Purification and characterisation of variants of Trp278
and Trp279 in the surface site of
barley -amylase
Ahmet Alsac
s002135
July 2005
1
CHAPTER 1 LIST OF ABBREVATIONS
3
CHAPTER 2
Summary
Preface
Introduction
4
4
5
6
CHAPTER 3 THEORY
7
CHAPTER 4 MATERIALS AND METHODS
Method Scheme
4.1 Expression of foreign genes in Pichia pastoris
4.2 Expression and purification of enzyme variants
4.2.1 Shake flask cultivation
4.2.2 Purification
4.3 Characterization of proteins
12
12
13
15
15
17
20
CHAPTER 5 RESULTS AND DISCUSSION
5.1 Expression
5.2 Purification
5.3. Characterization
33
33
35
39
CHAPTER 6 CONCLUSION
43
BIBLIOGRAPHY
44
CHAPTER 7 APPENDIX
Appendix 1 Expression and purification overview
Appendix 2 Affinity Chromatogram
Appendix 3 DP440
Appendix 4 Cl-pNPG7
Appendix 5 Starch Binding
Appendix 6 Stock Solutions for Media Preparation
Appendix 7 Coomassie blue and silver staining
46
46
49
51
53
55
57
58
2
Chapter 1 List of Abbrevations
A: Alanine
AMY1: barley -amylase low pI isozyme 1
AMY2: barley -amylase high pI isozyme 2
AMS: Ammonium sulfate
BMGY: Buffered glycerol complex medium
BMMY: Buffered methanol complex medium
Cl-pNPG7: 2-Chloro 4-nitrophenyl maltoheptaoside
DMSO: Dimethylsulfoxide
DP440: Degree of polymerization 440
DP17: Degree of polymerization 17
OD: Optical Density
MD: Minimal dextrose medium
MES: 2-morpholine ethansulfonicacid
rpm: revolutions per minute
Trp:Tryptophan
W: Tryptophan
wt: wild type
YPD: Yeast extract peptone dextrose medium.
YNB: Yeast nitrogen base with ammonium sulfate without amino acids
3
Chapter 2
Summary
Variants of barley -amylase isozyme AMY1 were expressed, purified and their kcat, Km
and Kd values were determined. Two single mutants one mutated at tryptophan at
position 278 (W278A) and one mutated at tryptophan at position 279 (W279A), the
double mutant (W278A/W279A) and wild type (wt) were expressed form the yeast
Pichia pastoris using shaker flasks. The variants of proteins were purified from the
culture supernatants by ultrafiltration and affinity chromatography on -cyclodextrinsepharose. For determination of kcat and Km the substrate amylose DP17, amylose DP440
and 2-chloro-4-nitrophenyl D--maltoheptaoside (Cl-pNPG7) were used. There were no
significant differences in these kinetic values between the wt and the single mutants. For
determination of the dissociation constant Kd the binding to barley granules was used.
Here was observed a difference between the wt and the single mutant. Thus single
mutants have higher Kd than the wt. W278A has a Kd of 2.67 (mg/ml), W279A has a Kd
of 3.14 (mg/ml) and wt has Kd of 0.70 (mg/ml). The double mutant gave the same kinetic
values as the wt. This was not an expected result and probably variant used was not
mutated at the right position.
4
Preface
This report is based on experimental research made in the Biochemistry and Nutrition
Group (BNG) of BioCentrum, Technical University of Denmark. The work for the report
corresponds to 30 ECTS points and it is the final project for the civil engineer study. This
study was limited and had a duration of 5 months, beginning 1. February 2005. My
supervisors were Henrik Næsted and Birte Svensson.
5
Introduction
-Amylases from cereals are involved in mobilization of seed storage starch. They are
essential in the production of beer and other beverages and in the baking industry.
Designer enzymes having improved specificity, activity, or synergy with enzyme partners
in the biotechnological area provide important prospects. Finding domains with influence
on -amylase binding to carbohydrates via mutation and engineering of the domain
surface binding site in barley -amylase would most probably enhance their catalytic
power on raw starch and related substrates. Combining the catalytic properties of, for
example psychrophilic -amylases, which hydrolyze polysaccharides at low temperature,
and the capacity of enhanced starch binding of barley -amylase isozyme 1(AMY1)
could result in an  -amylase with high efficiency on starch at low temperature. amylase has three binding sites for binding of starch; one at the active site, one at the so
called “old” starch granule binding surface site and one at the so called sugar tongs. I
have expressed and purified AMY1 wt and three variants, mutated at “old” starch granule
surface binding site, of which determination of kinetic profiles reveal the importance of
these mutations and how important the “old” starch granule surface binding site is for the
function of AMY1. The amino acids that are mutated are tryptophan at position 278
replaced with alanine, W278A, and tryptophan at position 279 replaced with alanine,
W279A, and the double mutat W278A/W279A. It is known that these tryptophans are the
functional residues that bind starch so it is expected that mutation of these residues will
result in a decrease in affinity towards starch.
6
Chapter 3 Theory
-amylases (1,4- -D-glucan glucanohydrolase) are monomeric enzymes widely occurring
in animals, plants, and microorganisms. They catalyze the hydrolysis of internal -D(1,4)-glucosidic linkages in starch [1] (amylose and amylopectin), glycogen, and related
oligo- and polysaccharides to produce maltodextrins, maltooligosaccharides, and glucose
(fig. 3.1). Most of the glucose residues in starch are unbranched and are linked by -D(1,4)-glucosidic bonds. The (1,4) linear molecule in starch is called amylose. The
branches are formed by -D-(1,6)- glucosidic bonds which occur for approximately every
thirty -D-(1,4)-glucosidic linkage, this molecule is called amylopectin. The branches
serve to increase the solubility and make its sugar easily accessible. For glycogen the -D(1,6)- glucosidic bonds occur about once in ten units. Therefore glycogen is highly
branched and more soluble than starch [1].
Seed germination [2] is triggered by an increase in temperature and humidity and causes
the embryo to synthesize gibberellic acid, which induces de novo synthesis of -amylase
and an array of other hydrolases from the living outer tissue of barley seeds called
aleurone layers(fig. 3.2). These activated hydrolases release sugar units from starch in
endosperm. The progressive release of sugars from the storage starch provides energy to
the growing embryo until the plant can feed itself by photosynthesis.
In germinating barley seeds, different  -amylase isozymes, encoded by two multigene
families and referred to as AMY1 and -amylase isozyme 2 (AMY2), are distinguished.
The two isozymes, which contain 414 and 403 amino acid residues, respectively, display
80% sequence identity, and are distantly related to -amylases from microorganisms and
animals. AMY1 has a molar weight at 45.5 kDa. Despite the high sequence identity,
AMY1 and AMY2 show distinctly different physicochemical and biochemical properties.
AMY1 is known as the low-pI (pI 4.9) isozyme, and AMY2 is known as the high-pI (pI
5.9) isozyme. AMY1 has highest affinity for calcium ions [3], is the most stable at acidic
pH, and is the least stable at elevated temperature.
7
Figure 3.1: Overview of the structure of starch. Above the building units and organization of
starch. Below a diagram showing how the side branching chains are clustered together
within the amylopectin molecule.
8
Figure 3.2: Mechanisms of release of sugar into the embryo from endosperm.
(1) The endosperm signals via gibberellic acid that it needs carbohydrates.
(2) The signal is received in aleurone which then activates carbohydrate degrading
enzyme. (3) Enzymes degrade the starch to sugar units in endosperm.
(4) Sugar units are consumed by embryo
While AMY2 is also most stable in urea at pH 6.7, AMY1 has highest stability in urea
below pH 6 or in the presence of NaCl. Moreover AMY1 is most stable in guanidinium
chloride. Charge screening thus destabilizes AMY2 but stabilizes AMY1. Isozyme
sequence comparison suggests that AMY1 lacks four of the 20 salt-bridges identified in
the crystal structure of AMY2. Moreover, AMY1 has the highest affinity and activity
toward starch granules, whereas on soluble substrates AMY1 still has the highest affinity,
9
but AMY2 has the highest turnover rate. Finally, a most remarkable difference is the
unique capacity of AMY2 in binding the endogenous bifunctional inhibitor BASI [4]
(barley -amylase/subtilisin inhibitor).
AMY1 contains three domains [5]. A major central domain with a parallel (/)8 barrel
super secondary structure called domain A. An irregular loop of 65 residues bulges in
domain A between 3 and 3, called domain B. Finally, the 61 residue long loop at the Cterminal is organized as a five – stranded antiparallel -sheet, called domain C
AMY1 has three binding site areas. The “sugar tongs” in domain C, the “old” starch
granule binding site on the surface of domain A and the binding site at the active site. It
has been shown by site-directed mutagenesis and crystallography, that AMY1 contains a
separate surface binding area on the basis of two contiguous tryptophans [5], Trp278 and
Trp279 (Trp276 and277 in AMY2) (fig. 3.3). This site has low affinity for acarbose
(Kd=5mM) and binds -cyclodextrin [5]. The binding of -amylase to -cyclodextrin is
used in affinity chromatography purification. No mutant was previously obtained at
Trp279Ala which is invariant in cereal -amylases. Sequence comparison suggests that
this starch binding site is unique to -amylases from higher plants, whereas other surface
binding sites are reported in certain microbial or mammalian enzymes [5]. Calcium ions
at the active site are critical for folding and conformational stability and hence for the
enzyme activity [3].
The starch granule binding surface binding site from the structure of AMY1/4I,4II,4IIItrithiomaltotetraoside (thio-DP4) is shown at fig. 3.3. It is seen that the two rings from the
thio-DP4 stack onto indole rings of a pair of tryptophans, Trp278 and Trp279. This site is
called the starch granule binding surface site. The two rings of thio-DP4 form six
hydrogen bonds to Trp278 and neighboring residues in the structure [5].The angle
between the planes formed by the two indole rings in Trp278 and Trp279 is around 135
and is constant between AMY1 and AMY2 in both native and complexed structures. The
closer the planes determined by the two indole rings and the plane of sugar rings the
stronger the forces in the hydrophobic stacking. The surface-binding site is suggested to
possess the special capacity of selecting substrates according to their geometrical
characteristics probably governed by the surroundings of the site. The two tryptophans
perform a locked conformation because of a very tight packing with neighboring
10
residues. This structure constitutes a geometric filter that favors binding of structurally
complementary molecules. Because the granular starch binding site and sugar tong are
distant to the active site they may play a more important role when the substrate is longer
and minor role when the substrate is shorter. When the substrate is long the two binding
sites force it in a loop structure around the enzyme.
Figure 3.3: Overall Structure of AMY1 in Complex with Thio-DP4. Calcium ions, spheres.
The three upper calcium ions are those found both in AMY1 and AMY2 (Ca500, Ca501, and
Ca502). The fourth Ca503 is located closer to the center, where it occupies the active site.
Thio-DP4 substrate analog fragments are shown as surface representations. To the left, the
starch granule binding surface site on domain A is shown with the two tryptophans residues
(Trp278 and Trp279). In domain C (bottom part of the figure), an entire thio-DP4 molecule is
curved around Tyr380 at the level of the sugar tongs.
11
Chapter 4 Materials and Methods
Method Scheme
Expression from P. pastoris in shake flask

Purification

Ultra filtration

Affinity chromatography

Protein characterisation
kcat Km :AmyloseDP17, AmyloseDP440 and Cl-pGNP7
Kd: Starch granule binding
12
4.1 Expression of foreign genes in Pichia pastoris
The insertion of gene in the host was not a part of my project, but I will tell the
background for how it is done.
The methylotrophic yeast Pichia pastoris is the yeast specie selected for its advantage
over S. cerevisiae. Two attributes are critical in its selection: the existence of
fermentation methods and presence of methanol regulated promoters [6].
The growth medium is a defined mixture of salts, trace elements, biotin and carbon. They
are basic and cheap sources. Biochemical studies revealed that methanol metabolism
required the induction of set of metabolic pathway enzymes. The most interesting is
alcohol oxidase (AOX), the first enzyme in the methanol utilization pathway. AOX is
undetectable in cells cultured on carbon sources like glycerol, but consists 30% of total
soluble proteins in methanol-grown cells. Under control of an AOX promoter, foreign
genes could be maintained in an expression-off mode on a non methanolic carbon source
to minimize selection for non-expressing mutant strains during cell growth, then
efficiently switched on by shifting to methanol. This expression system will be utilized to
produce -amylase. The goal of the expression effort is the secretion of the foreign
protein. Secretion requires the presence of a signal sequence to target the protein into the
secretory pathway. The secretion signal sequence from the Saccharomyces cerevisiae factor prepro peptide has been used most successfully.
The principle of insertion of a foreign gene in the host Pichia pastoris is shown at figure
4.1. The gene is inserted in the vector under control of AOX promoter. The vector
contains also a wild type gene for histidine. The host’s histidine gene is modified bysite
directed mutagenesis, so the host can not produce histidine and is therefore His−. If the
insertion is succeeded the host will be His+. The inserted gene will only be expressed
when there is methanol in the culture.
13
Figure 4.1: Vector insertion of foreign gene in Pichia pastoris where histidine
gene provides the selection control
For transferring the amylase gene to the host genome the plasmid pPICZA [7] is used. At
figure 4.2 the construction of the plasmid is seen.
14
pUC origin
ApaLI (4179)
AOX1 promoter
ApaLI (623)
CYC1 transcription terminator
ApaLI (3509)
5' AOX1 primer
HindIII (873)
Zeo(R)
SmaI (3254)
EcoRI (944)
pP I C ZA A M Y 1 delta9 novel
AvaI (3252)
NcoI (949)
4593 bp
XmaI (3252)
AvaI (3242)
NcoI (3162)
EM7 promoter
AMY1d9
AvaI (1616)
TEF1 promoter
BamHI (2678)
3' AOX1 primer
6xHis
AvaI (2246)
c-myc epitope
Figure 4.2: Construction of amylase gene (AMY19) in combination with AOX promoter in
the plasmid pPICZA.
4.2 Expression and purification of enzyme variants
4.2.1 Shake flask cultivation
The expression of recombinant proteins like amylase in Pichia pastoris requires the
preparation of several different media [8]. Recipes of these media start with preparation
of stock solutions (Appendix 6).
For the growth and selection of P. pastoris YPD agar plates are used. Agar plates are
done by 1 L of Yeast Extract Peptone Dextrose Medium (YPD) which is made by
dissolving 10 g yeast extract, 20 g of peptone and 20 g agar in 900 ml H2O. This solution
is autoclaved and then 100 ml of 10X dextrose is added. This medium is then
immediately transferred to agar plates and cooled at room temperature. Plates are stored
at +4C. Species of 3 mutants (Trp278Ala, Trp279Ala and Trp278Ala/Trp279Ala) and
wt of P. pastoris from -80C were streaked out on YPD plates and grown at 30C for 48
hours for best cultivation.
Streaks were taken from the plates after growth and inoculated in MD medium. Minimal
Dextrose Medium (MD) is composed of YNB, biotin and dextrose. The wt is histidine
15
mutant, because the insertion of wt of AMY1 gene into the host did not went well.
Therefore it can not grow in MD media hence BMGY media is preferred for wt. Cultures
are inoculated in 3 ml media for 24 hours at 30C at 190 rpm. MD is prepared by making
an 20 ml stock solution where 2 ml 10X YNB, 40 µl 500X biotin and 2 ml 10X dextrose
are mixed in autoclaved H2O with total volume up to 16 ml. The composition of BMGY
is given below. The culture from MD medium was next transferred to BMGY to get
higher cell densities. 1 ml of MD medium of every type is transferred to BMGY medium.
To get an idea of the growth profile of the metabolism of P. pastoris a pilot scale
experiment with small volumes is done. The culture was transferred to 25 ml fresh
BMGY medium in a 250 ml flask and grown at 30C for 24 hours at 190 rpm. The
composition of 25 BMGY is 0,25 g yeast extract and 0,5 g peptone autoclaved in 17,5 ml
H2O and then mixed with 2,5 ml 10X YNB, 2,5 ml 10X glycerol, 2,5 ml 1 M potassium
phosphate buffer pH 6 and 50 µl 500X biotin.
After 24 hours 5 ml of the cultures are transferred to 200 ml BMGY in 1 L flask and
grown at 30C with 190 rpm for 24 hours. After 24 hours a sample from the culture was
taken to check the OD600nm. The OD600nm should be around 1 when BMGY culture
resuspended in 100 ml BMMY (as BMGY except for 0.5 % methanol replacing
glycerol). The volume of BMGY culture, when resuspended in BMMY gives 1, was then
taken and the medium was replaced by 100 ml BMMY in 1 L flask for induction of the
recombinant protein for 24 hours at 30C. After growth the supernatant was isolated by
centrifugation the culture (9000 g, 15 min,+4C), while the pellet was kept for another
induction. During growth there were taken samples to measure OD600nm and the sample
was centrifuged so the supernatant could be used for measuring activity of -amylase by
Insoluble Blue Starch assay. The pellet was again resuspended in fresh 100 ml BMMY
for another induction up to 48 hours. Samples were taken at various times for activity and
OD600nm measuring. Insoluble Blue Starch activity was measured on Insoluble Blue
Starch suspended (6.25 mg/ml) in 20 mM sodium acetate buffer, 5 mM CaCl2, 5 mg/ml
BSA. The reaction was initiated by enzyme addition (around 10 µl) to the suspension
(800 µL) at 37C and stopped after 15 min by addition of 0.5 M NaOH (200 µL). After
centrifugation (4 min, 13 000 g) supernatants were transferred to a micro-titer plate (300
µL). A620 values (Ceres UV900 HDI micro-plate reader, Biotek Instruments,Inc., UK) in
16
the range 0.8-1.2 were used to calculate activity . One unit was defined as the amount of
enzyme that during 15 min reaction resulted in an increase in A620 of 1 in the
supernatant of the stopped reaction mixture. This formula to get the units of -amylase
per ml supernatant:
Units/ ml= ((A620 − Ablank)/ (5  volume sample))  dilution factor 1000
Units per ml can be converted to mg amylase per ml because we know the value of 2900
U/mg for the purified AMY1 wt [9].
According to the data for OD and activity versus time, the large volume production of
recombinant protein started. First all variants were grown on MD and BMGY for wt as in
pilot scale then transferred to 25 ml BMGY media after 1 day, after growth for one day
again, this culture was transferred to fresh 0.8 L BMGY media in 3 L flask and incubated
for 24 hours at 30C and shaken at 145 rpm. After end of culture period the OD600nm was
recorded and volume of BMGY was resuspended in 0.4 L BMMY media inoculated.The
inoculated BMMY should have an OD600 = 1. The culture was grown at 30C at 195 rpm
for 24 hours. After 24 hours the cultures were centrifuged at 9000 rpm and 4C for 15
min. The supernatant was kept, while the pellet was induced for another 48 hours in
BMMY. Like before the cultures were centrifuged, the supernatants were combined with
the previous supernatants and added 0.02% (w/v) sodium azide for germination
protection. The activity, OD280 and volume were recorded (See APPENDIX 1). The
pellets were discarded. The OD280 measures the aromatic residues of tryptophan and
tyrosine, thus representing the total content of protein in the sample. By dividing OD280nm
with extinction coefficient of amylase the content of amylase in a culture can be
estimated. The extinction coefficient of -amylase for OD280nm is 2.4 for 1 mg/ml [10], so
the concentration of -amylase can be found from OD280nm measurements.
4.2.2 Purification
After production of variants of amylase, they were purified from the culture supernatant.
First the supernatants were filtered through a 0.45 µm membrane to remove larger
particles. The first step in purification was ultra filtration (fig. 4.3). Using a membrane of
17
cut-off at 10.000 kDa. The sample was applied and collected in the retentate, while the
permeate was collected to check for content of -amylase. All purification steps were
carried out at 4C.
The second step in purification is affinity chromatography. First, the sample
Figure 4.3: Overview of the principle for ultra filtration
solution must be conditioned for to binding the enzyme to the gel material. This was done
by adding 5% (w/v) ammonium sulfate (AMS). The samples were purified on cyclodextrin-Sepharose (ÄKTA explorer automated chromatograph). Affinity
chromatography is a unique separation technique since it enables purification of almost
any biomolecule on the basis of its biological function or chemical structure. It is a type
of adsorption chromatography in which the molecule to be purified is specifically and
reversibly adsorbed by a complementary binding substance (ligand) immobilized on an
insoluble support (matrix). The dissociation constant (Kd) for the ligand binding
substance complex should ideally be in the range 10-4 to 10-8 M to avoid too weak or too
strong interactions [11]. It is also important that the immobilized ligand retains its
specific binding affinity for the substance of interest (S) and the methods are available for
selectively desorbing the bound substance in an active form, after washing away unbound
material. Purification is often of the order of several thousand fold and recoveries of
18
active material are generally very high [11]. After preparation of the ligand matrix
complex, affinity medium, the substance can be purified as follows. Affinity medium is
equilibrated in binding buffer. The absorbance will be stable baseline (see
chromatograms at Appendix 2). Sample is applied under conditions that favor the binding
of the target molecule to the ligand. This will result in increased absorbance. Target
molecules bind specifically but reversibly to the ligand and unbound material washes
through the column. Washing will result in decrease of absorbance to stable baseline.
Target protein is recovered by changing conditions to favor elution of the bound
molecules. If any target protein is recovered we will se a peak in absorbance. Elution is
performed non specifically by changing the pH, ionic strength, polarity or specifically by
a competing protein. Target protein is collected in a purified, concentrated form and the
affinity medium is re-equilibrated with binding buffer.
For practical work first buffers were prepared: The equilibration buffer is Buffer A, (20
mM sodium acetate, 25 mM CaCl2, pH 5.5, the washing buffer is Buffer B (20 mM
sodium acetate, 25 mM CaCl2, 200 mM NaCl, pH 5.5). The elution buffer is Buffer C (8
mg/ml -cyclodextrin in 20 mM sodium acetate buffer, 25 mM CaCl2, pH 5.5). All
buffers are filtered and degassed to avoid dirt and air in the chromatography.
Around 5 ml CNBr activated Sepharose coupled with -cyclodextrin is packed in a
column. Sepharose is a bead-formed agarose gel. The hydroxyl groups on the sugar
residues can be used for covalent attachment of a ligand. CNBr activated Sepharose
enables ligands containing amino groups to be immobilized. The column pressure limit
was set to 0.30 Mega Pascal and UV absorbance was done 280 nm.
This column is equilibrated with buffer A (4 column volumes) and the sample was
applied to the column at flow rate at 1 ml/min, followed by washing (4 column volumes
Buffer B) to get stable baseline. The bound protein was then eluted with buffer C and the
first peak appearing was collected in 12 ml tubes as 0.5 ml fractions and added 0.02% (w/v)
sodium azide. All purification steps were carried out at 4C and SDS-Page was made for
each purification step and stained by colloidal coomassie blue and silver (Appendix 7 for
detailed method description).
After purification -cyclodextrin was removed by dialysis. Bag tubes with molecular cut
off at 6000 – 8000 kDa were used. Variants of proteins in dialysis tubes were dialyzed
19
against (1 mM MES, 25 mM CaCl2 and pH 6.8) under gentle stirring for 3 days with
buffer changed every day. At fourth day the buffer was changed to 10 mM MES, 25 mM
CaCl2 and pH 6.8.
4.3 Characterization of proteins
Enzyme (E) catalyzed substrate (S) conversion reactions can be best described by the
following reaction scheme [12]:
k
ES
1

k2
k1
  

 ES  E  P ( 4.1)
The scheme shows that the reaction velocity will be proportional to the concentration of
the enzyme substrate ES complex as v = k2[ES]. To put these schemes in to mathematical
framework we use Henri-Menten-Michaelis approach, which assumes a rapid equilibrium
is established between (E + S) and the complex ES followed by slower conversion of ES
back to free enzymes and substrate, thus: k2 k-1. The free enzyme Ef combines with S
to form ES. The equilibrium dissociation constant for this complex is:
Ks =([E]f [S])/ [ES] (4.2)
where
[E]f = [E] − [ES] (4.3)
This can be arranged to:
[ES] =(E][S])/(Ks + [S]) (4.4)
A single chemical step defined by the first order rate constant k2 results in product
formation. More likely there will be a series of chemical reactions following ES complex
formation. These steps are described by the first order rate constant kcat:
20
Ks
E  S 
ES kcat

 E  P(4.5)
and the rate of product formation is given by the first order equation:
v = kcat[ES] (4.6)
Combining (4.4) and (4.6) we get:
v = (kcat[E][S])/( Ks + [S]) (4.7)
Equation 4.7 describes the reaction velocity as a hyperbolic function of [S] with a
maximum value of kcat[E] at infinite [S]. This value is the maximum reaction velocity or
Vmax:
Vmax = kcat[E] (4.8)
Combining this definition with equation 4.7 gives:
v =(Vmax[S])/( Ks + [S]) (4.9)
If the enzyme concentration is known the value of kcat can be calculated by dividing the
experimentally determined Vmax by [E]. The units of kcat are reciprocal time in s−1. It
provides a lower limit on the first order rate constant of the slowest step following
substrate binding that leads to product release.
Km is the substrate concentration that provides a reaction velocity that is half of the
maximal velocity obtained under saturating substrate conditions or it represents the
substrate concentration at which half of the enzyme active sites in the sample are filled or
saturated by substrate molecules in the steady state. It is usually measured in
concentrations in mM or mg/ml.
The catalytic efficiency of an enzyme is best defined by the ratio of the kinetic constants:
kcat/ Km. The ratio has units of a second order rate constant and is generally used to
compare the efficiencies of enzyme variants. The kinetic constants Vmax and kcat are
21
determined graphically with initial velocity measurements obtained at varying substrate
concentrations. The first way of graphing the data is plotting velocity as a function of [S].
For this purpose an excel calculation sheet is used to determine Km and Kcat from amylose
DP440 and DP17 assays (Appendix 3). The absorbency for each initial time and substrate
concentration is inserted in the sheet. Also a standard curve is determined and inserted in
the sheet. The standard curve ensures that we have the corresponding absorbency at
540nm for a given concentration of the product. The increase in absorbency in the
standard curve can be given as linear increase by determining the linear coefficients of
plotting absorbency as function of known product concentration. The standard curves
slope is used to determine product formation in the enzyme mix at various times. The
initial determined OD540 for each substrate concentration at various times are inserted,
then the slope of OD540/min can be determined. Dividing the slope with the slope of
standard curve gives us the velocity in mM/min. Plotting reciprocal value of velocity as
function of reciprocal value of concentration gives a linear line, which gives the
possibility to determine Km and Vmax, equation 4.11.This plott is called Lineweawer Burk
plot and was used before implementation of curve fitting programs. When enzyme kinetic
data is generated it can be linearized by using the method of Linewear and Burk:
v =(Vmax[S])/( Km + [S])
V max
(4.10)
Km
1
S
If reciprocal of this equation is taken we get:
1  Km 1 
1

(4.11)

v  V max Km  V max
We see that 4.11 is an equation for a straight line with slope of Km/Vmax and y intercept of
1/ Vmax, when the reciprocal of the velocity is plotted as function of the reciprocal of
[S]. Plotting velocity as a function of concentration gives us a hyperbolic curve a so
called Michaelis Menten plot. Usually curve fitting programs are used to generate line
through the data. The program Curve Expert is used and the function y = (ax)/ (b+x),
called growth saturation mode, is applied to curve fitting. The outcome of curve fitting is
22
the coefficients of the curve, where the coefficient a divided with enzyme concentration
is Kcat and b corresponds to Km.
Catalytic parameters were determined toward amylose DP440 (average degree of
polymerization 440). This is done by first making amylose stock solution. 100 mg
amylose is stirred for 30 min with 1.6 ml dimethylsulfoxide (DMSO). To this solution
H2O is added up to 12 ml and then 3.5 ml concentrated buffer (100 mM sodiumacetate
pH 5.5) and 18 µl 5% BSA (Bovine Serum Albumin) and 45 µl 2 M CaCl2. Finally H2O
is added up to 18 ml. Also buffer with DMSO and buffer without DMSO were
made.Buffer with DMSO were done by adding 62.5 µl 2 M CaCl2, 25 µl 5% BSA and
2.22 ml DMSO to 5 ml concentrated buffer and finally adding H2O to 25 ml. Buffer
without DMSO is made by adding 100 µl 2M CaCl2 and 40 µl 5% BSA to 8 ml
concentrated buffer and finally H2O up to 40 ml. After these solutions are made the series
of substrate solutions are prepared (tab. 4.1) Then a standard series is prepared from a
maltose stock containing 0.265 mM of maltose monohydrate (tab. 4.2) From amylose
substrate solution 990 µl is taken and every solution is incubated at 37C for 5 min. On
precise time 110 µl of enzyme solution (containing 8 nM variant of AMY1) is added to
substrates. Then 200 µl is taken out at exactly 2.5 min up to 12.5 min and added to 500 µl
reducing sugar assay reagents and 300 µl H2O, called STOP solution. The stop solution is
made by two reducing sugar assay reagents. Solution A is made by dissolving 27.4 g
Na2CO3 and 12.0 g NaHCO3 in 400 ml H2O then 971 mg disodium 2.2-bicinchionate and
added H2O to 500 ml.
23
Table 4.1: Amylose substrate solution
No Mg/ml
Amylose stock (ml)
Buffer with DMSO (ml)
Buffer without DMSO
1
2.5
3
0
3
2
2.0
2.4
0.6
3
3
1.5
1.8
1.2
3
4
1.0
1.2
1.8
3
5
0.75
0.9
2.1
3
6
0.5
0.6
2.4
3
7
0.4
0.48
2.52
3
8
0.3
0.36
2.64
3
9
0.2
0.24
2.76
3
10
0.1
0.12
2.88
3
Table 4.2: Maltose substrate solution
No Maltose mM
Maltose stock (µl)
µl H2O
1
0
0
2000
2
0.0158
120
1880
3
0.0211
160
1840
4
0.0317
240
1760
5
0.0528
400
1600
6
0.106
800
1200
This solution must be protected from light, Solution B is made by 620 mg CuSO4 5 H2O
and 630 mg L-serine dissolved in 500 ml H2O. This solution is kept at 4C in the dark.
Solution A and B are mixed in equal amounts before use. From maltose stock solution
500 µl aliquots are transferred into Eppendorf vials containing 500 µl reducing sugar
assay reagents. All samples are as quickly as possible heated at 80C for 30 min, then
quickly cooled and centrifuged at 14000 rpm for 5 min. 300 µl aliquots are transferred to
a micro titer plate and the absorbency is read at 540 nm in an ELISA reader.
24
It is also possible to determine catalytically parameters toward amylose substrates with
shorter degree of polymerization. It gives us the opportunity to compare the efficiency of
variants of -amylase towards shorter and longer amyloses. In this case amylose DP17
(average degree of polymerization 17) reducing sugar assay is used. The substrate is
made by dissolving 111.1 mg DP17 amylose (Amylose Ex-1, Hayashibara Bichemical
Lab) in 10 ml buffer. The buffer contains 20 mM NaOAc, 5 mM CaCl2 pH 5.5. This
solution is then heated in microwave for 30 second with effect of around 500 W to avoid
boiling. Then 10 µl 5% BSA is added. From this stock the following concentrations are
made by dilution in the mentioned buffer (Tab. 4.3). Then a standard series of maltose is
made.
Table 4.3: Amylose DP 17 substrate conc.
Final
No
Concentration (%)
1
1.0
2
0.7
3
0.5
4
0.3
5
0.2
6
0.1
7
0.08
8
0.05
9
0.03
10
0.01
A stock of maltose at 200 µg/ml is diluted with the mentioned buffer to get these
concentrations (Tab. 4.4). Every standard concentration is mixed with 500 µl reducing
sugar assay reagents as used for DP 440. From amylose substrate solution 900 µl is taken
and every solution is incubated at 37C for 5 min.
25
Table 4.4. Maltose conc. for amylose DP 17.
No
Conc. in µM
1
55.5
2
41.62
3
27.75
4
20.8
5
13.87
6
6.93
7
0
On precise time 100 µl enzyme solution (containing 20-50 nM variant of AMY1) is
added to the substrates. Then 10 / 50 µl samples are taken out to Eppendorf tubes with
490 / 450 µl plus 500 µl reducing sugar assay reagents with exactly 2.5 min intervals up
to 12.5 min (10 µl for conc. 0.2-1 % and 50 µl for conc. 0.01-0.1%). Both amylose and
maltose samples are heated for 30 min at 80C, then quickly cooled and centrifuged at
14000 rpm for 5 min. 300 µl aliquots are transferred to a micro titer plate and the
absorbency is read at 540 nm in an ELISA reader.
Another substrate of determining kcat and Km is pNPG7. The hydrolytic activity of the amylase for p-nitrophenyl maltoheptaoside (pNPG7) was determined for the variants of
enzymes using amylase assay kit. The parameters kcat and Km were calculated from the
initial rates at the 0.25 - 10 mM substrate concentration range. Buffers and enzymes are
made in concentrations so there is enough for kinetic determination in triplicate. First a
50 ml kit buffer is prepared by mixing 25 ml 0.1 M phosphate buffer with 0.1864 g KCl
and 500 l 2% NaN3 and H2O up to 50 ml. Then “buffer F12” is made by adding 15 l
5% BSA into 15 ml kit buffer. Than two enzyme solutions are made. 15 mg glucosidase (over 70 Units/ml) in 1.5 mg “F12 buffer”, and 0.89 mg -glucosidase (20-40
Units/ml) in 700 l “buffer F12”. Then a of Cl-pNPG7 series is made, as shown at table
4.5.
26
Tabel 4.5. Cl-PNG7 substrate solution concentration series.
No
mM
Cl-PNG7
-gluc.
-gluc.
Buffer F12
1
0.25
5 mM 36l
175
72
360
2
0.5
5 mM 72l
175
72
324
3
1
5 mM 144l
175
72
252
4
2
5 mM 288l
175
72
108
5
3
50 mM 43.2 l
175
72
353
6
5
50 mM 72 l
175
72
324
7
8
50 mM 115.2 l
175
72
280
8
10
50 mM 144 l
175
72
252
Then a standard solution concentration of Cl-pNP is made. 1.7 mg is transferred to 1 ml
F12 buffer to get a concentration at 10 mM, this solution is diluted 20 times to get a
concentration of 0.5 mM. The following concentrations are made, see tabel 4.6
Table 4.6. Standard curve for Cl-pNPG7
l Cl-pNP 0,5 mM
l buffer F12
Final conc. mM
0
200
0
40
160
0.1
80
120
0.2
120
80
0.3
160
40
0.4
200
0
0.5
These standard solutions are directly transferred (100 l) to microtiter plate. Each
substrate mixture (90 l) is transferred to a microtiter plate where they are pre incubated
for 2 min at 30 C. To this mixture 10 l enzyme solutions are added. Enzymes are
diluted in F12 buffer to get concentrations in 30- 50 mM. The p-nitrophenol release is
determined spectroscopically at 405 nm with 7 readings from 2 to 8 minutes. To
determine Km and Kcat an excel calculation sheet is used (Appendix 4). First the linearity
of OD405 as function of concentration of Cl-pNP in mM is plotted. The slope (OD/mM) is
27
determined and used for transform the inserted enzyme substrate mix OD values from
OD/min to the velocity in mM/min. Plotting velocity as a function of concentration gives
us a hyperbolic curve a so called Michaelis Menten plot. Usually curve fitting programs
are used to generate line through the data. The program Curve Expert is used and the
function y = (ax)/ (b+x), called growth saturation mode, is applied to curve fitting. The
outcome of curve fitting is the coefficients of the curve, where the coefficient a divided
with enzyme concentration is Kcat and b corresponds to Km.
Now a background for protein - ligand binding equilibrium is given for determination of
the equilibrium dissociation constant Kd. Enzymes catalyze the transformation of the
substrate to product. To ensure this enzyme and substrate must form binary complex with
each other. The macromolecular partner is called the receptor (R) and the smaller
molecular partner in this binding is called the ligand (L). The total concentrations of
ligand and receptor can be described as:
[R] = [RL] + [R]FREE (4.12)
[L] = [RL] + [L]FREE (4.13)
The position of the equilibrium between these partners is most commonly quantified in
terms of the dissociation constant, Kd, for the binary complex at equilibrium [13].
Kd = ([R]free[L]free )/ [RL] (4.14)
The tighter the ligand binds, the lower the value of the dissociation constant. The
dissociation constant can be related to the Gibbs energy of binding for the receptor –
ligand complex as follows:
Gbinding = RTln(Kd) (4.15)
The equilibrium constant can also be approached by determination of kinetic rates. The
second order rate constant for complex association can be defined as kon and the first
order rate constant for complex dissociation as koff.:
28
koff


R  L Kon RL (4.16)
The equilibrium dissociation constant for the complex is thus given by the ration of koff to
kon:
Kd = koff / kon (4.17).
At equilibrium the concentrations of the RL is complex constant. Hence the rates of
complex association and dissociation are equal.
d RL 
 k on R  f L  f (4.18)
dt
 d RL 
 k off RL  (4.19)
dt
combining eq. 4.18 and eq. 4.19 gives
kon[R]f[L]f = koff [RL] (4.20)
or
(kon /koff ) [R]f[L]f = [RL]
Knowing that kon/koff is equivalent to the equilibrium constant Ka thus Ka = 1/ Kd :
Ka [R]f[L]f = [RL] (4.21)
We prefer to work with an equation in terms of total added ligand and receptor that are
easier determined than concentrations of free ligand or receptor. If we apply eq. 4.11 on
eq. 4.21 and dividing both sides by 1+([RL]/[R]f we obtain:
29
R f

R (4.22)
RL 
1
R f
The fraction [RL]/[R] is referred to as the fractional occupancy of the receptor and is
often represented by the symbol B (for bound receptor). Using Ka=[RL]/[R]f[L]f on
eq.4.22 we obtain:
RL   K a L f
R
1  K a L f
(4.23)
or,
RL  
R L f
K d  L  f
(4.24)
Under most conditions the concentration of receptor is less than that of the ligand.
Therefore the formation of complex does not significantly diminish the concentration of
free ligand thus:
[L]f  [L] (4.25)
Eq. 4.24 can be rewritten as follows
RL   RL  RK (4.26)
K d  L 
1 d
L
Eq. 4.26 describes a hyperbola that is typical of saturable binding in biochemical
situations. The equation is known as the Langmuir isotherm equation. Plots of [RL] as a
function of total ligand concentrations are referred to as binding isotherms. By applying
non-linear least squares fit to the data estimates of Kd can be obtained.
For determination of the equilibrium dissociation constant the method of Starch Binding
assay is used. In this assay the absorbency of free unbound -amylase is read. The free
30
concentration of the enzyme will fall if it binds to starch and therefore we will se a
diminish in the absorbency. For this purpose the absorbency of enzyme mixed with
various concentrations of starch is read. First a buffer DB is made of 20 mM sodium
acetate, 5 mM CaCl2 0.005 % BSA at pH 5.5. Suspensions of barley starch granules are
prepared. First a Mother 1 suspension is made by dissolving (after two washes) 800 mg
barley starch granules in 10 ml DB buffer giving 80 mg/ml. Some of this suspension is
diluted to 40 mg/ml and called Mother 2. Again some of Mother 2 is diluted to 4 mg/ml
called Mother 3. Enzymes to be investigated are diluted in DB buffer to 100 nM in a total
volume of 1.5 ml. Then following (table 4.7) mixtures are made at 4C.
Table 4.7. Enzyme solution for starch binding analysis.
No
A
B
C
D
E
F
G
H
I
J
K
L
M
N
Starch
40
30
20
10
5
3
2
1.5
1
0.7
0.4
0.2
0.1
0
Enzm.
100
100
100
100
100
100
100
100
100
100
100
100
100
100
DB
400
525
400
650
775
825
400
525
650
725
800
850
875
900
500
375
500
250
125
75
500
375
250
175
100
50
25
0
mg/ml
buffer
Moth1
Moth2
Moth3
The enzyme solutions are incubated at 4C for 30 minutes by shaking to ensure binding
reaction. After time the tubes are centrifuged at 13.000 rpm for 5 minutes at 4C. 750 L
of the supernatant is transferred to new tubes on ice. To measure the activity a substrate
solution of Insoluble Blue Starch with 10 mg/ml in DB buffer is prepared. For each tube
at tabel 4.7 tubes with 500 l of substrate solution is prepared. Both the substrate and
enzyme solution are placed at 37 C in a heating block for 15 min. After time 300 L
enzyme solutions are added to the substrate solutions and mixed carefully. After exactly
15 minutes 200 L 0.5 M NaOH solution is added and mixed carefully to stop the
reaction. Centrifuged for 4 min at 13.000 rpm and transferred to wells of a micro titer
with volumes of 300 L and the absorbency is measured by ELISA reader at 620 nm. To
determine the KD and number of binding site an excel calculation sheet is used
31
(Appendix5). OD620 for every enzyme substrate concentration is inserted in the sheet. The
bound receptor to the ligand can be found by extracting the value of OD620 at zero
concentration of starch, where all enzyme is free called E0, from the initial substrate
concentrations OD620. These values can be transformed to fractional occupancy B by
dividing with the E0. Plotting B as function of ligand concentration will give a hyperbole
and applying the curve fitting with saturable growth model (y = a/(1+(b/x))) will give
data coefficients where the coefficient a corresponds to Kd and b corresponds to the
number of binding sites.
32
Chapter 5 Results and Discussion
5.1 Expression
To get an idea of the production of -amylase from Pichia pastoris a pilot scale
production was performed in shake flask. There were removed samples for activity
measurements towards Insoluble Blue Starch, the data was outlined vs. time, see table
5.1:
Table 5.1: Activity vs. time
Mutant
Time (h)
Activity Units /ml
W278A
0
0
W279A
0
0
W278A
24
24,34
W279A
24
42,64
W278A
48
1,50
W279A
48
2,74
From table 5.1 it is seen that maximum is reached about 24 hours and minimum level is
reached around 48 hours. It was from here decided that the first induction with methanol
should last between 24 hours up to 30 hours and the second induction should be around
40 hours and not longer than 48 hours.
Then the first induction at large scale volumes were done for W278A and W279A. The
result is shown on curve 5.2. After that a second induction at large scale was done. The
result is shown on curve 5.3.
33
Curve 5.2 Activity vs time first induction
30
Units/ml
25
20
W278A
15
W279A
10
5
0
3
23
25
27
29
Time (h)
Curve 5.3 Activity vs time second induction
50
Units/ml
40
30
W278A
20
W379A
10
0
1
20
22
24
43
45
47
Time in hours
I have only measured the progress of activity values for the mutants W279A and W278A.
From the curves it is observed that the concentration of -amylase peaks at around 24
hours for both variants than stabilize for the coming hours. Therefore for wt and the
double mutant the first induction should be stopped after 24 hours to obtain maximum
concentration. For wt and the double mutation variant I have only measured the final
values found to 1.45 and 20.66 Units/ml respectively (Appendix 1).
34
5.2 Purification
Table 5.4: Purification overview for Trp278Ala
Fraction
A280
Total activity in
Units
Recovery in (%)
Specific activity
Units/A280
Medium
15584.8
15225.6
Ultrafiltration
4035.0
12312.0
80.9
3.1
Affinity Chromat.
7.8
6547.8
53.2
836.5
Dialyze
4.46
4512
29.6
1012.4
Blue Starch
activity U/mg
1.0
2450
For detailed background of purifacation overviews see Appendix 1. From table 5.4 the
steps for expression and purification of the mutant W278A is seen. After ultra filtration
20% of the protein is lost. It is common to tolerate loss around 20% per purification step.
After affinity chromatography around 27% is lost while the specific activity increases.
The reason is that OD280 gets very lower because the only protein in the sample is amylase and affinity chromatography is thus a very efficient purification step. OD280 was
very high in medium and ultra filtration due to the high content of various proteins
peptides. After dialyzation around 23% is lost and that is much because dialyzation is not
really a purification step. It is a method to remove bound -cyclodextrin from the amylase. So perhaps the period of dialysis should be shortened from 4 days to 2 days and
using higher concentration of MES. The value of activity for Insoluble Blue Starch in
U/mg is obtained by multiplying specific activity with 2.4, which is the extinction
coefficient for -amylase.
35
Table 5.5: Purification overview for Trp279Ala
Fraction
A280
Specific activity
Blue Starch
Units
Units/A280
activity U/mg
1.6
Total activity in
Recovery in (%)
Medium
15648.2
24396.0
Ultra filtration
4727.5
19247.9
78.9
4.1
Affinity Chromat.
7.7
8894.3
46.2
1157.4
Dialyze
7.0
6870.5
28.2
978
2330
Mutant W278A and W279A were expressed and purified simultaneously and gave the
same patterns and profile for purification steps, though we get a little higher
concentration of W279A mutant. Yields were approximately 2 and 3 mg for W278A and
W279A respectively.
Table 5.6: Purification overview for Trp278Ala/Trp279Ala
Fraction
A280
Total activity in
Units
Recovery in (%)
Specific activity
Units/A280
Medium
20256.0
16528.0
Ultra filtration
4818.0
12526.8
75.8
2.6
Affinity Chromat.
14.4
11440.0
69.2
739.9
Dialyze
3.3
3048.8
18.4
935.3
Blue Starch
activity U/mg
0.8
2250
The double mutant and the wt were run simultaneously. But we do not see a similar
pattern for these two proteins as found for W278A and W279A. The double mutant has
the highest content of -amylase in the medium. The reason can be that it was my second
run and therefore I became better to do the practical work in expression by shake flask.
We see a loss of 25% after ultra filtration. It is a little more than for the other two mutants
but is steady in the sensible range. But the loss in affinity chromatograph is only 15 %
and is less than for the other mutants. The reason can again be that I got used to the
practical methods after using time and trials with affinity chromatography. The reason
why the loss in dialysis is so high is that I lost half the content of the dialysis tube.
36
Table 5.7: Purification overview for wt
Fraction
A280
Total activity in
Units
Recovery in (%)
Specific activity
Units/A280
Medium
3330
2602,8
Ultra filtration
1665
1998
76.8
1.3
Affinity Chromat.
1.6
1433
55.1
870.4
Dialyze
1.1
896.8
34.5
817.2
Blue Starch
activity U/mg
0,8
1960
The amount of -amylase wt is very low. Though I have expressed wt three times, the
content is not comparable with the other variants. The reason is as mentioned in Materials
and Method section that the molecular insertion of -amylase gene at the host’s genome
did not succeed well and therefore we get these low content. The molecular insertion
must be redone. But the final content after purification is enough for the kinetic analysis.
Yields were approximately 1.5 and under 0.5 mg for W278A/W279A and wt
respectively.
37
Figure 5.1: Coomassie colloidal blue gel for W279A
In figure 5.1 is seen that there appears several bands for medium, permeate and flow
through from affinity chromatography, while there is single band for the retentate and
elute fraction from affinity chromatography. This shows the capability of purification
steps to isolate -amylase.
38
Figure 5.2: Silver stained gel
From figure 5.2 it is seen for every variant the protein situation in medium (m), after ultra
filtration (u) and affinity chromatography (a). For (m) we see several proteins in form of
silver colouring all the column, but the band for -amylase is distinguished. The situation
for (u) is the same but the band for -amylase gets clearer. For (a) there is only the amylase. It shows that (a) is a very efficient purification step.
5.3. Characterization
For the variants of -amylase kinetic parameters were determined to see the effect of the
mutations. First the kcat and Km values are determined by DP440 assay. It gives us the
opportunity to see if the mutations at the surface binding sites affect the catalytic rate of
the enzyme and whether the Km values changes.
39
Tabel.5.7. Kinetic paramaeters determined for amylose DP440
Ref

-1
kcat (s )
[14]
Km(mM)
1.trial
W278A 162
W279A 183
212
Wt
W278A/
167
W279A

Ref
[14]
kcat/Km
Ref[14]
2.trial
229
47.38
156
19.09
1
nd
190
1.trial
0.28
0.27
0.25
2.trial
0.25 0.02
0.20 0.2
nd
0.435
1.trial
577
678
845
2.trial
923
768
177
0.10
0.15 0.04
1722
1188
7.07
437
This is seen that wt has the highest kcat value of 212 (s-1) which is around 20% higher
than the reference value. The kcat for W278A is up to 229 (s-1) but the standard deviation
is too high. The real value must be assumed to lie close to the W279A around 160 (s-1).
The double mutant has the lowest kcat. It can be concluded that the binding site mutations
do not effect the catalytic rate but it is expected that mutations effect Km so mutations
have higher values for Km than the wt. For the wt we got half of the value for Km of the
reference and the single mutants have values close to the wt. It can then be concluded that
single mutations at surface binding site do not effect the Km, but the double mutant has
even lower Km value than the wt. Dublicate values for Km values for each enzyme variant
are close and therefore the standard deviation is also low. There must be something
wrong the value of Km for double mutant. If I did the experimentals wrongly then I
should not get close results for duplicates. If the concentration of enzyme I used was too
high or too low I should not get a linear increase in absorbency with time or with
increasing substrate concentration, but my excel sheets and micro titer plate show that I
do. Also the concentration of enzyme has influence on kcat and not on Km. To compare the
results from amylose DP440 we use amylose DP17 to see if I get similar results.
1
No data
40
Tabel.5.8. Kinetic rates determined for amylose DP17.
Ref

kcat (s-1)
[14] Km (mg/ml)
1.trial
2.trial
1.trial
2.trial

Ref[14]
Ref
[14] kcat/Km
1.trial 2.trial
W278A
175
201 18.38
0.62
0.82 0.14
282
246
W279A
182
207 17.68
0.73
1.20 0.34
251
172
wt
227
211 11.31
0.71
0.52 0.13 0.57
320
404
W278A/
W279A
187
166 14.85
0.68
0.59 0.06
274
280
165
289.5
The value of kcat is highest for the wt while the others are not far from the wt value. The
wt kcat value is 35% higher than the reference value, but the standard deviation is not so
high. The wt’s Km value lies close to the reference, but the single mutants Km values are
a little higher than the wt’s. It is an expected result. The double determinations are close
to each other except for the W279A, which presumably to has a value close to the
W278A. The double mutant has again lower value of Km than wt. The problem why I got
this unexpected results can be that the assays for amylose DP440 and DP17 are very time
consuming, skill and concentration requiring experiments, and more trials are needed
before one gets reproducible results. But the project was time limited so the assays were
not repeated. The Cl-pPNG7 assay is much easier than the two other assays.
Tabel.5.9. Kinetic parameters determined for Cl-pNPG7.
kcat (s-1)
Ref Km (mg/ml)

[14]
1.trial
W278A
W279A
wt
W278A/
W279A
2.trial
1.trial
39
39
37
41 1.02
38 1.21
38 0.49
46
42 2.61
40

2.trial
0.55
0.85 0.21
0.50
0.56 0.04
0.76
0.80 0.03
0.86
Ref [14]
kcat/Km
1.trial
0.758
0.87 0.01
The double determinations of kcat for wt are very close to each other and to the reference
value, around 5% lower. But the mutants have a little higher kcat values and but it is not
significant higher. The Km value of wt perfectly matched with the reference and the
duplicates are close to each other. The single mutants have lower km values than the wt
with a decrease around 25%. An expected result is that the double mutant has higher Km
41
Ref
[14]
2.trial
72
48
78
67
50
48 52.8
53
48
value than the wt, but the difference is not significant. The pattern that the double mutant
has higher values of Km than the wt indicates an uncertainty in that if the double mutant
really is mutated at the right sites. Therefore there must be done an amino sequence of
this variant to verify the mutation site.
Tabel 5.10 Specific activity for Insoluble Blue starch
Variant
U/mg
W278A
2450
W2789A
2330
W278A/W279A
2250
Wt
1960
Reference for wt [9]
2900
From table 5.10 is seen that value wt of specific activity for Insoluble Blue starch is
approximately 30% lower than reference value. All mutants have higher activity than wt, where
W278A has the highest, approximately 25% more of wt. The mutants are therefore not effected
negatively in activity for Insoluble Blue Starch.
Tabel. 5.11 Dissociation constant determined by starch binding.
Variant
Kd (mg/ml)
2.672
W278A
3.136
W279A
0.702
Wt
0.706
W278A/W279A
2.0
Reference for W279A [10]
0.20
Reference for wt [10]
Binding sites (n)
1
1
1
1
From table 5.11 is seen that the value of Kd for wt is almost 3.5 higher than the reference
value. The double mutant has also the same Kd value as wt and it confirms that I have not
worked with a real double mutant. Thus amino sequence of the double mutant is needed.
The other single mutants have higher Kd values than wt, hence their binding to starch is
not so tight as for wt. This is an expected result. Also the Kd of W279A is comparable
with the reference value. It can be concluded that single mutations at the surface binding
site do not influence the values of kcat and Km but influence the dissociation constant Kd.
An amino acid sequence of every variant of -amylase was performed by lab technician
at BNG but the data was not comparable (data not shown) due to the contamination with
other proteins possibly skin proteins from my hands.
42
CHAPTER 6 Conclusion
The cloning construction and insertion of the wt must be redone, so it can be expressed in
required concentration for kinetic determination work. Another reason why it must be
redone is that the expression of wt requires three runs of expression whereas the mutants
require only one run, so wt can not be ran simultaneously.
The use of shake flask method for expression of variants of -amylase results in enough
amounts for determination of kinetic parameters. If one needs larger amount, a fermentor
should be used.
The purification protocol is good and gives -amylase in highly pure state. Especially the
affinity chromatography is an extreme efficient way of recovering the target protein. The
loss after dialysis is too high. The period of dialysis must be shortened.
The single mutants have not significantly different values of kcat and Km from wt
determined towards substrates amylose DP17 and DP440. The method of Cl-pNPG7 gave
the most comparable with reference value for the wt, maybe because this short substrate
is little affected by the surface site. There was a major difference in Kd between the single
mutants and the wt though the reference value of wt was higher than the reference value.
It can be concluded that single mutations at surface binding site did not influence Km and
kcat, but that both single mutations at surface binding site changing tryptophan at position
278 and tryptophan at position 279 increased the Kd value by making it higher meaning
that the mutants bind to starch less tighter than wt.
The double mutant gave the same results as the wt throughout the assays used. So
probably that the double mutant was not mutated at the planned positions. This missmutation could not be confirmed with amino acid sequencing due to contamination with
foreign proteins.
43
Bibliography
[1] L. Stryer, Biochemistry, 4. Edition, Chapter 18.
[2] D.C Baulcombr, R.A. Martiensen, A.M. Huttley, C.M. Lazarus, Hormonal
and development control of gene expression in wheat, JSTOR. 314, p 441-451, (1986)
[3] M.T. Jensen, T.E. Gottschalk, B. Svensson, Differences in conformational
stability of barley alpha-amylase Isozyme 1 and 2. Role of charged
groups and Isozyme 2 specific salt-bridges, Cereal Sci. 38, p289-300,(2003)
[4] P.K Nielsen, B.C. Brønsager, C.R Berland, B.W.Sigurskjold, B. Svensson, Kinetics
and Energetics of the binding between barley -amylase/subtilisin Inhibitor and Barley
-Amylase 2 Analyzed by Surface Plasmon Resonance and Isothermal Titration
Calorimetry, Biochemistry. 42, pp 1478-1487, (2002).
[5] X. Robert, R. Naser, T. E. Gottschalk, F. Ratajcak,
H. Driguez, B. Svensson, N. Aghajari, The structure of
barley -amylase Isozyme 1 reveals a novel role of domain c in substrate
recognition and binding, Strucure.11 973-984, (2003)
[6] J. M .Cregg, T. S. Vedvick, W. C. Raschenke, Recent
advances in the expression of foreign genes in Pitchia pastoris,
Biotechnology. 11, p 905-909,(1993)
[7] Easy select Pichia Selection Kit, Catalog no. K1740-01, Invitrogen.
[8] D. R. Higgins, Pichia Protocols, Methods in Molecular Biology, Chapter 9
Humana Press , Totowa New Jersey
44
[9] H. Mori, K. S. B.Jensen, T. E. Gottschalk, M. S. Motawia, I. Damager, B. L. Møller, B.
Svensson, Modulation of activity and substrate binding modes by mutation of single and double
subsites 11/12 and 25/26 of barley -amylase, Eur. J. Biochem. 268, p6545–6558, (2001).
[10] M. Søgård, A. Kadziola, R. Haser, B. Svensson, Site directed mutagenesis og histidine 93,
aspartic acid 180 glutamic acid 205, histidine 290 aspartic acid 291 at the active site and
tryptophan 279 at the raw starch binding site in barley -amylase, The Journal of Biological
Chemistry. 268, pp 22480-22484, (1993).
[11] Pharmacia, Affinity Chromatography Principles and Methods, Chapter 2.
[12] R. E. Copeland, Enzymes 2.ed., Chapter 5, WILEY
[13] R. E. Copeland, Enzymes 2. ed., Chapter 4, WILEY
[14] S. Bozonnet, K. Fukuda, B Kramhøft, B svensson, Mutational and gene shufling analysis of
specifity of barley alpha amylase , Biocatalysis Biotransformation 21, pp 209-214 (2003).
[15] Candiano et al., Electrophoresis 2004. 25, 1327-1333
45
Chapter 7 Appendix
Appendix 1 Expression and purification overview
1. Induction
2. Induction
Volume
Affinity Chrom.
U total
340
17.6
5984
W279A
320
28
8960
W278A
320
28.88
9241.60
W279A
340
45.4
15436
W278A / W279A
800
20.66
16528
1800
1.45
2602.8
Retentate W278A
150
82.08
12312
Filtrate W278A
510
5.10
2601
Retentate W279A
155
124.18
19247.9
Filtrate W279A
500
9.96
4980
Retentate W278A / W279A
330
37.96
12526.8
Filtrate W278A / W279A
720
1.78
1281.6
Retentate wt
370
5.4
1998
1650
0
0.
Pool 1
6.5
894
5811
Pool 2
3.5
210.5
736.75
1
82.08
82.08
Flow Through
140
14.76
2066.4
Pool 1
5.5
1466
8063
Pool 2
2.5
332.5
831.25
Residue
6.5
124.18
807.17
Wt
Ultrafiltration
Units/ml
W278A
Filtrate wt
W278A
Residue
W279A
46
Flow
W278A / W279A
150
Waste
Dialysis
11
1040
11440
Flow Through
320
1.72
550.4
Pool
6.1
235
1433.5
W278A
6.9
654
4512.6
W279A
9.1
755
6870.5
W278A / W279A
3.7
824
3048.8
Wt
5.9
152
896.8
A280
1. Induction
2. Induction
Ultrafiltration
1611
0
Pool
Wt
10.74
Dilution
A280/ml
U/A280
Total A280
W278A
2.02
10
20.20
37.80
6868
W279A
2.24
10
22.37
50.37
7158.4
W278A
2.72
10
27.24
1.06
8716.8
W279A
2.50
10
24.97
1.82
8489.8
W278A / W279A
2.53
10
25.32
0.82
20256
Wt
0.24
10
1.85
Retentate W278A
0.27
100
26.90
3.05
4035
Filtrate W278A
1.90
10
19.02
0.27
9700.2
Retentate W279A
0.31
100
30.50
4.07
4727.5
Filtrate W279A
2.67
10
26.73
0.37
13365
Retentate W278A / W279A
1.46
10
14.60
2.60
4818
Filtrate W278A / W279A
1.42
10
14.15
0.13
10188
Retentate wt
0.15
10
4.50
1.20
1665
Filtrate wt
0.22
10
2.24
0
3696
Pool 1
1.05
1
1.05
853.87
6.8
Pool 2
0.29
1
0.29
720.89
1
Residue
0.27
100
26.90
3.05
26.9
Flow Through
1.60
10
15.97
0.92
2235.8
3330
Affinity Chrom.
W278A
47
W279A
Pool 1
1.2
1
1.2
1226.78
6.57
Pool 2
0.45
1
0.45
747.19
1.11
Residue
0.31
100
30.50
4.07
198.25
Flow
1.77
10
17.73
0.61
2659.50
Waste
W278A / W279A Pool
Flow Through
Wt Pool
Dialysis
1.31
1.31
793.89
14.41
1.42
10
14.2
0.12
4544
0.4
1
0.27
870.37
1.65
W278A
0.39
1
0.65
6985.45
4.46
W279A
0.33
1
0.77
8899.61
7.03
W278A / W279A
0.65
1
0.88
3460.61
3.26
Wt
0.08
1
0.19
4821.51
1.10
48
Appendix 2 Affinity Chromatogram
49
Ahmet W278 W279 80405001:10_UV1_280nm
Ahmet W278 W279 80405001:10_Conc
Ahmet W278 W279 80405001:10_Fractions
Ahmet W278 W279 80405001:10_P960_Flow
Ahmet W278 W279 80405001:10_Cond
Ahmet W278 W279 80405001:10_pH
Ahmet W278 W279 80405001:10_Inject
Ahmet W278 W279 80405001:10_Cond%
Ahmet W278 W279 80405001:10_Flow
Ahmet W278 W279 80405001:10_Logbook
ml/min
0.8
0.6
0.4
0.2
0.0
F3
270.0
280.0
Waste A3 A5 A7 A9 A11 A14 B1 B3 B5 B7 B9 B11 B14 C1 C3
290.0
300.0
310.0
50
320.0
330.0
340.0
Waste
ml
Appendix 3 DP440
DP440
Date:
07-05-05
cells which have to be filled with data
Enzyme batch:
W278A
cells which have to be transferred to next sheet
[enz] in assay(µg/ml):
1,33
nmol/l
Maltose
conc (µg/ml)
conc (mM)
0
0,16
0,161
0,1605
0
0,0000
6
0,556
0,555
0,5555
0,395
0,0167
8
0,654
0,659
0,6565
0,496
0,0222
12
1
1
0,9305
0,77
0,0333
20
1,45
1,46
1,455
1,2945
0,0555
40
2,648
2,648
2,648
2,4875
0,1110
Standard curve
3
y = 22,427x + 0,015
2,5
OD 540 nm
=
Maltose
R2 = 0,9995
2
1,5
1
0,5
0
0,0000
0,0200
0,0400
0,0600
0,0800
0,1000
0,1200
conc (mM)
slope
22,4273093 UOD/mM
Time
Dilution factor:
conc
2,5
2,5
2,5
2
2,5
1,5
2,5
5
OD 540nm
0,737
OD 540nm
0,98
0,739
0,738
0,704
0,706
0,705
0,877
0,875
0,62
0,628
0,624
0,849
0,842
0,987
7,5
OD 540nm
1,254
1,256
0,876
1,175
0,8455
1,062
0,9835
10
12,5
1,255
OD 540nm
1,461
1,456
1,4585
OD 540nm
1,789
1,792
1,179
1,177
1,444
1,453
1,4485
1,731
1,061
1,0615
1,312
1,305
1,3085
1,598
UDO/min
1,7905
0,1032
1,733
1,732
0,10506
1,599
1,5985
0,09648
2,5
1
0,578
0,579
0,5785
0,725
0,721
0,723
0,979
0,978
0,9785
1,155
1,157
1,156
1,413
1,412
1,4125
0,08404
2,5
0,75
0,528
0,538
0,533
0,718
0,716
0,717
0,948
0,945
0,9465
1,144
1,142
1,143
1,4
1,404
1,402
0,08656
2,5
0,5
0,521
0,531
0,526
0,708
0,706
0,707
0,942
0,941
0,9415
1,139
1,138
1,1385
1,159
1,152
2,5
0,4
0,492
0,486
0,489
0,657
0,655
0,656
0,831
0,826
0,8285
1,026
1,024
1,025
1,026
1,018
2,5
0,3
0,496
0,491
0,4935
0,644
0,646
0,645
0,781
0,783
0,782
1,027
1,022
1,027
1,021
1,024
0,05266857
2,5
0,2
0,477
0,472
0,4745
0,617
0,624
0,6205
0,729
0,726
0,7275
0,96
0,963
0,96
0,954
0,957
0,04751429
2,5
0,1
0,436
0,446
0,441
0,491
0,495
0,493
0,548
0,545
0,5465
0,592
0,594
0,592
0,593
2
1,8
2,5
1,6
500
0,012
450
1,4
0,01
0,4
0,3
1/velocity
0,5
1
velocity
0,75
0,008
0,006
0,2
0,8
0,1
0,6
0,02038
350
1
1,2
0,07122
y = 35,59x + 55,775
400
2
1,5
OD 540 nm
0,014
0,593
0,08288
0,004
300
250
200
150
100
0,002
50
0,4
0
0,2
0
0
0,5
1
1,5
2
2,5
3
0
2
4
6
concentration (mg/ml)
0
0
2,5
5
7,5
10
12,5
15
1/conc
time (min)
51
8
10
12
11/27/2003
DP 440
W278A
1.33 nmol/l
std slope
0.0445885 22.42731
mg/mL
vol microL
OD/min
mM/min
sec-1
v
v/s
s
s/v
1/s
1/v
2.5
200
0.1032
0.0115
144.16
144.16
57.66
2.5
0.01734
0.40
0.00694
Use curve expert to fit the data
Data : mM/min = f(mg/ml)
2
200
0.10506
0.0117
146.76
146.76
73.38
2
0.01363
0.50
0.00681
1.5
200
0.09648
0.0108
134.77
134.77
89.85
1.5
0.01113
0.67
0.00742
1
200
0.08404
0.0094
117.39
117.39
117.39
1
0.00852
1.00
0.00852
0 .0
1
0.75
200
0.08656
0.0096
120.91
120.91
161.22
0.75
0.00620
1.33
0.00827
0 .0
1
0.5
200
0.08288
0.0092
115.77
115.77
231.55
0.5
0.00432
2.00
0.00864
0 .0
0
0.4
200
0.07122
0.0079
99.49
99.49
248.71
0.4
0.00402
2.50
0.01005
200
0.052668571
0.0059
73.57
73.57
245.24
0.3
0.00408
3.33
0.01359
0 .0
0
0.3
0.2
200
0.047514286
0.0053
66.37
66.37
331.86
0.2
0.00301
5.00
0.01507
0 .0
0
0.1
200
0.02038
0.0023
28.47
0 .0
0
Saturation Growth-Rate Model: y=ax/(b+x)
Curve fit #2-8
Coefficient Data:
a=
0.01295125
b=
0.28140147
slope
-3.07 slope
0.0062 slope
0.0019
b
520.74 b
0.0018 b
0.0060
R2=
0.8508 R2=
0.9942 R2=
0.9412
kcat
162 kcat
170 kcat
163 kcat
166.1
Km
0.281 Km
0.326 Km
0.288 Km
0.3
564.4 kcat/Km
538
kcat/Km
577 kcat/Km
Eadie-Hofstee
521 kcat/Km
Hanes-Woolf
Lineweaver-Burk
Different methods to linearize the
data: help to delete obviously wrong
experimental points
52
Y Axis (units)
S = 0.00021450
r = 0.98796502
0
0 .0 0.0
0.5
0.9
1.4
1.8
2.3
2.7
X Axis (units)
Copy/paste info from curve expert to cell A16
Appendix 4 Cl-pNPG7
Cl-pNP
mM Cl-pNP
0
0,1
0,2
0,3
0,4
0,5
UOD
UOD
slope standard =
0
0
0,414
0,823
1,205
1,575
2,055
4,04
UOD/mM
2,5
2
y = 4,04x + 0,002
R2 = 0,9989
1,5
1
0,5
0
0
0,1
0,2
0,3
0,4
0,5
53
0,6
02-08-02
Cl-pNPG7
cells which have to be filled with data
AMY 1
3 nmol/l
OD/min
mM/min
0,25
3,512
0,8693
0,5
12,143
3,0057
1
23
5,6842
2
25,381
3
26,286
5
std slope
0,24752475
sec-1
v/s
s
s/v
1/s
1/v
4829,48
4829,48
19317,93
0,25
0,00005
4,00
0,00021
16698,29
16698,29
33396,59
0,5
0,00003
2,00
0,00006
31578,66
31578,66
31578,66
1
0,00003
1,00
0,00003
6,2824
34902,37
34902,37
17451,18
2
0,00006
0,50
0,00003
6,5064
36146,86
36146,86
12048,95
3
0,00008
0,33
0,00003
28,905
7,1547
39748,35
39748,35
7949,67
5
0,00013
0,20
0,00003
8
24,226
5,9965
33314,08
33314,08
4164,26
8
0,00024
0,13
0,00003
10
22,714
5,6223
31234,87
31234,87
3123,49
10
0,00032
0,10
0,00003
Saturation Growth-Rate Model: y=ax/(b+x) Curve fit #2-8
slope
Coefficient Data:
b
a=
b=
4,04
v
7,11578800
R2=
0,544525560 ko
39532 ko
-0,46 slope
29128,97 b
Data : mM/min = f(mM)
0,0000 slope
0,0000
0,0000 b
0,0000
0,2119 R2=
0,9646 R2=
0,8907
63986 ko
34581 ko
94759,0
Km
0,545 Km
2,197 Km
ko/Km
72599 ko/Km
29129 ko/Km
Eadie-Hofstee
Use curve expert to fit the data:
Growth Models, Saturation Growth Rate
0,343 Km
100674,8 ko/Km
Hanes-Woolf
Copy/paste info from curve expert to cell A14
4,1
23087
S = 0 .0 0 0 5 7 8 2 0
r = 0 .9 7 9 0 7 8 9 2
Lineweaver-Burk
Values to report
1
0.0
Different methods to linearize the data: help to
delete obviously wrong experimental points
1
0.0
Y Axis (units)
mM
1
0.0
1
0.0
0
0.0
0
0.0
54
0
0.0 0.0
0.5
0.9
1.4
X Axis (units)
1.8
2.3
2.7
Appendix 5 Starch Binding
Starch binding assay:
27-04-05
cells to be filled
Enzyme: AMY1-W278A
to be transferred to other worksheets
concentration:
dilution before assay:
Results:
blind:
A
B
C
D
E
F
G
H
I
J
K
L
M
N
0,07
[starch]
40
30
20
10
5
3
2
1,5
1
0,7
0,4
0,2
0,1
0
0,07
Abs.
0,525
0,554
0,594
0,621
0,734
0,812
1,084
1,112
1,212
1,294
1,345
1,412
1,422
1,453
0,079
0,527
0,594
0,612
0,613
0,754
0,842
1,112
1,122
1,234
1,304
1,356
1,402
1,433
1
0,079
0
0,525
0,554
0,594
0,621
0,734
0,812
1,084
1,112
1,212
1,294
1,345
1,412
1,422
1,453
Mean Abs.
0,527
0,526
0,594
0,574
0,612
0,603
0,613
0,617
0,754
0,744
0,842
0,827
1,112
1,098
1,122
1,117
1,234
1,223
1,304
1,299
1,356
1,3505
1,402
1,407
1,433
1,4275
1
1,457
Efree
Ebound =
abs. Etot-Efree
0,526
0,931
0,574
0,883
0,603
0,854
0,617
0,84
0,744
0,713
0,827
0,63
1,098
0,359
1,117
0,34
1,223
0,234
1,299
0,158
1,3505
0,1065
1,407
0,05
1,4275
0,0295
1,457
0
B
0,638984
0,60604
0,586136
0,576527
0,489362
0,432395
0,246397
0,233356
0,160604
0,108442
0,073095
0,034317
0,020247
0
B/L
0,015975
0,020201
0,029307
0,057653
0,097872
0,144132
0,123198
0,155571
0,160604
0,154917
0,182739
0,171585
0,202471
1/B
1,564984
1,650057
1,706089
1,734524
2,043478
2,312698
4,058496
4,285294
6,226496
9,221519
13,68075
29,14
49,38983
1/L
0,025
0,033333
0,05
0,1
0,2
0,333333
0,5
0,666667
1
1,428571
2,5
5
10
L/B
62,59936
49,5017
34,12178
17,34524
10,21739
6,938095
8,116992
6,427941
6,226496
6,455063
5,4723
5,828
4,938983
L
40
30
20
10
5
3
2
1,5
1
0,7
0,4
0,2
0,1
1,6
1,4
B
0,7
0,7
1,2
0,6
1
0,5
0,8
0,4
0,6
0,3
0,6
0,5
0,4
0,3
0,2
0,2
0,4
0,1
0,1
0,2
55
0
0,1
0
0
0
0
10
20
30
40
50
5
10
15
20
25
30
35
40
45
1
10
100
Summary:
27-04-05
Use curve expert to fit the data:
Growth Models, Saturation Growth Rate
w278a
B
Scatchard
Hanes-Wolf
1/L
1/B
B/L
B
L
L/B
40
30
20
10
5
3
2
0,638678596
0,61871989
0,59119064
0,572608396
0,494838266
0,441156228
0,25395733
0,025
0,033333333
0,05
0,1
0,2
0,333333333
0,5
1,565732759
1,616240267
1,691501746
1,746394231
2,020862309
2,266770671
3,937669377
0,015966965
0,020623996
0,029559532
0,05726084
0,098967653
0,147052076
0,126978665
0,638678596
0,61871989
0,59119064
0,572608396
0,494838266
0,441156228
0,25395733
40
30
20
10
5
3
2
62,62931034
48,48720801
33,83003492
17,46394231
10,10431154
6,800312012
7,875338753
1,5
1
0,7
0,4
0,2
0,1
0,234686855
0,16586373
0,109428768
0,074328975
0,028217481
0,021335169
0,666666667
1
1,428571429
2,5
5
10
4,260997067
6,029045643
9,13836478
13,4537037
35,43902439
46,87096774
0,156457903
0,16586373
0,156326812
0,185822436
0,141087405
0,213351686
0,234686855
0,16586373
0,109428768
0,074328975
0,028217481
0,021335169
1,5
1
0,7
0,4
0,2
0,1
6,391495601
6,029045643
6,396855346
5,381481481
7,087804878
4,687096774
0 Kd/n =
1/n=
4,940730149 -Kd=
1,703616997 1/n=
-3,296061182 1/n=
0,710738213 Kd/n=
1,451348932
4,450430329
R2=
0,958888467 R2=
0,824010538 R2=
0,996280881
n=
Kd=
0,586986395 Kd=
2,900141379 n=
3,296061182 n=
1,406987808 Kd=
Data : B = f(starch)
S
r
Y A x is (u n i ts )
Langmuir
[starch]
User-Defined Model: y=a/(1+(b/x))
0,689014184 Coefficient Data:
3,066409621 a =
0,692941 =n
b=
2,6572012 =Kd
Different methods to linearize the data: help to delete
obviously wrong experimental points
56
0. 7
0
0. 5
8
0. 4
7
0. 3
5
0. 2
3
0. 1
2
0. 0
0
0.0
7.3
14.7
22.0
X Axis (units)
29.3
=
=
0 . 0 3 4 8 1 7 7 5
0 . 9 9 0 2 5 0 9 8
36.7
44.0
Appendix 6 Stock Solutions for Media Preparation
10X YNB (13.4% YNB):
For making 1 L of YNB, 134 g of YNB (Yeast nitrogen base with ammonium sulfate without amino acids) is dissolved in 1 L of H2O
and filtered for sterilization through 0.45 µm membrane and stored at +4C
500X Biotin (0,02% Biotin):
20 mg of biotin is dissolved in 100 ml H2O, filtered for sterilization and stored at +4 C.
10X Dextrose (20% Dextrose):
200g of glucose is dissolved in 1 L H2O, autoclaved and stored at room temperature.
10X Methanol (5% Methanol)
50 ml of methanol is mixed in 950 ml H2O, filter sterilized and stored at +4C
10X Glycerol (10% Glycerol):
100 ml glycerol is mixed with 900 ml H2O, filter sterilized and stored at +4C
1 M potassium phosphate buffer, pH 6.0 :
132 ml of 1 M K2HPO4 with 868 ml 1 M KH2PO4. The pH is adjusted with KOH to pH=6.0. This solution is autoclaved and stored at
room temperature.
57
Appendix 7 Coomassie blue and silver staining
Colloidal coomassie blue stain [15]: Samples of variant enzymes from the medium and purification steps were loaded on gradient gels
(Nupage Novex Bis Tris gels). The samples were mixed together with 7µl SDS Sample Buffer (3X) for coloring and 2.5µl DTT
(dithiotreitol) for reducing sulphite bindings. The total volume of sample should be 20 µl, at table 7.1 a list of volumes of various
enzymes is showed, rest volume is filled with H2O.The gel is fastened to its chamber and the chamber is filled with running buffer
which is prepared by mixing 50 ml 20X Nupage MES (2-morpholinethansulfosyre) with 950 ml H2O.
Table 7.1: Sample Volume for Staining
Sample
Volume in µl
Medium
5
Permeate
10
Retentate
3
Flow through
10
Pool
5 (100X)
Marker
6
Run conditions are done by setting the voltage to be constant at 200 V and choosing a run time at 35 min. After time the gel is fixed in
fixer solution for 30 min. The fixer solution consist of 30% ethanol and 2% phosphoric acid. Then the gel is washed twice with wash
solution which is 2% phosphoric acid. At last the gel is incubated over night in Blue silver stain. This stain can be made to 500 ml by
first adding 50 ml 85% phosphoric acid to 50 ml H2O, then adding 50 g ammonium sulfate and heat stirring until complete dissolve
58
and then adding 600 mg Coomassie Blue G-250. Adding H2O to 400 ml and methanol to 500 ml completes the stain. The stain is kept
from light. After time the gel is washed with deionized H2O and a digital photography is taken.
Silver staining: Samples of variant enzymes from the medium and purification steps were loaded on gradient gels (Nupage Noves Bis
tris Gels) as coomassie blue. The gel is fastened to its chamber and applied as in coomassie blue stain. The gel is the soaked in fixing
solution for 30 min. The fixing solution is made of fresh 40% methanol, 5%formaldehyd. The gel is then washed twice with H2O for 5
min and then soaked in 0, 02% Na2S2O3 for 1 min and washed for 20 sec. The gel is then soaked in 0, 1% silver nitrate for 1 min and
then soaked in fresh developer solution until bands appear. The developer solution is made of 0,3% Na2S2O3and 0,05% formaldehyde
and 0,0004% Na2S2O3. Process was stopped by adding 2.3M citric acid and shaken for 10 min. The gel was washed and dried and a
digital photography is taken.
59