Characterisation of Biocatalysts I

Purification of Biocatalysts

Katja.otto@bci.uni-dortmund.de

Reactants Process Products

Product Biocatalyst
Economics
Recovery Selection

DSP Screening

In situ Enzyme or
rescovery cells?

Application Biocatalyst
Characterization

Stability Biocatalyst
Engeneering Kinetics
Immobilisation
Cell
Reaction
Cofactor engineering
conditions
regeneration
Process
engineering Structural
Multi-Phase
information
systems
Enzyme
engineering

Enzyme Technology/Biocatalysis- Biocatalyst Characterisation 1 LS- BT

Whole Cells or Isolated Enzyme?

Enzyme Technology/Biocatalysis- Biocatalyst Characterisation 1 LS- BT

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 Isolated Enzyme Whole Cells
free immobilized resting growing
Biocatalysis Good Good Fair Fair
Substrate utilization +++ +++ ++ ++
Reaction specificity +++ +++ ++ +
Cofactor +/- +/- + +++
Inhibition by substrate or
++ ++ + +/-
product
Sensitivity to toxic
+++ +++ + +
substances
Byproducts +++ +++ + +
Process Good Better So-so So-so
Productivity +++ +++ + ++
Process control ++ +++ ++ ++
DSP ++ +++ + +
Process adaptability ++ +++ + +/-
Environmental effects ++ ++ + +/-
Reproducibility ++ +++ + +
Enzyme Technology/Biocatalysis- Biocatalyst Characterisation 1 LS- BT

Isolated Enzyme Whole Cells

free immob resting growing
Biocatalyst
effort effort easy easy
preparation
Cell growth +++ +++ +++ +++
Enzyme purification + + non non
Enzyme
non ++ non non
immobilization
Lifetime + ++ +/- +/-

Biocatalytic costs High Higher Low Low

+++ Good, easy, not a problem

+/- Poor, hard, problematic

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Reasons for studying (purified) enzymes

Application as Biocatalyst

Optimal reaction conditions Æ optimal space time yield

• side reactions
• kinetics
• structure

Enzyme Technology/Biocatalysis- Biocatalyst Characterisation 1 LS- BT

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 Objectives of protein purification

1) Separate one protein from a complex mixture of

proteins and cell debris, while maintaining biological
function

2) Separate one biological function from other from

other biological functions that interfere with the
intended application

Enzyme Technology/Biocatalysis- Biocatalyst Characterisation 1 LS- BT

BULK enzyme FINE enzyme

applications applications

• detergents (washing powder) • synthesis of pharmaceutical

• starch & sugar (modification, intermediates
hydrolysis, isomerization) • resolution of racemic mixtures
• baking and brewing • lens cleaning
• dairy (e.g. phytase in animal feed) • synthesis of biopolymers, organic
• fruit juice (clearing) acids, amino acids
• textile, leather • medical applications - diagnostics
• paper & pulp • scientific research (as tool: e.g.
• Novozymes molecular genetics)
• Genencore • DSM Fine Chemicals

Technically pure As pure as possible

(70 – 80 %) (90 - 99 %)
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A few basics…

•UV absorption (280nm): Tyrosine / Tryptophan

Concentration •Biuret method: interaction of Cu with peptide N
•Lowry method: interaction of Cu with peptide N
•Bradford: protein dye binding
•Gel electrophoresis
•Immunological methods (Western blot, ELISA)

Specific activity = activity (Int.Units)

Protein amount (mg) Protein Activity

Spectrophotometrically: absorption or fluorescence

Radiometric
HPLC or GC: Product analysis
Calorimetry
Electrochemical: Voltage

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 Calculation of purity from the specific activity
requires a reference number:

What is the specific activity of 100% pure, 100% active protein ?

degree of purification =
Enzyme purity spec. act. at step (N+1)
spec. act. at step N

Estimation of purity by sight from SDS PAGE

non-protein contaminants: DNA, RNA, Carbohydrates

Enzyme Technology/Biocatalysis- Biocatalyst Characterisation 1 LS- BT

Charge of an aminoacid depends on pH

H H H

R C NH3 + R C NH3+ R C NH2

COOH COO- COO -

pH 1 Isoelectric point pH
12

Isoelectric focussing (IEF)

Enzyme Technology/Biocatalysis- Biocatalyst Characterisation 1 LS- BT

Native Heterologous
Host Host

• Wildtype strain • Recombinant bacterial Host-

• Animal strains (Escherichia coli)
• Plant • Recombinant yeasts
(Saccharomyces cervicia)
• Plants: www.prodigene.com
• other eukaryotic hosts (HeLa,
insect cells)
• Low expression levels
• Low yields
• Laborious purification work
• Laborious cloning procedures
• Difficult cultivation conditions
• High expression levels
• Higher yields
• Easier purification work
• Easy cultivation conditions
Enzyme Technology/Biocatalysis- Biocatalyst Characterisation 1 LS- BT

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 Chemical & Physical Cellular
Properties Localisation

• Size • Membrane bound

• pI • Soluble in the cytoplasm
• Oxygen or protease sensitive
• Hydrophobicity

Or is nothing known except the enzyme activity?

How can we detect this activity?

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Protein Purification Strategy— General Rules I

• use as few steps as possible

• use the most selective step first
• reduce the volume of material to be handled
• avoid to many sample adaptation steps in between
chromatography steps as you loose too much material
• cheap and widely applicable (academic lab, many different
proteins)
• fast, reliable, automatable (industrial lab)

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Protein Purification Strategy— General Rules II
Parameters to retain protein integrity

• pH: determine optimal pH, use buffers

• Temperature: work at low temperatures (0 - 6 C°)
• Free radical formation: exclude oxygen
• Mechanical stress: avoid foam, interfaces
• Labile thiol groups: use DTT or b-mercaptoethanol
• Dilution effects: add BSA or other stabilizers
• Proteolysis: add protease inhibitors
• PMSF: phenylmethanesulfonyl fluoride (sunfonylation of serine)
• EDTA: ethylenediaminetetra acetic acid (binds metals)
• pepstatin (binding to active site)

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 U
1) Separation from source material

Mild: enzyme treatment, osmotic shock, homo-

Cell-lysis genizer
Moderately harsh: waring blender, grinding
vigorous: French pressure cell, bead mill, ultra-
sonication
Separation from Separation of extracellular enzymes from intact cells
solid materials Separation of intracellular enzymes from cellular
debris

Filtration: pressure filters, vacuum filters, cross-

Separation flow filtration
methods Centrifugation: sedimentation of solid particles in
a centrifugal field (density of the particles)

Enzyme Technology/Biocatalysis- Biocatalyst Characterisation 1 LS- BT

Methods— Precipitation
Fibrinogen
Hemoglobin
• salts decrease the 0.5 Myoglobin
solvating power of water
by “tying-up” water
Log solubility g/L

molecules 0

• hydrophobic patches on
-0.5
proteins are exposed
leading to aggregation

-1.0
• fractionated precipitation
0 2 4 6 8 10
Ionic strength, μis

Enzyme Technology/Biocatalysis- Biocatalyst Characterisation 1 LS- BT

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Methods— Chromatography: General Concept

Chromatography is the separation of molecules

according to their different reactions with two phases
(gas, liquid and solid)

Gas Chromatography, High Pressure Liquid

Chromatography, Fast Protein Liquid Chromatography

• The molecules are separated according to:

• physical properties (size, shape, charge, hydrophobic interactions)

• chemical properties (covalent binding)
• biological properties (biospecific affinity)

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 Affinity Chromatography
Specific Adsorption of the target molecule to the
chromatography matrix

Ion Exchange Chromatography

Adsorption of the proteins who have the opposite

charge of the chromatography matrix

Hydrophobic Interaction

Adsorption of molecules carrying exposed

hydrophobic goups
Gelfiltration or Size Exclusion
Chromatography

Molecules are separated

acording to their size

Enzyme Technology/Biocatalysis- Biocatalyst Characterisation 1 LS- BT

Affinity Chromatography
1 1 2 3

3 Elution of the target protein:

1) elution by simple buffer change
2)Harsh conditions in respect to pH or other parameters
are required. Denaturation of ligant or protein possible.
3+4) Replacement of ligant or binding group

Enzyme Technology/Biocatalysis- Biocatalyst Characterisation 1 LS- BT

IEC
1
Matrix

Positively charged
Equilibration of the
groups column with low ionic
strength buffer

2 Negatively charged
proteins Loading of the sample.
Negatively charged
Neutral charged proteins will bind
proteins

3 Elution of the protein

with increasing ionic
strength of the running
buffer

Enzyme Technology/Biocatalysis- Biocatalyst Characterisation 1 LS- BT

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 Hydrophobic Interaction Chromatography (HIC)

• Same principle as salting out: at high salt conc. proteins expose

hydrophobic patches and can bind to the column material

• With increasing water activity: protein dissolves better in water

• fewer hydrophobic patches exposed
• protein is released from the column material

• Typical hydrophobic adsorbents contain

• C4, C8, C18 linear aliphatic chains
• same alkyl chains with terminal amino-group

Enzyme Technology/Biocatalysis- Biocatalyst Characterisation 1 LS- BT

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Hydrophobic Interaction Chromatography (HIC)

Enzyme Technology/Biocatalysis- Biocatalyst Characterisation 1 LS- BT

Size Exclusion Chromatography (SEC)

• Also called gel filtration (GF)

• SEC material consists of
beads with large and/or small
pores
• Small molecules or salt ions
can enter the beads (occupy
the entire volume of the
column)
• Larger molecules are
excluded from (part of) the
beads, and therefore move
faster

Enzyme Technology/Biocatalysis- Biocatalyst Characterisation 1 LS- BT

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Purification table
Tot. Vol. Act./Vol. Prot. Conc. Tot. Act. Spec. Act. Recovery Pur.
Step
[mL] [U/mL] [mg/mL] [U] [U/mg] [%] Factor

100.
CE 55 2.1 13.5 115 0.15 1.0
0

IEC 13 6.5 13.5 85 0.48 73.9 3.2

HIC 3.6 10.3 5.0 37 2.06 32.2 13.7

SEC 0.6 15.2 3.8 9.1 3.96 7.9 26.4

CE: crude extract

Enzyme Technology/Biocatalysis- Biocatalyst Characterisation 1 LS- BT

Recommended Reading

Protein Purification: Principles and Practice

Robert K. Scopes
Springer Verlag New York

Protein Purification
Handbook Amersham Biosciences
http://www5.amershambiosciences.com
(pdf also on our website)

Enzyme Technology/Biocatalysis- Biocatalyst Characterisation 1 LS- BT