METHOD FOR OBTAINING MATERIAL FROM PLANT CELL SURFACES

Description

Field of the invention

[0001] The present invention relates to the isolation of proteins from cells. Background of the invention

[0002] Methods for isolating proteins from cells or cell aggregates include osmotic lysis, enzymatic or chemical lysis, sonication, and mechanical disruption. Mechanical disruption typically involves breaking up cells using a homogenizer or mixer.

[0003] Short treatment times can also be used to divide cell clusters and to suspend cells, resulting in only partial lysis of the entire cell population (Orellana-Escobedo et al., Plant Cell Rep. 2015, 34 (3) : 425-33) .

[0004] If a protein is isolated from individual organelles or cell compartments, the cell organelles are usually isolated before digestion and then digestion is carried out from the isolate.

[0005] Witzel et al., Plant Methods 2011, 7:48; Leary et al., J Vis Exp. 2014; (94): 52113; and Córdoba-Pedregosa et al., Plant Physiology 1996, 112(3):1119-1125, describe methods for extracting proteins from the apoplast of plant cells. The apoplast is the space outside the protoplasts. It consists of the cell walls and the intercellular space. The methods described in these publications include osmotic extraction with various infiltration solutions (e.g., salts) and centrifugation. However, this method has the disadvantage that only the materials accessible by infiltration can be extracted.

[0006] US 2015 / 0140644 A1 and AU 2017202473 B2 describe methods for obtaining proteins from the apoplast, including cell wall lysis, incubation, and extraction. Enzymatic or chemical modification of the proteins is possible.

[0007] It is an object of the invention to provide improved isolation or extraction possibilities of materials of the apoplast, in particular of material secreted in the apoplast. Summary of the invention

[0008] The present invention relates to a method for detaching expressed material from the surface or from the apoplast of plant cells, wherein the plant cells are treated in a liquid medium with a rotor-stator, wherein the specific heat of the rotor-stator introduced by rotation of the rotor is a maximum of 3 kJ per kg of the liquid medium and per g / l of dry mass of the plant cells and the specific heat output introduced into the medium is a maximum of 1.5 kJ per kg of the liquid medium per minute and per g / l of dry mass of the plant cells.

[0009] Likewise, in a further aspect, the invention relates to a method for detaching expressed material from the surface or the apoplast of one or more plant cells, wherein the one or more plant cells are treated in a liquid medium with a rotor-stator, wherein the heat of the rotor-stator introduced by rotation of the rotor is a maximum of 30 kJ per kg of the liquid medium and the heat output introduced into the medium is a maximum of 1.5 kJ per kg of the liquid medium and per minute.

[0010] The parameters of both aspects can be combined, especially since they are merely different reference values, while still fulfilling the essence of the invention. All detailed descriptions of the invention and preferred embodiments described herein relate to all aspects of the invention. Detailed description of the invention

[0011] The invention relates to a gentle rotor-stator treatment of plants or plant cells, which, in contrast to the otherwise conventional homogenization process, detaches material from the apoplast of the cells or plants. The protoplasts should remain largely intact to avoid contamination of the expressed material to be isolated by cell components from the interior of the protoplasts. Therefore, the intensity and duration of the treatment with the rotor-stator are limited according to the invention. It has been found that a rotor-stator treatment with low energy inputs—determined as specific heat or heat output—enables satisfactory detachment of the desired expressed material.Excellent results were achieved with a specific heat of the rotor-stator introduced by the rotation of the rotor of a maximum of 3 kJ per kg of the liquid medium and per g / l of dry mass of the plant cells, as well as with a specific heat output introduced into the medium by the rotation of the rotor of a maximum of 1.5 kJ per kg of the liquid medium per minute and per g / l of dry mass of the plant cells. Equivalent to the inventive concept, equally good results were obtained with a heat of the rotor-stator introduced by the rotation of the rotor of a maximum of 30 kJ per kg of the liquid medium and with a heat output introduced into the medium of a maximum of 1.5 kJ per kg of the liquid medium per minute. According to the invention, the plants or plant cells are not homogenized, but only treated to the extent that the expressed material is detached from the surface or from the apoplast.

[0012] Using the method according to the invention, expressed material is obtained from the surface of the cells or the apoplast (the entire cell wall and the intercellular space). Such material usually reaches these sites via a secretory pathway and is usually also found in the culture medium of the plant cells. However, in the course of the invention, it was discovered that large amounts of the secreted material adhere to the surface, in particular to the cell wall or the apoplast. This adhered material is obtained according to the invention, and production increases could be achieved using the method according to the invention. A 10-fold increase in yield compared to methods without rotor-stator treatment, i.e., isolation of the secreted expressed material, was observed.

[0013] The treatment intensity, duration, and capacity of the rotor-stator are selected within the maximum parameters according to the invention to ensure sufficient recovery of the expressed material (desired product). However, the treatment intensity, duration, and capacity, which represents the applied energy, quantified as heat or specific heat, are limited because product contamination occurs at higher energy inputs.

[0014] Preferably, the specific heat of the rotor-stator introduced by rotation of the rotor is a maximum of 3 kJ per kg of liquid medium and per g / l of dry mass of the plant cells (abbreviated as 3 kJ / kg / (g / l) or 3 kJ / kg / g / l). Particularly preferably, the specific heat can be kept lower in order to further reduce any residual contamination. Thus, the specific heat introduced by the rotor-stator is preferably a maximum of 2.75 kJ / kg / (g / l), or more preferably a maximum of 2.5 kJ / kg / (g / l), a maximum of 2.25 kJ / kg / (g / l), a maximum of 2 kJ / kg / (g / l), a maximum of 1.75 kJ / kg / (g / l), a maximum of 1.5 kJ / kg / (g / l), a maximum of 1.25 kJ / kg / (g / l), or a maximum of 1 kJ / kg / (g / l).

[0015] Independently of any reference to the plant quantity, the optional parameter of introduced heat was also determined according to the invention. This is relevant in some embodiments because the rotor-stator provides energy input into the cell medium independent of the plant cells, which can manifest itself as heating. Preferably, the heat introduced by the rotor-stator through rotation of the rotor is a maximum of 30 kJ per kg of liquid medium (abbreviated to kJ / kg), preferably a maximum of 25 kJ / kg, a maximum of 20 kJ / kg, a maximum of 15 kJ / kg, or a maximum of 10 kJ / kg.

[0016] This introduced specific heat or introduced heat can be adjusted, for example, by limited treatment times and / or the intensity of the treatment (as can the parameters of the specific heat output or the heat output): In the process according to the invention, the rotor-stator is operated at a low intensity, e.g. at a low speed. The heat introduced at a specific intensity (or heat output) of a specific process with selected parameters can be measured in a comparative test, e.g. by increasing the temperature of water or another medium with a known heat capacity. When determining the process heat, other temperature-influencing effects, in particular temperature losses, should be excluded or taken into account mathematically in order to arrive at the heat or heat output of the rotor-stator itself; the heat or heat output is preferably determined in a Dewar vessel.

[0017] Regardless of the device, the specific heat input or the heat output applied was recognized as relevant ("specific" refers, as above, to the optional reference to the amount of plant material). Preferably, the specific heat input into the medium is a maximum of 1.5 kJ per kg of liquid medium per minute and per g / l of dry mass of plant cells (abbreviated kJ / kg / min / (g / l) or kJ / kg / min / g / l). Particularly preferably, this specific heat output is a maximum of 1.25 kJ / kg / min / (g / l), a maximum of 1 kJ / kg / min / (g / l), a maximum of 0.8 kJ / kg / min / (g / l), a maximum of 0.6 kJ / kg / min / (g / l), a maximum of 0.5 kJ / kg / min / (g / l), a maximum of 0.4 kJ / kg / min / (g / l), a maximum of 0.3 kJ / kg / min / (g / l), a maximum of 0.2 kJ / kg / min / (g / l), a maximum of 0.15 kJ / kg / min / (g / l), a maximum of 0.125 kJ / kg / min / (g / l), or a maximum of 0.1 kJ / kg / min / (g / l). Analogously, the heat output introduced into the medium is preferably a maximum of 1.5 kJ per kg of the liquid medium and per minute (abbreviated as kJ / kg / min).Preferably, this heat output is a maximum of 1.25 kJ / kg / min, a maximum of 1 kJ / kg / min, a maximum of 0.8 kJ / kg / min, a maximum of 0.6 kJ / kg / min, a maximum of 0.5 kJ / kg / min, or a maximum of 0.4 kJ / kg / min.

[0018] The more intensive or prolonged the treatment, the greater the amount of material recovered. Preferably, the heat introduced by rotation of the rotor (of the rotor-stator) is at least 1 kJ per kg of liquid medium, particularly preferably at least 2 kJ / kg, and / or the specific heat of the rotor-stator is at least 0.1 kJ per kg of liquid medium and per g / l of dry mass of the plant cells, particularly preferably at least 0.2 kJ / kg / (g / l).

[0019] Preferably, the heat output introduced into the medium by rotation of the rotor is at least 0.2 kJ per kg of the liquid medium and per minute, preferably at least 0.4 kJ / kg / min, and / or the specific heat output into the medium is at least 0.02 kJ per kg of the liquid medium per minute and per g / l of dry mass of the plant cells, preferably at least 0.04 kJ / kg / min / (g / l).

[0020] The expressed material preferably contains proteins. Proteins, especially recombinantly expressed proteins, can be specifically directed to the secretory pathway using appropriate signal sequences and thus directed to accumulation at the surface or the apoplast. Preferably, the expressed material is in the apoplast of the plant cells, from which it can be obtained using the method according to the invention. Likewise, the expressed material is secreted material, preferably proteins secreted through the cell membrane or cell wall.

[0021] According to the invention, a rotor-stator is used to process the plant cells in the liquid medium in order to obtain the expressed material.

[0022] A rotor-stator comprises at least one rotor, which exerts shear forces on the plant cells through its rotational movement. These shear forces structurally loosen, mechanically influence, or abrade the surface of the plant cells, i.e., the apoplast and the cell walls, or partially erode them.

[0023] The rotor rotates in relation to a stator. The stator can be a housing shell or a counterpart to the rotor. The rotor can have cutting or shearing elements, with edges or shearing surfaces. A common design has a comb structure, with a plurality of shearing projections (e.g., teeth or prongs), usually arranged parallel to the rotation axis, exerting the shearing or cutting action. A "plurality" here can be, for example, 2, 3, 4, 5, 6, 7, 8, or more shearing projections.

[0024] The stator can be arranged as a counterpart to the rotor and, in particular, its cutting or shearing elements. Optionally, the stator can have its own cutting or shearing elements, similar to the rotor, e.g., also in a comb structure. Such designs are known in rod homogenizers, as described in DE 10 2005 031 459 A1.

[0025] In other embodiments, the stator may be a housing structure, such as is common for flow-through homogenizers. An example of a flow-through rotor-stator is described in WO 2009 / 062610 A1.

[0026] Examples of rotor-stators include a rod homogenizer or a shear pump. Shear pumps are particularly used in flow systems.

[0027] Preferably, there is a gap between the rotor and stator, e.g., at least the size of a plant cell or more, so that plant cells, at least in the form of protoplasts, can pass between the rotor and stator. Suitable gap sizes are 50 µm, 70 µm, 80 µm, 100 µm, 150 µm or more, as well as any range between these distances. Preferably up to a maximum of 500 µm, or up to 300 µm, or up to 200 µm.

[0028] As noted, the intensity is kept low to protect the plant cells in the form of the protoplast and to avoid or reduce contamination of the secreted expressed material to be harvested by the cell interior. In conventional rotor-stator models, the speed is reduced for this purpose, e.g., in embodiments where the rotor is operated at a maximum speed of 15,000 revolutions per minute, preferably 1,000 to 15,000 revolutions per minute. Possible speeds are 3,000 to 14,000, 4,000 to 13,000, 5,000 to 12,000, or 6,000 to 11,000 revolutions per minute.

[0029] The plant cells can be contained in a container into which the rotor-stator is inserted. For this purpose, the rotor-stator can be placed in a container containing the medium. This "batch" design (for a non-continuous process) is particularly used in rod homogenizers. For larger scales, flow-through rotor-stators are preferably used. According to this embodiment, the rotor-stator can have an interior space with at least one inlet and one outlet, through which the liquid medium is continuously transported through the interior space. An example of this is the shear pump for a continuous process.

[0030] The stator preferably defines a volume of 10 cm 3 (0.01 l) to 1 m 3 (1000 l). Preferred volumes are 0.1 l to 800 l, or 0.5 l to 600 l, or 1 l to 400 l, or 2 l to 200 l. Volumes up to 100 l are preferred, particularly preferably 0.65 l to 50 l, e.g., 1 l to 40 l. These volumes are particularly well-suited for the treatment of plant cell culture media.

[0031] Preferably, the amount of treated liquid medium is up to 50,000 kg, preferably 0.5 g to 50,000 kg, e.g., 1 g to 25,000, 2 g to 10,000 kg, 5 g to 5,000 kg, 10 g to 2,500 kg, 20 g to 1,000 kg, 30 g to 500 kg, 50 g to 250 kg, 100 g to 100 kg, 200 g to 50 kg, 500 g to 250 kg, 1 kg to 100 kg, 2 kg to 50 kg, or 4 kg to 20 kg. Such amounts are preferably used in the non-continuous process per pass.

[0032] The plant cells are preferably present in the liquid medium at a concentration of 0.2 g / l to 60 g / l (mass of plant cells as dry mass). Preferred plant cell concentrations (always dry mass) are 0.5 g / l to 50 g / l, 1 g / l to 40 g / l, 2 g / l to 30 g / l, 4 g / l to 20 g / l, and particularly preferably approximately 10 g / l, e.g., 5 g / l to 15 g / l. These plant cell concentrations can be processed particularly efficiently with the rotor-stator.

[0033] The plant cells are preferably treated with the rotor-stator for 2 minutes to 150 minutes. In a continuous process, these times refer to the average treatment time of the plant cells. Preferred times are, in particular, 3 minutes to 120 minutes, 5 minutes to 100 minutes, 8 minutes to 80 minutes, 10 minutes to 60 minutes, or particularly preferably 12 minutes to 40 minutes. For large culture volumes, longer rotor-stator treatments can also be carried out. Other possible times are preferably 1 hour to 24 hours, preferably 2 hours to 20 hours, 3 hours to 16 hours, or 4 hours to 12 hours. Thus, all preferred treatment times relate to the range 3 minutes to 24 hours, and any range between the stated treatment times or even longer.

[0034] Preferably, the plant cells can be cultivated in a suspension culture. This suspension can be processed directly as a liquid medium in the method according to the invention. Alternatively, moss can also be first isolated, e.g., from a solid culture, liquid culture, or suspension culture, and then suspended in an aqueous medium with suitable conditions for the use of the rotor-stator treatment. Plants that are particularly suitable for the method according to the invention are non-woody plants. Preferably, plants are algae and mosses, in particular bryophytes. Preferably, the bryophyte plant or cell is a moss, preferably P. patens. The bryophyte can be any bryophyte, but is preferably selected from moss, liverworts or hornworts, particularly preferably from the class Bryopsida or the genera Physcomitrella, Funaria, Sphagnum, Ceratodon, Marchantia and Sphaerocarpos. Especially preferred is Physcomitrella patens.Most preferably, the method according to the invention is carried out using cells from plant tissue such as protonema of the moss Physcomitrella patens Preferred algae are selected from the green algae, e.g. from the order Chlorellales, preferably the family Chlorellaceae, more preferably the genus Auxenochlorella or Chlorella, in particular Chlorella vulgaris, and the order of Volvocales, preferably the family Haematococcaceae, more preferably the genus Haematococcus, in particular Haematococcus pluvialis and the order Eustigmatales, preferably the families Loboceae, Chlorobothryaceae, Pseudocharaciopsidaceae, and Eustigmataceae. Other preferred plants are tobacco, beans, or lentils. The plant is preferably an aquatic plant, e.g., of the genera Lemna, Spirodela, Landoltia, Wolffia or Wolffiella.

[0035] The invention relates to plant cells. "Plant cell," as used herein, can refer to an isolated cell, a singularized cell, but also to a cell in or from a plant tissue, preferably a tissue selected from callus, protonema, phloem, xylem, mesophyll, stem, leaves, thallus, chloronema, rhizoid, or gametophore, or a cell in a plant organism.

[0036] In the process according to the invention, the medium preferably has a physiological pH, in particular to protect the protoplasts (cell components within the cell wall, especially from the cell membrane onwards), as already described, and to avoid contamination of the expressed material in the apoplast / on the surface of the cells. The pH of the liquid medium is preferably between 3.5 and 8.5, particularly preferably between 4 and 8, or between 4.5 and 7, or between 5 and 6.5, especially between 5.5 and 6, or combinations of these ranges, such as a pH of 5 to 8.

[0037] Also to protect the protoplasts, the osmolarity of the liquid medium is preferably physiological, in particular to avoid swelling stress, e.g. if the osmolarity is too low. If necessary, an upper limit of the osmolarity can also be provided in order to avoid osmotic shrinkage stress. The medium preferably has an osmolarity of at least 0.1 osmol / L, or preferably at least 0.150 osmol / L. The osmolarity can be adjusted by dissolved substances, such as salts or other medium components, such as sugars or sugar alcohols. An alkali metal salt such as Na +< and / or K +< is preferably provided. The anion is preferably a halide, such as Cl -< or F -< or I -< , a phosphate or an acetate. Buffer components such as Tris (tris(hydroxymethyl)aminomethane) are also possible.

[0038] Furthermore, to further protect the protoplasts, surface-active polymers can be added to the liquid medium for rotor-stator treatment. Surface-active polymers are described, for example, in WO 2013 / 156504 A1 and preferably comprise uncharged polymers, such as emulsifiers, e.g., polyalkyl glycol, especially polyethylene glycol. The polymer is, in particular, a non-ionic, water-soluble surface-active polymer. Preferably, it is not denaturing proteins. Examples are polymers or copolymers selected from polyethers such as polyalkyl glycols, polysorbates or polyvinylpyrrolidone, polyvinyl alcohol, water-soluble cellulose derivatives such as hydroxypropyl cellulose, hydroxypropylmethylcellulose, carboxymethylcellulose or hydroxyethylcellulose, vinylpyrrolidone-vinyl acetate copolymer (copovidone), polyvinyl acetate, partially hydrolyzed polyvinyl alcohol, polyvinyl alcohol-polyethylene glycol copolymers, and mixtures thereof. Other possibilities are polysorbate, e.g.,Polyoxyethylen-sorbitan-monolaurat, Polyoxyethylen-sorbitan-monooleat, Polyoxyethylen-sorbitan-monopalmitat,.

[0039] Polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan tristearate, preferably polysorbate 80 (polyoxyethylene(20) sorbitan monooleate, Tween®< 80), polyoxyethylene(40) stearate. The surface-active polymer is preferably present in the medium at a concentration of at least 0.05% by weight, especially preferably at least 0.08%, at least 0.1%, or at least 1.5% (all percentages in wt.%). The molecular weight of the surface-active polymer, e.g., PEG and the like, is preferably at least 500 Da, particularly preferably at least 1,000 Da, at least 1,500 Da, at least 2,000 Da, at least 3,000 Da, at least 4,000 Da, at least 6,000 Da, at least 8,000 Da, at least 10,000 Da, at least 20,000 Da, or at least 30,000 Da. The molecular weight is especially preferably between 500 Da and 2,000,000 Da, preferably between 1,000 Da and 200,000 Da, or between 1,200 Da and 80,000 Da.

[0040] The liquid medium is preferably aqueous, especially water or cell-compatible water mixtures. In particular, it can be a culture medium for (and containing) plant cells, provided the plants are not previously separated from it.

[0041] Preferably, the expressed material is expressed prior to treatment of the plant cells with the rotor-stator, so that it accumulates on the surface or in the apoplast of the plant cells. The plant cells can be cultured and / or grown, e.g., in a medium under plant growth conditions (nutrient media, light), as is generally known (see, e.g., Frank et al., Plant Biol 7, (2005):220-227). Expression or cultivation is preferably carried out for 13 minutes to 1 month (30 days) or longer, such as 2 months (60 days), e.g., 1 hour to 22 days, or 5 hours to 15 days, e.g., 10 hours to 7 days, or 20 hours to 3 days. In a continuous cell culture with regular cell removal for product recovery (expressed material), these time ranges or minimum times can correspond to an average time of a cell in the culture.

[0042] Further processes that damage, lyse, or homogenize the protoplast should be avoided. The cell wall is preferably not lysed, in particular not enzymatically and / or chemically and / or osmolytically and / or ultrasonically. The cell wall should preferably remain untouched or intact—apart from the rotor-stator treatment according to the invention. In particular, the cell membrane (protoplast) should remain intact. Cell vitality plays no particular role in the process according to the invention. However, contamination of the liquid medium by components of the cell interior, particularly the cytoplasm, should be avoided.

[0043] The present invention is further described by the following figures and examples, without being limited to these embodiments of the invention. Figures:

[0044] Fig. 1 :Determination of energy input into water using an Ultraturrax T25 homogenizer (IKA / Staufen). (A) Temperature profile in 1.5 l of water at a rotational speed of 10,000 rpm. (B) Calculated energy input [kJ]. (C) Calculated energy input [kJ / kg]. Fig. 2 : Determination of energy input into water using a homogenizer shear pump FSP712VC-2.2kW-FU (Fristram / Hamburg). (A) Temperature profile in 50 l of water at a speed of 2800 rpm. (B) Calculated energy input [kJ]. (C) Calculated energy input [kJ / kg]. Fig. 3 : Percent release of biomass-bound product (moss-aGal) during treatment with a shear pump (dashed line) and an Ultraturrax T25 rod (solid line). Shear pump treatment of the reactor culture was carried out in a volume of 50 l. Treatment of the reactor culture with a Turrax T25 rod was carried out in a volume of 0.65 l. Fig. 4 :Specific energy input by Ultraturrax T25 (A) and shear pump (B), both for 10 g / l dry biomass. Specific energy input according to the comparative example Ultraturrax T25 at 19,000 rpm with 1 g / l dry biomass (C). Fig. 5 : Percent release of biomass-bound product (moss-aGal) during treatment with a shear pump (dashed line) and an Ultraturrax T25 rod (solid line). Shear pump treatment of the reactor culture was performed in a volume of 50 l. Turrax T25 treatment of the reactor culture was performed in a volume of 0.65 l. Biomasses: 9.2 g / L (shear pump), 8.2 g / L (T25) Fig. 6 :Western blot analysis of product release (moss-aGal) compared to the release of intracellular marker proteins (Rubisco, large subunit). Intracellular proteins are detectable in minimal amounts in saline media (e.g., 20 mM Tris, 100 mM NaCl, pH 7) at energy inputs of up to 32.9 kJ / kg. Under osmotic stress conditions (demineralized water), they are detectable from approximately 10 kJ / kg. Target protein amounts (moss-aGal) increase in saline media and under osmotic stress conditions in accordance with the energy input. Fig. 7 : Microscopic analysis of the T25 process in demineralized water. Moss cells retain their integrity up to an energy input of 16.5 kJ / kg. At an energy input of 32.9 kJ / kg, cell integrity is maintained, but more particles are evident, indicating the onset of cell disruption. Fig. 8 :Microscopic analysis of the T25 process in saline buffer (20 mM Tris, 100 mM NaCl, pH 7). Mossy cells retain their integrity up to an energy input of 16.5 kJ / kg. At an energy input of 32.9 kJ / kg, cell integrity is maintained, but more particles are evident, indicating incipient cell disruption. Fig. 9 : Microscopic analysis of the shear pumping process in saline medium (reactor culture). Moss cells retain their integrity up to an energy input of 18.41 kJ / kg. Above an energy input of 27.20 kJ / kg, the integrity of the visible cells is maintained, but there are clearly more particles present, indicating the onset of cell disruption. Fig. 10 :Microscopic analysis of the shear pumping process in demineralized water. Up to an energy input of 9.62 kJ / kg, the moss cells retain their integrity. Starting at an energy input of 18.41 kJ / kg, the cells retain their integrity, but there are clearly more particles present, indicating the onset of cell disruption. Fig. 11 : Microscopic analysis of moss cells in an ultrasonic process as comparative images of a cell disruption. The cells lose their integrity after just a short time (1 minute of ultrasonic treatment). After just 3 minutes, only cell fragments and empty cell membranes are visible. (100% performance in a 50 ml batch). Fig. 12 : Comparative example: Determination of the energy input into water using a T25 homogenization tool (IKA / Staufen) at high energy and a speed of 19,000 rpm. (A) Temperature profile in 1 l of water. (B) Calculated energy input [kJ]. (C) Calculated energy input [kJ / kg]. Fig. 13 : Comparative example: Significantly faster product release at 19,000 rpm, especially up to an energy input of 7 kJ / kg. Starting at an energy input of 84 kJ / kg, product losses due to high temperature and shear stress occur. Examples: Example 1: Determination of energy input using a Turrax rod and shear pump

[0045] Temperature was used as a measurable measure of energy input into aqueous media. Using a Turraxstab T25-S25N-18G (IKA Staufen), 1.5 l of H2O were dispersed in a Dewar flask for 30 min at 10,000 rpm, and the temperature profile was measured. The room temperature during the experiment was between 20.5 and 20.7 °C. Energy input data were calculated using the specific heat capacity (4190 J kg -1< K -1< ) of H2O. Energy input data were compared with the literature (Orellana-Escobedo et al., Plant Cell Rep. 2015, 34(3), 425-433) and were carried out analogously at 19,000 rpm.

[0046] A shear pump (Shearpump FSP 712, Fristam Hamburg) pumped 50 l of H2O at 2800 rpm from a Nalgene reservoir vessel through fabric-stabilized PVC tubing. Temperature measurement in the Nalgene reservoir vessel was performed using a temperature sensor (precision thermometer, G002.1, Carl Roth). The experimental setup was carried out in a room maintained at 19 °C. Temperature losses to the environment were neglected in this experimental approach. Energy input data were calculated using the specific heat capacity (4190 J kg -1< K -1< ) of water. Example 2: Production of a moss culture

[0047] Moss production strains were cultured axenically for 3–4 weeks in 200 l single-use bioreactor bags (Cellbag 200, GE Healthcare) on Wave™< rocking motion bioreactors (Wave200, GE Healthcare). The cultivation parameters were shaking frequencies of 19 to 25 rpm, shaking angles of 9°, temperatures of 24–26 °C, a gassing rate of 2 L / min, and a 2% CO2 enrichment of the supply air. Four LED modules (serial numbers 120268 to 120282, Infors AG) with warm white LEDs installed above the bioreactor bag were used for illumination. Moss cultivation was carried out under 24 h of continuous illumination. SM07 (100 mM NaCl, 6.6 mM KCL, 2.0 mM MgSO 4 x 7H 2 O, 1.8 mM KH 2 PO 4 , 20.4 mM Ca(NO 3 ) 2 x 4H 2 O, 0.05 mM Fe Na-EDTA, 4.9 mM MES, 0.1% (w / v) PEG4000, 100.26 µM H 3 BO 3 , 0.11 µM CoCl 2 x 6H 2 O, 0.1 µM CuSO 4 x 5H 2 O, 5 µM KI, 85.39 µM MnCl 2 x 4H 2 O, 1.03 µM Na 2 MoO 4 x 2H 2 O, 0.11 mM NiCl 2 x 6H 2 O, 0.04 Na 2 SeO 3 x 5H 2 O, 0.039 Zn-acetate x 2H 2 O) supplemented with 1000x Nitsch vitamins (Nitsch vitamin mixture, Duchefa, corresponding. Manufacturer's information) served as Mineral salt medium. The pH value of 5–6 was controlled via WAVEPOD I and Pump20 (GE Healthcare) with automated addition of 0.25 M MH 2 SO 4 and 0.25 M NaOH. Expression of recombinant α-galactosidase (aGal or α-Gal A) was performed as described in WO 2016 / 146760 A1 ("moss-aGal"). Example 3: Release of moss-bound product and analytical methods

[0048] To analyze the temporal course of the release of moss-bound product, the culture obtained from Example 2 was exposed to different energy inputs using a Turrax rod T25-S25N-18G and a shear pump FSP 712. c PL product concentrations (c PL ) for released product were determined using a moss-aGal ELISA (Biogenes / Germany). The degree of cell disruption was detected by microscopic image analysis of the moss cells (microscope: Axiovert 200 oper Stemi SV11 with AxioCam camera, AxioSoft software, and KL 1500 LCD cold light source (Carl Zeiss). A comparison to microscopic images of a total disruption was possible by microscopic analysis of a 50 ml ultrasonic disruption (probe: UW2070, Bandelin; amplifier: HD 2070, Bandelin) at 100% power for 20 min. At the molecular level, Western blotting was used to qualitatively analyze released product and the intracellular marker protein Rubisco.Anti-aGal (H00002717-D01P, abnova) and anti-Rubisco (AS03037, Agrisera) were used as primary antibodies, and anti-Rabbit HRP (abcam, AS03037) was used as secondary antibodies.

[0049] To analyze the ratio between released (cPL) and releasable moss-bound product (cPX), the untreated culture was subjected to cell disruption using a ball mill (steel balls: RB-3 / G20W, Schleer; ball mill: MM300, Retsch). After separation of cell debris, the product concentration was determined using an ELISA (Biogenes). This assay measured αGal (α-galactosidase, also known as "moss-αGal"), a protein expressed in the apoplastic space. Example 4: Results and discussion

[0050] The energy inputs (as heat in kJ / kg, kg related to the liquid medium) of two different homogenizers - a Turrax rod T25-S25N-18G and with Shearpump FSP 712 - into a culture medium (kg) were determined by temperature measurements ( Fig. 1-5). The homogenizers were set to a low rotation speed so that a low heat output (in kJ / kg / min) was achieved. The total heat over a certain period of time (0-60 min) could thus be determined. The energy input into water was calculated using the specific heat coefficient of H 2 O [4.182 KJ / kg*K] based on the measured temperature. The heat ( Fig. 1-3 ) or the specific heat related to the dry mass of the plant parts ( Fig. 4-5 ) calculated.

[0051] Fig. 3 and 5 show the product release of the desired protein (recombinant aGal from moss, "moss-aGal"), which accumulates on the surface or in the apoplast. The release also increases with increasing treatment.

[0052] In comparative tests, the Turrax rod was operated at high heat output, namely at 19000 rpm ( Fig. 4C , 12-13). The heat output was approximately a factor of 100 greater than with the gentle treatment at 10,000 rpm (cf. Fig. 4 A and 4 C). Operation at high heat output leads to rapid product release but also to the destruction of the cells (protoplasts), so that extracellular products are contaminated by components of the cell interior. These effects occur after just 1 minute of treatment.

[0053] Figure 6Shows the quality of the product release of the extracellular protein (moss aGal) and the intracellular protein Rubisco. Rubisco occurs in high concentrations in plant cells and was therefore used as a highly sensitive marker for the release of cell contents. In the experiments in physiological saline solution, an increasing release of Rubisco was observed at higher energy inputs (heat) of over 32.9 kJ / kg. In a comparative experiment with demineralized water (DI water), Rubisco contamination occurred earlier, at approximately 10 kJ / kg, due to osmolytic effects in addition to the shear stress from the homogenizer. The different heat inputs (energy inputs) were controlled by the treatment time.

[0054] The microscopic analysis of the cell clusters after treatment reflects these results. Fig. 7 shows the results after treatment with the Ultraturrax rotor-stator in demineralized water; Fig. 8after treatment with the Ultraturrax rotor-stator under physiological conditions (20 mM Tris, 100 mM NaCl, pH=7). Fig. 9 shows the cell aggregates after treatment with the shear pump under physiological conditions. With increasing treatment time (heat), the number of particles, presumably formed by destroyed cells, increases. From about 30 kJ / kg, increased cell disruption is observed. For comparison, Fig. 10 The tests in demineralized water using the shear pump. Here, comparable particles already occur at approximately 20 kJ / kg.

[0055] In comparison to the Fig. 7-10 shows Fig. 11 Cell disruption using ultrasound. After just 1 minute, the cells lose their integrity.

[0056] This means that both the energy input per unit of time (intensity of rotation of the rotor-stator; heat output) and the absolute energy input (heat) should be limited – here in the experiment controlled by the treatment duration. These parameters can be used as such or in relation to the biomass (dry biomass, TBM). Useful parameters for a gentle process that removes as many of the absorbed or apoplast-bound products as possible while keeping the protoplasts largely intact are a maximum of 3 kJ / kg per g / l of dry mass and a maximum of 1.5 kJ / kg / min per g / l of dry mass, or a maximum of 30 kJ / kg and a maximum of 1.5 kJ / kg / min. Possible treatment times with such low heat outputs are 2 minutes to 150 minutes – depending on the intensity of the rotation, generally longer than the short but intensive treatments commonly used.

Claims

1. A method for detaching expressed material from the surface of or from the apoplast of plant cells, wherein the plant cells are treated with a rotor-stator in a liquid medium, wherein the heat introduced from the rotor-stator by rotation of the rotor is a maximum of 3 kJ per kg of the liquid medium and per g / L dry weight of the plant cells and the heat introduced into the medium is a maximum of 1.5 kJ per kg of the liquid medium per minute and per g / L dry weight of the plant cells.

2. The method as claimed in claim 1, characterized in that the expressed material is in the apoplast of the plant cells.

3. The method as claimed in claim 1 or claim 2, characterized in that the heat from the rotor-stator introduced by rotation of the rotor is at least 1 kJ per kg of the liquid medium, and / or the heat from the rotor-stator is at least 0.1 kJ per kg of the liquid medium and per g / L of dry weight of the plant cells.

4. The method as claimed in one of claims 1 to 3, characterized in that the heat introduced into the medium by rotation of the rotor is at least 0.2 kJ per kg of the liquid medium and per minute, and / or the heat into the medium is at least 0.02 kJ per kg of the liquid medium per minute and per g / L of dry weight of the plant cells.

5. The method as claimed in one of claims 1 to 4, characterized in that the expressed material contains proteins, and / or in that the expressed material is secreted material, preferably proteins secreted through the cell membrane.

6. The method as claimed in one of claims 1 to 5, characterized in that the rotor-stator is introduced into a container with the medium.

7. The method as claimed in one of claims 1 to 6, characterized in that the rotor-stator has an interior which has at least one inlet and outlet via which the liquid medium is continuously fed through the interior.

8. The method as claimed in one of claims 1 to 7, characterized in that the stator delimits a volume of 10 cm3 to 1 m3, and / or in that the quantity of the treated liquid medium is up to 50 kg, preferably 0.5 g to 50 kg.

9. The method as claimed in one of claims 1 to 8, characterized in that the plant cells are in a concentration of 0.2 g / L to 60 g / L in the liquid medium (mass of plant cells as dry weight).

10. The method as claimed in one of claims 1 to 9, characterized in that the plant cells are moss cells, preferably P. patens cells.

11. The method as claimed in one of claims 1 to 10, characterized in that the rotor is operated at a maximum rotational speed of 15,000 revolutions per minute, preferably 1,000 to 15,000 revolutions per minute.

12. The method as claimed in one of claims 1 to 11, characterized in that the rotor-stator is a rod homogenizer or a shear pump, and / or wherein the stator has a comb structure.

13. The method as claimed in one of claims 1 to 12, characterized in that the heat from the rotor-stator introduced by rotation of the rotor is a maximum of 30 kJ per kg of the liquid medium and the heat introduced into the medium is a maximum of 1.5 kJ per kg of the liquid medium and per minute.

14. The method as claimed in one of claims 1 to 13, characterized in that the medium has a pH in the range 5 to 8 and / or an osmolarity of at least 0.1 osmol / L.

15. The method as claimed in one of claims 1 to 14, characterized in that the plant cells are treated with the rotor-stator for 2 min to 150 min.