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Process Biochemistry 39 (2004) 889–896 Large-scale separation and production of engineered proteins, designed for
Process Biochemistry 39 (2004) 889–896 Large-scale separation and production of engineered proteins, designed for

Process Biochemistry 39 (2004) 889–896

Process Biochemistry 39 (2004) 889–896 Large-scale separation and production of engineered proteins, designed for

Large-scale separation and production of engineered proteins, designed for facilitated recovery in detergent-based aqueous two-phase extraction systems

Klaus Selber a , Folke Tjerneld b , Anna Collén b , Teppo Hyytiä c , Tiina Nakari-Setälä c , Michael Bailey c , Richard Fagerström c , John Kan d , Joop van der Laan d , Merja Penttilä c , Maria-Regina Kula a,

a Institute of Enzyme Technology, Heinrich-Heine University, Düsseldorf, D-52426 Juelich, Germany b Department of Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, S-22100 Lund, Sweden c VTT Biotechnology and Food Research, P.O. Box 1500, FIN-02044 VTT, Espoo, Finland d Genencor International, B.V., Archimedesweg 30, 2333 CN Leiden, The Netherlands

Received 11 July 2002; received in revised form 7 April 2003; accepted 22 May 2003

Abstract

The feasibility and scalability of extraction in detergent-based aqueous two-phase systems for the separation of proteins from culture broth is demonstrated. At the same time the large-scale production of a fusion protein and the influence of cultivation scale on the efficiency

of separation were investigated. An amphiphilic fusion protein EGIcore-HFBI was chosen, consisting of the catalytic core of the cellulase

endoglucanase I and the small protein hydrophobin I, expressed homologously in Trichoderma reesei. Using the technical nonionic detergent Agrimul NRE 1205 the separation was successfully scaled up to 1200 l. No differences in yield or in partition coefficient were observed at 10

ml and 1200 l scale. Changes in the fermentation temperature and scale, however, can influence the properties of the protein and thus alter

partition coefficient and yield. The decreased separation efficiency appears to be correlated with changes in glycosylation at lower cultivation temperatures. © 2003 Published by Elsevier Ltd.

Keywords: Detergent-based aqueous two-phase systems; Large-scale extraction; Endoglucanase I; Hydrophobin fusion protein

1. Introduction

Extraction in aqueous two-phase systems (ATPS) is a reliable method to clarify, purify and preconcentrate pro- teins and other biomolecules [1–3]. Only PEG/salt systems are reported to be in industrial use today. In such systems, PEG and certain salts (phosphates, sulphates) are needed in moderate concentrations to accomplish phase formation. Economically, ATPS have certain advantages especially with respect to costs of labour, energy and investment. One drawback, however, is the relatively high costs of chemicals, if no recycling is applied [4,5]. In the meantime, sewage costs are increasing heavy and have to be considered. A newer development in ATPS could possibly overcome these

Corresponding author. E-mail address: mrk3372002@yahoo.de (M.-R. Kula).

0032-9592/$ – see front matter © 2003 Published by Elsevier Ltd.

doi:10.1016/S0032-9592(03)00198-5

drawbacks through the use of only one single-phase form- ing component at lower concentrations. These so-called

detergent-based systems or cloud-point extraction systems form two phases in solutions of nonionic detergents above

a certain temperature, referred to as the cloud-point tem-

perature. These systems are suitable for the separation of hydrophobic and amphiphilic molecules. Most applications of the detergent-based ATPS target the solubilisation and separation of membrane bound sub- stances. This has been done in a broad variety of laboratory- scale experiments. Membrane proteins have been solubilised

from cells including plants and neural cells, archaebacteria, eubacteria and yeasts, as summarized by Sánchez-Ferrer

et al. [6] and Hinze and Pramauro [7].

Few large- or pilot-scale experiments and applications of ATPS are reported in the literature. In 1982, Kroner et al. [8] showed that scale up of extraction processes is easy and

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K. Selber et al. / Process Biochemistry 39 (2004) 889–896

predictable for PEG/salt systems. Kim et al. [9] report an amylase extraction on a 50,000 l scale and Heinsohn et al. [10] report a large-scale industrial chymosin process. These and other processes are referenced in a review focussing on the application of ATPS [3]. Concerning detergent-based systems, Minuth et al. [11] report a 5 kg scale experiment isolating the membrane bound cholesterol oxidase from Nocardia rhodochrous using a

technical detergent (C 1218 E 5 ). In the present investigation,

a similar nonionic detergent Agrimul NRE 1205 was used

as phase forming polymer. It is a polyoxyethylene with a carbon chain distribution consisting to a large extent of C12 chains and minor fractions of C14, C16, and C18, which is derived from plant oil rich in C12 lipids. The cloud-point (the point where the dehydration of the detergent and thus phase separation occurs) at a concentration of 2% Agrimul

NRE 1205 in water is at 22 C. The phase diagram and the physical properties of the detergent are discussed in detail by Selber et al. [12]. Several recent studies to facilitate separation by geneti-

cal engineering of target proteins can be found in the liter- ature. Investigations published before 1999 are summarized in Berggren et al. [13]. These studies indicate that tagging of proteins using small molecules can be a versatile tool, which enables the directed partition in ATPS for the separa- tion of a target protein. This has also been demonstrated by additions of small peptide tags to the two-domain cellulase endoglucanase [14]. However, difficulties in the expression of fusion proteins with hydrophobic peptide tags have been observed [15]. The target protein chosen for this investigation was a ge- netically engineered protein consisting of the fungal cel- lulase endoglucanse I (EGI) and the amphiphilic protein hydrophobin I (HFBI) from Trichoderma reesei. Since, a part of EGI (the cellulase-binding domain) was replaced by HFBI the protein is called EGIcore-HFBI. HFBI is a class

II hydrophobin with a molecular mass of 7 kDa. The fusion

protein is expressed homologously under the control of the strong cbh1 promoter. An important application of cellulases is the treatment of cotton fabric, e.g. for the production of stone-washed jeans or in the wash. Cellulases are bulk products, which are usually applied as cocktails without purification after the clarification and concentration of the culture broth. In order to be able to profit from a specific application of a single cellulase a separation procedure must be inexpensive and applicable on a large-scale. The detergent employed for phase separation in detergent-based systems could in principle be removed from the protein and recycled [16]. However, in some applications the detergent may provide a perfect formulation and does not need to be removed. The goal of this work was to examine the technical feasibility of large-scale purification of enzymes using detergent-based ATPS. For this aim a direct scale up from 10 ml to 1200 l was done in the downstream processing while the cultivation was scaled up from 7 to 1500 l.

Influences arising in extraction of EGIcore-HFBI in detergent-based systems due to the scale of the fungal cultivations were studied and are also discussed.

2. Materials and methods

The following characteristics describe the partitioning of molecules in ATPS. The partition coefficient K is defined as the ratio of con- centration in the top (c i,T ) and bottom phase (c i,B )

K = c i,T

.

c i,B

The yield in the top phase Y T is defined as

Y T =

n T

n T + n B

or consequently as

Y T =

1

1 + ((V B /V T )(1/K))

If no index is used for the yield it refers to the yield in the detergent-rich phase. The volumes of the phases can be described by the volume ratio R

R = V T

V B

and by the concentration factor in the light phase c f

c f = V total

V

T

= V T + V B

V

T

where V is the volume of the phase denoted.

2.1. Separation and preparation of ATPS

2.1.1. Separation of phase systems on a laboratory scale

The detergent-based systems in tube scale were produced by weight using undiluted detergent. The detergent was filled first into graduated centrifuge tubes (Assistent Hecht, Merck KgaA, Darmstadt, Germany). The tubes were then filled to 10 g with culture supernatant. If salt was added, it was first solubilised in the culture supernatant before addition of the detergent. The tubes were closed and mixed thoroughly in an overhead shaker (Heidolph, Kehlheim, Germany) at room temperature. After mixing, the tubes were heated to the separation temperature and either separated by gravity settling or by centrifugation at 3000 rpm at constant temper- ature. After phase separation, the phase volumes could be determined with a reproducibility of approximately 0.1 ml.

2.1.2. Separation of phase systems on a pilot scale

The separation was performed in two steps. 1st step: To 1160 l culture supernatant with a density of

1005 g/cm 3 50 kg of detergent and 20 kg of NH 4 H 2 PO 4 were added resulting in 4.1% concentration of detergent

K. Selber et al. / Process Biochemistry 39 (2004) 889–896

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and a 0.15 mol/l NH 4 H 2 PO 4 solution. Phase separation was accomplished by gravity settling, 840 kg of bottom phase

were removed in the 2nd step and 410 kg water and 12 kg

NH 4 H 2 PO 4 were added to the remaining original top phase so that the salt was solubilised in 300 l of water at 30 C and added to the bioreactor as well as the remaining 110 l of water. Mixing of the phase system during the first and second ex-

traction was accomplished by stirring gently for 30 min at 60 rpm. In small-scale experiments, the systems have not shown any tendency to form stable emulsions and they also showed a very fast mass transport as already described by Fauquex et al. [17]. Therefore, no problem on a large-scale was ex- pected. Nevertheless, mixing time and speed were chosen

very conservatively to ensure proper mass transport while

avoiding the creation of microemulsions, which would not allow a separation by gravity settling. The experiment was carried out in a bioreactor equipped with a 330 mm Rushton turbine (New Brunswick Scientific Company, Edison, NJ) and temperature control set to 24.7 and 30 C, respectively.

2.2. Target protein

The EGIcore-HFBI fusion protein contained the catalytic domain and the linker region of EGI fused to the gene en- coding the 75 residue long mature HFBI, thus the cellulose binding domain of EGI was replaced by HFBI.

2.3. Glycosidase treatment

To 200 l of culture supernatant 200 l of sodium citrate

buffer (buffer 65), 200 l of buffer NP-40, 10 l PNGase F 704S and 10 l Endo H f 703S (both hydrolases purchased

from Biolabs, Inc., New England, USA) were added. The

samples were incubated at 36 C for 16 h. A reference sam-

ple was treated likewise but omitting enzyme addition. The separation was carried out using 4% detergent at 30 C.

2.4. Determination of enzymic activity

Endoglucanase I (EGI) was assayed using p-nitrophenyl- -d-cellobioside (Sigma N-5759) as substrate, dissolved in 50 mM sodium acetate buffer, pH 5.0. EGI hydrolyses the -glycosidic bond and p-nitrophenol is released, which

was measured at 400 nm. Since, CBHI (cellobiohydrolase I) also hydrolyses the substrate it was inhibited using 4.6

mM cellobiose. The reaction is performed at 50 C and

under the cbhI promoter. The following cultivation media was used. 20.0 g/l lactose, 4.0 g/l peptone, 1.0 g/l yeast extract, 15.0 g/l KH 2 PO 4 , 2.8 g/l (NH 4 ) 2 SO 4 , 0.6 g/l MgSO 4 ·7H 2 0, 0.8 g/l CaCl 4 ·2H 2 0, 2.0 ml/l trace element solution [18] (5.0 mg/l FeSO 4 ·7H 2 O, 1.6 mg/l MnSO 4 ·H 2 O, 1.4 mg/l ZnSO 4 ·7H 2 O and 3.7 mg/l CoCl 2 ·7H 2 O). In the pilot-scale cultivation the amount of lactose was increased to 40 g/l, all other ingredients remained the same. One 200 ml shake flask was inoculated from agar slants and incubated on a rotary shaker at 200 rpm at 29 C for 2 days. The culture broth was then distributed between three shake flasks and incubated for one more day under the same conditions. After inoculation of a 15 l bioreactor the culti- vation was continued for about 4 days at a pH between 4.0 and 5.0 at an agitation rate of 400–600 rpm. The pH was au- tomatically adjusted with sodium hydroxide and phosphoric acid. For other bioreactors, the volume of the inoculum was adapted accordingly. On a 1200 l scale, the temperature at the beginning was set to 27 C, agitation at 160 rpm and aeration rate at 12 m 3 /h. The cultivation inoculum was first in a 15 l cultivation, then in 180 l each for 24 h. Growth time in the pilot-scale reactor was 4 days. Antifoam was injected automatically on foaming.

2.6. Chemicals

All compounds used were of analytical quality. Agrimul NRE 1205 (C 1218 E 5 ) was a gift from Henkel/ Cognis, Düsseldorf, Germany. The antifoam DOW Corning 1500 (EU), Edegem, Bel- gium was employed. Celite 535 (diatomaceous earth) from Celite France SA, Rueil, Malmeson, France was used as filter aid for rotating vacuum drum filtration. 12 kg of Celite were suspended in 250 l water and deposited equally on the drum prior to filtration. The vacuum drum filter was constructed by Larox OY, Lappeenranta, Finland and has a surface area of 1 m 2 .

3. Results

3.1. Scale up of the downstream process

terminated by addition of 12.5% sodium carbonate solution

Before the start of the scale up, cultivation was investi-

after

10 min.

gated in shake flasks and 7 l bioreactors. Downstream pro-

4-Methylumbelliferone (Sigma M-1508) was used to gen-

cessing was carried out in 10 ml tube scale. On this small

using culture supernatant. The influence of cells is reported

erate

a standard curve.

scale, the basic process was optimised as described in Sel-

2.5.

Cultivations

ber et al. [19]. All experiments discussed were carried out

Production of the target protein EGIcore-HFBI was car- ried out with a recombinant T. reesei strain VTT D-99702

by Selber et al. [19]. The scale up of cultivation and downstream processing was done using culture broth obtained in large cultivation for

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K. Selber et al. / Process Biochemistry 39 (2004) 889–896

K. Selber et al. / Process Biochemistry 39 (2004) 889–896 Fig. 1. Comparison of extraction experiments

Fig. 1. Comparison of extraction experiments in 1200 l and 10 ml scale. Phase system composition: detergent (Agrimul NRE 1205) (w/w) = 0.041 and 0.15 mol/l (NH 4 )H 2 PO 4 . The extraction temperature for the phase system was 24.7 C and the pH was 5.5.

the pilot-scale downstream process. The quality of the down- stream process was judged by a parallel experiment on a test tube scale, starting with the identical culture supernatant. At the end of the cultivation, the mycelium was separated by means of a rotating vacuum drum filter using Celite 535 diatomaceous earth as filter aid. 1160 l was obtained out of 1500 l whole broth. The supernatant was transferred into the cleaned biore- actor, adjusted to the separation temperature of 24.7 C and mixed with salt and detergent to give 0.15 mol/l ammonium dihydrogen phosphate and 4.1% of detergent Agrimul NRE 1205. The phases were left to separate by gravity settling for 15 h. The target protein should stay in the light, the detergent-rich phase. The light phase had a volume of 250 l or a concentration factor of 3.3. The heavier phase was removed through the bottom valve. The yield, partition coefficient and concentration factor from this extraction are shown in Fig. 1. The partition co- efficient and the concentration factor are equal in the 10 ml and 1200 l scale separation within the measurement error. The yield is slightly different due to the small deviations of partition coefficient and volume ratio. Scale up factor is 1.2 × 10 5 .

3.2. Scale up of the cultivation and downstream process in a two-step procedure

A second-phase separation can be performed to increase

the purity of the fusion protein. Water (additionally salt) can be added to the detergent-rich phase from the first separation step.

In this investigation, a re-extraction was of special in-

terest since endogeneous EGI is expressed in low amounts by the recombinant host. The activity assay cannot distin- guish between fusion protein and endogeneous EGI. Endo- geneous EGI remains almost quantitatively in the bottom phase. The cultivation conditions and, therefore, the scale up of the cultivation have an impact on the overall result as could be anticipated from the moderate partition coefficients of the fusion protein and yields presented so far. The cultivation had to be scaled up from 7 to 1500 l (the difference to the above mentioned volume of 1200 l is due to the separation of the mycelia). Even if no major difficul- ties were expected and encountered during the 11-day cul- tivation period, in this investigation the influence of cultiva- tion can only be judged by the following phase separation. The results for the extraction from 7 and 1500 l cultiva- tion are compared in Fig. 2. In a two-step-procedure, new water and salt were added to the light phase of 250 l to give a final volume of 750 l which was stirred and left for gravity settling for 5 h. The target protein partitioned to the detergent-rich light phase. The heavy phase was removed through the bottom valve. As the first step of the pilot sep- aration, this second step did not reach the same yield and partition coefficient as found after cultivation ona7l scale. The apparent K value of 11 and 33 (first and second sep- aration, respectively, 7 l) and 3.8 and 7.7 (first and second separation, respectively, 1500 l) demonstrate the significant differences. Several factors which could cause experimentally ob- served differences were investigated: insufficient mass transport, high entrainment, influence of antifoam, genetic

transport, high entrainment, influence of antifoam, genetic Fig. 2. Separation behaviour of EGIcore-HFBI obtained from 7

Fig. 2. Separation behaviour of EGIcore-HFBI obtained from 7 l cultivation and pilot-scale (1500 l) cultivation. Extraction was performed with 10 ml culture supernatant each. Phase system composition: detergent (Agrimul NRE 1205) concentration (w/w) = 0.041 and 0.15 mol/l (NH 4 )H 2 PO 4 . The extraction temperature for the phase system was 24.7 C and the pH was pH 5.5.

K. Selber et al. / Process Biochemistry 39 (2004) 889–896

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instability of the strain, differences in the cultivation proce- dure and glycosylation of the fusion protein.

3.3. Limiting mass transport/insufficient mixing

While stirring the culture supernatant slowly, salt was solubilised in the bioreactor and detergent was added. The bioreactor used as mixer and settler was stirred at 60 rpm for 30 min while the temperature was maintained at 24.7 C. The long mixing time should ensure a sufficient mass trans- fer while a slow rotational speed was chosen to prevent the formation of stable microemulsions which would lead to a very slow separation. Parallel tube scale investigations showed that mass transport was satisfactory.

3.4. Entrainment/insufficient separation

High entrainment, especially entrainment in the bottom phase lowers the apparent partition coefficient and yield. Entrainment was monitored in the first and second separa- tion step. In the first step, the entrainment (incomplete sep- aration) could be observed mainly in the top phase, which included 16% of bottom phase. In the second step, no en- trainment could be monitored in the top phase while 4.6% of top phase remained in the bottom phase. Assuming the entrained phases were entrapped after the transport of fu- sion protein was completed the amount of fusion protein in the entrainment can be considered as high as its amount in the purified phases. The theoretical separation results in the absence of entrainment can be calculated. The results are shown in Table 1. The increase in concentration factor from 3.4 to 3.8 and in K of 3.8 to 4.2 and no difference in yield cannot explain the big difference between the separation experiments shown in Fig. 2. The yields in the presence of entrainment and in the absence of entrainment are almost identical since mainly heavy phase was entrapped in the light phase. It has to be mentioned that, while performing the mea- surement of the entrainment volume, no entrainment could be measured after 10 min of centrifugation in 10 ml cen- trifuge tubes at 3000 rpm. It appears that the emulsion is stabilised by ingredients from the culture broth. This inter- pretation is enforced by samples taken from the very top (and from the very bottom) of the phases in the large-scale experiment which show no higher (lower) activity than the phase average (data not shown).

Table 1 The calculated effect of the entrainment on the concentration factor, partition coefficient and yield of EGI activity in the 1200 l separation experiment

 

Concentration factor, c f

Partition

Yield,

coefficient, K

Y (%)

Measured values

3.4

3.8

59

with entrainment

Calculated values

3.8

4.0

58

without entrainment

3.5. Influence of antifoam

Antifoam was automatically injected into the bioreactor during cultivation. No device for precise measurement of the injected antifoam volume was available. Therefore, the amount of antifoam could only be estimated. In the pilot-scale cultivation a different antifoam than in the 7 l cultivation was used, however, no influence on the sep- aration data was expected from shake flask controls where partition experiments were performed with separately added antifoam in different concentrations (not shown).

3.6. Genetic instability

This cultivation was the first in which the producing strain was grown in such a large volume. The total growth period from the original agar slant was an uninterrupted 7 days. At the end of the cultivation, the strain was routinely plated on potato dextrose agar to monitor the growth and especially the sporulation of the fungus. Two different phe- notypes of the spores could be observed on the plates. Plates were prepared using different dilutions of the mycelium. They show a usual dark green and an unusual light green colour. In other cultivations only one phenotype occurred. To investigate a possible influence the spores were iso- lated, germinated, grown in shake flasks and the culture broth extracted. The resulting partition coefficients for EGIcore-HFBI under the same extraction conditions as in the pilot-scale experiment were K = 37 and 26, respectively.

3.7. Protein instability

The hydrophobin directs the partitioning of the fusion protein [20,15] Hydrophobin could potentially be cleaved from the active endoglucanase core thus altering the partition results since endoglucanase activity is measured. However, the fusion protein was not degraded in the culture broth, as controlled by SDS-PAGE and Western blot.

3.8. Extraction of samples during cultivation

Samples were taken routinely during the cultivation pro- cess after inoculation of the 1500 l bioreactor. These sam- ples were extracted at 30 C with 4% detergent. Due to the higher temperature the values were different to the values in the pilot-scale experiment ([20,15]). At 13 h of growth a partition coefficient of 10 was observed. After 25 h of cultivation, the partition coefficient dropped significantly to values lower than 4 and decreased to 2.5 after 94 h. A sig- nificant lag phase was not detected as seen, e.g. by protein concentration measurements during cultivation (not shown).

3.9. Glycosylation

To investigate the glycosylation of the fusion protein and its influence on the partitioning, culture supernatant

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K. Selber et al. / Process Biochemistry 39 (2004) 889–896

Table 2 Extraction experiments using culture supernatant produced in different scale and at different temperatures

 

Cultivation temperature ( C)

c f

K

Y (%)

7

l cultivation

29

3.3

11

81

1st pilot scale

22–27

3.4

3.8

59

7

l cultivation

22–27

3.3

7.9

78

2nd pilot scale

29

2.8

8.3

81

EGI activity is measured.

from the pilot-scale fermentation was treated with the gly- cosidases PNGase F and Endo H f . Because of the high protein content in the supernatant and the small amount of glycosidases available only partial deglycosylation could be expected. Separating the treated supernatant using 4.0% detergent without adding salt resulted in a more than dou- ble partition coefficient compared to the untreated sample (K(deglycosylated) = 5.9; K(not treated) = 2.5). SDS-PAGEs of large and small scale cultivations do not show large difference in glycosylation, however, there was a large distribution in glycosylation (data not shown). An additional small influence on the partition coefficient by the deglycosylation of endogenous EGI is possible. How- ever, the amount of endogeneous EGI is relatively small and was calculated to 1/7 of total EGI [20].

3.10. Scale and temperature of the cultivation

Cultivation on a large-scale was conducted slightly dif- ferent from the cultivations which served to optimise the separation procedure, leading to differences in pH, dissolved oxygen and growth characteristics. Thus, the cultivation on a larger scale was conducted at a starting temperature of 27 C and reduced in two steps to increase protein pro- ductivity. To exclude the influence of these profiles a very similar profile was run ina7l cultivation. The tube scale extraction results of this cultivation are shown in Table 2. The measured partition coefficient of 7.9 is about twice as high as in the pilot-scale experiment. A second pilot-scale cultivation was carried out in the same experimental set up, but this time 29 C was chosen as cultivation temperature. With regard to temperature the cultivation was similar to the cultivations carried out during optimisation [19]. The resulting partition coefficient of 8.3 is again smaller than in the optimised case (K = 11), but significantly higher than the first pilot-scale cultivation (K = 3.5).

4. Discussion

4.1. Scale up

Kroner et al. [4,21] showed that scale up of extraction processes in ATPS is easy and well predictable for PEG/salt systems. This was proven by gravity settling experiments

as well as by centrifugal separation using disk stack sepa-

rators. Patents report successful operation in large-scale in

industry [3]. The largest detergent-based gravity settling experiment found in the literature was conducted on a 5 kg scale. No comparison to the tube scale experiment is reported [11]. This investigation demonstrates the ease of up scale of detergent-based systems with respect to gravity settling. The unusual high scale up factor of 1.2 × 10 5 for a single step emphasises the cheap, reliable and unproblematic scale up of the downstream process. Centrifugation has been demonstrated not to be the optimal means for separation of detergent-based systems due to the low interfacial tension and the high friction in the separator, which hinders the essential coalescence of droplets [12].

4.2. Limited mass transport

Mass transfer in detergent-based systems is very fast

resulting from a very low interfacial tension and, there- fore, generation of small droplets and large surface area

at low-energy input [17] According to Minuth et al. [11]

the interfacial tension is about 0.01–0.004 mN/m. This is

a much smaller value than, for example, the interfacial

tension of PEG/phosphate systems (0.04–0.1 mN/m [17]).

A series of separation experiments were conducted on a

tube scale to ensure complete mass transfer. Since, paral-

lel experiments on a 10 ml scale led to the same result as

the large-scale experiment incomplete mass transfer can be ruled out as a reason for the low values found.

4.3. Entrainment/insufficient separation

The entrainment observed is undesired, however, it does

not contribute significantly to lowering of the experimen-

tal values and can easily be overcome. The reason for the

entrainment in the bottom phase in the second extraction step is the fact that the required settling time could not be monitored in the bioreactor, because an observation window was lacking. In a large-scale process this could be over- come by separation of the total volume or re-separation of the smaller volume close to the interface in an appropriate vessel geometry including an observation window or using a transparent vessel. If desired, the separation time can be shortened signifi- cantly by the use of static mechanical devises like coalesces. Due to friction problems leading to redispersion dynamic devices have to be treated with caution as indicated above. It has to be mentioned that detergent-based ATPS can cause problems in phase separation if microemulsions are formed. This was demonstrated by Minuth et al. [11] and Selber [20] on a smaller scale. The reason for formation of microemulsion was ascribed to the presence of additional de- tergent or amphiphilic compounds under cultivation condi- tions. Hydrophobins like HFBI and HFBII, expressed by the recombinant host, may have influenced the large-scale exper- iment, but no phase separation problems were encountered.

K. Selber et al. / Process Biochemistry 39 (2004) 889–896

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4.4. Influence of antifoam

Antifoams are amphiphilic detergents themselves. They can interact with the micelles of the phase building de- tergent and they can interact with the amphiphilic protein EGIcore-HFBI. Little is known about the interactions with proteins, but the influence on the binodal has been described. By addition, especially of amphiphilic substances the phase diagram is altered [22,23] and the partitioning of proteins can become less efficient. Here, the amount of antifoam employed was in the range 0.2% of total volume. Even if only small additions of sub- stances can have a significant influence, the amount of an- tifoam was too small to cause the differences in extraction.

4.5. Genetic instabillity

The partitioning values of re-grown T. reesei cultures were much higher than in the initial pilot-scale experiment, cor- responding to previous shake flask cultivations. However, there was a significant difference in colour between the two phenotypes found. This could be due to different sporulation environment, but it could also be a result of the screening procedure since the strain is not monoclonal. Due to the dif- ficult biochemical and genetic work necessary to understand the molecular differences in the strain the investigation of this topic was not pursued further. Besides for partition coefficients >20 small analytical de- viations determining the concentration/activity in the heavy phase will lead to a larger error, and the difference found in K values of 37 and 26 may not be significant.

4.6. Glycosylation

Glycosylation in general can have a significant influence on the partitioning of proteins as described by Pryde and Philipps [24] for membrane proteins or by Terstappen et al. [25] for lipases. The fusion protein EGIcore-HFBI includes the linker region connecting the catalytic core with HFBI replacing the cellulose-binding domain. The linker of wild type EGI of T. reesei is highly glycosylated [26], while the hydrophobins are not glycosylated. The glycosylation of the linker is known to have a broad distribution, as glycosylation in general. Obviously, the glycosylation reduces the driving force of partitioning to the detergent-rich phase. This can be true for different reasons: the sugar chains extend into the solvent and hinder the enclosure of the protein by a detergent mi- celle. Glycosylation can hinder the approach of micelles to a protein or affect the agglomeration behaviour of proteins. This can additionally lead to a change of the optimal param- eter for separation. The other possibility is that glycosylation might hide the hydrophobic parts of the enzyme which play an important role in the driving force for separation [25]. A change in activity of highly glycosylated enzymes is not expected as

the glycans do not influence enzyme–substrate interaction

[27].

The glycosylation distribution can in principal be influ- enced and narrowed by the cultivation procedure. Therefore, the negative influence of excess glycosylation on the parti- tioning can be overcome by an well-adapted and controlled cultivation process. Another possibility would be to delete the most glycosylated region of EG1-the linker-completely or partially. Theoretically, a procaryotic host could be used for production to cancel out any glycosylation. Unfortu- nately, bacterial systems do not match Trichoderma in their secretion capacity. The change in partitioning can also not be related to a change in growth rate. The growth rate has an influence e.g. on the glycosylation. The growth in pilot-scale was very steady and no defined lag phase could be observed. A long time of unlimited growth, starting from the pre-cultures and taking up to 11 days until the end of the pilot-scale cultivation, could cause the difference in parti- tioning, due to a change of the characteristics of the protein, e.g. by a change in glycosylation.

4.7. Scale and temperature of the cultivation

From these experiments it is obvious that the size and temperature of the cultivation must have a significant influ- ence, even if it might not be the only one. The temperature has a strong influence as could be demon- strated in the second pilot-scale experiment which showed significantly higher values for the partition coefficient and the yield in comparison to the first pilot-scale experiment at lower temperature. Since, the partition coefficient of the first 7 l cultivation at 25 C was higher than in the corresponding and second pilot-scale cultivation (the yields were similar due to the dif- ferent concentration factor) the temperature cannot be the only effect. This is emphasised by the equivalent investiga- tion at low temperatures. The second reason must be linked to the scale of the cultivation. If the size of the cultivation had a strong effect, it would indicate that most likely either the total growth time or the transport mechanism of metabolic substances, e.g. the oxygen transfer towards the cells causes the change in separation behaviour of the fusion protein. This can additionally be emphasized through very high values of partition coefficients and yields in experiments form shake flask cultivations.

5. Conclusion

The technical feasibility of large-scale extractions in detergent-based ATPS was proven, however, care must be taken in the scale up of the cultivation process that prop- erties of the target protein are not altered. This appears especially important for glycosylated protein.

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K. Selber et al. / Process Biochemistry 39 (2004) 889–896

Acknowledgements

We would like to thank our colleagues from VTT, Finland who supported the experimental work. We also like to thank Hans Christian Raths, Horst Wollenweber and Jean-Pierre Moiltor Henkel/Cognis for the generous supply of Agrimul NRE 1205. The work has been supported by a grant from the EU-Project BI04-CT96-0435.

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