Method for obtaining material from plant cell surfaces

Abstract

The invention relates to a method for detaching expressed material from the surface or the apoplast of plant cells, the plant cells being treated in a liquid medium using a rotor-stator. The specific heat of the rotor-stator introduced by the rotation of the stator is at most 3 kJ per kg of the liquid medium and per g / l dry mass of the plant cells and the specific heat output introduced into the medium is at most 1.5 kJ per kg of the liquid medium per minute and per g / l dry mass of the plant cells.

Description

[0001] Methods for obtaining material from plant cell surfaces

[0002] Field of invention

[0003] The present invention relates to the isolation of proteins from cells.

[0004] Background of the invention

[0005] Methods for isolating proteins from cells or cell clusters include osmotic lysis, enzymatic or chemical lysis, ultrasound treatment, and mechanical disruption. Mechanical disruption typically involves breaking down cells using a homogenizer or mixer.

[0006] Short treatment times can also be used to divide cell clusters and to suspend cells, resulting in only partial lysis of the total cell count (Orellana-Escobedo et al., Plant Cell Rep. 2015).

[0007] 34(3):425-33) .

[0008] If a protein is isolated from individual organelles or cell compartments, the cell organelles are usually isolated before digestion, and then the isolate is digested.

[0009] Witzei et al., Plant Methods 2011, 7:48; Leary et al., J Vis Exp. 2014; (94): 52113; and Cördoba-Pedregosa et al., Plant

[0010] Physiology 1996, 112(3):1119-1125, describe methods to extract proteins from the apoplast of plant cells.

[0011] The apoplast is the space outside the protoplasts. It consists of the cell walls and the intercellular space. The methods described in these publications involve osmotic extraction with various infiltration solutions (e.g., salts) and centrifugation. However, this method has the disadvantage that only materials accessible through infiltration can be extracted.

[0012] US 2015 / 0140644 Al and AU 2017202473 B2 describe methods for obtaining proteins from the apoplast, involving cell wall lysis, incubation, and extraction. Enzymatic or chemical modifications of the proteins are possible.

[0013] An objective of the invention is to provide improved isolation or extraction possibilities of materials from the apoplast, in particular of material secreted in the apoplast.

[0014] Summary of the invention

[0015] 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 introduced by rotation of the rotor-stator is a maximum of 3 kJ per kg of the liquid medium and per g / l dry mass of the plant cells and the specific heat power introduced into the medium is a maximum of 1.5 kJ per kg of the liquid medium per minute and per g / l dry mass of the plant cells.

[0016] In another aspect, the invention relates to a method for detaching expressed material from the surface or 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 introduced by rotation of the rotor-stator is a maximum of 30 kJ per kg of the liquid medium and the heat power introduced into the medium is a maximum of 1.5 kJ per kg of the liquid medium per minute.

[0017] The parameters of both aspects can be combined, particularly since they are merely different reference values, thereby fulfilling the essence of the invention. All detailed descriptions of the invention and preferred embodiments described herein relate to all aspects of the invention.

[0018] Detailed description of the invention

[0019] The invention relates to a gentle rotor-stator treatment of plants or plant cells, which, contrary to the otherwise conventional homogenization process, detaches material from the apoplast of the cells or plants. The protoplasts are intended to remain largely intact in order to avoid contamination of the expressed material to be isolated by cell components from the interior of the protoplasts. Therefore, according to the invention, the intensity and duration of the rotor-stator treatment are limited. It has been found that a satisfactory detachment of the desired expressed material is possible through rotor-stator treatment with low energy inputs – defined as specific heat or thermal power.Excellent results were obtained with a specific heat input of the rotor-stator of up to 3 kJ per kg of liquid medium and per g / l dry mass of plant cells, and with a specific heat input of up to 1.5 kJ per kg of liquid medium per minute and per g / l dry mass of plant cells. Equally good results were obtained with a heat input of up to 30 kJ per kg of liquid medium and with a heat input of up to 1.5 kJ per kg of liquid medium per minute, demonstrating the invention's principles. 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.

[0020] The inventive method recovers expressed material from the surface of the cells or the apoplast (the entirety of the cell walls and the intercellular space). Such material typically reaches these locations via a secretory pathway and is usually also found in the culture medium of plant cells. However, it was discovered during the course of the invention that large quantities of the secreted material adhere to the surface, especially to the cell wall or the apoplast. This adhering material is recovered according to the invention, and the inventive method has resulted in production increases. A tenfold increase in yield compared to methods without rotor-stator treatment, i.e., the isolation of the secreted expressed material, has been observed.

[0021] The treatment intensity, duration, and capacity of the rotor-stator are selected within the maximum parameters according to the invention to obtain sufficient yield of the expressed material (desired product). However, the treatment intensity, duration, and capacity, which represent the input energy quantified as heat or specific heat, are limited because product contamination occurs at higher energy inputs.

[0022] Preferably, the specific heat introduced by the rotation of the rotor-stator is a maximum of 3 kJ per kg of the liquid medium and per g / l of dry mass of the plant cells (abbreviated 3 kJ / kg / (g / l) or 3 kJ / kg / g / l). Particularly preferably, the specific heat can be kept lower to further reduce any residual contamination. Thus, the introduced specific heat of 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).

[0023] Independently of any reference to the amount of plant material, the optional parameter of heat input was also determined according to the invention. This is relevant in some embodiments because the rotor-stator provides energy input into the cell medium independently of the plant cells, which can manifest as heating. Preferably, the heat input by the rotor-stator through rotation is a maximum of 30 kJ per kg of the liquid medium (abbreviated 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.

[0024] This applied specific heat or heat can be adjusted, for example, by limiting the treatment duration and / or the intensity of the treatment (as well as the parameters of the specific heat output or the heat output):

[0025] In the method according to the invention, the rotor-stator is operated at a low intensity, e.g., at a low rotational speed. The heat (or thermal power) introduced at a specific intensity by a particular 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, especially temperature losses, should be excluded or computationally accounted for in order to arrive at the heat or thermal power of the rotor-stator itself; preferably, the heat or thermal power is determined in a Dewar flask.

[0026] Regardless of the device, the specific heat input or the heat input 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 the liquid medium per minute and per g / l dry mass of the plant cells (abbreviated kJ / kg / min / (g / l) or kJ / kg / min / g / l). Particularly preferred is a specific heat output of at most 1.25 kJ / kg / min / (g / 1), at most 1 kJ / kg / min / (g / 1), at most 0.8 kJ / kg / min / (g / 1), at most 0.6 kJ / kg / min / (g / 1), at most 0.5 kJ / kg / min / (g / 1), at most 0.4 kJ / kg / min / (g / 1), at most 0.3 kJ / kg / min / (g / 1), at most 0.2 kJ / kg / min / (g / 1), at most 0.15 kJ / kg / min / (g / 1), at most 0.125 kJ / kg / min / (g / 1), or at most 0.1 kJ / kg / min / (g / 1). Similarly, the heat input into the medium is preferably at most 1.5 kJ per kg of the liquid medium per minute (abbreviated 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.

[0027] The more intensive or prolonged the treatment, the greater the amount of material obtained. Preferably, the heat introduced by rotation of the rotor (rotor-stator) is at least 1 kJ per kg of the 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 the liquid medium and per g / l dry mass of the plant cells, particularly preferably at least 0.2 kJ / kg / (g / l).

[0028] Preferably, the heat input into the medium by rotation of the rotor is at least 0.2 kJ per kg of the liquid medium per minute, preferably at least 0.4 kJ / kg / min, and / or the specific heat input into the medium is at least 0.02 kJ per kg of the liquid medium per minute and per g / l dry mass of the plant cells, preferably at least 0.04 kJ / kg / min / (g / l).

[0029] The expressed material preferably contains proteins. Proteins, particularly recombinantly expressed proteins, can be selectively directed to the secretory pathway by appropriate signal sequences and thus directed to accumulation at the cell surface or the apoplast. Preferably, the expressed material is located in the apoplast of plant cells, from which it can be obtained using the method according to the invention. Equally preferred is the expressed material being secreted material, preferably proteins secreted through the cell membrane or cell wall.

[0030] 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.

[0031] A rotor-stator comprises at least one rotor, which exerts shear forces on the plant cells through its rotation. These shear forces loosen, mechanically affect, or abrade the surface of the plant cells, specifically the apoplast and cell walls (structurally), or cause partial surface abrasion.

[0032] The rotor rotates relative to a stator. The stator can be a housing casing or a counterpart to the rotor. The rotor can have cutting or shearing elements with edges or shear surfaces. A common design features a comb-like structure, with numerous shear projections (e.g., teeth or tines), usually arranged parallel to the axis of rotation, exerting the shearing or cutting action. A "numerous" number here could be, for example, 2, 3, 4, 5, 6, 7, 8, or more shear projections.

[0033] The stator can be arranged as a counterpart to the rotor and, in particular, to 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 102005 031 459 A1.

[0034] In other embodiments, the stator can be a housing structure, as is common for flow homogenizers. An example of a flow rotor stator is described in WO 2009 / 062610 A1.

[0035] Examples of rotor stators include a rod homogenizer or a shear pump. Shear pumps are used particularly in flow systems.

[0036] 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 the protoplast, can pass between the rotor and stator. Suitable gap sizes are 50 g / m.

[0037] 70 gm, 80 gm, 100 gm, 150 gm or more, and any range between these distances. Preferably up to a maximum of 500 gm, or up to 300 gm, or up to 200 gm.

[0038] As noted, the intensity is kept low to protect the plant cells, specifically 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, this is achieved by reducing the rotational speed, for example, 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.

[0039] The plant cells can be placed in a container into which the rotor-stator is inserted. For this purpose, the rotor-stator can be placed in a container with the medium.

[0040] This batch design (for a non-continuous process) is used particularly in rod homogenizers. For larger scales, flow-through rotor stators are preferred. 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 conveyed. An example of this is the shear pump for a continuous process.

[0041] Preferably, the stator limits a volume of 10 cm³ 3 (0.011) to 1 m 3 (10001). Preferred volumes are 0.11 to 800 l, or 0.51 to 600 l, or 11 to 400 l, 21 to 200 l. Volumes up to 100 l are preferred, in particular 0.651 to 50 l, e.g. 11 to 40 l. These volumes are particularly well suited for the treatment of plant cell culture media.

[0042] Preferably, the quantity of the treated liquid medium is up to 50,000 kg, preferably 0.5 g to 50,000 kg, e.g., lg 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. Such quantities are preferably used in a non-continuous process per pass.

[0043] 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 mass). Preferred concentrations of plant cells (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 about 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.

[0044] Preferably, the plant cells are treated with the rotor-stator for 2 to 150 minutes. In continuous processes, these times refer to the average treatment time of the plant cells. Preferred times are, in particular, 3 to 120 minutes, 5 to 100 minutes, 8 to 80 minutes, 10 to 60 minutes, or, most preferably, 12 to 40 minutes. Longer rotor-stator treatments are also possible for large culture volumes. Other preferred treatment times are 1 to 24 hours, preferably 2 to 20 hours, 3 to 16 hours, and 4 to 12 hours. Thus, all preferred treatment times cover the range of 3 minutes to 24 hours, and any range between the aforementioned treatment times or even longer.

[0045] Preferably, the plant cells can be cultivated in a suspension culture. This suspension can be processed directly as a liquid medium in the process according to the invention. Alternatively, moss can first be isolated, e.g., from a solid culture, liquid culture, or suspension culture, and then suspended in an aqueous medium with suitable conditions for the rotor-stator treatment. Plants particularly suitable for the process according to the invention are non-woody plants. Preferably, the plants are algae and mosses, especially bryophytes. Preferably, the bryophyte plant or cell is a moss, preferably *Physicospermum patens*. The bryophyte can be any bryophyte, but is preferably selected from mosses, liverworts, or hornworts, particularly preferably from the class *Bryopsida* or the genera *Physcomitrella*, *Funaria*, *Sphagnum*, *Ceratodon*, *Marchantia*, and *Sphaerocarpos*. *Physcomitrella patens* is especially preferred.The inventive process is most preferably carried out using cells from plant tissue such as the protonema of the moss *Physcomitrella patens*. Preferred algae are selected from the green algae, e.g., 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*. Other preferred plants are tobacco, beans, or lentils. Preferably, the plant is an aquatic plant, e.g., of the genera *Lemna*, *Spirodela*, *Landoltia*, *Wolffia*, or *Wolffiella*.

[0046] 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.

[0047] In the process according to the invention, the medium preferably has a physiological pH value, particularly to protect the protoplasts (cell components within the cell wall, especially from the cell membrane onwards), as already described, in order to avoid contamination of the expressed material in the apoplast / on the surface of the cells. The pH value 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, particularly between 5.5 and 6, or combinations of these ranges, such as a pH value of 5 to 8.

[0048] To protect the protoplasts, the osmolarity of the liquid medium is preferably physiological, particularly to avoid swelling stress, e.g., at excessively low osmolarity. An upper limit for the osmolarity may also be provided to prevent osmotic shrinkage stress. Preferably, the medium has an osmolarity of at least 0.1 osmol / L, or preferably at least 0.150 osmol / L. The osmolarity can be adjusted by solutes, such as salts, or other medium components, such as sugars or sugar alcohols. Preferably, an alkali metal salt such as Na₂ is used. + and / or K +The anion is preferably a halide, such as Ci, F, or I, a phosphate, or an acetate. Buffer components, such as Tris (tris(hydroxymethyl)aminomethane), are also possible. Furthermore, to protect the protoplasts, surfactant polymers can be added to the liquid medium for rotor-stator treatment. Surfactant polymers are described, for example, in WO 2013 / 156504 Al and preferably comprise uncharged polymers, such as emulsifiers, e.g., polyalkyl glycols, especially polyethylene glycol. The polymer is, in particular, a nonionic, water-soluble surfactant polymer. Preferably, it is non-denaturing to 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, hydroxypropyl methylcellulose, carboxymethyl cellulose, or hydroxyethyl cellulose, vinylpyrrolidone-vinyl acetate copolymer (copovidone), polyvinyl acetate, partially hydrolyzed polyvinyl alcohol, polyvinyl alcohol-polyethylene glycol copolymers, and mixtures thereof. Other possibilities include polysorbate, e.g., polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, and polyoxyethylene sorbitan mono-imitation.

[0049] Polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan tristearate, preferably polysorbate 80 (polyoxyethylene (20) sorbitan monooleate, tween ®80), polyoxyethylene (40)stearate. The surfactant polymer is preferably present in the medium at a concentration of at least 0.05 wt.%, particularly preferably at least 0.08%, at least 0.1% or at least 1.5% (all percentages in wt.%). The molecular weight of the surfactant 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, at least 30,000 Da. A molecular weight between 500 Da and 2,000,000 Da is particularly preferred, preferably between 1,000 Da and 200,000 Da or between 1,200 Da and 80,000 Da.

[0050] The liquid medium is preferably aqueous, in particular water or water mixtures compatible with cells. In particular, it can be a culture medium for (and with) plant cells, provided the plants are not separated from it beforehand.

[0051] Preferably, the expressed material is expressed before the plant cells are treated with the rotor-stator, so that it accumulates on the surface or in the apoplast of the plant cells. The plant cells can then be cultured and / or grown, e.g., in a medium under plant growth conditions (nutrient media, light), as is generally known (see 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 the average time a cell spends in culture.

[0052] Further processes that damage, lyse, or homogenize the protoplast are to be avoided. The cell wall is preferably not lysed, in particular not enzymatically, chemically, osmolytically, or by ultrasound. 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. While cell viability is not a particular factor in the process according to the invention, contamination of the liquid medium by components of the cell interior, especially the cytoplasm, should be avoided.

[0053] The present invention is further described by the following figures and examples, without being limited to these embodiments of the invention.

[0054] Figures:

[0055] Fig. 1: Determination of the energy input into water using the Ultraturrax T25 homogenizer (IKA / Staufen). (A)

[0056] Temperature profile in 1.51 liters of water at a rotational speed of 10,000 rpm. (B) Calculated energy input [kJ]. (C) Calculated

[0057] Energy input [kJ / kg].

[0058] Fig. 2: Determination of the energy input into water by homogenizer Shearpump FSP712VC-2.2kW-FU (Fristram / Hamburg).

[0059] (A) Temperature profile in 501 ml water at a rotational speed of 2800 rpm. (B) Calculated energy input [kJ]. (C) Calculated energy input [kJ / kg].

[0060] Fig. 3: Percentage release of biomass-bound product (moss-aGal) during treatment with a shear pump (dashed line) and Ultraturrax rod T25 (solid line). The shear pump treatment of the reactor culture was carried out in a volume of 501 liters. The treatment of the reactor culture with the Turrax rod T25 was carried out in a volume of 0.651 liters.

[0061] Fig. 4: Specific energy input by Ultraturrax rod T25 (A) and shear pump (B), both for 10 g / L dry biomass. Specific energy input according to the comparative example Ultraturrax rod T25 at 19,000 rpm with 1 g / L dry biomass (C). Fig. 5: Percentage release of biomass-bound product (moss-aGal) during treatment with shear pump (dashed line) and Ultraturrax rod T25 (solid line). The shear pump treatment of the reactor culture was carried out in a volume of 50 L. The treatment of the reactor culture with the Turrax rod T25 was carried out in a volume of 0.65 L. Biomass: 9.2 g / L (shear pump), 8.2 g / L (T25)

[0062] 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 up to 32.9 kJ / kg. Under osmotic stress conditions (demineralized water), they are detectable from approximately 10 kJ / kg. Target protein levels (moss-aGal) increase in saline media and under osmotic stress conditions in proportion to the energy input.

[0063] Fig. 7: Microscopic analysis of the T25 process in demineralized H2O. 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, cell integrity is still present, but there are clearly more particles, indicating the onset of cell disruption.

[0064] Fig. 8: Microscopic analysis of the T25 process in saline 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, cell integrity is maintained, but there are clearly more particles present, indicating the onset of cell disruption.

[0065] Fig. 9: Microscopic analysis of the shear pump process in saline medium (reactor culture). Up to an energy input of 18.41 kJ / kg, the moss cells retain their integrity. From an energy input of 27.20 kJ / kg, the integrity of the visible cells is preserved, but there are clearly more particles present, indicating the onset of cell disruption. Fig. 10: Microscopic analysis of the shear pump process in demineralized H₂O. Up to an energy input of 9.62 kJ / kg, the moss cells retain their integrity. From an energy input of 18.41 kJ / kg, the integrity of the cells is preserved, but there are clearly more particles present, indicating the onset of cell disruption.

[0066] Fig. 11: Microscopic analysis of moss cells in an ultrasonic process as comparative images of cell disruption. The cells lose their integrity after only a short time (1 minute of ultrasonic treatment). After just 3 minutes, only cell fragments and empty cell walls are visible. (Performance 100% in 50 ml solution).

[0067] Fig. 12: Comparative example: Determination of the energy input into water by homogenization tool T25 (IKA / Staufen) at high energy and a rotational speed of 19,000 rpm. (A) Temperature profile in 1 L water. (B) Calculated energy input [kJ]. (C) Calculated

[0068] Energy input [kJ / kg].

[0069] Fig. 13: Comparative example: Significantly faster product release at 19,000 rpm. Especially up to an energy input of 7 kJ / kg. Above an energy input of 84 kJ / kg, product losses occur due to high temperature and shear stress.

[0070] Examples:

[0071] Example 1: Determination of energy input using a Turrax rod and shear pump

[0072] Temperature was used as a measurable quantity to measure the energy input into aqueous media. For Turraxstab T25-S25N-18G (IKA Staufen), 1.51 L of H₂O was dispersed in a Dewar flask for 30 minutes at 10,000 rpm, and the temperature profile was measured. The ambient temperature during the experiment was between 20.5 and 20.7 °C. The specific heat capacity (4190 J / kg) was used to determine the temperature. _1 K _1 Energy input data were calculated for H2O. Energy input data were compared to the literature (Orellana-Escobedo et al., Plant Cell Rep. 2015, 34(3), 425-433) and performed analogously at 19000 rpm.

[0073] Using a shear pump (Shearpump FSP 712, Fristam Hamburg), 501 liters of H2O were circulated at 2800 rpm from a Nalgene reservoir through fabric-reinforced PVC tubing. The temperature was measured in the Nalgene reservoir using a temperature sensor (precision thermometer, G002.1, Carl Roth). The experimental setup was conducted in a room heated to 19 °C. Temperature losses to the environment were neglected in this experiment. The specific heat capacity (4190 J / kg) was used to determine the temperature. _1 K _1 Energy input data were calculated for water.

[0074] Example 2: Creating a moss culture

[0075] Moss production strains were axenically cultivated for 3-4 weeks in 2001 single-use bioreactor bags (Cellbag 200, GE Healthcare) on Wave™ Rocking Motion bioreactors (Wave200, GE Healthcare). Cultivation parameters included shaking frequencies of 19 to 25 rpm, a shaking angle of 9°, temperatures of 24-26°C, a gassing rate of 2 L / min, and an incoming air enrichment of 2% CO2. Four warm white LED modules (serial numbers 120268 to 120282, Infors AG) were installed above the bioreactor bag for illumination. Moss cultivation was carried out under continuous 24-hour lighting. SM07 (100 mM NaCl, 6.6 mM KCL, 2.0 mM MgS04x 7H20, 1.8 mM KH2P04, 20.4 mM Ca(N03)2x 4H20, 0.05 mM Fe Na-EDTA,

[0076] 4.9 mM MES, 0.1% w / v PEG4000, 100.26 mM H3BO3, 0.11 mM CoCl2x 6H2O, 0.1 mM CuS04x 5H2O, 5 mM KI, 85.39 mM MnCl2x 4H2O, 1.03 mM Na2MoO4x 2H2O, 0.11 mM NiCl2x 6H2O, 0.04 mM Na2SeO3x 5H2O, 0.039 mM Zn-acetate x 2H2O) supplemented with 1000 x Nitsch vitamins (Nitsch vitamin mixture, Duchefa, according to manufacturer's instructions) served as the mineral salt medium. The pH of 5-6 was controlled via WAVEPOD I and Pump20 (GE Healthcare) by automated addition of 0.25 M H₂SO₄ and 0.25 M NaOH. Expression of recombinant α-galactosidase (αGal or αGal A) was performed as described in WO 2016 / 146760 Al (“moss-αGal”).

[0077] Example 3: Release of moss-bound product and analytical methods. To analyze the time 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 Shearpump FSP 712. PL - Product concentration determinations (cPL) The released product was analyzed using moss-aGal-ELISA.

[0078] (Biogenes / Germany). The degree of cell disruption was detected via microscopic image analysis of moss cells (microscope: Axiovert 200 oper Stemi SV11 with camera AxioCam, software AxioSoft and cold light source KL 1500 LCD (Carl Zeiss)). A comparison to microscopic images of total disruption was performed via microscopic analysis of a 50 ml sample.

[0079] Ultrasound lysis (probe: UW2070, Bandelin; amplifier: HD 2070, Bandelin) at 100% power for 20 minutes was possible. At the molecular level, qualitative analysis for released product and the intracellular marker protein Rubisco was performed via Western blotting. Anti-aGal (H00002717-D01P, abnova) and anti-Rubisco (AS03037, Agrisera) were used as primary antibodies, and anti-Rabbit HRP (abcam) as a secondary antibody.

[0080] AS03037).

[0081] For an analysis of the relationship between released (c PL) and releasable moss bound product (c PX) 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 fragments, the product concentration was determined using ELISA (Biogenes).

[0082] In this study, aGal (α-galactosidase, also known as "moss-αGal"), a protein expressed in the apoplastic space, was determined.

[0083] Example 4: Results and Discussion

[0084] The energy inputs (as heat in kJ / kg, kg based on the liquid medium) of two different homogenizers – a Turrax rod T25-S25N-18G and a shear pump FSP 712 – into a culture medium (kg) were determined by temperature measurements (Figs. 1-5). The homogenizers were set to a low rotational speed to achieve a low heat output (in kJ / kg / min). The total heat over a specific period (0-60 min) was thus determined. The energy input into water was calculated using the specific heat capacity of H₂O [4.182 kJ / kg*K] based on the measured temperature. The heat (Figs. 1-3) and the specific heat based on the dry mass of the plant parts (Figs. 4-5) were then calculated.

[0085] Figures 3 and 5 show the product release of the desired protein (recombinant aGal from moss, "moss-aGal"), which binds to the surface and within the apoplast, respectively. The release increases with increasing treatment.

[0086] In comparative tests, the Turrax rod was operated at high heat output, specifically at 19,000 rpm (Fig. 4C, 12-13). The heat output was approximately 100 times greater than that of the gentler treatment at 10,000 rpm (see Fig. 4A and 4C). Operation at high heat output leads rapidly to product release but also to the destruction of cells (protoplasts), resulting in extracellular products being contaminated by components from the cell interior. These effects occur after just 1 minute of treatment.

[0087] Figure 6 shows the quality of the product release of the extracellular protein (moss aGal) and the intracellular protein Rubisco. Rubisco is present in plant cells at high concentrations and was therefore used as a highly sensitive marker for the release of cell contents. In experiments with physiological saline solution, an increasing release of Rubisco was observed at higher energy inputs (heat) above 32.9 kJ / kg. In a comparative experiment with demineralized water (DI water), Rubisco contamination occurred earlier, at approximately 10 kJ / kg, because osmolytic effects were added to the shear stress from the homogenizer. The different heat inputs were controlled by the treatment time.

[0088] Microscopic analysis of the cell aggregates after treatment reflects these results. Fig. 7 shows the results after treatment with the Ultraturrax rotor-stator in demineralized water; Fig. 8 after 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 shows the experiments in demineralized water with the shear pump. Here, comparable particles appear at about 20 kJ / kg.

[0089] For comparison with Figures 7-10, Figure 11 shows cell disruption by ultrasound. The cells lose their integrity after just 1 minute.

[0090] This implies that both the energy input per unit of time (intensity of rotor-stator rotation; heat output) and the absolute energy input (heat) – controlled in this experiment by the treatment duration – should be limited. These parameters can be used individually or in relation to the biomass (dry biomass, TBM). Suitable parameters for a gentle process that removes as many absorbed or apoplast-bound products as possible while largely preserving the protoplasts 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 at such low heat outputs range from 2 minutes to 150 minutes – depending on the rotation intensity – and are generally longer than the previously used short but intensive treatments.

Claims

Claims:

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

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

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

4. The method according to one of claims 1 to 3, characterized in that the heat input into the medium by rotation of the rotor is at least 0.2 kJ per kg of the liquid medium per minute, and / or the specific heat input into the medium is at least 0.02 kJ per kg of the liquid medium per minute and per g / l dry mass of the plant cells.

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

6. The method according to one of claims 1 to 5, characterized in that the rotor-stator is placed in a container with the medium.

7. The method according to any one of claims 1 to 6, wherein characterized in that the rotor-stator has an interior space which has at least one inlet and one outlet through which the liquid medium is continuously conveyed through the interior space.

8. The method according to any one of claims 1 to 7, characterized in that the stator has a volume of 10 cm³ 3 up to 1 m 3 limited, and / or that the amount of treated liquid medium is up to 50 kg, preferably 0.5 g to 50 kg.

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

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. The method according to any one of claims 1 to 10, characterized in that the rotor is operated at a speed of at most 15000 revolutions per minute, preferably 1000 to 15000 revolutions per minute.

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 wherein the stator has a comb structure.

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

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

15. The method according to any one of claims 1 to 14, wherein characterized by the fact that the plant cells are treated with the rotor-stator for 2 min to 150 min.

Images