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Chemical Biotechnology
Protein Engineering
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Enzyme
engineering
Chemical Biotechnology― Protein Engineering LS- BT
Protein Engineering : content
• objectives
• tools
• site-directed mutagenesis
• modeling, prediction
• examples
• subtilisin
• glucose isomerase
• thermostabile enzymes
1
The Goals of Protein Engineering
• increased understanding of the mechanisms which determine:
• structural stability (pH, T, solvents)

• reaction mechanism and kinetics (kcat, Km , KI )
• allosteric regulation
• effector recognition (receptors); protein-protein interactions
• DNA binding and gene regulation; protein-DNA interactions
• protein folding
Protein Engineering in Biotechnology
• industrial applications require properties that are not selected

for in nature
• searching in nature (screening), for example isolating

thermostable enzymes from extremophiles, is not a general
solution
• some (combinations of) properties are not selected for in nature
• enzymes may have to work with unnatural substrates
=> engineering of industrial enzymes is useful
Improvement of Enzymes by Protein

Engineering
• alter KM & kcat -> increased catalytic efficiency (kcat/KM)

• increased thermal stability
• changed pH optimum
• modification of reactivity, stability, and solubility in organic solvents
• create a cofactor independent enzyme from a cofactor dependent
one (e.g. H2O2 as electron-donor)
• change NADPH-preference to NADH-preference
• reduced protease-sensitivity
• altered allosteric regulation of an enzyme to abolish feedback inhibition
• changed post-translational modifications
• reduction of folding problems (inclusion bodies)
2
Modification of Protein Structure
protein
engineering
genetic chemical
techniques techniques
modification of
site-specific localized random
a.a. side-
mutagenesis mutagenesis
groups
chemical synthesis of
hybrid proteins or parts of
formation proteins
Protein Engineering Cycle
purification
improved
enzyme properties protein
3-D structure expression
molecular
mutagenesis
modelling
predictions sequence
purification
improved
molecular
mutagenesis
modelling
3
Mutagenesis Techniques
• site directed mutagenesis techniques (in vitro ) by PCR
• random and semi-random approaches:

• generation of mutants (in vivo) and selection of a desired phenotype
• saturation mutagenesis of a gene (segment) and selection in vivo
• directed evolution / gene shuffling
• gene synthesis
• difficult-to-clone genes
• unusual (non-optimal) codon usage
• source organism is not accessible
Site-Directed Mutagenesis: Requirements
• cloned gene
• method to introduce single or multiple mutations

• QuikChange (Stratagene)
• knowledge about structure, function and structure-function

relationships
Quikchange Site-Directed Mutagenesis
plasmid DNA a PCR reaction with primers that a restriction enzyme (DpnI - cleaves at
template is isolated contain the mutation is run over GAmetTC sequences) digests the
from a DAM+ strain: the whole plasmid template methylated wild type template DNA
the unmethylated DNA is transformed
DNA is methylated at the PCR generates un- into E. coli where the nicks are repaired
GATC seqs methylated plasmids with two colonies can be screened for the
nicks correct mutation
4
purification
improved
molecular
mutagenesis
modelling
SDM: Selection of Target Position
• known 3-D structure (> 10000, many of which closely related

to each other)
• known structure - function relationships
• obtain pdb-file (Protein Data Bank: www.rcsb.org/pdb/)

• view structure e.g. in SwissPdbViewer (freeware program for
PC/PowerMac: http://www.expasy.org/swissmod)
• identify amino acid residues to modify
• model changes in SwissPdbViewer
• make changes by site-directed mutagenesis
Subtilisin: a Protease
5
• known 3-D structure but unknown structure-function relationships
• make (educated) guesses to identify regions or residues to modify based on

• structure homology
• sequence homology
• biochemical data
• localized random mutagenesis (or SDM)
• unknown 3-D structure (by far most cases)

• compare sequence with Database sequences using the BLAST sequence
similarity search tool (www.ncbi.nlm.nih.gov)
• homology with proteins for which 3-D structure data are

available
• submit aa sequence to Swiss-Model: An Automated Comparative

Protein Modelling Server
• compares protein sequence to the subset of Swiss-Prot database
sequences for which 3-D structures are available
• calculates and optimizes a 3-D model based on sequence homology
• view structure in SwissPdbViewer --> etc. as above

• no homology with proteins for which 3-D structure data are
available
• compare sequence using the BLAST sequence similarity search tool

• identify sequence motifs for which data linking sequence to
function are available
• identify highly conserved residues
• make changes by site-directed mutagenesis
• or random mutagenesis (screen or selection method)
=> Random mutagenesis can generate data linking sequence to

function
6
• PROSITE: Database of protein families and domains

(http://www.expasy.org/prosite/)
• proteins share motifs or domains, e.g. NAD-binding fold (G X G X X G)
• proteins share post-translational modifications
• cleavage site for signal peptidase
• ribosylation or phosphorylation sites
• biochemical characterization is necessary, but sequence motifs

allow site-directed mutagenesis to modify potential binding sites
or post- translational modification sites

purification
improved
molecular
mutagenesis
modelling
• structure determination or prediction
• X-ray diffraction
• NMR techniques (multidimensional NMR: 1H, 13C, 15N)
• de novo structure prediction based on primary structure

(prediction of 3D structure from the primary structure) requires
• description of the folded state

• the kinetic pathway of folding (not very successful yet)
7
Prediction of Altered Proteins
• hydrophobic non-polar
aromatic: Phe (F), Trp (W)
aliphatic: Pro (P), Gly (G), Ala (A), Val (V),
Leu (L), Ile (I), Met (M)
• uncharged polar: Ser (S), Thr (T), Tyr (Y),
Cys (C), Asn (N), Gln (Q)
• charged positive: Lys (K), Arg (R), His (H)
negative: Asp (D), Glu (E)
• special cases
• Cys - disulfide bonds (covalent)
• Pro - imino acid, restricted mobility, cis and trans
• Gly - not chiral
Industrial Applications of PE
• most major companies producing industrially applied enzymes are

active in protein engineering
• most patents and publications deal with
• methods
• medically important proteins
• proteases and lipases (washing powders)
• glucose isomerases
• laccases (e.g. lignin modification, paper strengthening, phenol
polymerization, hair dyeing, textile bleaching and waste-water
treatment)
CASE STUDIES
•subtilisin: used in detergents
•glucose isomerase: high-fructose corn-syrup

production (HFCS)
•thermostabile enzymes
8
Enzymes in washing powder
composition of a heavy duty washing powder
group ingredient content (w/w)

surfactants anionic 2-10%
non-ionic 0.5-6%
soap 1-5%
sequestering polyphosphate 30-50%
agents or zeolites
bleaching sodium perborate 20-30%
agent activator (TAED)
enzymes proteases/lipases 0.3-0.6%
sodium sulphate to make up to 100%
Subtilisin: Ca++-Dependence
• builders have a negative influence of subtilisin (protease)

activity and stability
• builders (sodium triphosphate, EDTA) promote autolysis

• autolysis is a persistent problem in performance, storage and
production
• autolysis is strongly Ca++ dependent
• subtilisin is Ca++ dependent
• protein engineering to create a more stable Subtilisin BPN'
• some homologous proteases are NOT Ca++ dependent

• structure of BPN' is available
Subtilisin Stabilization
• Strategy
• Cys instead of Ser (thiosubtilisin; chemical modification; less autolysis)

• delete Ca++ loop
• restabilize the enzyme w/o Ca++ loop (disulfide bond)
• X-ray structure of thio-subtilisin BPN'
• examine regions of high mobility and introduce stabilizing mutations
• random mutagenesis in regions of high mobility & screening
• assessment of enzyme stability
• combination of stabilizing mutations (∆75-83, Q2K, S3C, P5S, K43N,
M50F, A73L, Q206C, Y217K, N218S, Q271E)
9
Subtilisin: Ca++-Dependence
Half-lives and kinetic parameters of BPN’ and a loop

deleted variant at T = 65°C
Enzyme 10 mM EDTA 10 mM Calcium
native BPN’ < 1 second 60 minutes

loop deleted 35 minutes 90 minutes
Enzyme kcat (sec-1) Km (mM) kcat/Km x 105

(M-1sec-1)
native BPN’ 52 0.16 3.2
loop deleted 84 0.32 2.7
Subtilisin: Oxidation
• protein engineering of a protease stable to bleach
• Proctor and Gamble noted in 1969 (before SDM!) that in their

subtilisin (a protease from Bacillus licheniformis) methionine 222 was
oxidized by a bleach, thereby severely inactivating the enzyme.
• At that time no remedy was available.
• in the early ‘80-ties SDM was applied to this enzyme
10
Subtilisin: Oxidation―Saturation Mutagenesis
100
M222A
M222S
wild
type
0
0 1 2 3 4 5 6
week
Storage stability of the alkaline protease and its mutants
in bleach-containing detergent
Subtilisine: Substrate Specificity
Gly166 is in the
substrate binding
pocket P1 close to
the catalytic triad
--> likely to affect
substrate specificity
Succinyl-Ala-Ala-Pro-X-p-nitroanilide: X is P1 amino acid

11
Subtilisin: Substrate Specificity
pos. charged a.a. --> higher activity against P-1 = Glu

acidic a.a. --> poor activity against P-1 = Phe, Ala
Kcat values vary as much as Km values
Glucose Isomerase
• industrially important (one

of the three highest
tonnage enzymes with
amylase and protease)
• many properties can be
improved
• 3-D structure available
Glucose Isomerase
•substrate specificity: the natural substrate of GI is

D-xylose (hemicellulose degradation)
•stability to glycation (Maillard reaction)
•alteration of pH-optimum
• most GIs have a high pH optimum
• starch liquefaction runs at a low pH
•thermostability
•GI sensitivity to calcium (necessary for starch
liquefaction Æ additional process step)
12
Glucose Isomerase: Substrate spec.
CH 2 OH
OH HOCH2 O OH
H D-glucose (xylose)
H
OH H H HO
isomerase H CH2 OH
HO OH
H OH OH H
D-glucose D-fructose
H
OH H O OH
H
D-glucose (xylose)
H
OH H H HO
isomerase H CH 2 OH
HO OH
H OH OH H
D-xylose D-xylulose
Thr141
The C. thermo-
sulfurogenes GI
was modelled
based on the
Arthrobacter GI
Trp139 and
Val186 were
Val186 Trp139 changed by SDM
U
=> double mutants have up to 6-fold increased catalytic

efficiency (kcat/Km) for glucose
13
K
Glucose Isomerase

•thermostability
K
Glucose isomerase: Maillard reaction
NHR
HC O HC NR
• stability to
HCOH
glycation HCOH HCOH
+RNH2 HCOH -H2O
• under process HO CH HO CH
conditions GI is HO CH
HCOH HCOH
exposed to high HCOH
HCOH HCOH
conc. of glucose HCOH
(3M) and high CH2OH CH2OH
CH2OH
temperatures D-glucose CH3NHR
(60°C)
HCOH
CH2OH
N-substituted
HO CH 1-amino-1-deoxy
O
OH -D-fructose
glucosylamine NHR HCOH
OH HCOH
OH
CH2OH
Æ lysine residues react with glucose

Glucose Isomerase: Maillard Reaction
14
U
Glucose Isomerase: Maillard Reaction
• glycation of Lys 253 of Actinoplanes missouriensis GI

(located at the interface of the active dimer) leads
to inactivation
• mutagenesis of Lys 253 to Arg increases the half life
of GI
Glucose Isomerase
•thermostability
U
Alteration of pH-Optimum
•starch liquefaction runs at a low pH (esp. glucoamylase

has a low pH-optimum)
•most GIs have a high pH optimum
• Æ screening for low pH GIs

• Æ SDM of Glu-186 to Gln lowers the pH-optimum from 7 to
6.25
15
K
Glucose isomerase: Thermostability

•a more thermostable GI would allow the process to
run for a longer time
• thermal inactivation limits operating time
•or at a higher temperature

• reduced risk of microbial infection
• decreased viscosity, fewer by-products
• higher conversion because DG = 5 kJ/mol Æ equilibrium is
T-dependent:
25°C 43.5% fructose
65°C 51.5%
90°C 55.6%
U
Glucose Isomerase: Thermostability
Glucose Isomerase: Thermostability
• synergistic effect of mutations

• replacement of the glycines or the alanine alone causes a
loss in stability
• the triple mutant is stabilised, probably due to sidechain
interactions
16
K
PE towards thermostable enzymes
• enzymes from (hyper)thermophilic microorganisms

• high denaturation temperature
• high rigidity
• optimal activity at high T (process requirement)
• flexible at high temperature, but rigid at low T --> can be
stored at room temperature
• purification by heat treatment (in E. coli only the ferredoxins
are heat stable)
• easily crystallized
• easily overproduced in mesophiles (often protease resistant)
K
Causes of Thermostability
• no 'general rule' to account for the stability and rigidity, but
• molecular determinants that increase thermostability:
• fewer aminoacids that are vulnerable to heat

• deamination: Asn, Gln
• disulfide bond destruction: Cys
• peptide bond hydrolysis: Asp
• more densely packed interior (intramolecular packing)
• water excluding hydrophobic interactions (more Phe, Ile, Leu, Val, ...)
K
Causes of Thermostability
• no deep spaces (smaller surface area to volume ratio)

• compact structures
• increase in helix-forming amino acids
• more ion pairs on the surface
• internal salt bridge networks
• restriction of C- and N-terminal mobility
• stabilization of α-helix dipoles
• smaller surface loops
• less surface loops
• more Pro residues in loops
17
U
Intrinsic Thermostability
-galactosidase: free energy of stabilization
Source Relative Approx. ∆Gstab

(Topt in ˚C) half lives at Tm (˚C) relative to
85˚C E. coli
E. coli (37˚C) 1 60 0
T. aquaticus (75˚C) 1080 75 -20 kJ / mole
S. solfataricus (85˚C) 2880 88 -43 kJ / mole
Free Energy and Stabilization
•the change in free energy responsible for a 10˚C

increase in Tm of a protein is -20 to -30 kJ / mole.
•for comparison:
• H-bonds 2 - 10 kJ / mole
• Salt bridges 4 - 20 kJ / mole
• Hydrophobic interactions 0.5 - 3 kJ / mole
Stabilization by Metal Binding
Enzyme mole Ca++ half-life ∆Gstab ∆Gstab ∆Gstab due

per mole t1/2 (h) at total intrinsic to Ca++
enzyme 80˚C (kJ/mole) apoenzyme (kJ/mole)
(kJ/mole)
Subtilisin 1 <1 -107 -94 -13

Thermolysin 4 1 -120 -86 -34
Caldolysin 6 >30 -169 -88 -80
Archaelysin 0 >>30 -200 -200 0
18
K
Protein Engineering: Summary
•to test knowledge about protein structure and

function
•to make specific changes that affect the

properties of an enzyme
K
PE: Limitations & Perspectives
•limitations
• reality often doesn’t match prediction

• it has proven very difficult to change substrate specificity in a
predictable manner
• method is labor intensive
• requires extensive prior knowledge
•perspectives
• structure prediction from primary sequence data

• molecular modelling
• rapid increase in primary and 3-D structure information
pK1 pK2
Amino Acid Symbol Structure* pK R Group
(COOH) (NH2)
Amino Acids with Aliphatic R-Groups
Glycine Gly - G 2.4 9.8
Alanine Ala - A 2.4 9.9
Valine Val - V 2.2 9.7
Leucine Leu - L 2.3 9.7
Isoleucine Ile - I 2.3 9.8
Non-Aromatic Amino Acids with Hydroxyl R-Groups
Serine Ser - S 2.2 9.2 ~13
Threonine Thr - T 2.1 9.1 ~13
Amino Acids with Sulfur-Containing R-Groups
Cysteine Cys - C 1.9 10.8 8.3
Methionine Met-M 2.1 9.3

19
pK1 pK2
Amino Acid Symbol Structure* pK R Group
(COOH) (NH2)
Acidic Amino Acids and their Amides
Aspartic Acid Asp - D 2.0 9.9 3.9
Asparagine Asn - N 2.1 8.8
Glutamic Acid Glu - E 2.1 9.5 4.1
Glutamine Gln - Q 2.2 9.1
Basic Amino Acids
Arginine Arg - R 1.8 9.0 12.5
Lysine Lys - K 2.2 9.2 10.8
Histidine His - H 1.8 9.2 6.0
Amino Acids with Aromatic Rings
Phenylalanine Phe - F 2.2 9.2
Tyrosine Tyr - Y 2.2 9.1 10.1
Tryptophan Trp-W 2.4 9.4
Imino Acids
Proline Pro - P 2.0 10.6

Prediction of Altered Proteins
• the PAM250 matrix (or Dayhoff matrix)
• statistical evaluation of naturally occurring mutations between

related proteins
• indicates how often an amino acid is replaced by another in
known structures
• examples
• Asp is often replaced by Glu (both negatively charged) whereas

Asp is less frequently replaced by Phe (hydrophobic, aromatic)
• Trp is very often conserved in homologous proteins
• Ser, Thr, or Ala are not well conserved
U
PAM250 matrix (Dayhoff)
PAM (percent accepted mutations): a matrix of weights that is
derived from how often different amino acids replace other
amino acids in evolution
ORIGINAL AMINO ACID
Ala Arg Asn Asp Cys Gln Glu Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val
A R N D C Q E G H I L K M F P S T W Y V
Ala A 13 6 9 9 5 8 9 12 6 8 6 7 7 4 11 11 11 2 4 9
Arg R 3 17 4 3 2 5 3 2 6 3 2 9 4 1 4 4 3 7 2 2
Asn N 4 4 6 7 2 5 6 4 6 3 2 5 3 2 4 5 4 2 3 3
Asp D 5 4 8 11 1 7 10 5 6 3 2 5 3 1 4 5 5 1 2 3
Cys C 2 1 1 1 52 1 1 2 2 2 1 1 1 1 2 3 2 1 4 2
Gln Q 3 5 5 6 1 10 7 3 7 2 3 5 3 1 4 3 3 1 2 3
Glu E 5 4 7 11 1 9 12 5 6 3 2 5 3 1 4 5 5 1 2 3
Gly G 12 5 10 10 4 7 9 27 5 5 4 6 5 3 8 11 9 2 3 7
His H 2 5 5 4 2 7 4 2 15 2 2 3 2 2 3 3 2 2 3 2
Ile I 3 2 2 2 2 2 2 2 2 10 6 2 6 5 2 3 4 1 3 9
Leu L 6 4 4 3 2 6 4 3 5 15 34 4 20 13 5 4 6 6 7 13
Lys K 6 18 10 8 2 10 8 5 8 5 4 24 9 2 6 8 8 4 3 5
Met M 1 1 1 1 0 1 1 1 1 2 3 2 6 2 1 1 1 1 1 2
Phe F 2 1 2 1 1 1 1 1 3 5 6 1 4 32 1 2 2 4 20 3
Pro P 7 5 5 4 3 5 4 5 5 3 3 4 3 2 20 6 5 1 2 4
Ser S 9 6 8 7 7 6 7 9 6 5 4 7 5 3 9 10 9 4 4 6
Thr T 8 5 6 6 4 5 5 6 4 6 4 6 5 3 6 8 11 2 3 6
Trp W 0 2 0 0 0 0 0 0 1 0 1 0 0 1 0 1 0 55 1 0
Tyr Y 1 1 2 1 3 1 1 1 3 2 2 1 2 15 1 2 2 3 31 2
Val V 7 4 4 4 4 4 4 4 5 4 15 10 4 10 5 5 5 2 4 17
20
U
Industrial applications of PE
• Patents 1982-1998: 1267
• Patents 1998: 127

• Patents 1999: 70
• Patents 2000: 95 ----> slightly decreasing trend
21

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Chemical Biotechnology

Reactants Process Products

Chemical Biotechnology― Protein Engineering LS- BT

Protein Engineering : content

Chemical Biotechnology― Protein Engineering LS- BT

• structural stability (pH, T, solvents)

Chemical Biotechnology― Protein Engineering LS- BT

Protein Engineering in Biotechnology

• industrial applications require properties that are not selected

• searching in nature (screening), for example isolating

=> engineering of industrial enzymes is useful

Chemical Biotechnology― Protein Engineering LS- BT

Improvement of Enzymes by Protein

• alter KM & kcat -> increased catalytic efficiency (kcat/KM)

Chemical Biotechnology― Protein Engineering LS- BT

Chemical Biotechnology― Protein Engineering LS- BT

Protein Engineering Cycle

3-D structure expression

Chemical Biotechnology― Protein Engineering LS- BT

Protein Engineering Cycle

3-D structure expression

Chemical Biotechnology― Protein Engineering LS- BT

• site directed mutagenesis techniques (in vitro ) by PCR

• random and semi-random approaches:

Chemical Biotechnology― Protein Engineering LS- BT

Site-Directed Mutagenesis: Requirements

• method to introduce single or multiple mutations

• knowledge about structure, function and structure-function

Chemical Biotechnology― Protein Engineering LS- BT

Quikchange Site-Directed Mutagenesis

Chemical Biotechnology― Protein Engineering LS- BT

3-D structure expression

Chemical Biotechnology― Protein Engineering LS- BT

SDM: Selection of Target Position

• known 3-D structure (> 10000, many of which closely related

• obtain pdb-file (Protein Data Bank: www.rcsb.org/pdb/)

Chemical Biotechnology― Protein Engineering LS- BT

Chemical Biotechnology― Protein Engineering LS- BT

• known 3-D structure but unknown structure-function relationships

• make (educated) guesses to identify regions or residues to modify based on

• unknown 3-D structure (by far most cases)

Chemical Biotechnology― Protein Engineering LS- BT

SDM: Selection of Target Position

• homology with proteins for which 3-D structure data are

• submit aa sequence to Swiss-Model: An Automated Comparative

• view structure in SwissPdbViewer --> etc. as above

Chemical Biotechnology― Protein Engineering LS- BT

SDM: Selection of Target Position

• compare sequence using the BLAST sequence similarity search tool

=> Random mutagenesis can generate data linking sequence to

Chemical Biotechnology― Protein Engineering LS- BT

• PROSITE: Database of protein families and domains

• biochemical characterization is necessary, but sequence motifs

Chemical Biotechnology― Protein Engineering LS- BT

Protein Engineering Cycle

3-D structure expression

Chemical Biotechnology― Protein Engineering LS- BT

SDM: Selection of Target Position

• structure determination or prediction

• de novo structure prediction based on primary structure

• description of the folded state

Chemical Biotechnology― Protein Engineering LS- BT

Chemical Biotechnology― Protein Engineering LS- BT