4.1.1 Biological material
The following strains were obtained from the culture collection of the Institute of Food Biotechnology, University of Natural Resources and Life Sciences, Vienna:
Table 5 Biological Material
Institutional code Host Plasmid Gene Reference
B230 Escherichia
coli, BL21* pET21a PcPOx Pisanelli et al.,
2009
Y58
Pichia pastoris,
X33
pPICZB BaLac Kittl et al., 2012
4.1.2 Chemicals
All chemicals used for the preparation of media, buffers and various working solutions were of the highest grade available and purchased from Sigma Aldrich Chemie (Steinberg, Germany);
Carl Roth GmbH (Karlsruhe, Germany) and Merck (Darmstadt, Germany), unless otherwise mentioned for certain applications.
4.1.3 Media
All components were weighed in according to the specifications below, but for liquid media preparation no Agar-Agar was added. The appropriate volume of RO-H2O was added to dissolve the components and finally, the medium was autoclaved.
4.1.4 Antibiotics
The following antibiotics were added to autoclaved media for selection purposes whenever necessary in the following concentrations:
Table 6 Antibiotics in Media
Antibiotic substance Concentration [mg/L]
Ampicillin 100
Kanamycin 50
Zeocin for Yeast: 100
for Bacteria: 25
4.1.5 Media for E. coli cultivation 4.1.5.1 LB medium
Table 7 Composition of LB medium
Component Concentration [g/L]
Peptone f. Casein 10
Yeast Extract 5
NaCl 10
5 (for media supplemented with Zeocin)
Agar Agar 15
4.1.5.2 TB medium
Table 8 Composition of TB medium
Component Concentration [g/L]
Peptone f. Casein 12
Yeast Extract 24
Glycerol 5
Agar Agar 15
dissolved in 900 mL RO-H2O
1 M potassium phosphate buffer, pH 7.5, was autoclaved separately, prior to use 100 mL buffer per Liter TB-medium were added.
4.1.6 Media for P. pastoris cultivation 4.1.6.1 YPD
Table 9 Composition of YPD medium
Component Concentration [g/L]
Peptone f. casein 20
Yeast Extract 10
D-Glucose 4
Agar Agar 15
4.1.6.2 BMM medium
Table 10 Composition of BMM medium
Component Concentration
Yeast extract 10 g/L
Peptone f. caseine 20 g/L
1 M potassium phosphate buffer, pH 6.0 100 mL/L
10 x YNB 100 mL/L
500 x Biotin 2 mL/L
4.1.6.3 Production Stage Medium (MPCGlyc-Medium)
Table 11 Composition of MPCGlyc Medium
Component Concentration
Peptone f. caseine 10 g/L
Glycerol 10 g/L
5x M9 Salts (autoclaved separately) 200 mL/L
4.1.6.4 Seed Stage medium
Table 12 Composition of Seed Stage Medium
Component Concentration [g/L]
Glycerol 5
Peptone f. casein 5
Yeast extract 5
NaCl 5
Ampicillin 0.1
4.1.6.5 Basal Salts Medium
Table 13 Composition of Basal Salts Medium
Component Concentration
H3PO4, 85% 26.7 mL/L
CaSO4.2H2O 0.93 g
K2SO4 18.2 g/L
MgSO4.7H2O 14.9 g/L
KOH 4.13 g/L
Glycerol 40.0 g/L
4.1.6.6 BMD1, BMM2 and BMM10 media
Following Sygmund et al. (2012) the buffered minimal media BMD1, BMM2 and BMM10 contained 1.34% yeast nitrogen base, 4*10-5 % biotin, 200 mM potassium phosphate and 1%
glucose, 1% methanol or 5% methanol respectively.
4.1.7 Buffers
4.1.7.1 Enzyme activity buffer
50 mM sodium citrate buffer at pH 5.5 was used as a standard buffer for enzyme activity measurements, if not stated differently. This special pH was chosen in accordance with the intended use of all enzymes in sourdough (see above). A 500 mM buffer stock was prepared by dissolving 500 mM citric acid in water, adjusting to pH 5.5 with NaOH and then filling up with RO-H2O to the final volume. Whenever fresh buffer was needed, it was prepared by diluting this
stock 1:10.
4.1.7.2 Buffer for rheological measurements
McIlvaine buffer was prepared by mixing solutions of 0.1 M citric acid and 0.2 M Na2HPO4 to yield the pH values given below:
Table 14 Preparation of McIlvaine buffer
pH 0.1 M citric acid [mL] 0.2 M Na2HPO4 [mL]
4.0 30.7 19.3
5.0 24.3 25.7
5.5 (approximately) 21.8 28.2
6.0 17.9 32.1
4.1.7.3 Purification buffers
“Buffer A” is another term for loading buffer, “Buffer B” for elution buffer. When E.coli-expressed protein had to be purified, buffer A was also used as cell disruption buffer.
4.1.7.3.1 Hydrophobic interaction chromatography (HIC) buffers
HIC Buffer A: 50 mM sodium citrate buffer, pH 5.5, 40% (NH4)2SO4 saturation HIC Buffer B: 50 mM sodium citrate buffer, pH 5.5, 0% (NH4)2SO4 saturation
4.1.7.3.2 Immobilized metal ion affinity chromatography (IMAC) buffers according to Pisanelli et al. (2009) and Spadiut et al. (2010)
IMAC Buffer A: 20 mM or 50 mM phosphoric acid, 0.5 M NaCl, 0.02 M imidazole, adjusted to pH 6.5 with NaOH
IMAC Buffer B: 20 mM or 50 mM phosphoric acid, 0.5 M NaCl, 0.5 M imidazole, adjusted to pH 6.5 with H3PO4
4.1.7.3.3 Ion exchange chromatography (IEC) buffers according to Meng et al. (2013)
IEC Buffer A: 20 mM potassium phosphate buffer, pH 7.0
IEC Buffer B: 20 mM potassium phosphate buffer, pH 7.0, 0.5 M NaCl
4.1.7.4 Other buffers
4.1.7.4.1 1 M potassium phosphate buffer, pH 7.5 for TB medium
For a 1 M potassium phosphate buffer at pH 7.5 23.5 g/L KH2PO4 and 144.1 g/L K2HPO4 were dissolved in water. Any deviation from the desired pH was corrected with either H3PO4 or KOH.
4.1.7.4.2 TAE buffer
50 x TAE buffer contains 40 mM TRIS, 20 mM acetic acid and 1 mM EDTA.
4.1.7.4.3 SDS running buffer
The SDS running buffer for Mini-PROTEAN® TGX™ Precast Gels from BIORAD contained 25 mM Tris, 192 mM glycine, 0.1% SDS, and had to be stored at 4°C.
4.1.8 Other Solutions
4.1.8.1 500x Biotin – Stock solution
Table 15 Composition of 500x biotin solution
Component Concentration [g/L]
Biotin 0.2
500x Biotin solution was filter sterilized and stored at 4°C.
4.1.8.2 10x YNB - Stock solution
Table 16 Composition of 10x YNB solution
Component Concentration [g/L]
Yeast nitrogen base
(w/o ammonium sulfate and amino acids) 34
(NH4)2SO4 100
10x YNB solution was filter sterilized stored at 4°C.
4.1.8.3 5x M9 salts – Stock solution
Table 17 Composition of 5x M9 Salts solution
Component Concentration [g/L]
Na2HPO4.2H2O 42.5
KH2PO4 15.0
NaCl 2.5
NH4Cl 5.0
4.1.8.4 PTM1 Trace Salts
The PTM1 Trace Salts solution is filter sterilized and stored at room temperature.
Table 18 Composition of PTM1 Trace Salts solution
Component Concentration
CuSO4.5H2O 6 g/L
NaI 0.08 g/L
MnSO4.H2O 3 g/L
Na2MoO4.2H2O 0.2 g/L
H3BO3 0.02 g/L
CoCl2 0.5 g/L
ZnCl2 20 g/L
FeSO4 65 g/L
Biotin 0.2 g/L
H2SO4 5 mL/L
4.1.8.5 Rye Arabinoxylan Standard Solution, 2% (w/v)
Rye arabinoxylan with the following properties was purchased from Megazyme:
the arabinose: xylose ratio was 38:62, the viscosity of a 1 % (w/v) solution at 30°C was stated to be 36 cSt and the molecular weight of AX to be 440 kDa. Impurities include 4.0 % ash, 2.5 % protein, 0.2 % starch and 0.1 % β-glucan. The purity was indicated to be approximately 90%, but Ms. Bender found it to be 75%. A phenolic acid content of 48.48 ± 2.39 mg /100 g arabinoxylan was measured. For PnGOx and PcPOx measurements D-Glucose was added in concentrations suiting their published Km values.
Table 19 Preparation of 2%(w/v) AX solution
Rye Arabinoxylan (g) Volume of McIlvaine buffer at various pH steps (mL)
0.1 5
After addition of McIlvaine buffer to rye arabinoxylan the solution was put into the ultrasonic bath for about one hour.
4.1.8.6 Phusion Master Mix
Table 20 Pipetting scheme Phusion Master Mix
Component Volume [µL]
5x HF buffer 5
10 mM dNTP mix (10 mM of each dATP,
dCTP, dGTP, dTTP in UHQ-water) 0.5
Phusion polymerase 0.25
UHQ-H2O 6.75
Final volume 12
4.1.9 Vectors
The expression vectors pET21a, pPICZαA, as well as pGAPZαA were provided by the Institute of Food Biotechnology of the University of Applied Life Sciences, Vienna.
4.1.10 Primers
The primers used for the site-directed mutagenesis experiment were synthesized on request by Microsynth (Balgach, Switzerland).
Table 21 Primers for site-directed mutagenesis
Name Sequence (5’-3’) Length
(bp) Tm [°C]
5GOXmut1 GAG AGG CTG AAG CTT ACG TAG AAT TCT ACT CCC CAG CCG 39 80.0
3GOXmut2 AGC TTC AGC CTC TCT TTT CTC G 22 62.1
Standard primers for sequencing were available in stock at the Institute of Food Biotechnology and added to each sequencing sample at a DNA concentration of 10 pmol/µL.
Table 22 Standard primers for sequencing
Name Sequence (5’-3’) Application with
vector Producer
T7-fw GTA ATA CGA CTC ACT ATA GGG C pET21a(+) VBC Biotech
T7termrev CCC AAG GGG TTA TGC TAG TTA TTG CTC A pET21a(+) VBC Biotech
pGAPFw GTC CCT ATT TCA ATC AAT TGA A pGAPZ A VBC Biotech
AOX1 Fw GAC TGG TTC CAA TTG ACA AGC pPICZαA VBC Biotech
3AOX GCA AAT GGC ATT CTG ACA TCC pPICZαA, pGAPZ A Microsynth 4.1.11 Genes
Invitrogen synthesized the genes for PnGOx and Cgl1 on request by making use of its GeneArt® Gene Synthesis service, relying on the following sequences:
Cgl1 http://www.ncbi.nlm.nih.gov/nuccore/58036263/?from=3169415&to=3170896 PnGOx http://www.ncbi.nlm.nih.gov/nuccore/JN809250
The documents from Invitrogen showing both full sequences are added to the appendix of this thesis. Cgl1 was ordered with bases additional to the given sequence to ensure adequate restriction sites, namely CATATG (NdeI; with ATG already part of the protein sequence) and GCGGCCGC (NotI). Moreover, the Cgl1 sequence was ordered with E.coli-optimized codons to increase the protein expression level. PnGOx was also ordered with bases additional to the given sequence to ensure adequate restriction sites, namely GAATTC (EcoRI) and GCGGCCGC (NotI). Both genes were delivered inserted into vector pMK, which encodes a kanamycin resistance gene. The plasmid map of both delivered genes is given below.
Figure 12 Plasmid map Cgl1 in pMK-RQ (Invitrogen)
Figure 13 Plasmid map of PnGOx in pMK (Invitrogen)
4.1.12 Enzymes
Table 23 Enzymes for various applications
Enzyme Producer
EcoRI Thermo Scientific
DpnI Thermo Scientific
Horseradish peroxidase Sigma Aldrich
NdeI Thermo Scientific
NotI Thermo Scientific
PagI (BspHI) Thermo Scientific
SacI Thermo Scientific
T4-DNA-Ligase Thermo Scientific
4.1.13 Kits
Table 24 Kits
Kit Producer Application
PureYield™ Plasmid Miniprep
System Promega (Madison, USA) Purification of plasmid DNA
from E.coli cells illustra™ GFX™ PCR DNA
and Gel Band Purification Kit
GE Healthcare Life Sciences (Little Chalfont, UK)
DNA purification from agarose gels
4.1.14 Equipment
Table 25 Equipment
Tool Producer and Specification
96 Well Cell Culture Plate greiner bio-one, CELLSTAR®
Autoclaves Advantage Lab, AL02-03
Thermo Scientific, VARIOKLAV®
Balances Sartorius, analytical balance AW-224
Sartorius, precision balance AW-4202
Buffer exchange & protein concentration tools
Merck Millipore, Amicon® Ultra Centrifugal Filters, 30 000 MWCO
Spectrum, KrosFlo Research IIi TFF System with
Sartorius, Vivaflow 50 module
or MiniKros® Plus Tangential Flow Filtration Module
Bunsen burner WLD-TEC, gasprofi 1 SCS micro
Cell disruption tools
AMINCO, French Pressure Cell Press APV Systems, Homogenizer
Bandelin, Sonopuls HD 60 Ultrasonic Homogenizer
Centrifuges
Beckman, L-70 Ultracentrifuge Beckman-Coulter, Avanti J-26XP
eppendorf, Centrifuge 5415 R, 5804, 5804 R, 5424 Kendro Laboratory Products, SORVALL® Evolution RC
Conductivity meter Thermo Scientific, Orion 4-Star pH/Conductivity Meter
Electrophoresis Equipment Agarose gel electrophoresis:
SDS-PAGE:
BIO-RAD, PowerPac™ 300 power supply BIO-RAD, PowerPac™ HC power supply BIO-RAD, Mini-PROTEAN™ Tetra Cell
BIO-RAD, Mini-PROTEAN® TGX™ Precast Gels
Electroporation Equipment
BIO-RAD, MicroPulser™
BIO-RAD, E.coli Pulser® Cuvette, 0.1 cm electrode gap
Fermenter applikon®, bioreactor type NAT’L BD. 758 (70 liters) and bioreactor type NAT’L BD. 828 (42 liters)
Imager BIO-RAD, Molecular Imager® Gel Doc™ XR+
Laminar flow working bench Thermo Scientific, Safe 2020
PCR Thermocycler BIO-RAD, C1000™ Thermal Cycler
pH meter Metrohm, pH Meter 744
Pipettes Gilson® PIPETMAN Pipettes
Purification equipment Amersham Pharmacia, ÄKTAexplorer Amersham Pharmacia, ÄKTApurifier
Rheometer Malvern Instruments, Kinexus pro
Stirring and shaking devices IKA® Labortechnik, lab dancer
IKA® Labortechnik, RCT basic magnetic stirrer Sterile filtration equipment
Syringe, Needle & Filter:
Vacuum pump & attachment
B.Braun, Injekt® and Omnifix® Single-use syringes B.Braun, Sterican®, Single-use needles
Carl Roth, Rotilabo®-syringe filters, PVDF, sterile Vacuubrand, Diaphragm vacuum pump
Merck Millipore, Stericup® Filter Unit UV/Vis spectrometer Perkin Elmer, Lambda 35 UV/Vis
Beckman-Coulter, DU® 800
Ultrasonic bath Bandelin, SONOREX™
Water bath JULABO, TW12 Water bath