Method for obtaining materials from plant cell surfaces

Abstract

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 rotor-stator in a liquid medium, and the rotor-stator-derived specific heat introduced by the rotation of the rotor is at most 3 kJ per kg of liquid medium and per g / L of dry weight of plant cells, and the specific heat capacity introduced into the medium is at most 1.5 kJ per kg of liquid medium and per g / L of dry weight of plant cells per minute.

Description

[Technical Field]

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

[0002] Methods for isolating proteins from cells or cell complexes include osmotic lysis, enzymatic or chemical lysis, sonication, and mechanical digestion. Typically, cells are disrupted using a homogenizer or mixer for mechanical digestion.

[0003] Also, short treatment times can be used to break up cell complexes and suspend cells, resulting in only partial lysis of the total cell count (Orellana-Escobedo et al., Plant Cell Rep. 2015, 34(3):425-33).

[0004] When proteins are isolated from individual organelles or cellular compartments, the organelles are usually isolated before digestion, followed by digestion using the isolates.

[0005] Witzel et al., Plant Methods 2011, 7:48; Leary et al., J Vis Exp. 2014; (94):52113; and Cordoba-Pedregosa et al., Plant Physiology 1996, 112(3):1119-1125 describe methods for extracting proteins from the apoplast of plant cells. The outer space of a protoplast is defined as the apoplast. The apoplast is composed of the cell wall and the intercellular space. The methods described in these publications include osmotic extraction with various infiltration solutions (e.g., salts) and centrifugation. However, these methods suffer from the disadvantage that only material accessible by infiltration can be extracted.

[0006] US 2015 / 0140644 A1 and AU 2017 202473 B2 describe methods for obtaining proteins from the apoplast using cell wall lysis, incubation, and extraction steps, in which enzymatic or chemical modifications to the proteins are possible. Summary of the Invention [Problem to be solved by the invention]

[0007] One of the aims of the present invention is to provide improved options for the isolation or extraction of materials from the apoplast, in particular materials secreted into the apoplast. [Means for solving the problem]

[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 rotor-stator in a liquid medium, and the rotor-stator-derived specific heat introduced by the rotation of the rotor is at most 3 kJ per kg of liquid medium and per g / L of dry weight of plant cells, and the specific heat capacity introduced into the medium is at most 1.5 kJ per kg of liquid medium and per g / L of dry weight of plant cells per minute.

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

[0010] The parameters of both aspects may be combined, especially since only reference values ​​are concerned and the spirit of the invention is provided in both cases. The detailed description of the invention and all preferred embodiments described therein refer to all aspects of the invention. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention relates to conservative rotor-stator treatment of plants or plant cells, which, in contrast to homogenization commonly used in other contexts, detaches material from the cell or plant apoplast. In this regard, the protoplasts remain substantially intact, avoiding contamination of the expressed material to be isolated with cellular components from the interior of the protoplast cells. In this regard, according to the present invention, the intensity and duration of rotor-stator treatment are limited. It has been found that rotor-stator treatment using low energy input, determined as specific heat or heat capacity, allows satisfactory detachment of the desired expressed material. Excellent results can be obtained using a rotor-stator-derived specific heat introduced by rotor rotation of up to 3 kJ per kg of liquid medium and per g / L of dry weight of plant cells per minute, and a specific heat capacity introduced by rotor rotation of up to 1.5 kJ per kg of liquid medium and per g / L of dry weight of plant cells per minute. Similarly, good results are obtained, and the concept of the invention remains exactly the same, when the rotor-stator heat introduced by the rotation of the rotor is at most 30 kJ per kg of liquid medium and when the heat capacity introduced into the medium is at most 1.5 kJ per kg of liquid medium and per minute. According to the invention, the plants or plant cells are not homogenized in this case, but are treated only to the extent that the expressed material is detached from the surface or out of the apoplast.

[0012] Using the method of the present invention, expressed material is obtained from the surface of the cell or apoplast (the cell wall and the entire intercellular space). This type of material usually reaches these sites by secretion and, for plant cells, is also usually found in the culture medium. However, during the course of this invention, it was discovered that a large amount of secreted material is attached to the surface, particularly to the cell wall or apoplast. This attached material is obtained by the present invention, and increased production can then be achieved using the method of the present invention. An approximately 10-fold increase in yield was observed compared to a method not using rotor-stator processing, i.e., isolation of secreted expressed material.

[0013] The rotor-stator treatment intensity, duration and volume are selected according to the present invention to be within maximum parameters to obtain a sufficient amount of expressed material (desired product). However, since product contamination occurs with higher energy inputs, treatment intensity, duration and volume, which represent the energy introduced, quantified as heat or specific heat, are limited.

[0014] Preferably, the rotor-stator specific heat introduced by the rotation of the rotor is at most 3 kJ per kg of liquid medium and per g / L of dry weight of plant cells (abbreviated as 3 kJ / kg / (g / L) or 3 kJ / kg / g / L). Particularly preferably, the specific heat can be kept lower to further reduce contamination of all residues. Thus, preferably, the specific heat introduced by the rotor-stator is at most 2.75 kJ / kg / (g / L), or more preferably at most 2.5 kJ / kg / (g / L), at most 2.25 kJ / kg / (g / L), at most 2 kJ / kg / (g / L), at most 1.75 kJ / kg / (g / L), at most 1.5 kJ / kg / (g / L), at most 1.25 kJ / kg / (g / L), or at most 1 kJ / kg / (g / L).

[0015] Furthermore, according to the present invention, the appropriate parameters of the heat introduced are independent of any reference to plant mass. In some embodiments, this is important because the rotor-stator delivers energy to the cell culture medium independent of the plant cells, which can manifest as a temperature increase. Preferably, the rotor-stator-derived heat introduced by the rotation of the rotor is at most 30 kJ per kg of liquid medium (abbreviated as kJ / kg), preferably at most 25 kJ / kg, at most 20 kJ / kg, at most 15 kJ / kg, or at most 10 kJ / kg.

[0016] This introduced specific heat or introduced heat can be adjusted, for example, by means of a time-limited treatment duration and / or treatment intensity (and likewise the specific heat capacity or heat capacity parameter):

[0017] In the method according to the present invention, the rotor-stator is operated at a low intensity, for example, at a low rotation speed. The heat (or heat capacity) of a particular method introduced at a specific intensity using selected parameters can be measured in a comparative experiment, for example, by increasing the temperature of water or another medium with a known heat capacity. When determining the heat of the method, other effects that affect the temperature, especially heat losses, can be excluded or taken into account in the calculation to obtain the heat or heat capacity of the rotor-stator in this way. Preferably, the heat or heat capacity is determined in a Dewar flask.

[0018] Regardless of the equipment, the specific heat capacity introduced or the heat capacity introduced is recognized to be important (as above, "ratio" refers to an appropriate reference to the amount of plant material). Preferably, the specific heat capacity introduced into the medium is at most 1.5 kJ per kg of liquid medium per minute and per g / L of dry weight of plant cells (abbreviated as kJ / kg / min / (g / L) or kJ / kg / min / g / L). Particularly preferably, this specific heat capacity is at most 1.25 kJ / kg / min / (g / L), at most 1 kJ / kg / min / (g / L), at most 0.8 kJ / kg / min / (g / L), at most 0.6 kJ / kg / min / (g / L), at most 0.5 kJ / kg / min / (g / L), at most 0.4 kJ / kg / min / (g / L), at most 0.3 kJ / kg / min / (g / L), at most 0.2 kJ / kg / min / (g / L), at most 0.15 kJ / kg / min / (g / L), at most 0.125 kJ / kg / min / (g / L) or at most 0.1 kJ / kg / min / (g / L). In a similar manner, the heat capacity introduced into the medium is at most 1.5 kJ per kg of liquid medium per minute (abbreviated as kJ / kg / min). Preferably, this heat capacity is at most 1.25 kJ / kg / min, at most 1 kJ / kg / min, at most 0.8 kJ / kg / min, at most 0.6 kJ / kg / min, at most 0.5 kJ / kg / min, or at most 0.4 kJ / kg / min.

[0019] The more intensive or longer the treatment, the greater the amount of material obtained. Preferably, the heat introduced by the rotation of the rotor (rotor-stator) is at least 1 kJ per kg of liquid medium, particularly preferably at least 2 kJ / kg, and / or the specific heat from the rotor-stator is at least 0.1 kJ per kg of liquid medium and per g / L of dry weight of plant cells, particularly preferably at least 0.2 kJ / kg / (g / L).

[0020] Preferably, the heat capacity introduced into the culture medium by the rotation of the rotor is at least 0.2 kJ per kg of liquid medium and per minute, preferably at least 0.4 kJ / kg / min, and / or the specific heat capacity into the culture medium is at least 0.02 kJ per kg of liquid medium per minute and per g / L of dry weight of plant cells, preferably at least 0.04 kJ / kg / min / (g / L).

[0021] The expressed material preferably contains a protein. Proteins, particularly recombinantly expressed proteins, can be directed specifically down the secretory pathway by an appropriate signal sequence and thus concentrated on the surface or in the apoplast. Preferably, the expressed material is present in the apoplast of the plant cell, from where it can be obtained using the method according to the invention. More preferably, the expressed material is a secreted material, preferably a protein secreted through the cell membrane or cell wall.

[0022] According to the present invention, a rotor-stator is used to process plant cells in a liquid medium to obtain expressed material.

[0023] The rotor-stator comprises at least one rotor which, due to its rotational movement, exerts shear forces on the plant cells, such that the surface or apoplast and cell walls of the plant cells are (structurally) loosened and mechanically affected, scraped or partially removed from the surface.

[0024] The rotor rotates relative to the stator. The stator can be a containment sheath or a counterpart to the rotor. The rotor can have cutting or shearing elements with blades or shearing surfaces. A common structure has a comb-like structure, in which multiple shearing projections (e.g., teeth or tines), usually arranged parallel to the axis of rotation, exert a shearing or cutting action. By way of example, "multiple" can be 2, 3, 4, 5, 6, 7, 8 or more shearing projections.

[0025] The stator can be configured as a counterpart to the rotor, in particular as its cutting or shearing element. Like the rotor, the stator can have its own cutting or shearing elements, for example, also having a comb-shaped structure. This type of construction is known in rod homogenizers, as described in DE 10 2005 031 459 A1.

[0026] In other embodiments, the stator may be a housed structure, as is conventional in, for example, a flow-through homogenizer. An example of a flow-through rotor-stator is described in WO2009 / 062610 A1.

[0027] Examples of rotor-stators are rod homogenizers or shear pumps, which are used especially in connection with flow-through systems.

[0028] Preferably, there is a gap between the rotor and stator, e.g., the size of at least one or more plant cells, 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 larger, and any range between these distances is preferably at most 500 μm, or at most 300 μm, or at most 200 μm.

[0029] As described above, to preserve plant cells in the form of protoplasts, the strength is kept low to prevent or reduce contamination of the secreted expressed material to be collected with intracellular substances. In a typical rotor-stator model, the rotation speed is reduced for this purpose, for example, in embodiments where the rotor is operated at a maximum rotation speed of 15,000 revolutions per minute, preferably 1,000 to 15,000 revolutions per minute. Possible rotation 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.

[0030] The plant cells may be present in a vessel into which the rotor-stator is introduced. For this purpose, the rotor-stator may be introduced into a vessel containing the culture medium. This "batch" configuration (for discontinuous operation) is used in particular with rod homogenizers. On a larger scale, it is preferred to use a flow-through rotor-stator. According to this embodiment, the rotor-stator may have an interior with at least one inlet and outlet, by means of which the liquid culture medium is continuously supplied from the interior. An example in this case is a shear pump for a continuous process.

[0031] Preferably, the stator is 10 cm 3 (0.01L)~1m 3 The range is set to a volume of up to (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 particularly preferred, with 0.65 L to 50 L being particularly preferred, for example 1 L to 40 L. These volumes are particularly suitable for treating culture media for plant cells.

[0032] Preferably, the amount of liquid medium to be processed is up to 50,000 kg, preferably 0.5 g to 50,000 kg, for example 1 g to 25,000 kg, 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. Quantities of this type are preferably those used in a single run in a discontinuous process.

[0033] Preferably, the plant cells are present in the liquid medium at a concentration of 0.2 g / L to 60 g / L (mass of plant cells as dry weight). In this regard, preferred concentrations for plant cells (always as dry weight) 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 preferred are around 10 g / L, for example 5 g / L to 15 g / L. These plant cell concentrations are processed particularly efficiently in a rotor-stator.

[0034] Preferably, the plant cells are treated in the rotor-stator for 2 minutes to 150 minutes. In continuous processes, these times refer to the average treatment time for the plant cells. Particularly preferred times are 3 minutes to 120 minutes, 5 minutes to 100 minutes, 8 minutes to 80 minutes, 10 minutes to 60 minutes, or especially preferred 12 minutes to 40 minutes. When using large culture volumes, longer rotor-stator treatments can be performed. Further preferred possible times are 1 hour to 24 hours, preferably 2 hours to 20 hours, 3 hours to 16 hours, or 4 hours to 12 hours. In this manner, preferred treatment times are all within the range of 3 minutes to 24 hours, and any range between the recited treatment times, or indeed longer.

[0035] Preferably, plant cells can be cultured in suspension culture. This suspension can be directly processed as a liquid medium in the method according to the invention. Alternatively, mosses can be similarly first isolated, for example, from a solid, liquid or suspension culture, and then suspended in an aqueous medium under conditions suitable for carrying out rotor-stator treatment. Plants particularly suitable for the method according to the invention are non-woody plants. Preferred plants are algae and mosses, in particular bryophytes. Preferably, the bryophyte plant or cell is a moss, preferably P. patens. The bryophyte may be any bryophyte, but is preferably selected from the mosses, liverworts, or hornworts, particularly preferably from the class Bryopsida or the genera Physcomitrella, Funaria, Sphagnum, Ceratodon, Marchantia, and Sphaerocarpos. Physcomitrella patens is particularly preferred. Most preferably, the method according to the invention is carried out using cells from plant tissue, such as protonemata from the liverwort Physcomitrella patens. Preferred algae are selected from green algae from the order Chlorellales, preferably from the family Chlorellaceae, more preferably from the genus Auxenochlorella or Chlorella, in particular Chlorella vulgaris, and from the order Volvocales, preferably from the family Haematococcaceae, more preferably from the genus Haematococcus, in particular Haematococcus pluvialis, and from the order Eustigmatales, preferably from the families Loboceae, Chlorobothryaceae, Pseudocharaciopsidaceae and Eustigmataceae.Further preferred plants are tobacco, bean or lentil. Preferably, the plant is an aquatic plant, for example from the genera Lemna, Spirodela, Landoltia, Wolffia or Wolffiella.

[0036] Plant cells are a component of the subject matter of the present invention. The term "plant cell", as used herein, can refer to an isolated cell, an individualized cell, but also to a cell in or derived from a plant tissue, preferably a tissue selected from callus, protonema, phloem, xylem, mesophyll, stem, leaf, thallus, chloronema, rhizoid or stem phylloth, or a cell in a plant organism.

[0037] In the method according to the invention, the medium preferably has a physiological pH to prevent contamination of the expressed material in the apoplast / on the cell surface, in particular as described above, in order to preserve the protoplasts (cell components inside the cell wall, in particular from the cell membrane). The pH of the liquid medium is preferably 3.5 to 8.5, particularly preferably 4 to 8, or 4.5 to 7, or 5 to 6.5, in particular 5.5 to 6, or a combination of these values, for example pH 5 to 8.

[0038] Furthermore, for the preservation of protoplasts, the osmolality of the liquid medium is preferably physiological, for example, to avoid swelling stresses, especially if the osmolality is too low. Where appropriate, an upper limit on the osmolality can be set to avoid osmotic contraction stresses. Preferably, the medium has an osmolality of at least 0.1 osmol / L, or preferably at least 0.150 osmol / L. The osmolality can be adjusted by salts or other medium components, for example, dissolved substances such as sugars or sugar alcohols. Preferably, Na + and / or K + Alkali metal salts such as Cl are provided. - or F - or I -Halides, phosphates, or acetates such as HCl, etc., are provided as anions. Buffer components such as Tris (tris(hydroxymethyl)aminomethane) are also possible.

[0039] Additionally, surfactant polymers may be added to the liquid medium for rotor-stator treatment to further preserve the protoplasts. Surfactant polymers are described, for example, in WO 2013 / 156504 A1, and preferably include emulsifiers, such as uncharged polymers such as polyalkyl glycols, especially polyethylene glycols. In particular, the polymer is a nonionic water-soluble surfactant polymer. Preferably, the polymer does not denature proteins. Examples include polymers or copolymers selected from polyethers such as polyalkyl glycols, polysorbates or polyvinylpyrrolidone, polyvinyl alcohol, water-soluble cellulose derivatives such as hydroxypropyl cellulose, hydroxypropylmethyl cellulose, carboxymethyl cellulose, or hydroxyethyl cellulose, vinylpyrrolidone-vinyl acetate copolymers (copovidone), polyvinyl acetate, partially hydrolyzed polyvinyl alcohol, polyvinyl alcohol-polyethylene glycol copolymers, and mixtures thereof. Further possibilities include polysorbates such as polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan tristearate, preferably polysorbate 80 (Tween® 80, which is polyoxyethylene (20) sorbitan monooleate), polyoxyethylene (40) stearate. The surfactant polymer is preferably present in the medium at a concentration of at least 0.05% by weight, particularly preferably at least 0.08%, at least 0.1% or at least 1.5% (all percentages by weight). The molecular weight of the surfactant polymer, such as PEG, 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.Particularly preferably, the molecular weight is 500 Da to 2,000,000 Da, preferably 1,000 Da to 200,000 Da or 1,200 Da to 80,000 Da.

[0040] The liquid medium is preferably aqueous, in particular water or a water mixture that is compatible with the cells. In particular, the liquid medium can be a culture medium for (and with) plant cells, as long as the plant has not previously been separated from the liquid medium.

[0041] Preferably, the expressed material is expressed before treating the plant cells with a rotor-stator, so that the plant cells collect on the surface or in the apoplast. In this regard, the plant cells may be cultured and / or grown, for example, in a medium (nutrient medium, light) under plant growth conditions, as is generally known (see, for example, Frank et al., Plant Biol. 7, (2005):220-227). Expression or culturing is preferably carried out for 13 minutes to 1 month (30 days) or longer, for example, 2 months (60 days), for example, 1 hour to 22 days, or 5 hours to 15 days, for example, 10 hours to 7 days, or 20 hours to 3 days. In continuous cell cultures involving regular removal of cells to obtain the product (expressed material), these time ranges or minimum times may correspond to the average period for cells in culture.

[0042] Other methods that damage the protoplasts or lyse or homogenise them should be avoided. The cell walls are preferably not lysed, in particular not enzymatically and / or chemically and / or osmolytically and / or using ultrasound. Preferably, apart from the rotor-stator treatment according to the invention, the cell walls should remain intact or unharmed. In particular, the cell membrane (protoplast) should remain intact, and while cell vitality does not play any particular role in the method according to the invention, contamination of the liquid medium with internal cell components, in particular cell cytoplasm, should be avoided.

[0043] The invention will now be described in more detail with the aid of the figures and the following examples, without limiting the invention to these embodiments. [Brief explanation of the drawings]

[0044] [Figure 1] Determination of energy input in water using a T25 Ultra Turrax rod homogenizer (IKA / Staufen). (A) Temperature profile in 1.5 L of water at a rotation speed of 10,000 rpm. (B) Calculated energy input [kJ]. (C) Calculated energy input [kJ / kg]. [Figure 2] Determination of energy input in water using an FSP712VC-2.2kW-FU shear pump homogenizer (Fristram, Hamburg). (A) Temperature profile in 50 L of water at a rotation speed of 2,800 rpm. (B) Calculated energy input [kJ]. (C) Calculated energy input [kJ / kg]. [Figure 3] Percent release of biomass-bound product (moss-aGal) during treatment with a shear pump (dashed line) and a T25 Ultra Turrax rod (solid line). Shear pump treatments of reactor cultures were carried out in a volume of 50 L. Treatments of reactor cultures with a T25 Turrax rod were carried out in a volume of 0.65 L. [Figure 4] Specific energy input using a T25 Ultra Turrax rod (A) and shear pump (B), both for 10 g / L dry biomass. Specific energy input with a comparative T25 Ultra Turrax rod at 19,000 rpm with 1 g / L dry biomass (C). [Figure 5]Percent release of biomass-bound product (moss-aGal) during treatment with a shear pump (dashed line) and a T25 Ultra Turrax rod (solid line). Shear pump treatment of reactor culture was carried out in a volume of 50 L. Treatment of reactor culture with a T25 Turrax rod was carried out in a volume of 0.65 L. Biomass: 9.2 g / L (shear pump), 8.2 g / L (T25). [Figure 6] Similar Western blots for product release (moss-aGal) compared to the release of an intracellular marker protein (Rubisco, large subunit). Intracellular protein detectable in salt-containing medium (e.g., 20 mM Tris, 100 mM NaCl, pH = 7) with minimal energy input of up to 32.9 kJ / kg. Under osmotic stress conditions (demineralized water), detectable from approximately 10 kJ / kg. Target protein abundance (moss-aGal) increases in salt-containing medium and under osmotic stress conditions as a function of energy input. [Figure 7] Microscopic analysis of the T25 process in demineralized H2O. 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 still present, but more particles are clearly present, which is a sign of the beginning of cell digestion. [Figure 8] Microscopic analysis of the T25 process in a salt-containing buffer (20 mM Tris, 100 mM NaCl, pH=7). Up to an energy input of 16.5 kJ / kg, the moss cells retain their integrity. At an energy input of 32.9 kJ / kg, the cell integrity is still there, but more particles are clearly present, which is a sign of the beginning of cell digestion. [Figure 9] Microscopic analysis of the shear pumping process in salt-containing medium (reactor culture). Up to an energy input of 18.41 kJ / kg, the moss cells retain their integrity. Above an energy input of 27.20 kJ / kg, the cell integrity is still there, but more particles are clearly present, which is a sign of the beginning of cell digestion. [Figure 10]Microscopic analysis of the shear pumping process in demineralized H2O. Up to an energy input of 9.62 kJ / kg, moss cells retain their integrity. Above an energy input of 18.41 kJ / kg, cell integrity is still present, but more particles are clearly present, which is a sign of the beginning of cell digestion. [Figure 11] Microscopic analysis of moss cells in the ultrasonic process as a comparative image for cell digestion. After only a short period of time (1 minute of sonication), the cells lose their integrity. After just 3 minutes, cell fragments and empty cell debris can be observed (100% in 50 mL sample). [Figure 12] Comparative example: Determination of energy input in water using a T25 homogenization tool (IKA / Staufen) at high energy, 19,000 rpm rotation speed. (A) Temperature profile in 1 L of water. (B) Calculated energy input [kJ]. (C) Calculated energy input [kJ / kg]. [Figure 13] Comparative example: significantly faster product release at 19,000 rpm, especially up to an energy input of 7 kJ / kg. Product loss due to high temperature and shear stress above an energy input of 84 kJ / kg. [Example]

[0045] [Example 1] Determining energy input using Turrax rod and shear pump The parameter used for energy input in the aqueous medium was temperature as a measurable variable. 1.5 L of HO was dispersed in a Dewar flask with a T25-S25N-18G Turrax rod (IKA Staufen) at 10,000 rpm for 30 min, and the temperature profile was measured. The room temperature during the experiment was 20.5°C to 20.7°C. Energy input data was calculated based on the specific heat capacity of HO (4,190 J kg -1 K -1Energy input data, comparable to literature (Orellana-Escobedo et al., Plant cell Rep. 2015, 34(3), 425-433), were also acquired at 19,000 rpm.

[0046] For the shear pump (shear pump FSP 712, Fristam, Hamburg), 50 L of H2O was circulated at 2,800 rpm from a Nalgene container using reinforced PVC tubing. Temperature measurements in the Nalgene container were performed using a temperature sensor (G002.1 precision thermometer, Carl Roth). The experiment was set at 19 °C in a temperature-controlled chamber. Heat losses to the environment were neglected in this experimental setup. Energy input data were calculated using the specific heat capacity (4,190 J kg -1 K -1 ) was calculated using

[0047] [Example 2] Production of moss cultures Axenic cultivation of the moss production strain was carried out in a Wave™ Rocking Motion bioreactor (Wave200, GE Healthcare) in 200 L disposable bioreactor bags (Cellbag 200, GE Healthcare) for 3-4 weeks. The cultivation parameters were a shaking frequency of 19-25 rpm, a shaking angle of 9°C, a temperature of 24-26°C, and a gas flow rate of 2 L / min and enriched air supply with 2% CO2. Illumination was provided by four LED modules (reference numbers 120268-120282, Infors AG) mounted on the bioreactor bag using "warm white LEDs." Moss cultivation was carried out under 24-h light. SM07 (100 mM NaCl, 6.6 mM KCl, 2.0 mM MgSO4 x 7H2O, 1.8 mM KH2PO4, 20.4 mM Ca(NO3)2 x 4H2O, 0.05 mM Fe2O3, Na-EDTA, 4.9 mM MES, 0.1% (w / v) PEG4000, 100.26 μM H3BO3, 0.11 μM CoCl2 x 6H2O, 0.1 μM CuSO4 x 5H2O, 5 μM KI, 85.39 μM MnCl2 x 4H2O, 1.03 μM Na2MoO4 x 2H2O, 0.11 mM NiCl2 x 6H2O, 0.04 μM NaCl) supplemented with 1000× Nitsch vitamins (Nitsch vitamin mixture, Duchefa, see manufacturer's specifications). The mineral salt medium was Na2SeO3 x 5H2O, 0.039 Zn-acetate x 2H2O. The pH was set to 5-6 using a WAVEPOD I and Pump20 (GE Healthcare) to automatically add 0.25 M H2SO4 and 0.25 M NaOH. Recombinant α-galactosidase (aGal or α-GalA) was expressed as described in WO 2016 / 146760 A1 ("moss-aGal").

[0048] [Example 3] Method for the release and analysis of moss-bound products To analyze the time profile of the release of moss-bound products, the cultures obtained from Example 2 were exposed to a T25-S25N-18G Turrax rod and to a shear pump FSP 712 at various inputs of energy. PL Product concentration determination (C PL ) was performed using the moss-aGal ELISA (Biogenes, Germany). Detection of the degree of cell digestion was performed using microscopic image analysis of the moss cells (microscope: Axiovert 200 oper Stemi SV11 (Carl Zeiss) equipped with an AxioCam camera, AxioSoft software, and a KL 1500 LCD cold light source). Comparison with the microscopic images of total digestion was possible by microscopic analysis of 50 mL of a 20-minute ultrasonic digestion using 100% powder (probe: UW2070, Bandelin; amplifier: HD 2070, Bandelin). At the molecular level, qualitative analysis was performed using Western blots on the released products as well as the intracellular marker protein Rubisco. The primary antibody used was anti-aGal (H00002717-D01P, Abnova), and the secondary antibodies were anti-Rubisco (AS03037, Agrisera) and anti-rabbit HRP (Abcam, AS03037).

[0049] The released product (C PL ) and releasable moss-bound products (C PX To analyze the relationship between the moss-derived α-galactosidase and the moss-derived α-galactosidase, untreated cultures were subjected to cell digestion using a ball mill (steel balls: RB-3 / G20W, Schleer; ball mill: MM300, Retsch). After separating the cell debris, product concentrations were determined using ELISA (Biogenes). For this, aGal (α-galactosidase, also known as "moss-aGal"), a protein expressed in the apoplastic cavity, was assayed.

[0050] [Example 4] Results and Discussion The energy input (as heat, in kJ / kg, in kg of liquid medium) for two different homogenizers, T25-S25N-18G Turrax rod and FSP 712 shear pump, was established in the culture medium (kg) using temperature measurements (Figures 1-5). In this regard, the homogenizers were set at a low rotation speed to generate a low heat capacity (in kJ / kg / min). The total heat over a specific period (0-60 min) was determined in this manner. In this regard, the energy input in water was calculated based on the measured temperatures using the specific heat coefficient of HO [4.182 kJ / kg * K]. For this, the heat (Figures 1-3) or specific heat with respect to the dry weight of the plant parts (Figures 4-5) was calculated.

[0051] Figures 3 and 5 show the product release of a desired protein (moss-derived recombinant aGal, "moss-aGal") deposited on the surface or in the apoplast. The release increases with increasing treatment.

[0052] In a comparative experiment, the Turrax rod was operated at a higher heat capacity, in fact at 19,000 rpm (Fig. 4C, Figs. 12-13). The heat capacity was approximately 100 times higher than with the conservative treatment at 10,000 rpm (compare Fig. 4A and Fig. 4C). Operation at a higher heat capacity resulted in rapid product release, but also in cell disruption (protoplasts), resulting in contamination of the extracellular product with internal cell components. These effects occurred already after 1 min.

[0053] Figure 6 shows the amount of product release from the extracellular protein (moss aGal) and the intracellular protein Rubisco. Rubisco is present in plant cells at high concentrations and is therefore considered a highly sensitive marker for leakage of cellular contents. In experiments using physiological salt solutions, increased release of Rubisco was observed at higher energy inputs (heat) above 32.9 kJ / kg. In experiments using demineralized water (DM water), contamination with Rubisco occurred as quickly as approximately 10 kJ / kg, due to osmotic effects occurring in addition to shear stress from the homogenizer. The varying heat (energy input) was controlled using treatment time.

[0054] Microscopic analysis of the cell complexes after treatment mirrors these results. Figure 7 shows the results after treatment with an Ultraturrax rotor-stator in demineralized water, and Figure 8 shows the results after treatment with an Ultraturrax rotor-stator under physiological conditions (20 mM Tris, 100 mM NaCl, pH = 7). Figure 9 shows the cell complexes after treatment with a shear pump under physiological conditions. As the treatment time (heat) increased, the number of particles formed, presumably due to disrupted cells, increased. From approximately 30 kJ / kg, increased cell digestion occurred. For comparison, Figure 10 shows an experiment in demineralized water using a shear pump. Here, comparable particles appeared at approximately 20 kJ / kg.

[0055] In comparison to Figures 7-10, Figure 11 shows cell digestion using ultrasound: the cells lose their integrity after just one minute.

[0056] This indicates, on the one hand, that the energy input per time unit (intensity of rotor-stator rotation; heat capacity) should be limited, and, on the other hand, that the absolute energy input (heat) in the experiments here is controlled by the treatment time. This parameter can be included as is or linked to the biomass (dry biomass, TBM). Suitable values ​​for a conservative method that releases as many 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 weight and a maximum of 1.5 kJ / kg / min per g / L of dry weight, or a maximum of 30 kJ / kg and a maximum of 1.5 kJ / kg / min per g / L of dry weight. Possible treatment times with this type of low heat capacity range from 2 to 150 minutes, depending on the intensity of rotation, and are generally longer than the short-term but intensive treatments used so far.

Claims

1. 1. A method for detaching expressed material from the apoplast of plant cells, wherein the apoplast comprises the outer cell wall and intercellular spaces, and the plant cells are processed in a rotor-stator in a liquid medium, wherein the rotor-stator-derived specific heat introduced by the rotation of the rotor is at most 3 kJ per kg of the liquid medium and per g / L of dry weight of the plant cells, and the specific heat introduced into the medium is at most 1.5 kJ per minute per kg of the liquid medium and per g / L of dry weight of the plant cells.

2. 10. The method of claim 1, wherein the expressed material is present in the apoplast of the plant cell.

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

4. 4. The method according to claim 1, wherein the specific heat introduced into the culture medium by the rotation of the rotor is at least 0.2 kJ per kg of liquid medium per minute and per minute, and / or the specific heat introduced into the culture medium is at least 0.02 kJ per kg of liquid medium per minute and per g / L of dry weight of the plant cells.

5. 5. The method according to any one of claims 1 to 4, characterized in that the expressed material contains a protein and / or the expressed material is secreted material, preferably a protein secreted through the cell membrane.

6. 6. The method according to claim 1, wherein the rotor-stator is introduced into a vessel containing the culture medium.

7. 7. The method according to claim 1, wherein the rotor-stator has an interior with at least one inlet and outlet, and the liquid medium is continuously supplied from the interior via the at least one inlet and outlet.

8. The stator is 10 cm ３ ~1m ３ and / or the amount of liquid medium to be treated is up to 50 kg, preferably between 0.5 g and 50 kg.

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

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

11. 11. A method according to any one of the preceding claims, characterized in that the rotor is operated at a maximum rotational speed of 15,000 revolutions per minute, preferably between 1,000 and 15,000 revolutions per minute.

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

13. 13. The method according to claim 1, wherein the rotor-stator specific heat introduced by the rotation of the rotor is at most 30 kJ per kg of the liquid medium, and the specific heat introduced into the medium is at most 1.5 kJ per kg of the liquid medium per minute.

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

15. 15. The method according to any one of claims 1 to 14, characterized in that the plant cells are treated in the rotor-stator for between 2 minutes and 150 minutes.

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