Urease enzyme compositions with increased activity

Abstract

The invention relates to methods and compositions for improving urease activity in plants or parts thereof. A nickel-enriched culturing method is provided, which can yield plants or parts thereof with markedly increased urease activity. The urease can be isolated for various applications. The invention also encompasses further products related to this method.

Description

[0001] Urease enzyme compositions with increased activity

[0002] Field of the invention

[0003] The invention relates to methods and compositions for improving urease activity in plants or parts thereof. A nickel-enriched culturing method is provided, which can yield plants or parts thereof with markedly increased urease activity. The urease can be isolated for various applications. The invention also encompasses further products related to this method.

[0004] Background art

[0005] Patients with end stage kidney disease (ESKD) or severe acute kidney failure can undergo dialysis to replace kidney function. In conventional dialysis, patient fluids are generally dialysed against a dialysis fluid (referred to as dialysate), which is then discarded. During this process waste solutes from the patient fluid move towards the dialysate by diffusion and / or convection, often through a semipermeable membrane. Opposed to this “single pass” use of dialysate, home-dialysis devices wherein dialysis-fluid is recycled are available for patients with kidney-failure. The use of home-dialysis devices reduces the volume of dialysis fluid, as it is repeatedly regenerated and reused by removal of waste solutes from used dialysate. Efficient regeneration of dialysate reduces the need for large volumes of dialysis fluid, making dialysis more practically implemented, less resource-dependent, and reducing waste streams. Furthermore, home-dialysis is less burdensome for patients.

[0006] An enzyme that plays an important role in the regeneration of dialysate is urease (urea amidohydrolase, EC 3.5.1.5), a nickel-containing enzyme produced by plants, fungi, and bacteria that catalyzes the hydrolysis of urea into ammonia and carbon dioxide. To regenerate the dialysis fluid in a home-dialysis setup, a disposable filter system is used with included immobilized urease to hydrolyze urea. To commercialize this product, the efficiency of urease must be sufficient, and it should be producible in a cost effective way, on a scale that can meet demand.

[0007] Urease can be isolated from plants such as Jack Bean (Canavalia ensiformis) using known methods. Metal ions within the active site of ureases were found to contribute to the enzymic activity. Carter et al. (2009) achieved in vitro activation of bacterial ureases with manganese and cobalt. Dalton et al. (1985) showed that addition of nickel to a low-nickel soil that was pre-incubated with urea and glucose increased the activity of microbial ureases. This soil did not result in increased growth of plants.

[0008] There is an ongoing need of improved means of urea removal. There is a need of enzymes and enzyme compositions that are more efficient in removal of urea. There is a need of urease enzymes with increased activity. There is a need of cost-effective urease sources. There is a need of improved sources from which urease can be purified or isolated. Summary of the invention

[0009] The inventors found that beans of Canavalia plants with a strongly increased urease activity could be obtained from plants that were cultured in a nickel-supplemented environment. Other means for obtaining such improved beans, such as treatment of Canavalia beans by soaking them in nickel-holding media, were not successful. Accordingly the invention provides a method for providing a Canavalia plant or part thereof, the method comprising the steps of: i) providing a growth substrate, wherein the growth substrate comprises 0.01 - 0.4 mmol Ni2+per litre; ii) culturing a Canavalia plant on the growth substrate provided in step i); iii) optionally harvesting the plant of step ii) or a part thereof.

[0010] Preferably the method comprises the steps of: i) providing soil, wherein the soil comprises 0.01 - 0.4 mmol Ni2+per kg soil; ii) culturing a Canavalia plant in the soil provided in step i); iii) optionally harvesting the plant of step ii) or a part thereof. Preferably the Canavalia plant is a Canavalia ensiformis plant. Preferably step i) comprises mixing soil with a source of Ni2+, wherein the source of Ni2+is preferably a nickel salt such as NiSC . Preferably step ii) comprises planting a Canavalia seed below the surface of the soil, preferably 1 -10 cm below the surface of the soil, more preferably 2-5 cm below the surface of the soil. Preferably the culturing of step ii) occurs at a temperature of 16-26°C, preferably at 20-22°C and / or encompasses exposure to light for 12-20 hours per day, preferably for 15-17 hours. In preferred embodiments no fertilizer is applied to the soil during step ii) before the Canavalia plant develops flowers. Preferably the plant or part thereof in step iii) is harvested and is a seed, a flower, a leaf, a stem, a root, a shoot, a nodule, or a pod, preferably a seed.

[0011] Preferably step ii) comprises applying a fertilizer to the soil after 10 - 14 weeks of culturing, preferably after 12 weeks of culturing. Preferably the fertilizer comprises about 0.5 mM / L NH4+and / or about 3 mM / L K+and / or about 1 .8 mM / L Ca2+and / or about 1 mM / L Mg2+and / or about 5.5 mM / L NOs- and / or about 1 .3 mM / L SO42-and / or about 1 mM / L H2PO4- and / or about 9 pMol / L Mn and / or about 1.8 pMol / L Zn and / or about 44 pMol / L B and / or about 0.5 pMol / L Cu and / or about 0.5 pMol / L Mo and / or about 25 pMol / L Fe.

[0012] Also provided is a method for preparing urease or flour or meal comprising urease, the method comprising the steps of: a) providing soil comprising 0.01 - 0.4 mmol Ni2+per kg soil; b) culturing a Canavalia plant in the soil provided in step a); c) harvesting beans of the cultured Canavalia plant of step b); d) processing the beans of step c) into flour or meal; e) optionally extracting urease from the flour or meal of step d).

[0013] Also provided is soil comprising 0.01 - 0.4 mmol Ni2+per kg soil, preferably 0.038 - 0.38, more preferably 0.152 - 0.342, even more preferably 0.19 - 0.304 mmol Ni2+per kg soil. Preferably the soil comprises at most 1.8 gram glucose per kg dry soil, preferably at most 1.5 gram glucose per kg dry soil. Also provided is the use of such soil, for culturing a plant. Also provided is a plant or part thereof, wherein the plant was cultured in such soil. Also provided is bean meal or bean flour obtainable by a method as described above, or derived from a plant or part thereof as described above.

[0014] Description of embodiments

[0015] The inventors found that beans of Canavalia plants with a strongly increased urease activity could be obtained from plants that were cultured in a nickel-supplemented environment. Other means for obtaining such improved beans, such as treatment of Canavalia beans by soaking them in nickel-holding media, were not successful. Accordingly the invention provides a method for providing a Canavalia plant or part thereof, the method comprising the steps of: i) providing a growth substrate, wherein the growth substrate comprises 0.01 - 0.4 mmol Ni2+per litre; ii) culturing a Canavalia plant on the growth substrate provided in step i); iii) optionally harvesting the plant of step ii) or a part thereof.

[0016] Such a method can be referred to herein as a culturing method according to the invention.

[0017] Growth substrates for plants are commonly known. Examples of a growth substrate are soil or a hydroponics system. A hydroponics system generally comprises a water-based mineral nutrient solution and optionally a growing support material for hydroponics. In some embodiments the substrate is a hydroponics system comprising a growing support material for hydroponics. In hydroponics a growing support material is provided in combination with water-based mineral nutrient solutions in an artificial environment. Plants may grow freely with their roots exposed to the nutrient solutions, or the roots may be mechanically supported by a growing support material.

[0018] A growing support material for hydroponics generally is an inert medium. Growing support materials are known in the art, and a skilled person is able to select a suitable material. Examples are mineral wool (such as rock wool), expanded clay aggregate, growstones (generally made from glass waste), coconut coir, rice husks, perlite, vermiculite, pumice, sand, gravel, wood fiber, excelsior (wood wool), sheep wool, brick shards, and polystyrene shapes such as polystyrene packing peanuts. Mineral wool is preferred, in particular rock wool.

[0019] Water-based mineral nutrient solutions are known in the art, and a skilled person is able to select a suitable solution. In the present invention the hydroponics system is characterized in that it is enriched in Ni2+per litre. It is convenient when the water-based mineral nutrient solution comprises this nickel, so that conventional growing support materials can be used. Furthermore it is convenient to prepare nickel-enriched solutions, as compared to preparing nickel-enriched growing support material.

[0020] Accordingly in preferred embodiments the growth substrate is a hydroponics system wherein the growing support material for hydroponics is preferably as defined above, wherein the water-based mineral nutrient solution comprises 0.001 - 0.5 mmol Ni2+per litre, more preferably 0.005 - 0.4 mmol Ni2+per litre, even more preferably 0.01 - 0.4, still more preferably 0.015 - 0.3, still more preferably 0.02 - 0.2, even more preferably 0.025 - 0.25, still more preferably 0.03 - 0.2 mmol Ni2+per litre. In preferred embodiments the growth substrate is soil. In these embodiments is provided a method for providing a Canavalia plant or part thereof, the method comprising the steps of: i) providing soil, wherein the soil comprises 0.01 - 0.4, preferably 0.038 - 0.380 mmol Ni2+per kg soil; ii) culturing a Canavalia plant in the soil provided in step i); iii) optionally harvesting the plant of step ii) or a part thereof.

[0021] In some embodiments step iii) is not performed. In some embodiments the harvesting of step iii) is not optional.

[0022] Canavalia plant

[0023] Plants of the Canavalia genus form a genus within the family of Fabaceae (legumes). Canavalia are commonly referred to as jack-beans, Jack beans, or similar. Jack-beans have a pantropical distribution and are native to many regions across the world. Several species of Jack-bean are legume crops, including common jack-bean (C. ensiformis), sword bean (C. gladiata) and C. cathartica. Jack-beans such as C. ensiformis can also make a beneficial weed- and pathogensuppressing living mulch. Jack-beans are used as a source of various specialty substances, such as the lectin concanavalin A, or urease enzyme. Thus plants of the Canavalia genus are commonly cultivated.

[0024] Examples of plants of the Canavalia genus are Canavalia acuminata, Canavalia africana, Canavalia altipendula, Canavalia aurita, Canavalia bicarinate, Canavalia boliviana, Canavalia bonariensis, Canavalia brasiliensis, Canavalia campylocarpa, Canavalia cathartica, Canavalia centralis, Canavalia concinna, Canavalia dolichothyrsa, Canavalia dura, Canavalia ensiformis, Canavalia eurycarpa, Canavalia forbesii, Canavalia galeata, Canavalia glabra, Canavalia gladiata, Canavalia grandiflora, Canavalia haleakalaensis, Canavalia hawaiiensis, Canavalia hirsutissima, Canavalia iaoensis, Canavalia kauaiensis, Canavalia kauensis, Canavalia lineata, Canavalia macrobotrys, Canavalia macropleura, Canavalia madagascariensis, Canavalia makahaensis, Canavalia mattogrossensis, Canavalia matudae, Canavalia microsperma, Canavalia mollis, Canavalia molokaiensis, Canavalia munroi, Canavalia napaliensis, Canavalia nitida, Canavalia nualoloensis, Canavalia obidensis, Canavalia oxyphylla, Canavalia palmeri, Canavalia papuana, Canavalia parviflora, Canavalia peninsularis, Canavalia picta, Canavalia piperi, Canavalia plagiosperma, Canavalia pubescens, Canavalia raiateensis, Canavalia ramosii, Canavalia regalis, Canavalia rockii, Canavalia rosea, Canavalia rutilans, Canavalia sanguinea, Canavalia saueri, Canavalia septentrionalis, Canavalia sericea, Canavalia sericophylla, Canavalia stenophylla, and Canavalia villosa. Preferred examples are Canavalia brasiliensis, Canavalia cathartica, Canavalia ensiformis, Canavalia gladiata, Canavalia molokaiensis, Canavalia napaliensis, Canavalia plagiosperma, Canavalia pubescens, and Canavalia rosea. More preferred plants of the Canavalia genus are Canavalia cathartica, Canavalia ensiformis, Canavalia gladiata, Canavalia molokaiensis, Canavalia napaliensis, Canavalia pubescens, and Canavalia rosea. Even more preferred plants of the Canavalia genus are Canavalia cathartica, Canavalia ensiformis, and Canavalia gladiata. The most preferred plant of the Canavalia genus is Canavalia ensiformis, and accordingly in preferred embodiments of the culturing method according to the invention the Canavalia plant is a Canavalia ensiformis plant.

[0025] The method is for providing a Canavalia plant or part thereof. The plant can be a whole plant, which can for instance be cultured further. The whole plant can also be processed further, such as into parts. Parts of a plant can be a seed, a flower, a leaf, a stem, a root, a shoot, a nodule, or a pod, preferably a seed, which for legumes such as Canavalia plants are also called beans. In preferred embodiments of the method, the plant or part thereof in step iii) is harvested and is a seed, a flower, a leaf, a stem, a root, a shoot, a nodule, or a pod, preferably a seed.

[0026] A particularly preferred part of a plant is a bean. In such embodiments the method is for providing a Canavalia bean. It is highly preferred that the method is for providing Canavalia beans, more preferably Canavalia ensiformis beans, which can be provided in pods. Beans can be processed into meal or flour, which is of particular interest. Accordingly the culturing method according to the invention can be for providing bean meal or flour of Canavalia beans, preferably of Canavalia ensiformis beans. In such a method step iii) is not an optional step, and comprises harvesting beans, and further comprises the subsequent step of processing said beans into bean meal or flour.

[0027] As another aspect, the invention is also directed to a method of producing a pod comprising obtaining a plant according to the instant invention and harvesting a pod from the plant. In embodiments, obtaining a plant of the invention comprises growing the plant to produce a pod. In one embodiment, the method further comprises processing the pod to obtain a bean.

[0028] The inventors found that beans of Canavalia plants with a strongly increased urease activity could be obtained from plants that were cultured in a nickel-supplemented environment. The invention therefore provides a plant or part thereof, wherein the plant was cultured in the soil as described later herein. Preferably, the plant is a Canavalia plant as described above, more preferably a Canavalia ensiformis. Highly preferred are beans of such plants, particularly of such Canavalia plants, more preferably of Canavalia ensiformis plants. Also highly preferred is bean meal or flour obtained from beans of such plants.

[0029] Preferred beans weigh at least 800 mg, more preferably at least 900, 1000, 1100, 1200,

[0030] 1300, or 1400 mg. Beans weiging at least 1200, preferably at least 1250 mg were obtained when

[0031] 30-70 mg / kg Ni was used during culturing. Beans weighing at least 1300 mg were obtained when

[0032] 50-70 mg / kg Ni was used during culturing. Beans weighing at least 1400 mg were obtained when about 50 mg / kg Ni was used during culturing.

[0033] Also preferred are pods of such plants. The pods comprise beans. Preferred pods have a total specific urease activity per pod of at least 30.000, preferably at least 32.000, more preferably at least 33.000, still more preferably at least 34.000, more preferably at least 36.000, more preferably at least 38.000, more preferably at least 40.000, more preferably at least 42.000, more preferably at least 44.000, most preferably at least 46.000.

[0034] Such plants, and particularly such beans, and also such bean meal or flour, can be identified by high urease activity. Urease activity can be determined using any known urease assay. A preferred method for determining the specific activity of urease is by quantification of the amount of ammonia formed in time (via the Berthelot reaction) when the material is placed in a aqueous 100 mM potassium phosphate buffer at pH 7.5 in the presence of 15 mM urea at room temperature (about 20 °C), more preferably it is as described in Example 2. It is to be understood that urease refers to enzymes of EC 3.5.1 .5, belonging to the superfamily of amidohydrolases and phosphotriesterases. Urease catalyzes the hydrolysis of urea into carbon dioxide and ammonia. The urease is plant urease, more particularly Canavalia urease, or as otherwise specified.

[0035] Plants or parts thereof preferably have a specific activity of urease per mg of dry weight of at least 2.25 U / mg, more preferably at least 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1 , 3.2, 3.25, 3.3,

[0036] 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or more preferably at least 4 U / mg, even more preferably at least 4.2, still more preferably at least 4.5, even more preferably at least 5, most preferably at least 5.4 U / mg. More preferably said activity is activity in jack bean beans, more preferably in Jack bean bean meal or flour. Most preferably it is specific urease activity in Units / mg dry weight seed. In some embodiments is provided bean meal or flour of Canavalia ensiformis beans with a specific urease activity per mg of dry weight of at least 2.25 U / mg, more preferably at least 3 U / mg, even more preferably at least 4 U / mg, even more preferably at least 4.2, still more preferably at least

[0037] 4.5, even more preferably at least 5, most preferably at least 5.4 U / mg.

[0038] Also provided is a method for preparing urease or flour comprising urease or meal comprising urease, the method comprising the steps of: a) providing a growth substrate, preferably soil, comprising 0.01 - 0.4 mmol Ni2+per kg soil; the soil is preferably as defined elsewhere herein; b) culturing a Canavalia plant in the soil provided in step a); the culturing is preferably as defined elsewhere herein; c) harvesting beans of the cultured Canavalia plant of step b); d) processing the beans of step c) into flour or meal; e) optionally extracting urease from the flour of step d).

[0039] The growth substrate is preferable soil and more preferably soil comprising 0.038 - 0.380 mmol Ni2+per kg soil; even more preferably 0.152 - 0.380 mmol Ni2+per kg soil. Harvesting of the beans can be done per bean or per pod, a skilled person is well capable of harvesting beans as described in step c). Processing the beans of step c) into flour or meal can also be performed using any known method. Preferably beans are ground or milled, such as using mill stones or a burr grinder. After grinding the resulting powder, generally meal, can be sieved, such as on a 1 mm raster, to obtain a flour.

[0040] Extraction of the urease as in step e) can be performed using water, such as de-ionized water. Extraction can be performed for any suitable amount of time, such as 0.2-5 hours, preferably 0.5-3 hours, more preferably 1 hour. Extraction can be performed at any suitable temperature, preferably room temperature, such as about 10 to about 30 °C, more preferably about 15 to about 25, such as about 20 °C. Preferably the mixture is settled after extraction, after which solids are discarded. The resulting urease solution can be used as such, or can be lyophilised to provide a dry urease composition. The urease has high Ni-occupation, preferably having at least 25, 30, 35, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100% of its theoretical binding sites occupied by a nickel atom.

[0041] The invention thus provides bean meal or bean flour obtainable by a method as described above, or derived from a plant or part thereof as described above. This bean meal or bean flour preferably comprises urease as described above. It preferably has a specific urease activity per mg dry weight of at least least 4 U / mg, even more preferably at least 4.2, still more preferably at least 4.5, even more preferably at least 5, most preferably at least 5.4 U / mg. Also provided are Canavalia seeds or beans with a specific urease activity per mg dry weight of at least least 4 U / mg, even more preferably at least 4.2, still more preferably at least 4.5, even more preferably at least 5, most preferably at least 5.4 U / mg.

[0042] Nickel-supplemented soil

[0043] In step i) soil is provided, wherein the soil comprises 0.01 - 0.4, preferably 0.038 - 0.380 mmol Ni2+per kg soil. Soil is commonly known and commonly available, and a skilled person can select suitable soil for any purpose, including for culturing plants such as Canavalia plants. Soil is sometimes referred to as earth, and is a mixture of organic matter, minerals, gases, liquids, and organisms that can together support the life of plants.

[0044] A preferred soil is potting soil. Any suitable potting soil can be used, as well as any other suitable soil, which may be referred to as horticultural soil or as cultivation soil. Preferably the pH of the soil is about 5-7. More preferably about 5.1-6.5, still more preferably about 5.1 -6.4, 5.2-6.3, 5.3-6.2, 5.4-6.1 , 5.5-6, 5.5-5.9, 5.6-5.8, most preferably about 5.7. EC is preferably about 0.6-1 , more preferably about 0.7-0.9, most preferably about 0.8. Soil preferably comprises some amount of peat, such as about 0.5-1 ENm3, more preferably 0.7-0.9, most preferably 0.8. Soil preferably comprises a structural component such as bark or perlite, for instance 0.1 -0.3, preferably 0.15- 0.25, such as about 0.2 ENm3. Soil can comprise clays, such as about 30-50 kg / ENm3, more preferably about 35-45, most preferably about 40 kg / ENm3. Soil preferably has an EN-factor 1 .23. Soil preferably comprises lime +MgO such as about 2-3 kg / ENm3, preferably about 2.2-2.7, more preferably about 2.45 kg / ENm3. Soil preferably comprises PG-mix 15-10-20 + trace such as about 0.6-1 kg / ENm3, preferably 0.7-0.9, most preferably about 0.8 kg / ENm3.

[0045] The soil comprises 0.01 - 0.4, preferably 0.038 - 0.380 mmol Ni2+per kg soil. Nickel content can be determined using any known method, such as ICP-AES, or an assay can be used based on the reaction of Ni2+with mercaptoethanol in borate buffer to form a complex with strong absorbance bands from about 300 to 600 nm. Preferably the soil comprises 0.038 - 0.38, more preferably 0.152 - 0.342, even more preferably 0.19 - 0.304 mmol Ni2+per kg soil. Such soil is provided as part of the invention. Such soil is useful for application in the cultivation method according to the invention.

[0046] The soil preferably comprises at least 0.01 , 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1 , 0.105, 0.11 , 0.115, 0.12, 0.125, 0.13, 0.135, 0.14, 0.145, 0.15, 0.155, 0.16, 0.165, 0.17, 0.175, 0.18, 0.185, 0.19, 0.195, 0.2, 0.21 , 0.22, 0.23, 0.24, 0.25, O.,26 0.27, 0.28, 0.29, 0.3, 0.31 , 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, or 0.39 mmol Ni2+per kg soil. The soil preferably comprises at most 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1 , 0.105, 0.11 , 0.1 15, 0.12, 0.125, 0.13, 0.135, 0.14, 0.145, 0.15, 0.155, 0.16, 0.165, 0.17, 0.175, 0.18, 0.185, 0.19, 0.195, 0.2, 0.21 , 0.22, 0.23, 0.24, 0.25, O.,26 0.27, 0.28, 0.29, 0.3, 0.31 , 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39 or 0.4 mmol Ni2+per kg soil.

[0047] In preferred embodiments the soil comprises 0.01 - 0.4, more preferably 0.02 - 0.38, more preferably0.035 - 0.36, more preferably 0.038 - 0.34, more preferably 0.04 - 0.32, more preferably 0.06 - 0.3, more preferably 0.08 - 0.28, more preferably 0.1 - 0.26, more preferably 0.12 - 0.24, more preferably 0.14 - 0.22, more preferably 0.16 - 0.2, most preferably about 0.18 - 0.19 mmol Ni2+per kg soil. Further preferred soil comprises 0.035 - 0.35 mmol Ni2+per kg soil, more preferably 0.1 - 0.36, even more preferably 0.1 - 0.28, still more preferably 0.175 - 0.28 mmol Ni2+per kg soil. Further more preferred soil comprises 0.038 - 0.342 mmol Ni2+per kg soil, more preferably 0.114 - 0.342, even more preferably 0.114 - 0.266, still more preferably 0.19 - 0.266 mmol Ni2+per kg soil.

[0048] Provision of such soil can be achieved through any suitable means. It can be convenient that step i) comprises mixing soil with a source of Ni2+, wherein the source of Ni2+is preferably a nickel salt such as NiSO4. Nickel salts are particularly useful as sources of Ni2+, especially Ni(ll) salts. Nickel(ll) forms compounds with all common anions, including sulfide, sulfate, carbonate, hydroxide, carboxylates, and halides. Such salts such as nickel(ll) sulfate can be hydrates, such as hexa- and heptahydrate. Common salts of nickel, such as chloride, nitrate, and sulfate, readily dissolve in water. Examples of nickel(ll) salts are NiF2, NiCh, NiBr2, Nil2, Ni(CN)2, Ni(SCN)2, NiO, Ni(OH)2, NiCO3, NiSO4, Ni3(PO4)2, NiCrO4, NiTiO3, NiSeO4, NiS, NiSe, Ni(CIO4)2, Ni(NO3)2, Ni(NO2)2, and Ni(acac)2. Preferred are nickel salts with a horticulturally acceptable anion, such as NiCh, Ni(OH)2, NiCO3, Ni(NO3)2, and NiSO4, more preferably NiCh, Ni(NO3)2, and NiSO4. NiSO4is particularly preferred. Salts can be used as hydrates, such as NiSO4hexahydrate.

[0049] Additional nickel can be added during cultivation, for instance through addition of additional soil containing a source of Ni2+, through addition of a source of Ni2+to the water used for irrigation, or through spraying of the biomass with water comprising a source of Ni2+.

[0050] It can be preferred that the soil essentially consists of soil and the source of Ni2+. For instance, in a preferred embodiment is provided soil as described above, wherein the soil comprises at most 2, preferably at most 1 .8 gram glucose per kg dry soil, more preferably at most 1 .5 gram glucose per kg dry soil, even more preferably at most 1 .3, still more preferably at most 1 gram glucose per kg dry soil. Most preferably the soil does not comprise glucose.

[0051] Also provided is the use of soil as described above, for culturing a plant. The plant is preferably as described above. The use is preferably as described below.

[0052] Culturing plants

[0053] In step ii) of the cultivation method, a Canavalia plant is cultured, on the growth substrate such as in the soil provided in step i). This culturing can be performed in any suitable way. To culture, seedlings can be planted, which can then be further cultivated. Alternatively, beans can be planted, which can then be cultivated into plants. Cultivation can be performed in any suitable system, such as in fields or in greenhouses, in an open field or in pots. For convenience it can be preferred to culture in pots, preferably in a greenhouse, which can also be referred to as a glasshouse.

[0054] Because beans, which are the seeds of legumes such as Canavalia plants, are so easily handled, it is preferred to perform step ii) wherein it comprises planting a Canavalia seed below the surface of the soil, preferably 1 -10 cm below the surface of the soil, more preferably 2-5 cm below the surface of the soil. Most preferably the seeds are planted about 3-4 cm below the surface of the soil. In preferred embodiments the Canavalia seed is soaked in water prior to its planting, preferably at least 4 hours, more preferably at least 12 hours, even more preferably at least 24 hours. Soaking for more than about 24 hours is not preferred.

[0055] Good culturing conditions for Canavalia plants are commonly known. Preferably culturing occurs at a temperature of 16-26°C, preferably at 20-22°C and / or encompasses exposure to light for 12-20 hours per day, preferably for 15-17 hours. In some embodiments the culturing occurs at a temperature of 16-26°C, preferably at 20-22°C or as indicated below. In some embodiments the culturing encompasses exposure to light for 12-20 hours per day, preferably for 15-17 hours, or as indicated below.

[0056] The culturing temperature is preferably kept within the indicated window throughout cultivation, and the temperature is preferably not below 16 °C, more preferably not below 17 °C, even more preferably not below 18 °C, still more preferably not below 19 °C, most preferably not below 20 °C. Preferably the temperature is not above 27 °C, more preferably not above 26 °C, still more preferably not above 25 °C, still more preferably not above 24 °C, still more preferably not above 23 °C, most preferably not above 22 °C.

[0057] Exposure to light is beneficial to Canavalia plant growth. Exposure is preferably for 12-20 hours per day, more preferably for 13-19, even more preferably 14-18, still more preferably 15-17 hours per day, such as about 16 hours per day. Exposure is preferably in contiguous time periods, alternating light and dark. Exposure can be conveniently provided by any suitable horticultural lighting equipment. Average light is preferably about 250-550 W / m2, more preferably 300-500 W / m2, even more preferably 350-450 W / m2, most preferably about 375-425 W / m2, such as about 400 W / m2.

[0058] The plants may reach a height of more than 3 m in a glasshouse environment. Good results were obtained when plants were shortened, preferably shortened periodically such as every four weeks, every three weeks, every two weeks, every week, every 5 days, every 4 days, every 3 days, every 2 days, or daily. Plants are preferably shortened to 2 meters, more preferably to 1 .5 meters.

[0059] Fertilizer can be used to promote plant growth. Fertilizer can be mixed in with the soil prior to culturing, or can be added to the soil during culturing, for instance by watering or automated dripping. In some embodiments no fertilizer is used. It was found that good results were obtained when no fertilizer is applied to the soil before the Canavalia plant develops flowers. Accordingly in preferred embodiments no fertilizer is applied to the soil during step ii) before the Canavalia plant develops flowers. More preferably no fertilizer is applied to the soil during step i) or and during step ii) before the Canavalia plant develops flowers. Preferably, soil is nitrogen-poor soil. More preferably soil is nitrogen-poor soil at least before the Canavalia plant develops flowers.

[0060] Good results were obtained when fertilizer was applied after the Canavalia plant develops flowers. Fertilizer was not applied before. Accordingly in preferred embodiments step ii) comprises applying a fertilizer to the soil after the Canavalia plant develops flowers. In this light, in preferred embodiments step ii) comprises applying a fertilizer to the soil after 9 - 15, preferably 10 - 14, more preferably 11 -13 weeks of culturing, most preferably after 12 weeks of culturing. More particularly in preferred embodiments step ii) comprises applying a fertilizer to the soil for the first time after the Canavalia plant develops flowers. In other preferred embodiments step ii) comprises applying a fertilizer to the soil for the first time after 4 - 15, preferably 8 - 14, more preferably 12 - 13 weeks of culturing, most preferably after 12 weeks of culturing. In other embodiments step ii) comprises applying a fertilizer to the soil for the first time after 4 - 12, preferably 8 - 12, more preferably 12 weeks of culturing. Accordingly, it is highly preferred that no fertilizer is applied during the first 4, preferably 8, more preferably 12 weeks of culturing. In these embodiments, fertilization can continue until harvest of the plant or part thereof.

[0061] Fertilizer is preferably provided by watering or automated dripping. Suitable fertilizer compositions are commonly known. There is a preference for fertilizers with a low nitrogen content such as for instance a Hoagland solution, more preferably Hoagland 0.5. A fertilizer preferably comprises at most 10 mM / L nitrogen, more preferably at most 8 mM / L nitrogen, even more preferably at most 6 mM / L nitrogen.

[0062] Preferably the fertilizer comprises about 0.5 mM / L NH4+and / or about 3 mM / L K+and / or about 1 .8 mM / L Ca2+and / or about 1 mM / L Mg2+and / or about 5.5 mM / L NOs- and / or about 1 .3 mM / L SO42- and / or about 1 mM / L H2PO4- and / or about 9 pMol / L Mn and / or about 1 .8 pMol / L Zn and / or about 44 pMol / L B and / or about 0.5 pMol / L Cu and / or about 0.5 pMol / L Mo and / or about 25 pMol / L Fe. More preferably the fertilizer comprises about 0.5 mM / L NH4+, about 3 mM / L K+, about 1.8 mM / L Ca2+, about 1 mM / L Mg2+, about 5.5 mM / L NOs-, about 1.3 mM / L SO42-, about 1 mM / L H2PO4', about 9 pMol / L Mn, about 1.8 pMol / L Zn, about 44 pMol / L B, about 0.5 pMol / L Cu, about 0.5 pMol / L Mo, and about 25 pMol / L Fe.

[0063] Harvesting plants

[0064] In step iii) the plant of step ii) or a part of the plant of step ii) is harvested. Step iii) is optional. In preferred embodiments step iii) is not optional and the plant or part thereof is harvested. Accordingly in preferred methods the plant or part thereof in step iii) is harvested and is a complete plant, a seed, a flower, a leaf, a stem, a root, a shoot, a nodule, or a pod, preferably a pod or a seed, most preferably a seed.

[0065] Typically after 12-14 weeks the plant develops pink flowers in bunches of about 6-8 flowers. Per bunch 2 or 3 flowers develop into pods which develop over a period of 6-8 weeks into green pods up to a length of 30-35 cm containing 11-16 seeds per pod. After full ripening of the pods, the pods color to yellow-brown. It was found that the seeds have a high specific urease activity, and these seeds, or pods comprising these seeds, are provided as part of the invention.

[0066] The plants, or the parts of the plants, can be further processed as described earlier herein, preferably to prepare bean meal or bean flour. Harvesting of the beans can be done per bean or per pod, a skilled person is well capable of harvesting beans or pods. Processing the beans into flour or meal can also be performed using any known method. Preferably beans are ground or milled, such as using mill stones or a burr grinder. Grinding or milling is preferably at a low temperature, such as at room temperature, preferably about 15 to about 25, such as about 20 °C. After grinding the resulting powder, generally meal, can be sieved, such as on a raster such as a 1 mm raster or a 3.5 mm raster, to obtain a flour.

[0067] An urease composition can be provided by extraction from the meal or flour. Extraction of the urease can be performed using water, such as de-ionized water. Extraction can be performed for any suitable amount of time, such as 0.2-5 hours, preferably 0.5-3 hours, more preferably 1 hour. Extraction can be performed at any suitable temperature, preferably room temperature, such as about 10 to about 30 °C, more preferably about 15 to about 25, such as about 20 °C. Preferably the mixture is settled after extraction, after which solids are discarded. The resulting urease solution can be used as such, or can be lyophilised to provide a dry urease composition. The urease has high Ni-occupation, preferably having at least 25, 30, 35, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100% of its theoretical binding sites occupied by a nickel atom.

[0068] Examples

[0069] Example 1 - Introduction

[0070] Introduction

[0071] Jack Bean (Canavalia ensiformis) is grown mainly as green manure and as cover crop in soil erosion control programs, because its deep root system can find water during drought. It is mostly intercropped with bananas, cacao, cassava, citrus, coconut, coffee, maize, pineapple, sweet potatoes, and tobacco, to improve the soil. It can serve as supplementary food for ruminants (cudchewing animals), though it should be introduced gradually as a small percentage of the total diet because of toxins. Some Asian cultures eat the young green pods and seeds, but only after thorough cooking. In the open field, Jack Bean grows up to 2 m high with 8-20 cm long trifoliate leaves and a strong root system. The flowers are pink, mauve or white with a red base. The pods are up to 36 cm long and typically contain about 12 to 16 ellipsoid seeds, 1-2 cm long. Geographic location of Jack Bean

[0072] The native origin of this crop lies in Mexico, Ecuador and Dominican Republic, but has been introduced in a diversity of countries for cultivation in tropical areas on both hemispheres. Although in general there is no commercial market for this crop, as it is mainly used to replenish the soil in between other crops in areas where the soil is generally poor. The produced biomass is mostly used as fodder. The main regions where Jack Bean can be acquired nowadays can be found in Brazil (Minas Gerais), Kenia (Garissa), China (Guanxi), Malaysia (Pahang), Papua New Guinea (Morobe), and Australia (Queensland).

[0073] Urease

[0074] One of the interesting applications of the Jack bean seeds is the abundant presence of the enzyme urease. Urease (urea amidohydrolase, EC 3.5.1.5) is a nickel-containing enzyme produced by plants, fungi, and bacteria and catalyzes the hydrolysis of urea into ammonia and carbon dioxide. Urease is one of the most proficient enzymes known because the uncatalyzed hydrolysis of urea is undetectably low. Urease is of historical importance in biochemistry as it was the first enzyme ever to be crystallized. The observation of nickel in urease’s active site was the first indication of a biological role for this metal ion. The active site of urease consists, besides the two nickel atoms, of one carbamylated lysine, four histidine residues and one aspartate residue. It is the coordination of urea to these nickel atoms that allow hydrolysis of urea, which makes these metal ions important for the enzyme activity. Next to the amino acid residues that compose the active site itself, other residues, including a conserved cysteine, form the “mobile flap”, which works as a spatial gate for the substrate to enter the cleft where the actual hydrolysis occurs. This flap is composed by a helix-turn-helix motif and is responsible for substrate influx and product efflux in urease. A conserved histidine residue controls the motion of the gate. In the catalysis, amino acid residues of the mobile flap participate in the substrate binding, mainly through H- bonds, thereby stabilizing the catalytic transition state and accelerating the reaction.

[0075] Application of urease

[0076] One of the commercial applications of the enzyme urease is in regenerating dialysis-fluids that are recycled in for example home-dialysis devices for patients with kidney-failure. Dialysis removes formed urea as major nitrogen degradation product from the blood. To regenerate the dialysis fluid in a home-dialysis setup, a disposable filter system is used with included immobilized urease to hydrolyze urea. To commercialize this product the urease needs to be produced in a cost effective way and on a scale that meets the demand.

[0077] Relevant for the application of urease as essential component in a disposable for homedialysis of kidney patients is that the activity of the enzyme is sufficiently high to achieve the timely break-down of urea adequately to allow regeneration of the dialysis-fluid. Growing Jack beans with high specific activity would contribute to making the urease source more cost effective. However, the specific activity of the enzyme depends strongly on the geographic origin of the Jack bean, as is illustrated in Table 1. To establish these activities the protein was generally extracted from the seeds using the protocol essentially described in Example 2. The specific activity of the extracted protein was measured as described in Example 2, and the dry weight was also determined as described in Example 2.

[0078] Table 1 . Ranges of typical specific activities of urease per mg of dry weight of a Jack Bean seed extract derived from different geographic origins.

[0079] Also a commercially available Jack Bean seed (Sigma-Aldrich) was analyzed in a similar way, showing a specific activity in the range of 2.0-2.2 Units / mg dry weight. The origin of these seeds is not disclosed.

[0080] The present invention describes a method of increasing the specific activity of Jack Bean urease by introducing additional Ni2+-ions in the fertilization of the plant during growth. In doing so, an enzyme activity can be obtained which is significantly higher than can be found in natural sources, and thereby makes the application of this enzyme for the home-dialysis disposable more cost effective.

[0081] It was previously reported that when Jack bean seeds were grown hydroponically in the absence of added Ni2+the seeds had a low in urease activity (<10% of parent seeds). As the regular potting soil that was used contained trace elements of the normally abundant metal ions, it was suggested that metal ions other than Ni2+cannot substitute for Ni2+in the formation of normally active urease (Dixon et al., 1980).

[0082] Ni2+is the 17th element recognized as essential for plant growth and development (Liu, 2001). In most plant species, Ni2+deficiency is rarely observed because only very minute amounts of this metal are needed for normal metabolism, and the adequate range between limiting and toxic concentrations is exceptionally large compared to other heavy metals. Nickel requirement is the lowest of all essential elements at < 0.5 mg kg1of dry weight soil (Patra et al., 2020). However, we hypothesized that for applications where the product of crop growth is active urease, the availability of nickel might be a controlling factor.

[0083] Example 2 - Material and methods

[0084] Crop growth

[0085] Jack bean seeds from the Guanxi province (China) harvested in October 2022 were soaked in water for 24 hours prior to planting each seed in a 12L pots containing 4.3 kg of soil. In the soil (0, 10, 50, 100 or 250 mg nickel sulfate hexahydrate (respectively 0, 0.038, 0.19, 0.38, or 0.95 mmol Ni2+) per kg soil was thoroughly mixed prior to the planting of the seed. Seeds were planted 3-4 cm below the surface. Common potting soil was used, with a composition roughly as follows: sphagnum peat 0.2 ENm3, baltic peat 0.3 ENm3, garden peat 0.3 ENm3, bark 0.2 ENm3, lime +MgO 2,45 kg, PG-mix 15-10-20 + trace 0.81 kg, clays 40.61 kg, EC 0.8, pH 5.7, EN-factor 1.23. The pots were placed in a glasshouse at a controlled temperature of 20-22°C and exposed to 16 hour artificial light (400W / m2) per day.

[0086] The plants were moistened by an automated drippling system. Three different procedures of supplemental fertilization was performed. Either (i) prior to seeding 24 g of the fertilizer Osmocote Pro (compositional details below) was mixed through the soil. This fertilizer are coated granules and yields a slow release of its components during 5-6 months. In (ii) another procedure Hoagland 0.5 was included into the moistening system containing the elements and their final concentrations as described in detail below. In a final (iii) procedure no extra fertilizer was added during the first 12 weeks. After that the Hoagland 0.5 procedure was applied.

[0087] The plants may reach a height of more than 3 m in a glasshouse environment, but are shortened to 1.5 m to stimulate the development of flowers. Typically after 12-14 weeks the plant develops pink flowers in bunches of about 6-8 flowers. Per bunch 2 or 3 flowers develop into pods which develop over a period of 6-8 weeks into green pods up to a length of 30-35 cm containing 11-16 seeds per pod. After full ripening of the pods, the pods color to yellow-brown.

[0088] Osmocote Pro (see (i) above):

[0089] Total Nitrogen (N) = 19% (6.3% as nitrate; 8.2% as ammonium; 4.5% as urea)

[0090] Fosforpentoxide (P2O5) = 8% (6.8% of the 8% water soluble)

[0091] Potassiumoxide (K2O) = 10% (all water soluble)

[0092] Magnesiumoxide (MgO) = 2% (1 .3% of the 2% water soluble)

[0093] Borium (B) = 0.01 % (all water soluble)

[0094] Cupper (Cu) = 0.037% (0.023% of the 0.037% water soluble)

[0095] Iron (Fe) = 0.3% and 0.06% complexed iron

[0096] Manganese (Mn) = 0.04%

[0097] Molybdeen (Mo) = 0.015% (0.01 % of the 0.015% water soluble)

[0098] Zinc (Zn) = 0.011 %

[0099] Hoagland 0.5 (see (ii) above): pH 5.8 pH is adjusted by: K2O or H2SO4

[0100] Macro-elements in mM / L

[0101] NH4+0.5 K+3 Ca2+1 .8 Mg2+1

[0102] NO3- 5.5 SO42- 1.3 H2PO4- 1

[0103] Micro-elements in pMol / L

[0104] Mn 9 Zn 1.8 B 44 Cu 0.5 Mo 0.5 Fe 25 (50% Fe-DTPA 3% 50% Fe- EDDHSA 3%)

[0105] Urease extraction

[0106] 8 Jack Bean seeds are grinded using mill stones (or for instance a burr coffee grinder). In a 50 mL Falcon tube 3.0 g of coarse Jack Bean flour is weighted and 27 mL of de-ionized water is added to extract the urease. The Falcon tube is placed in a vertically rotating spinner for 60 minutes at room temperature, after which the mixture is allowed to settle for at least 15 minutes. The urease activity from the upper layer is determined as described below in the section “Determination of the activity of urease”.

[0107] Determination of the activity of urease

[0108] The specific activity of urease in a urease-containing solution is determined by quantification of the amount of ammonia formed in time (via the Berthelot reaction) when the material is placed in a aqueous 100 mM potassium phosphate buffer at pH 7.5 in the presence of 15 mM urea at RT. The following solutions are prepared for determination of the specific activity of urease:

[0109] Reagent A: A solution of sodium salicylate (4.80 g, 30 mmol), sodium nitroprusside dihydrate (0.54 g, 1 .8 mmol), EDTA (0.373 g, 1 .28 mmol) in 500 mL de-ionized water.

[0110] Reagent B: A solution of sodium hydroxide (3.0 g, 75 mmol) and sodium hypochlorite 5- 15% (10.2 g, 8.4 mL) in 500 mL de-ionized water.

[0111] Buffered 15 mM urea solution: A solution of dibasic potassium phosphate (7.26 g, 41.7 mmol), monobasic potassium phosphate (1.13 g, 8.3 mmol) and urea (0.45 g, 7.5 mmol) in 500 mL de-ionized water.

[0112] The solution containing urease (15-50 pL) is pipetted into a 50 mL Falcon tube. A buffered 15 mM urea solution (10 mL) was added to the Falcon tube (at t=0 min), after which the Falcon tubes were capped and placed on a roller bank at 55 rpm. At several timepoints (4, 8 and 16 minutes) the ammonia concentration of the solution in the Falcon tubes was determined by pipetting 50 pL of the solution in a 4 mL polystyrene cuvette. To each cuvette 3000 pL of a 1 :1 (v / v) mixture of reagent A and B (which reacts with ammonia and form a dye with a green color) kept on ice was added and the mixture was incubated for 20 minutes at RT after which the absorption was measured at 620 nm to quantify the ammonia concentration. For each timepoint 50 pL of a blanc solution (the 15 mM urea buffer) and 50 pL of a 10 mM ammonia solution were also incubated with 3000 pL of 1 :1 (v / v) mixture of reagent A and B for 20 minutes. The absorbance of these cuvettes were used for the calibration curve for determination of the ammonia concentration in the other samples. The ammonia concentration in the Falcon tube was plotted against time and the slope of the three timepoints was calculated with linear regression. The specific activity of the urease sample was determined with the following formula:

[0113] Activity = 1000 * (Total volume * Slope) / (Volume urease solution) Wherein Activity is the specific enzymatic activity of urease in U / mL;

[0114] Total volume is the volume of the 15 mM urea buffer, which is 10 mL;

[0115] Slope is the slope in the plot of ammonia concentration (mM) versus time (minutes); and Volume urease solution is the amount of the urease solution in pL in the Falcon tube.

[0116] Determination of the dry weight of the urease solution

[0117] The dry weight of a solution is the weight of a solution after all the water (and other volatiles) are evaporated, which is used to quantify to total amount of solutes in the solution. The dry weight is determined as described below.

[0118] The weight of an aluminum pan is recorded, after which a solution containing urease (IQ- 20 mL) was added in the pan. The pan was placed in an oven which is set to maintain the temperature at 80 °C for 12-16 hours and 105 °C for 2 hours, during which all the water in the sample was evaporated. The weight of the pan and the residue of the solution were determined and the dry weight of the urease solution was calculated using the following formula.

[0119] Dry weight= (Weight pan + residue - Weight pan) / Weight urease solution) *100%

[0120] Wherein Dry weight is the total amount of non-volatile components in the solution in %;

[0121] Weight pan + residue is the weight recorded after all the water is evaporated in gram;

[0122] Weight pan is the weight of the pan in gram before the urease solution is added, typically 1.55- 1 .60 g; and

[0123] Weight urease solution is the amount of urease solution that was added to the aluminum pan in gram, typically 10-20 g.

[0124] Example 3 - Influence of nickel sulfate on the growth of Jack Bean when nickel sulfate is mixed through the soil upon seeding

[0125] In the presence of the fertilizer Osmocote with 1 , 10 and 50 mg nickel sulfate per kg of soil (resp. 0.0038, 0.038 and 0.190 mmol Ni2+ / kg soil, nickel sulfate hexahydrate was used) mixed through the soil upon seeding the Guanxi Jack Bean, the plants reach a height of about 1.5 m within 10 weeks. After that the plants were cut back every two weeks to a height of 1 .5 m. These plants developed large leaves with a bright green color. After 12 weeks budding was apparent in small bunches of 7-9 buds and subsequent flowers were developed. Pods were formed after week 15 that were matured in the consecutive 6-7 weeks.

[0126] The plants grown in the presence of Osmocote and 100 mg nickel sulfate per kg soil were struggling to grow compared to those at lower nickel concentrations. In 10 weeks the plant reached only heights of about 80-100 cm and the developed leaves were dark green and more irregular shaped. The plants did develop flowers and in some cases small pods were formed which did not further develop than 20 cm and did not show significant production of seeds upon ripening of the pod.

[0127] The seeds that were exposed to Osmocote and 250 mg nickel sulfate per kg soil did germinate slowly but developed only marginally to plants higher than 1 m. The leaves were irregular shaped and colored. The plant did develop some buds and pods were eventually formed, but seed formation inside the pod was negligible.

[0128] Our findings shows that the plants’ appearance after 24 weeks differs depending on the amounts of nickel sulfate supplemented to the soil. It can be observed that the pods from plants grown in the presence of 0-50 mg Nickel sulfate / kg soil have ripened, while the ones at higher amounts of nickel are still in the early green phase and not swollen. It can be concluded that with additional amounts up to 50 mg nickel sulfate per kg soil, the Jack Bean can develop well as crop, while at higher amounts of nickel the plants are struggling or not viable as a result of phytotoxicity.

[0129] Pods of these plants were collected after 25 weeks. Every pod contained about 11-16 seeds that were collected and analyzed for the specific urease activity. The results are shown in Table 2.

[0130] Table 2. Specific urease activity of Jack Bean seeds grown in glasshouses as a function of the amount of nickel-sulfate added to the soil upon planting the seed.

[0131] Table 2 shows that the urease in the seeds has a low activity in the absence of additional nickel to the soil. As nickel is known to speed up the hydrolysis of urea by urease, it could be that the low specific activity at 0 mg of added nickel-sulfate / kg soil (Table 2) is caused by the limited nickel- uptake during the development of the seed to incorporate this trace-element in the protein upon synthesis. This could resemble the geographic situation in the far-east (Malaysia and Papua New Guinea; see Table 1). However, by upfront addition of 10 mg nickel-sulfate / kg of soil, already a strong increase in specific urease activity can be observed. The observed activity is comparable to that found in the province of Guanxi (China) and higher than found in seeds from Australia, Kenia or Brazil (see Table 1). Most remarkable is that when 50 mg of nickel sulfate is added per kg of soil an almost 45% increase of urease activity is obtained, higher than globally can be found in natural sources. At 100 or 250 mg nickel-sulfate no results are presented, as the plants did not develop seeds. These data show that even though a diversity of Jack bean plant proteins require trace elements of heavy metals for its viability, too high concentrations are lethal for proper growth.

[0132] In a further trial Jack Beans were cultivated under similar conditions, with results shown in Table 2.1. For this trial some variations in fertilizer use were tested. Seeds where 250 mg nickel sulfate / kg soil was used were eventually harvested in small number from poorly developed plants. Notably these seeds have lower specific activity than seeds where only 10 mg of nickel per kg soil was used.

[0133] Table 2.1. Specific urease activity of Jack Bean seeds grown in glasshouses as a function of the amount of nickel-sulfate added to the soil upon planting the seed.

[0134] In a further trial Jack Beans were cultivated under similar conditions, with results shown in Table 2.2. For this trial additional variations in fertilizer use were tested: fertilizer (Osmocote Pro) was only added four weeks after the seed was planted, unless otherwise indicated.

[0135] Table 2.2. Specific urease activity of Jack Bean seeds grown in glasshouses as a function of the amount of nickel-sulfate added to the soil upon planting the seed.

[0136] At 50 mg / kg nickel sulfate, beans with delayed fertilization showed increased urease activity. Doses in the range of 30 to 90 mg each yielded good results.

[0137] It can be concluded that providing nickel-sulfate in a soluble form has a positive effect on the inclusion of nickel in the catalytic site of the enzyme urease, thereby providing a source of urease that has a significantly higher activity than can be expected from Jack beans from a natural geographic origin.

[0138] Example 4 - Addition of nickel to post-harvested Jack Bean seeds

[0139] \Ne assessed the effects on urease activity following the addition of nickel to Jack Bean seeds after harvesting. Seeds obtained from Jack Bean plants grown in Guanxi (China) were soaked for 24 hours in a nickel-nitrate containing solution. During this soaking period, the average weight of the beans increases by about a factor 2.1 and the outer shell turned from smooth and hard to silky and elastic. The beans were ground, and the resulting Jack Bean flour was extracted and analyzed for the urease activity as described in Example 2. The results are summarized in Table 3. It can be concluded from these data that post-harvest exposure of the seed to water-soluble nickel has no effect on the urease activity after extraction.

[0140] Table 3. Adding nickel to post-harvested Jack Bean (JB) seeds does not affect urease activity.

[0141] To verify that adding nickel to post-harvested Jack Bean seeds does not affect urease activity, seeds obtained from an original Guanxi Jack Bean were re-seeded on soil without any nickel (see JB grown in absence of Ni2+in Table 3). The seeds obtained from Jack Beans grown in the absence of nickel were soaked in a nickel-nitrate containing solution, but did still not display significant urease activity. This is in line with the lack of increase in urease activity in the original seeds from Guanxi upon soaking in a Ni2+-containing solution. It can be concluded that adding nickel to post-harvested Jack Bean seeds does not affect urease activity.

[0142] Example 5 - Influence of nutritional soil quality on the urease specificity

[0143] As the natural role of urease is to make nitrogen accessible for biological processes in plants related to seed germination and growth, the nutritional state of the plant may affect the urease activity developed in seeds. To test this, three different fertilization conditions were studied. When applying coated Osmocote granules in the soil during germination of the Jack bean seed during a period of 5-6 months there is a slow release of the fertilizer components. In the presence of the 50 mg / kg soil this resulted in an urease activity in the developed seeds of around 3.3 units / mg dry weight. Using the fertilizer Hoagland 0.5 that contains a lower nitrogen level, dissolved in the moistening fluid, a slightly elevated urease activity was observed (Table 4). When no fertilizer was added during the first 12 weeks of seed germination and plant growth, a remarkable increase in urease activity was observed (Table 5). Thus, surprisingly, growing the plants on a poor soil results in plants with higher urease activity in the developed seeds. This might ensure plant viability under stringent growth conditions.

[0144] Table 4. Nutritional state of the Jack Bean plant affects urease activity in developed seeds. Soil contains 50 mg nickel-sulfate / kg soil.

[0145] Table 5. No fertilization of Jack Bean plant during the first 12 weeks increases urease activity in developed seeds. Soil contains 50 mg nickel-sulfate / kg soil, fertilization was done by moistening the soil with Hoagland 0.5.

[0146] Fertilization starting after 8 weeks or after 12 weeks was further assessed at different Ni concentrations. Nick sulfate was mixed with soil at the indicated concentrations, and fertilization (Osmocote Pro) was commenced after the indicated number of weeks. Table 5.1 shows the resulting urease activity of the beans (U / mg).

[0147] Table 5.1. Delayed fertilization improves urease activity

[0148] These data further confirm that an initial growth strategy without fertilization is advantageous for the resulting specific urease activity in the beans, tested with different types of fertilizer.

[0149] Example 6 - Nickel can improve bean weight and pod properties

[0150] Different fertilization and Ni supplementation conditions were tested for their effect on bean weight. Soil contains an amount of nickel-sulfate / kg soil as indicated in Table 6, fertilization was with Osmocote and was performed starting at the beginning of the indicated week after planting the beans (for instance “week 5” indicates that cultivation was for the initial 4 weeks without fertilizer, and that fertilizer was added starting after those 4 weeks).

[0151] Table 6. Nickel improves bean weight, particularly with fertilizer added after 12 weeks

[0152] A similar experiment was performed wherein specific urease activity was determined for complete bean pods. It was found that with fertilizer after 12 weeks (so starting at week 13) as described above, activity per pod was as shown in table 7. This is consistent with the conclusion from table 5, that omission of fertilizer during the first 12 weeks (approximately three months) increases specific urease activity (Units / mg dry weight).

[0153] Table 7. Nickel improves total urease activity of complete bean pods. References

[0154] Dixon N.E., et al. (1980) Canadian Journal of Biochemistry 58(6): 474-80.

[0155] Carter E. L. et al. (2009) Metallomics, 1 (3): 207-221.

[0156] Dalton et al. (1985) Plant and Soil, 88, 245-258.

[0157] Liu, G. D. (2001) Plant Nutrition and Fertilizer Science, 7(1), 101-103. Patra A., et al. (2020) Food and Chemical Toxicology vol 1 : 35-37.

Claims

Claims1 . A method for providing a Canavalia plant or part thereof, the method comprising the steps of: i) providing soil, wherein the soil comprises 0.01 - 0.4 mmol Ni2+per kg soil; ii) culturing a Canavalia plant in the soil provided in step i); iii) optionally harvesting the plant of step ii) or a part thereof.

2. The method according to claim 1 , wherein the Canavalia plant is a Canavalia ensiformis plant.

3. The method according to claim 1 or 2, wherein step i) comprises mixing soil with a source of Ni2+, wherein the source of Ni2+is preferably a nickel salt such as NiSC .

4. The method according to any one of claims 1 -3, wherein step ii) comprises planting a Canavalia seed below the surface of the soil, preferably 1 -10 cm below the surface of the soil, more preferably 2-5 cm below the surface of the soil.

5. The method according to any one of claims 1 -4, wherein the culturing of step ii) occurs at a temperature of 16-26°C, preferably at 20-22°C and / or encompasses exposure to light for 12-20 hours per day, preferably for 15-17 hours.

6. The method according to any one of claims 1 -5, wherein no fertilizer is applied to the soil during step ii) before the Canavalia plant develops flowers.

7. The method according to any one of claims 1 -5, wherein step ii) comprises applying a fertilizer to the soil after 4 - 14 weeks of culturing, preferably after 12 weeks of culturing.

8. The method according to claim 7, wherein the fertilizer comprises about 0.5 mM / L NH4+and / or about 3 mM / L K+and / or about 1 .8 mM / L Ca2+and / or about 1 mM / L Mg2+and / or about 5.5 mM / L NOs- and / or about 1.3 mM / L SO42-and / or about 1 mM / L H2PO4- and / or about 9 pMol / L Mn and / or about 1 .8 pMol / L Zn and / or about 44 pMol / L B and / or about 0.5 pMol / L Cu and / or about 0.5 pMol / L Mo and / or about 25 pMol / L Fe.

9. The method according to claim any one of claims 1 -8, wherein the plant or part thereof in step iii) is harvested and is a seed, a flower, a leaf, a stem, a root, a shoot, a nodule, or a pod, preferably a seed.

10. A method for preparing urease or flour or meal comprising urease, the method comprising the steps of: a) providing soil comprising 0.01 - 0.4 mmol Ni2+per kg soil; b) culturing a Canavalia plant in the soil provided in step a); c) harvesting beans of the cultured Canavalia plant of step b);d) processing the beans of step c) into flour or meal; e) optionally extracting urease from the flour or meal of step d).

11. Soil comprising 0.01 - 0.4 mmol Ni2+per kg soil, preferably 0.038 - 0.38, more preferably 0.152 - 0.342, even more preferably 0.19 - 0.304 mmol Ni2+per kg soil.

12. Soil according to claim 1 1 , comprising 0.02 - 0.4 mmol Ni2+per kg soil.

13. Soil according to claim 11 or 12, wherein the soil comprises at most 1.8 gram glucose per kg dry soil, preferably at most 1 .5 gram glucose per kg dry soil.

14. Use of the soil according to any one of claims 1 1 -13, for culturing a plant.

15. A plant or part thereof, wherein the plant was cultured in the soil according to any one of claims 11-14.

16. The plant or part thereof according to claim 15, wherein the plant or part thereof has a specific urease activity per mg dry weight of at least least 3.5 U / mg, preferably 3.8 U / mg.

17. The plant or part thereof according to claim 16, wherein the plant or part thereof has a specific urease activity per mg dry weight of at least least 4 U / mg.

18. Bean meal or bean flour obtainable by a method according to claim 10, or derived from a plant or part thereof according to any one of claims 15-17.

19. The bean meal or bean flour according to claim 18, wherein the plant or part thereof has a specific urease activity per mg dry weight of at least least 3.5 U / mg.

20. The bean meal or bean flour according to claim 18, wherein the plant or part thereof has a specific urease activity per mg dry weight of at least least 3.8 U / mg.21 . The bean meal or bean flour according to claim 18, wherein the plant or part thereof has a specific urease activity per mg dry weight of at least least 4 U / mg.

22. The bean meal or bean flour according to any one of claims 18-21 , wherein bean meal or bean meal flour has a specific urease activity per mg dry weight of at least least 3.5 U / mg.

23. The bean meal or bean flour according to any one of claims 18-21 , wherein bean meal or bean meal flour has a specific urease activity per mg dry weight of at least least 3.8 U / mg.

24. The bean meal or bean flour according to any one of claims 18-21 , wherein bean meal or bean meal flour has a specific urease activity per mg dry weight of at least least 4 U / mg.

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