage

l Leakage of enzyme l Non-specific binding

l Over-loading on the support

(6)

Enzyme Immobilization ; Covalent binding

l Formation of covalent bond between enzyme and support

l -NH2 of lysine or arginine

-COOH of aspartic acid or glutamic acid -OH of serine or threonine

-SH of cystein

l Hydrophilicity is the most important factor for maintaining enzyme activity in a support environment.

→ polysaccharide polymers (-OH)

(7)

Enzyme Immobilization ; Covalent binding

l

EDC (1-Ethyl-3-(3dimythylaminopropyl)) activation

Ref.) http://piercenet.com/files/0475as4.pdf

l Nanotube – protein conjugate

Ref.) JACS, 124 (2002) 12664 - 12665

(8)

Enzyme Immobilization ; Covalent binding

Ref.) Biosensors and Bioelectronics 15 (2000), 43-52

(9)

Enzyme Immobilization ; Entrapment

l Entrapped enzymes into lattice by polymer structure synthetic polymer ; polyacrylamide, polyvinyl alcohol natural polymer ; k-carageenan, Ca-alginate

l Restricted in movement by lattice structure of gel

l The porosity of gel lattice is tight enough to prevent leakage of enzyme, yet the same time allow free movement of substrate and product.

(10)

(Ex) Sol-gel Immobilization Method

l One of entrapment method that stabilizes the enzyme by silica structure

Additives polymers, surfactant,

protein stabilizer

Biomolecules Proteins, DNA, cells

Precursor sol

Gelation

Sol-gel formation with entrapped biomolecules

Aging

Polycondensation by solvent evaporation and sol-gel shrinkage

(11)

Sol-gel Immobilization Method

l Colloidal suspension of silica particles that is gelled to form a solid

l Prevent the enzymes to leak out and easily mass transfer because of porous structure.

l Advantages

; Entrapped enzymes with beneficial microenvironments High enzyme activity

Easy mass transfer because of the porous structure Optically transparent silicate materials

Ideal for the development of chemical / biochemical sensors

(12)

Sol-gel Immobilization Process

(a) precursor solution (TMOS, TEOS), sol phase (b) addition of proteins solution

(c) networking

(d) immobilized proteins in porous silica gel

(13)

Enzyme Immobilization ; Encapsulation

l Enveloping the enzymes within various form of semi-permeable membranes such as nylon and cellulose nitrate

l Microcapsules varying from 10-100μm in diameter

l Large enzymes cannot pass out or into the capsule, but small substrates and products can pass freely across the semi-permeable membrane.

(14)

Enzyme Immobilization ; Cross-linking

l Covalent bond formation between enzymes by means of a bi- or multi- functional reagent

l Glutaraldehyde, toluene diisocyanate

l Form a large, three-dimensional complex structure l Toxicity of reagent or limiting factor.

l Multiple- binding of enzymes

(15)

Surface Display

Storaging Time(hr)

0 20 40 60 80 100

Relative Activity(%)

0 20 40 60 80 100

□ : surface displayed β-glucosidase l : β-glucosidase

Surface display of b-glucosidase Degradation of cellobiose Uptake of hydrolyzed glucose Ethanol production

G G

G G G G G G

G

G

Cellobiose

Glucose Surface displayed

b-glucosidase

E E E E 1

3 2

4

1 2 3 4

Ethanol

ß-glucosidase

yeast

(16)

(Ex) Application to Protein-Chip

l Ag - Ab reaction

l Protein – protein interaction / Protein – DNA interaction

l Analysis of protein function

l Proteomics

l Medical diagnosis / drug discovery

(17)

Immobilization type

Adsorption /

Absorption Covalent crosslinking Affinity attachment

Advantage

Disadvantage

ŸGenerally simple

Ÿ No manipulation of the protein sample

Ÿ Reproducibility and stability of protein layer Ÿ The possibility of controlling the density and environment of the immobilized species

Ÿ Identical orientation by site specific

immobilization

Ÿ Direct immobilization by high affinity

Ÿ Easy protein

purification and array fabrication

Ÿ Reproducibility and stability of protein layer Ÿ The possibility of controlling the density and environment of the immobilized species Ÿ Some protein denature

and inactive

Ÿ Unstable binding Non- specific random and multi-oriented protein immobilization: activity decreased

Ÿ Irreproducibility of results

Ÿ Non-specific random orientation: activity decreased

Ÿ Some protein denature and inactive

Ÿ Additional chemical reaction for modification in vitro

Ÿ Difficult application in multi-subunit proteins Ÿ The possibility of elusion of some protein

(18)

Immobilization of Coenzyme

; Entrapment in Membrane Module

[Schematic representation of the PEG-NAD=

regeneration system. (FDH, formate

dehydrotenase; SHL, salicylate hydroxylase)]

l NAD+ immobilized to macromolecular carriers like polyethylene glycol l The coenzyme is continuously available for the enzyme activity

l Some biosensors based on the co-entrapment of NAD(P)+-dependent dehydrogenase and NAD+-dextran of PEG-NAD+ have been reported.

(19)

Immobilization of Coenzyme

; Entrapment in Membrane Module

l Recent developments demonstrate the use of nanofiltration membranes for coenzyme retention in continuous enzyme synthesis.

l In this method, a native coenzyme could be used instead of a chemically modified coenzyme.

[Comparison of the production of L-tert-leucine in continuous working enzyme membrane

reactors either equipped with an ultrafiltration or a nanofiltration membrane]

(20)

Immobilization of Coenzyme

; Entrapment in Membrane Module

l

The N6-(2-aminomethyl) –functionalized NAD+, was covalently linked to a pyrroloquinoline quinone monolayer associated with a gold electrode.

* The enzyme-electrode stimulates the

biocatalytic oxidation of lactate and act as an amperometric biosensor for lactate.

[The assembly of an integrated lactate

dehydrogenase monolayer-electrode by the

crosslinking of an affinity complex formed between the enzyme and a PQQ_NAD+ monolayer-

functionalized gold electrode.]

PQQ : Pyrroquinolinequinone

(21)

(Ex) Immobilization of Whole Cells

Carbon nanopowder-Neutral red

Ca-alginate matrix Permeabilized cell

Carbon nanopowder-linker-Neutral red

(22)

(Ex) Hybrid Immobilization

Entrapment

- Bead strong

- Limitation of high cell loading

- Cell leakage

Encapsulation

- No cell leakage - High cell loading -Weak mechanical

strength

Hybrid Immobilization

- Encapsulation technique was applied to entrapment method.

- Increased cell loading - Decreased cell leakage Improvement of Biological Denitrification Efficiency

(23)

Hybrid Immobilization

Matrix Membrane

Entrapment Encapsulation

Hybrid Immobilization

Biocatalyst

Biocatalyst

(24)

Reactors for Immobilized Enzymes

(A) Packed-bed reactor (B) Suspended reactor (C) Panel type reactor (D) Filter type reactor (E) Membrane reactor

(25)

Characteristics of Bioreactors

lImmobilized Bioreactor

Reactor Characteristics

Batch Separation is required

Small amounts of fine chemicals

CSTR

Ease of control, operation Shear effect

Substrate inhibition – favorable

Packed-bed

Most widely used reactor type Conversion – length of the reactor Product inhibition – favorable

Fluidized-bed

Packed-bed< < CSTR Hinimal pressure drop Ease of control – pll, temp.

Oxygen supply, gas removal - better Hollow-fiber Economy of immobilization

Lack of proper control of cell loading

(26)

Engineering Considerations

lBiocatalysts : Engineering Consideration

l1. Critical fluid velocity l

- shear rate

l

- tensile strength (CO₂)

l2. External and internal mass transfer l3. Operational stability (half-life)

l4. Compression behavior, pressure drop

(27)

Considerations for commercialization

l Immobilization yield (Ex: Eupergit) l Long-term stability

l Cost

l Contamination by microorganisms l Impurity treatment in raw materials

(ex) Production of high fructose corn syrup

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