Protein expression and purification

• MiniPrep according to Kit specifications

• Linearization of mutated plasmid by restriction digest with SacI

• Transformation by electroporation into competent Pichia pastoris X-33 cells

BMM10 medium. The plate was then centrifuged for 15 minutes at 3000 rpm and the supernatants were subjected to a carbohydrate oxidase activity assay using ABTS.

4.2.3.1.4 PnGOx - Expression in P. pastoris using pPICZαA-transformed cells in deep-well plates after site-directed mutagenesis

Site-directed mutagenesis was performed to equalize differences between the vector pPIC9 used by Gao et al. (2012) and pPICZαA, in other words to introduce the sequence “TAC GTA”

to pPICZαA which was located between the EcoRI restriction site and the S. cerevisiae α- secretion signal sequence of pPIC9. This step was carried out to evaluate if those additional 2 amino acids were the reason for an approximately 300 times higher activity reached by Gao et al. (2012) when compared to the present expression experiment (see below). The screening for PnGOx activity of transformed yeast cells was conducted exactly as already stated for pPICZα A-transformed cells without site-directed mutagenesis in 4.2.3.1.3.

4.2.3.2 Expression on a medium scale for preliminary rheological tests

4.2.3.2.1 PcPOx - Expression in E.coli in 1 L shaking flasks

Following Pisanelli et al. (2009), the starter culture was prepared by adding a colony of E.coli (B230) to 100 mL of TB medium, supplemented with ampicillin, and by subsequent incubation overnight at 37°C and 150 rpm. 5 baffled shaking flasks with overall 200 mL TB medium were each inoculated with 10 mL of starter culture. After two hours at 25°C and 150 rpm, the expression was induced by addition of IPTG to a final concentration of 0.1 mM. The fermentation continued over night at the same temperature and agitation. Cells were then harvested by centrifugation at 6000 rcf and 4°C for 30 minutes, and disrupted by using the French Press.

4.2.3.2.2 PnGOx - Expression in P. pastoris using pPICZαA-transformed cells in 5L shaking flasks (Fed-Batch)

600 mL of inoculum were prepared by transferring one colony to each of four 1L shaking flasks filled with YPD medium The inoculum was incubated overnight at 30°C and 125 rpm. The main culture was started by adding 150 mL of inoculum to each of four 5 L baffled shaking flasks, each filled with 1500 mL BMM medium. Antifoam was added whenever necessary. The main fermentation was cultivated at 110 rpm and 30°C at a constant feed of 50% (v/v) methanol. A pump regulated the methanol feed rate via a timer switch. The pump setting was 0.78 rpm for the whole fermentation process, whereas regulation via the timer switch was changed in accordance with cell growth throughout the process as follows:

Table 31 Methanol feed variations during medium-scale PnGOx expression

Time [h] Gilson® pump setting [rpm]

Timer switch setting [/day]

Corresponding feed rate [mL* L^-1* h^-1] 0

0.78

24 x 15 minutes 12.5

77.5 48 x 15 minutes 25

168.5 72 x 15 minutes 37.5

174.5 66 x 15 minutes 34.4

The fermentation broth was finally centrifuged at 6000 rpm and 4°C for 30 minutes, and then the supernatant was subjected to diafiltration with 20 mM potassium phosphate buffer, pH 7.0, and concentrated for purification.

4.2.3.3 Expression on a large scale for constructive rheological tests and baking trials 4.2.3.3.1 PcPOx - Expression by E.coli on a large scale in an applikon® fermenter for baking

trials (Batch)

Following Spadiut et al. (2010), the inoculum was prepared by picking an E.coli (B230) colony from an LB-Amp plate, by resuspending it in 500 µL LB-Amp medium and by transferring the resuspended colony to a 1 L-baffled flask with 250 mL Seed Stage medium. After incubation at 30°C and 140 rpm overnight, the main fermentation was started by adding the inoculum to 30 L of pre-sterilized Production Stage medium, which was supplemented with M9 salts and diluted antifoam. The main fermentation was carried out as a batch process in an applikon® bioreactor with an overall volumetric capacity of 70 L at 25°C, 530 rpm and an airflow of 40 L/h. The expression of the target protein was induced with 0.5% (w/v) lactose. After 28.5 hours, the fermentation was stopped as the cell growth started to decrease and as the dO2 remounted to 100% due to declining oxygen consumption by bacteria. Cells were then harvested by centrifugation at 6000 rpm and 4°C for 20 minutes, and disrupted by using the Homogenizer.

4.2.3.3.2 BaLac - Expression by P. pastoris (Y58) in an applikon® fermenter (Fed-Batch)

The process was planned and conducted on the basis of Pichia Fermentation guidelines (Invitrogen). The fermentation was divided into three phases: the glycerol batch phase and the glycerol fed-batch phase at 30°C for the generation of biomass, and the methanol fed-batch phase at 25°C, where the expression of BaLac was induced. Both glycerol and methanol feed contained an appropriate amount of PTM1 trace salts and were provided at feed rates from 0.3

to 1 mL/min to keep the dissolved oxygen concentration at around 2 %. The whole fermentation was run at an agitation of 500 to 600 rpm and a pH of 4.5 to 4.9. DO2 levels were maintained by supplying filtered air (10-40 L/h) and oxygen (2-4 L/h), pH was adjusted automatically by addition of ammonium hydroxide. Antifoam and methanol pulses of 100% methanol with PTM1 trace salts were injected manually whenever necessary, the latter in a way to not exceed a methanol concentration of 1-2 % (v/v) which would be toxic. At the beginning, an inoculum was prepared by transferring one P. pastoris (Y58) colony from a YPD-Zeocin plate to each of 5 baffled shaking flasks, each filled with 200 mL YPD medium. The inoculum was incubated overnight at 30°C and 250 rpm. To start the glycerol batch phase the inoculum was transferred to an applikon® fermenter with an overall volumetric capacity of 42 L, which contained 9L of Basal Salts medium supplemented with PTM1 trace salts. The end of this phase was announced after 31 h by the increase in dO₂ to 100 %, which indicated the depletion of glycerol; at the same time, the Glycerol Fed-Batch Phase was initiated by setting a glycerol feed of 0.26 mL/min. When a biomass of 117.5 mg/mL was reached, a methanol pulse of 50 mL was added for induction while the glycerol feed was reduced gradually to allow the cells an adaption to methanol. The methanol fed-batch phase started after 55 hours; methanol pulses were added and the methanol feed rate was varied throughout the rest of the fermentation in accordance with current feed requirements. After 72h an additional 4 L of Basal Salts medium with PTM1 trace salts, but without glycerol, were added to increase the fermentation volume in order to facilitate oxygenation by reaching another stirrer blade. The fermentation was ended after 170 hours as soon as the cell growth started to decrease.

4.2.3.4 Cell harvesting and cell disruption

E.coli cells were harvested by centrifugation at 6000 x g and 4°C for 30 minutes. As the expressed protein accumulates inside the cell during E.coli fermentation the cell membrane has to be disrupted to get access to the desired protein. Smaller volumes of cell pellets were disrupted using the French press, whereas for larger volumes the Homogenizer was the method of choice. Prior to use of each apparatus the cell pellet had to be diluted approximately 1:3 to reduce its viscosity. During disruption the collected sample was stored on ice.

4.2.3.4.1 French pressure cell press

The cell cylinder was filled with at most 35 mL, assembled and placed in the armature. Cell lysis occurs as cells are transported through a narrow valve under high pressure at 1000 bar. One batch was treated a second or even third time to decrease the solutions viscosity that increased due to release of DNA.

4.2.3.4.2 Homogenizer

The method principle is the same as for the French pressure cell press, but volumes up to one liter per batch can be handled. During circulation of the sample the pressure was increased to 800 to 900 bar.

4.2.3.5 Buffer exchange and Concentration

The concentration of Pichia supernatants or purified enzyme solutions as well as buffer exchange (diafiltration) was achieved by using either Amicon^® centrifugal filters or a tangential flow filtration module. Amicon^® centrifugal filters with a molecular weight cut-off of 30 kDa enable the removal of molecules smaller than 30 kDa, whereas a polyethersulfone hollow fiber module for tangential flow filtration removes molecules smaller than 10 kDa. An Amicon^® centrifugal filter tube can comprise 15 mL, whereas the Vivaflow 50 module was used for up to 1 L, and the MiniKros^® Plus hollow fiber module for volumes larger than 1 L. This procedure took place in the 4°C room and was especially useful to dispose of undesirable adjuvants, like ammonium sulfate or imidazole. The transmembrane pressure was always set at slightly below 100 mbar.

4.2.3.6 Purification

The execution principle of chromatography is the same for all of the three given chromatography techniques: column-washing with buffer A, sample binding, protein collection during elution with a gradient to 100 % of buffer B, and finally washing of the column. The flow rate of each buffer or sample was adjusted manually in a way that did not lead to exceeding a pressure limit of 0.25 MPa, and the UV/VIS detector was set to 280 nm in order to monitor proteins. Purification factor and yield, which are given in all purification tables in the result section, were calculated as follows:

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4.2.3.6.1 Immobilized metal ion affinity chromatography

Immobilized metal ion affinity chromatography (IMAC) is based on affinity interaction between a His-Tag exposed on the surface of the protein and chelated Ni²⁺.

Cell harvesting and disruption were performed as described in 4.2.3.4. As the final E.coli pellet after expression on a larger scale was quite big, it was divided into five smaller pellets and therefore five purification steps were performed with IMAC. Following Pisanelli et. al., PcPOx was purified by one-step IMAC on a 65 mL column packed with Profinity IMAC Ni-Charged

Resin (Bio-Rad Inc., Hercules, CA). The crude extract was applied to the column at a flow rate of 8 to 10 mL/min and eluted by using a linear gradient of 20 mM to 1 M imidazole in approximately 10 column volumes. Active fractions were pooled (220 mL in total), desalted and concentrated in 20 mM potassium phosphate buffer (pH 7.0) using the Vivaflow 50 module (Sartorius), filter sterilized, and stored at -30°C.

4.2.3.6.2 Hydrophobic interaction chromatography

The purification of BaLac is based on hydrophobic interactions between non-polar amino acid side chains of the enzyme and hydrophobic ligands. These interactions are promoted by a high salt concentration of the loading buffer.

Following Kittl et al. (2012), the fermentation broth was harvested by centrifugation at 6000 rpm for 20 min at 4°C. After reduction of the supernatant volume to 5 L, solid ammonium sulfate was slowly added to the supernatant to 40% saturation in a way that no layer of 100% saturated ammonium sulfate emerged. Ammonium sulfate was added at 4°C within 2.5 h. Afterwards, the solution was centrifuged at 4°C and 6000 rpm for 20 min to remove protein precipitate. The supernatant was divided into two batches of 2.5 L, which were each applied to a 500 mL phenyl sepharose column and eluted with a linear gradient from 40 to 0% ammonium sulfate saturation in 50 mM sodium citrate buffer (pH 5.5). Fractions of 50 to 100 mL were analyzed in terms of laccase activity and the most active fractions were pooled. Finally, the pooled fractions were concentrated by means of the Vivaflow 50 module (Sartorius), sterile-filtered and stored at -30°C.

4.2.3.6.3 Ion exchange chromatography

PnGOx was purified following Meng et al. (2014), who purified glucose oxidase derived from Aspergillus niger, using a 500 mL Q-Sepharose FF column. The column matrix is a strong anion exchanger, therefore positively charged at any practical pH, and the interaction between charged amino acids of a protein and the matrix is of electrostatic nature. (Carta & Jungbauer, 2010)

At first, the Pichia fermentation broth was harvested by centrifugation at 6000 rpm for 30 min at 4°C. The supernatant was concentrated and dialyzed against 20 mM potassium phosphate buffer, pH 7.0, using the Vivaflow 50 module (Sartorius). Then, the supernatant was applied to the column and eluted using a linear gradient from 0 to 0.5 M NaCl. As PnGOx did not bind to the column, fractions of 100 mL of the flow-through were analyzed in terms of PnGOx activity and all fractions of more than 0.12 U/mL were pooled (3100 mL in total), concentrated to 200 mL by means of the Vivaflow 50 module (Sartorius) and stored at 4°C.

4.2.3.7 SDS PAGE

SDS-PAGE, more precisely SDS polyacrylamide gel electrophoresis, is a discontinuous electrophoresis method for analysis of proteins. Dividing the gel into a resolving gel with smaller pores and a stacking gel with larger pores is the reason for discontinuity. Proteins form stacks in the stacking gel in order of their mobility, but later they destack and separate according to electrophoresis principles in the resolving gel. Addition of SDS, sodium dodecyl sulphate, to the polyacrylamide gel renders all proteins negatively charged, therefore prevents the migration of positively charged proteins towards the cathode and allows the proteins separation to be based exclusively on molecular weight. (Westermeier, 2005) In this project SDS-PAGE was performed to evaluate the purity of the produced enzyme solutions before and after chromatographic purification. This was accomplished by exposing ready-to-use Mini-PROTEAN^® TGX™ Precast Gels from BIO-RAD to 200 V and 0.3 A. The standard used to provide a reference for the molecular weight of proteins was BIO-RAD Precision Plus Protein™ Unstained Protein Standard (Figure 15).

Figure 15 Precision Plus Protein Unstained Protein Standard, BIO-RAD

4.2.4 Characterization of Enzymes