WO2019185519A1 - Procédé de réduction de la répulsion d'eau du sol - Google Patents

Procédé de réduction de la répulsion d'eau du sol Download PDF

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Publication number
WO2019185519A1
WO2019185519A1 PCT/EP2019/057395 EP2019057395W WO2019185519A1 WO 2019185519 A1 WO2019185519 A1 WO 2019185519A1 EP 2019057395 W EP2019057395 W EP 2019057395W WO 2019185519 A1 WO2019185519 A1 WO 2019185519A1
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Prior art keywords
soil
amino acid
water
acid sequence
polypeptide
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PCT/EP2019/057395
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English (en)
Inventor
Alexander Wissemeier
Cristina POP
Michael LISZKA
Wolfgang Weigelt
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Basf Se
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Publication of WO2019185519A1 publication Critical patent/WO2019185519A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F11/00—Other organic fertilisers
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
    • C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
    • C05G3/80—Soil conditioners
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K17/00—Soil-conditioning materials or soil-stabilising materials
    • C09K17/14—Soil-conditioning materials or soil-stabilising materials containing organic compounds only
    • C09K17/18—Prepolymers; Macromolecular compounds
    • C09K17/32—Prepolymers; Macromolecular compounds of natural origin, e.g. cellulosic materials
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/20—Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2
    • Y02P60/21—Dinitrogen oxide [N2O], e.g. using aquaponics, hydroponics or efficiency measures

Definitions

  • the present invention relates to a method for improving water availability in soil using a lipase.
  • Soil water repellency is a condition where soil does not spontaneously wet when a drop of water is applied to the soil surface, i.e. the soil is too hydrophobic (Muller and Deuer (2011 ) Agric., Ecosystems and Environ. 144: 208-221). Hydrophobic soils occur in many countries on various lands, such as agricultural, pasture, coastal dune sands, forest, shrub lands, parks, turfgrass soils, no-till agriculture, and soils irrigated with treated wastewater. A substantial inter- est in soil water repellency soils has grown in recent times (WO 2013/181240; Dekker et al. (2005) Aust. J. of Soil Res. 43: 403-441 ).
  • Soil water repellency can cause undesirable consequences such as environmental deterioration and considerable losses in crop production. Soil water repellency becomes especially problem- atic on water relationships and can cause associated environmental issues, such as, but not limited to, reduction in soil water intake, uneven wetting patterns, reduced irrigation efficiency and effective precipitation, increased preferential flow that can have adverse effects on aquifer contamination, greater runoff and erosion, limited seed and vegetative establishment, and re-umbled plant growth and quality (Doerr et al. (2000). Earth-Sci. Reviews 51 : 33-65; Muller and Deuer (201 1 )).
  • soil water repellency across large areas of crop-producing fields leads to reduction or complete loss of already planted crops as well as reduction in soil quality and wa- tering problems for the next set of seeds.
  • soil water repellency is a reoccurring problem called“localized dry spot” (LDS).
  • LDS soil water repellency appears as irregular dry areas from a few centimeters to several meters diameter with the repellency usually extending from the surface of the soils into 5-10 cm depth.
  • the primary cause of soil water repellency is the formation of a coating of hydrophobic, organic material on soil particles.
  • This hydrophobic organic material can include surface waxes, fatty ac- ids, and other organics such as lignin, a phenolic polymer. These materials originate from plant leaves and other decomposing organic matter, plant root exudates, fungal hyphae/exudates, and volatized organic materials condensing on soil particles following forest or grassland fires (Atanassova and Doerr (2010) Europ. J. of Soil Sci. 61 : 298-313). Sandy soils are especially susceptible to soil water repellency due to a lower particle surface area.
  • the present inventors have surprisingly found that the application of a lipase to soil leads to a reduced soil water repellency and an increased water holding capacity of non-wetting soils. Ad- ditionally, the application of said lipase also improves the water availability in wetting soils.
  • the present invention relates to the use of an isolated polypeptide having lipase ac- tivity which is selected from the group consisting of:
  • polypeptide comprising an amino acid sequence with at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 1 or an enzymatically active fragment thereof;
  • the soil water repellency of non-wetting soil is reduced and/or the water holding capacity of non-wetting soils is increased. In another embodiment the water holding capacity of wetting soil is increased and/or the evapo- ration from wetting soil is decreased.
  • the variant comprises at least one amino acid insertion, amino acid deletion or amino acid substitution with respect to the sequence according to SEQ ID NO:1.
  • the at least one amino acid insertion, amino acid deletion or amino acid substitution may be at an amino acid residue position selected from the group of positions 23, 33, 82, 83, 84, 85, 160, 199, 254, 255, 256, 258, 263, 264, 265, 268, 308 and 31 1.
  • the at least one amino acid substitution may be selected from the group consisting of: Y23A, K33N, S82T, S83D, S83H, S83I, S83N, S83R, S83T, S83Y, S84S, S84N, I85A, I85C, I85F, I85H, I85L, I85M, I85P, I85S, I85T, I85V, I85Y, K160N, P199I, P199V, I254A, I254C, I254E, I254F, I254G, I254L, I254M, I254N, I254R, I254S, I2454V, I254W, I254Y, I255A, I255L, A256D, L258A, L258D; L258E, L258G, L258H, L258N, L258Q, L258R, L258S, L258T, L258V,
  • the variant comprises a combination of amino acid substitutions with re- spect to the sequence according to SEQ ID NO:1.
  • the combination of amino acid substitutions may be selected from the group consisting of: a) I85L, D265S, T268G;
  • the polypeptide having lipase activity is used in combination with at least one soil additive, preferably selected from the group consisting of surfactants, fertilizers, nitrifi- cation inhibitors and/or pesticides, preferably fungicides.
  • soil additive preferably selected from the group consisting of surfactants, fertilizers, nitrifi- cation inhibitors and/or pesticides, preferably fungicides.
  • the polypeptide having lipase activity is applied in a concentration of be- tween 0.01 kg to 600 kg of polypeptide per hectare, preferably of between 0.16 kg to 100 kg per hectare, more preferably of between 0.6 kg to 20 kg per hectare, most preferably of between 1 kg to 5 kg per hectare.
  • the soil may be selected from agricultural land, pasture, coastal dune sands, forest, shrub lands, parks, turfgrass soils, no-till agriculture, and soils irrigated with treated wastewater.
  • the present invention also relates to a method for increasing the water availability in soil corn- prising treating an area of groundcover with an isolated polypeptide having lipase activity which is selected from the group consisting of: (i) a polypeptide comprising the amino acid sequence according to SEQ ID NO: 1 or an enzy- matically active fragment thereof;
  • polypeptide comprising an amino acid sequence with at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 1 or an enzymatically active fragment thereof;
  • the isolated polypeptide having lipase activity is as defined above.
  • the soil water repellency of non-wetting soil is reduced and/or the water holding capacity of non-wetting soil is increased.
  • the water holding capacity of wetting soil is increased and/or the water infiltration time of wetting soil is decreased.
  • the isolated polypeptide having lipase activity may be used in combination with at least one soil additive, preferably selected from the group of surfactants, fertilizers, nitrification inhibitors and/or pesticides, preferably fungicides.
  • the polypeptide having lipase activity may be applied in a concentration of between 0.01 kg to 600 kg of polypeptide per hectare, preferably of between 0.16 kg to 100 kg per hectare, more preferably of between 0.6 kg to 20 kg per hectare, most preferably of between 1 kg to 5 kg per hectare.
  • Figure 1 and Figure 2 show the results for assessing water droplet penetration time (WDPT) for lipases LIP120 and LIP173.
  • WDPT water droplet penetration time
  • steps of a method or use or assay there is no time or time interval coherence between the steps, i.e. the steps may be carried out simultaneously or there may be time intervals of sec- onds, minutes, hours, days, weeks, months or even years between such steps, unless other- wise indicated in the application as set forth herein above or below.
  • the present invention is based on the finding that the application of a li- pase to soil increases the water availability in soil.
  • water availability refers to the amount of water which is present in a given volume of soil. This amount of water can be taken up by plants and used for growing. Hence, by improving the water availability in soil, the soil is more suitable for growing plants.
  • the water availability in soil is increased by at least 3% or 5%, preferably by at least 8% or 10%, more preferably by at least 12% and most preferably by at least 15%.
  • the amount of water which can be taken up by plants treated in accordance with the present invention is increased by at least 3% or 5%, pref- erably by at least 8% or 10%, more preferably by at least 12% and most preferably by at least 15% compared to a soil which has not been treated in accordance with the present invention.
  • the application of the lipase reduces soil water repellency and/or increases the water holding capacity of non-wetting soil.
  • soil water repellency is a condition where soil does not spontaneously wet when a drop of water is applied to the surface. It reduces the affinity of soils to water such that they resist wetting for variable periods. It may be present in a large area and therefore affect agriculture. Such large areas of water-repellent soil predominantly occur in Australia as well as in North and South America.
  • soil water repellency may present as "localized dry spots” which are irregular dry areas from a few centimeters to several meters diameter and mainly occur on sandy turfgrass soils and grasslands.
  • soil water repellency may present as "fairy rings" which are caused by fungi, in particular by basidiomycetes and which have a round shape. The fairy rings can range from a few centimeters to 20 meters in diameter, particularly from 0.5 m to 5m in diameter.
  • the organic matter contributing to hydrophobic organic coatings in soils with soil water repel- lency arises from sources such as, but not limited to: a) plant vegetation and root exudates with certain plant species especially prone to causing SWR, such as pine, gum, and oak trees and grasses; b) decomposition products from soil microbial activity, soil microorganism biomass such as fungal hyphae, and root exudates; c) applied organic amendments; d) ashed or volatized organic materials condensing on soil particles following forest or grassland fires; and e) organic matter in treated waste water used for irrigation. Regardless of soil type or organic matter source, soil drying increases repellency with air-drying greatly enhancing SWR severity.
  • the typical types of organic compounds suggested to be involved in soil water repellency in- clude: a) high molecular weight, polar fatty acids and their esters (alkanes that are derived from plant and cuticular waxes); b) other alkanes (paraffin-like compounds), microbial derived waxes, alkanols, phytanols, phytanes; c) amphiphilic (partially hydrophobic) lipids, stigmasterols and plant derived sterols that have polar (hydrophilic) and non-polar (hydrophobic) groups; d) other polar molecules such as sugars, gylocsides, aromatic acids, and low molecular weight organic acids; e) humic and fulvic acids from soil microbial activity or possibly added as amendments; and f) hydrophobins, cysteine rich proteins expressed only by filamentous fungi.
  • the soil water repellency can be measured and classified by a variety of methods.
  • the water droplet penetration time (WDPT) test is based on the time taken for a drop of water to infiltrate into an air- or oven-dried soil sample (Dekker et al. (1998) Soil Sci. 163: 780-796). A description of this method is provided in the examples section herein.
  • the molarity of ethanol droplet (MED) or ethanol test uses a series of aqueous ethanol solutions prepared in concentrations ranging between 0% and 36%.
  • the degree of soil water repellency is then defined as the ethanol percentage or molarity of the least concentrated ethanol solution that is absorbed by the soil in a mean time of ⁇ 10 s (DeBano et al. (2000) J. Hydrol. 231 :4-32).
  • sorptivity of wa- ter which is influenced by repellency is compared to the sorptivity of ethanol which is not influ enced by repellency (Tillman et al. (1999) Austr. J. Soil Res. 27: 637-644).
  • the contact angle between water and soil may be measured e.g. by the capillary rise method (Woche et al. (2005) Eur. J. Soil Sci. 56: 239-251 ).
  • the soil water repellency of non-wetting soils is reduced by at least 10% or 20%, preferably by at least 30% or 40%, more preferably by at least 50% or 60% and most preferably by at least 70% or 75%. If the soil water repellency is meas- ured using the WDPT test, the time taken for a drop to infiltrate the soil is reduced by at least 10% or 20%, preferably by at least 30% or 40%, more preferably by at least 50% or 60% and most preferably by at least 70% or 75%.
  • water holding capacity refers to the amount of water that a given amount of soil can hold. It has an impact on the water supply of any crops planted on the soil, but also on the re- tention of nutrients and pesticides by the soil.
  • the water holding capacity can be determined by applying a defined amount of water onto a defined amount of soil and after a certain period of time such as two hours weighing the soil. The difference between the weight of the soil after ap- plying the water and before applying the water is the amount of water which is held by the soil.
  • the water holding capacity can be determined by placing a water saturated soil sample on a porous ceramic plate which is then placed in closed chambers and a known amount of pressure is applied to the chamber which forces water out of the soil sample and into the porous plate and out of the chamber.
  • the water holding capacity can also be determined by measuring the amount of water leached through the soil after a defined amount of soil has been watered with an amount of water which exceeds the water holding capacity. A lower amount of water which has leached from the soil indicates an increased water holding capacity.
  • the water holding capacity of non-wetting soil is increased by at least 3% or 5%, preferably by at least 8% or 10%, more preferably by at least 12% and most preferably by at least 15%.
  • the amount of water which is present in the soil after a certain period of time after application is increased by at least 3% or 5%, pref- erably by at least 8% or 10%, more preferably by at least 12% and most preferably by at least 15% compared to a soil which has not been treated in accordance with the present invention.
  • the soil water repellency of non-wet- ting soil is reduced and the water holding capacity of non-wetting soil is increased.
  • the soil wa- ter repellency of non-wetting soil is reduced by at least 10% and the water holding capacity of non-wetting soil is increased by at least 3%, preferably the soil water repellency of non-wetting soil is reduced by at least 30% and the water holding capacity of non-wetting soil is increased by at least 8%, more preferably the soil water repellency of non-wetting soil is reduced by at least 50% and the water holding capacity of non-wetting soil is increased by at least 12% and most preferably the soil water repellency of non-wetting soil is reduced by at least 70% and the water holding capacity of non-wetting soil is increased by at least 15%.
  • evaporation refers to the process of losing water over a surface, particularly a soil surface.
  • the evaporation can be determined by adding water to a soil sample to exceed the wa- ter holding capacity and leaving the soil sample for a period of several days, for example eight days, without watering. After this period the soil sample is weighed and the difference in weight at the timepoint after adding the water and the weight after the period of leaving the soil is deter- mined. This difference is defined as evaporation.
  • the water holding capacity of wetting soil is in- creased by at least 5% or 8%, preferably by at least 10% or 12%, more preferably by at least 15% or 18% and most preferably by at least 20%.
  • the evaporation from wetting soil is reduced by at least 1 %, preferably by at least 2%, more preferably by at least 3% and most preferably by at least 4%.
  • soil refers to material forming the surface of the earth and including a mixture of or- ganic material and minerals. Soil includes materials such as mud, sand, silt, and clay. It may it- self form the surface of the earth in some areas, and in other areas it may underlie other types of groundcover, such as grass and other plants and vegetation, gravel, pebbles, and the like. Preferably, the soil is sandy soil characterized by a low particle-surface area.
  • the soil may be agricultural land, pasture, coastal dune sands, forest, shrub lands, parks, turfgrass soils, potting mixes and soils irrigated with treated wastewater.
  • the soil is agricultural land. This means that the soil is used for planting crops.
  • the agricultural land has not been treated by tillage (so-called no-till agricul- ture). This means that the soil has not been disturbed by tillage.
  • the agricultural land may corn- prise huge areas of non-wetting soil of several square kilometers as contrasted to localized dry spots which have a size of at most several meters. In Western Australia the total area of agricul- tural land having water repellent soil is about 5 million hectares.
  • the soil is not a turfgrass soil.
  • Turfgrass refers to any vegetative ground covering such as, but not limited to, various species of grasses used for lawns, fields, golf course grounds, and the like.
  • the soil is turfgrass soil, for example turfgrass soil from a golf course.
  • the turfgrass soil may have one or more localized dry spots (LDS). LDS are irregular dry areas from a few centimeters to several meters diameter with the repellency usually extending from the surface into a depth of 5 to 10 cm.
  • the turfgrass soil may have one or more "fairy rings" which are caused by fungi, in particular by basidiomycetes and which have a round shape.
  • the fairy rings can range from a few centimeters to 20 meters in diameter, particularly from 0.5 m to 5 m in diameter.
  • the turfgrass soil has at least one localized dry spot and at least one fairy ring.
  • the soil is non-wetting soil.
  • non-wetting soil refers to a soil which does not spontaneously wet when a drop of water is applied to its surface.
  • the non-wetting soil can be characterized by the water droplet penetration time test as explained above.
  • the non- wetting soil has a water droplet penetration time of at least 5 seconds, preferably of at least 20 or at least 100 seconds, more preferably of at least 300 or 500 seconds, even more preferably of at least 700 or 1000 seconds and most preferably at least 1 ,200 seconds.
  • the non-wetting soil can also be defined by the MED test discussed above.
  • the non- wetting soil requires a molarity of ethanol of at least 0.2, preferably of at least 0.5, more prefera- bly of at least 1.0 and most preferably of at least 2.0 to absorb the solution into the soil.
  • the non-wetting soil can also be defined by the contact angle between wa- ter and soil as determined for example by the capillary rise method.
  • the non-wetting soil is de- fined by a contact angle of greater than 90°.
  • the soil is wetting soil.
  • the term "wetting soil” refers to a soil which sponta- neously wets when a drop of water is applied to its surface.
  • the wetting soil can be character- ized by the water droplet penetration time test as explained above.
  • the wetting soil has a water droplet penetration time of less than 5 seconds.
  • the wetting soil can also be defined by the contact angle between water and soil as determined for example by the capillary rise method.
  • the wetting soil is defined by a contact angle of less than 90°.
  • the wetting soil can also be defined by the MED test discussed above.
  • the non-wetting soil requires a molarity of ethanol of 0 to absorb the solution into the soil.
  • isolated polypeptide refers to a polypeptide that has been separated from its biologi cal source such as a bacterium or fungus producing the polypeptide, for example by centrifuga- tion or filtration. After separating the polypeptide from the biological source it may be purified to remove other components from the biological source or medium components. Hence, the term “isolated polypeptide” includes both purified polypeptides and polypeptides which are present in a cell culture supernatant or the like.
  • the isolated polypeptide used according to the present invention has lipase activity.
  • Lipases (E.C. 3.1.1.3) are hydrolytic enzymes that are known to cleave ester bonds in lipids. Lipases in- clude phospholipases, triacylglycerol lipases, and galactolipases.
  • the enzyme used in the method of the present invention is preferably a triacylglycerol lipase which cleaves the ester bond between glycerol and fatty acids, resulting in the release of fatty acids from the glycerol.
  • the lipase used in the context of the present invention is selected from the group consisting of:
  • a lipase comprising the amino acid sequence according to SEQ ID NO: 1 or an enzymatically active fragment thereof;
  • a lipase comprising an amino acid sequence with at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 1 or an enzymatically active fragment thereof;
  • an "enzymatically active fragment" of a lipase is understood to refer to a smaller part of the li- pase which consists of a contiguous amino acid sequence found in the amino acid sequence of the lipase and which has lipase activity.
  • the skilled person knows that for a fragment to be en- zymatically active the fragment has to comprise at least the amino acids present in the catalytic centre of the lipase. These amino acids are either known for a given lipase or can easily be identified by the skilled person, for example by homology screening or mutagenesis.
  • a "fragment" of a nucleic acid sequence encoding an enzymatically active fragment of a lipase is understood to refer to a smaller part of the nucleic acid sequence which consists of a contigu- ous nucleic acid sequence found in the nucleic acid sequence of the lipase and which encodes a protein having lipase activity.
  • the skilled person knows that for a fragment of an enzyme to be enzymatically active the fragment has to comprise at least the amino acids present in the cata- lytic centre of the lipase. These amino acids are either known for a given lipase or can easily be identified by the skilled person, for example by homology screening or mutagenesis.
  • Sequence Identity means a com- parison of a first amino acid sequence to a second amino acid sequence, or a comparison of a first nucleic acid sequence to a second nucleic acid sequence and is calculated as a percentage based on the comparison. The result of this calculation can be described as“percent identical” or “percent ID.”
  • a sequence alignment can be used to calculate the sequence identity by one of two different approaches.
  • first approach both, mismatches at a single position and gaps at a single position are counted as non-identical positions in final sequence identity calculation.
  • second approach mismatches at a single position are counted as non-identical positions in final sequence identity calculation; however, gaps at a single position are not counted (ignored) as non-identical positions in final sequence identity calculation.
  • gaps at a single position are not counted (ignored) as non-identical positions in final sequence identity calculation.
  • ap- proach gaps are ignored in final sequence identity calculation.
  • sequence identity is determined by a program, which pro- Jerusalem an alignment, and calculates identity counting both mismatches at a single position and gaps at a single position as non-identical positions in final sequence identity calculation.
  • EMBOS Needle
  • Needleman and Wun- sch Needleman and Wunsch (1970) J. Mol. Biol.
  • sequence identity can be calculated from a pairwise alignment showing only a local region of the first sequence or the second sequence (“Local Iden- tity”).
  • program Blast NCBI
  • % sequence identity (# of identical residues / length of alignment) x 100)].
  • a sequence alignment is calculated with mismatches at a single po- sition being counted as non-identical positions in final sequence identity calculation; however, gaps at a single position are not counted (i.e. they are ignored) as non-identical positions in final sequence identity calculation.
  • the sequence alignment is generated by using the algorithm of Needleman and Wunsch (J. Mol. Biol. (1979) 48, p. 443-453).
  • the program“NEEDLE” The European Molecular Biology Open Software Suite (EMBOSS)
  • EMBOSS European Molecular Biology Open Software Suite
  • the nucleic acid sequence hybridizing under stringent conditions with a complementary se- quence of a nucleic acid sequence according to SEQ ID No. 2 or an enzymatically active frag- ment thereof encodes a protein having lipase activity.
  • hybridizing under stringent conditions denotes in the context of the present invention that the hybridization is implemented in vitro under conditions which are stringent enough to en- sure a specific hybridization.
  • Stringent in vitro hybridization conditions are known to those skilled in the art and may be taken from the literature (e.g. Sambrook and Russell (2001 ) Molec ular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY).
  • specific hybridization refers to the circumstance that a mole- cule, under stringent conditions, preferably binds to a certain nucleic acid sequence, i.e. the tar- get sequence, if the same is part of a complex mixture of, e.g. DNA or RNA molecules, but does not, or at least very rarely, bind to other sequences.
  • Stringent conditions depend on the circumstances. Longer sequences hybridize specifically at higher temperatures. In general, stringent conditions are chosen such that the hybridization temperature is about 5°C below the melting point (T m ) of the specific sequence at a defined ionic strength and at a defined pH value. T m is the temperature (at a defined pH value, a defined ionic strength and a defined nucleic acid concentration), at which 50% of the molecules comple- mentary to the target sequence hybridize to the target sequence in the state of equilibrium.
  • stringent conditions are conditions, where the salt concentration has a sodium ion con- centration (or concentration of a different salt) of at least about 0.01 to 1.0 M at a pH value be- tween 7.0 and 8.3, and the temperature is at least 30°C for small molecules (i.e. 10 to 50 nucle- otides, for example).
  • stringent conditions may include the addition of substances, such as, e. g., formamide, which destabilise the hybrids.
  • substances such as, e. g., formamide, which destabilise the hybrids.
  • said stringent conditions are chosen such that sequences which are about 65%, preferably at least about 70%, and especially preferably at least about 75% or higher homologous to each other, normally remain hybridized to each other.
  • a preferred but non-limiting example of stringent hybridization conditions is hybridizations in 6 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washing steps in 0.2 x SSC, 0.1 % SDS at 50 to 65°C.
  • the temperature depends on the type of the nucleic acid and is between 42°C and 58°C in an aqueous buffer having a concentration of 0.1 to 5 x SSC (pH value 7.2).
  • the tem- perature is about 42°C under standard conditions.
  • the hybridisation conditions for DNA:DNA hybrids are, for example, 0.1 x SSC and 20°C to 45°C, preferably 30°C to 45°C.
  • the hybridisation conditions for DNA:RNA hybrids are, for example, 0.1 x SSC and 30°C to 55°C, preferably between 45°C and 55°C.
  • the above-mentioned hybridization temperatures are determined, for example, for a nucleic acid which is 100 base pairs long and has a G/C con- tent of 50% in the absence of formamide.
  • Typical hybridization and washing buffers for example have the following composition:
  • Hybridization solution pre-hybridization solution
  • Pre-hybridization at least 2 h at 50 - 55 °C
  • proteins encoded by the nucleic acid sequence hybridizing under stringent conditions to the complement of the sequence according to SEQ ID No. 2 preferably comprise the amino acids present in the catalytic site of the lipase.
  • proteins having the abovementioned sequence identity to the sequence according to SEQ ID NO: 1 and the proteins encoded by the nucleic acid sequence hybridizing under stringent conditions to the complement of the sequence according to SEQ ID NO: 2 are called variants of this protein.
  • variant polypeptide refers to a polypeptide which differs from its parent polypeptide in its amino acid sequence.
  • variants of the lipase according to SEQ ID No. 1 are those that comprise at least one amino acid residue insertion, deletion or substitution compared to the sequence according to SEQ ID No.1 and which have lipase activity.
  • amino acid substitution is described by providing the original amino acid followed by the number of the position within the amino acid sequence.
  • a substitution of amino acid residue 24 means that the amino acid of the parent at position 24 can be substituted with any of the 19 other amino acid residues.
  • a substitution can be described by provid- ing the original amino acid followed by the number of the position within the amino acid se- quence and followed by the specific substituted amino acid.
  • the substitution of iso- leucin at position 254 with serine is designated as“Ne254Ser” or“I254S”.
  • Combinations of sub- stitutions are described by inserting commas between the amino acid residues, for example: I85L, D265S, T268G; represent a combination of three different amino acid residues substitu- tions when compared to a parent polypeptide.
  • Variants having substitutions in the context of amino acid changes may also be applied to nucleic acid modifications, e.g. by substitutions.
  • the amino acid substitution may be a conservative amino acid substitution.
  • A“conservative amino acid substitution” or related amino acid” means replacement of one amino acid residue in an amino acid sequence with a different amino acid residue having a similar property at the same position compared to the parent amino acid sequence.
  • Some examples of a conservative amino acid substitution include but are not limited to replacing a positively charged amino acid residue with a different positively charged amino acid residue; replacing a polar amino acid resi- due with a different polar amino acid residue; replacing a non-polar amino acid residue with a different non-polar amino acid residue, replacing a basic amino acid residue with a different basic amino acid residue, or replacing an aromatic amino acid residue with a different aromatic amino acid residue.
  • a list of related amino acids is given in the Table below (see for example Creighton (1984) Proteins. W.H. Freeman and Company (Eds)).
  • amino acid insertion is described by providing the number of the position within the amino acid sequence behind which the amino acid is inserted followed by an apostrophe and the spe- cific inserted amino acid. For example, the insertion of serine behind position 84 is designated as“84'S”. Variants having insertions in the context of amino acid changes may also be applied to nucleic acid modifications, e.g. by insertions.
  • amino acid residue insertion, deletion or substitution may be at an amino acid residue posi- tion selected from the group of positions 23, 33, 82, 83, 84, 85, 160, 199, 254, 255, 256, 258, 263, 264, 265, 268, 308 and 311 of SEQ ID No. 1.
  • the at least one amino acid substitution is selected from the group consisting of Y23A, K33N, S82T, S83D, S83H, S83I, S83N, S83R, S83T, S83Y, S84S, S84N, I85A, I85C, I85F, I85H, I85L, I85M, I85P, I85S, I85T, I85V, I85Y, K160N, P199I, P199V, I254A, I254C, I254E, I254F, I254G, I254L, I254M, I254N, I254R, I254S, I2454V, I254W, I254Y, I255A, I255L, A256D, L258A, L258D; L258E, L258G, L258H, L258N, L258Q, L258R, L258S, L258T, L258V
  • the at least one amino acid substitution is D265G.
  • the numbering refers to the numbering of amino acids in the sequence according to SEQ ID No. 1.
  • the at least one amino acid insertion is selected from the group consisting of 84 ⁇ , 84’L and 84’S.
  • the numbering refers to the numbering of amino acids in the sequence accord- ing to SEQ ID No. 1.
  • the variant may also comprise a combination of two, three, four, five, six or more amino acid substitutions as described above, preferably of two or three amino acid substitutions as de- scribed above.
  • the variant comprises one of the following combinations of amino acid substitutions: a) I85L, D265S, T268G; b) S83H, I85L, T268G; and
  • the numbering refers to the numbering of amino acids in the sequence according to SEQ ID No. 1 .
  • lipase activity means that a protein has the ability to cleave a suitable lipid substrate.
  • the variants of the lipase have essentially the same lipase activity as the wild-type lipase characterized by the specific sequence according to SEQ ID No. 1 , i.e. the activity of the variants is at least 50% or 60%, preferably at least 70% or 80%, more preferably 85% or 90% and most preferably at least 95% or 98% of the activity of the wild-type lipase characterized by the specific sequence according to SEQ ID No. 1 .
  • the activity of the variants can be compared with the activity of the corresponding wild-type li- pase by incubating the variant and the wild-type lipase with a suitable substrate under suitable conditions and detecting the amount of the cleavage products of the variant and the wild-type lipase.
  • 1 include triacylglycerol, monoglactosyl diglyceride, digalactosyl diglyceride, N-acylphosphatidyl ethanolamine, phospha- tidyl choline, monoacyl glycerol, diacyl glycerol, free fatty acid, monogalactosyl monoglyceride and digalctosyl monoglyceride.
  • the lipase or an enzymatically active variant thereof as described herein is preferably recombi- nantly produced using a bacteria, fungi, or yeast expression system.“Expression system” also means a host microorganism, expression hosts, host cell, production organism, or production strain and each of these terms can be used interchangeably for this disclosure.
  • expression systems include but are not limited to: Aspergillus niger, Aspergillus oryzae, Han- senula polymorpha, Thermomyces lanuginosus, Fusarium oxysporum, Fusarium heterosporum, Escherichia coH, Bacillus, preferably Bacillus subti/is, or Bacillus Hcheniformis, Pseudomonas, preferably Pseudomonas fluorescens, Pichia pastoris (also known as Komagataella phaffii), My- celiopthora thermophile ( C1 ' ), Schizosaccharomyces pombe, Trichoderma, preferably Tricho- derma reesei and Saccharomyces, preferably Saccharomyces cerevisiae.
  • the lipase or an enzymatically active variant thereof as described herein is produced using one of the expression systems listed above.
  • Suitable vectors for expressing the lipase and cultivation con- ditions for producing the lipase are known to the skilled person.
  • the lipase or an enzymatically active variant thereof as described herein may be isolated from the host microorganism by well-known methods including centrifugation and filtration which re- move most of the host cell components. If a higher degree of purity is desired, the lipase may be subjected to further purification steps such as anion or cation exchange chromatography, hy- drophobic interaction chromatography, mixed mode chromatography or hydroxyapatite chroma- tography.
  • the isolated lipase may be used directly or it may be subjected to a drying step such as a spray-drying step. If the isolated lipase is dried, e.g. spray-dried or lyophilized, it has to be dissolved in a suitable solvent, before it is applied to the soil.
  • the lipase or an enzymatically active variant thereof as described above may be used in combi- nation with another enzyme which may be useful in reducing soil water repellency and/or en- hancing water holding capacity.
  • This other enzyme may be selected from the group consisting of amylases, cellulases, chitinases, esterases, beta-glucosidases, laccases, pectinases, prote- ases and xylanases.
  • the lipase or a variant thereof is used in combination with a chi- tinase, a laccase, a pectinase and/or a protease.
  • the chitinase may be from Streptomyces griseus.
  • the laccase may be from Pycnoporus sp. SYBC-L3.
  • the pectinase may be from Asper gillus niger
  • the protease may be from Aspergillus oryzae.
  • the lipase may be used in a composition with at least one auxiliary.
  • the at least one auxiliary may be a liquid carrier which may be selected from the group of water and organic solvents.
  • the at least one auxiliary may be a solid carrier selected from the group of phytogels, hydro- gels, mineral earths, meal, cellulose powder, fumed silica or precipitated silica, polysaccharides, or compost.
  • the lipase is dissolved in water.
  • the lipase may be used in combination with at least one soil additive.
  • the soil additive may be selected from the group consisting of surfactants, fertilizers, nitrification inhibitors, urease inhibi- tors and/or pesticides.
  • surfactant (synonymously used herein with“surface active agent” and “wetting agent”) means an organic chemical that, when added to a liquid, changes the properties of that liquid at an in- terface. According to its ionic charge, a surfactant is called non-ionic, anionic, cationic, or am- photeric. Other examples of surfactants include block polymers and polyelectrolytes.
  • Non-limiting examples of surfactants are disclosed McCutcheon's 2016 Detergents and Emulsi fiers, and McCutcheon's 2016 Functional Materials, both North American and International Edi- tion, MC Publishing Co, 2016 edition. Further useful examples are disclosed in earlier editions of the same publications which are known to those skilled in the art.
  • Non-ionic surfactant means a surfactant that contains neither positively nor negatively charged (i.e. ionic) functional groups. In contrast to anionic and cationic surfactants, non-ionic surfac- tants do not ionize in solution. Mixtures of two or more different non-ionic surfactants may also be used.
  • Amphoteric surfactants are those, depending on pH, which can be either cationic, zwitterionic or anionic. Mixtures of two or more different amphoteric surfactants may also be used.
  • Anionic surfactant means a surfactant with a negatively charged ionic group.
  • Anionic surfactants include, but are not limited to, surface-active compounds that contain a hydrophobic group and at least one water-solubilizing anionic group, usually selected from sulfates, sulfonate, and car- boxylates to form a water-soluble compound. Mixtures of two or more different anionic surfac- tants may also be used. Mixtures of non-ionic and/or amphoteric and/or anionic surfactants may also be used.
  • Cationic surfactant means a surfactant with a positively charged ionic group.
  • these cationic moieties are nitrogen containing groups such as quaternary ammonium or protonated amino groups.
  • the cationic protonated amines can be primary, secondary, or tertiary amines.
  • Suitable block polymers are block polymers of the A-B or A-B-A type comprising blocks of poly- ethylene oxide and polypropylene oxide or of the A-B-C type comprising alkanol, polyethylene oxide and polypropylene oxide.
  • the surfactant is an ethylene oxide-propylene oxide block copolymer (EO/PO) or a blend of alkyl polyglycoside and ethylene oxide-propylene oxide block copolymer (APG-EO/PO).
  • EO/PO ethylene oxide-propylene oxide block copolymer
  • APG-EO/PO ethylene oxide-propylene oxide block copolymer
  • Such surfactants are marketed as ACA 1853 (EO/PO) and ACA 1848 (APG-EO/PO), respectively.
  • Suitable polyelectrolytes are polyacids or polybases.
  • polyacids are alkali salts of polyacrylic acid or polyacid comb polymers.
  • polybases are polyvinylamines or poly- ethyleneamines.
  • surfactants are commercially available for reducing soil water repellency. These in- clude, but are not limited to, Kick ® (available from Compo), Clearing (available from Collier Turf Care), Primer ® 604 (available from Plant Products) and Aqueduct ® , Revolution ® , ACA 1853 and ACA 1848 (all available from Aquatrols).
  • solvents or liquid carriers, solid carriers, surfactants, adjuvants, dispersants, emulsifiers, wetters, adjuvants, solubilizers, penetration enhancers, protective col- loids, adhesion agents, thickeners, humectants, compatibilizers, bactericides, anti-freezing agents, anti-foaming agents, colorants, tackifiers, binders, preservatives, antioxidants, and odorants may be used in addition to the lipase.
  • Suitable solvents and liquid carriers are water and organic solvents, such as mineral oil frac- tions of medium to high boiling point, e.g. kerosene, diesel oil; oils of vegetable or animal origin; aliphatic, cyclic and aromatic hydrocarbons, e. g. toluene, paraffin, tetrahydronaphthalene, alkyl- ated naphthalenes; alcohols, e.g. ethanol, propanol, butanol, cyclohexanol; glycols; DMSO; ke- tones, e.g. cyclohexanone; esters, e.g.
  • mineral oil frac- tions of medium to high boiling point e.g. kerosene, diesel oil
  • oils of vegetable or animal origin oils of vegetable or animal origin
  • aliphatic, cyclic and aromatic hydrocarbons e. g. toluene, paraffin, tetrahydronaphthalene, al
  • lactates carbonates, fatty acid esters, gammabutyrolac- tone; fatty acids; phosphonates; amines; amides, e.g. N-methylpyrrolidone, fatty acid dimethyla- mides; and mixtures thereof.
  • the solvent is water.
  • Suitable solid carriers or fillers are mineral earths, e.g. silicates, silica gels, talc, kaolins, lime- stone, lime, chalk, clays, dolomite, diatomaceous earth, bentonite, calcium sulfate, magnesium sulfate, magnesium oxide; polysaccharides, e.g. cellulose, starch; fertilizers, e.g. ammonium sulfate, ammonium phosphate, ammonium nitrate, urea; products of vegetable origin, e.g. ce- real meal, tree bark meal, wood meal, nutshell meal, and mixtures thereof.
  • mineral earths e.g. silicates, silica gels, talc, kaolins, lime- stone, lime, chalk, clays, dolomite, diatomaceous earth, bentonite, calcium sulfate, magnesium sulfate, magnesium oxide
  • polysaccharides e.g. cellulose, starch
  • Suitable thickeners are polysaccharides (e.g. starch, xanthan gum, carboxymethylcellulose), anorganic clays (organically modified or unmodified), polycarboxylates, superabsorbent poly- mers and silicates.
  • Suitable bactericides are bronopol and isothiazolinone derivatives such as alkylisothiazolinones and benzisothiazolinones.
  • Suitable anti-freezing agents are ethylene glycol, propylene glycol, urea and glycerin.
  • Suitable anti-foaming agents are silicones, long chain alcohols, and salts of fatty acids.
  • Suitable colorants are pigments of low water solubility and water- soluble dyes. Examples are
  • inorganic colorants such as iron oxide, titan oxide, iron hexacyanoferrate,
  • metal-complex dyes such as chromium-complex dyes, for example Orasol Yellow 141 ,
  • organic colorants such as alizarin-, azo- and phthalocyanine colorants.
  • Preferred colorants are metal-complex dyes, more preferably chromium-complex dyes
  • Suitable tackifiers or binders are polyvinylpyrrolidones, polyvinylacetates, polyvinyl alcohols, polyacrylates, biological or synthetic waxes, and cellulose ethers.
  • Suitable preservatives include e.g. sodium benzoate, benzoic acid, sorbic acid, and derivatives thereof.
  • Suitable antioxidants include sulfites, ascorbic acid, tocopherol, tocopherol acetate, tocotrienol, melatonin, carotene, beta-carotene, ubiquinol, and derivatives thereof. Tocophercol acetate is preferred as antioxidant.
  • compounds which stabilize the lipase enzyme may be used, such as buffers, chela- tors, anti-oxidants, non-ionic surfactants, sugars, proteins (e.g. BSA) and heavy metal and phe- nol scavengers.
  • fertilizers and/or nitrification inhibitors and/or urease inhibitors may be used in addition to the lipase or a variant thereof as defined herein.
  • fertilizer includes any chemical compound that improves the levels of available plant nutrients and/or the chemical and physical properties of soil, thereby directly or indirectly promoting plant growth, yield, and quality. Fertilizers are typically applied either through the soil (for uptake by plant roots) or by foliar feeding (for uptake through leaves).
  • the term “fertilizer” can be subdivided into two major categories: a) organic fertilizers (composed of decayed plant/animal matter) and b) inorganic fertilizers (composed of chemicals and minerals).
  • Inorganic fertilizers are usually manufactured through chemical processes (such as the Haber- Bosch process), also using naturally occurring deposits, while chemically altering them (e.g. concentrated triple superphosphate). Naturally occurring water soluble inorganic fertilizers in- clude Chilean sodium nitrate.
  • the fertilizer is preferably a urea-containing fertilizer, and/or P-containing fertilizer, and/or a K fertilizer (potassium-containing fertilizer), and/or a N fertilizer (nitrogen-containing fertilizer), and/or a NK fertilizer (nitrogen-potassium fertilizer), and/or a NPK (nitrogen-phosphorous-potas- sium fertilizer), and/or a single or dual element fertilizer containing S, Ca, Mg, Fe, Mn, Cu, Zn, Mo, B, Ni, Cl, or a combination thereof.
  • a“urea-containing fertilizer” is defined as a fertilizer comprising at least one component selected from the group consisting of urea, urea ammonium nitrate (UAN), and sus- pensions of isobutylidene diurea (IBDU), crotonylidene diurea (CDU) and urea formaldehyde (UF), urea-acetaldehyde, and ureaglyoxal condensates.
  • IBDU isobutylidene diurea
  • CDU crotonylidene diurea
  • UF formaldehyde
  • urea-acetaldehyde ureaglyoxal condensates.
  • the urea-containing fertilizer is urea or urea ammo- nium nitrate (UAN).
  • the urea In customary commercial fertilizer quality, the urea has a purity of at least 90%, and may for ex- ample be in crystalline, granulated, compacted, prilled or ground form.
  • the urea-containing fertilizer may be used together with a urease inhibitor.
  • Urease is an en- zyme which hydrolyzes urea to ammonia and carbon dioxide.
  • high urease activity during treatment with urea-containing fertilizers causes significant environmental and economic problems due to the release of ammonia which may be toxic to the plants and which deprives the plants of urea. Accordingly, it is desirable to inhibit the action of urease.
  • Inhibitors of urease activity may comprise (i) substrate structural analogs of urea as e.g. hydroxyurea or hydroxamic acid or (ii) inhibitors which affect the mechanism of the urease reaction.
  • the later may be di- vided in the four groups of (i) phosphorodiamidates or phosphorotriamidiates as e.g. N-(n-bu- tyl)thiophosphoric triamide, (ii) thiols as e.g. cysteamine, (iii) hydroxamic acids and its deriva- tives as e.g. acetohydroxamic acid, and (iv) ligands and chelators of the nickel ion in the active center of ureases as e.g. fluoride ions.
  • Urease inhibitors are also discussed in Upadhyay (2012) Ind. J. Biotechnol. 1 1 : 381-388 and in "Improving Efficiency of Urea Fertlizers by Inhibition of Soil Urease Activity" by Kiss and Simihaian (2002), Springer Netherlands, ISBN 978-1-4020- 0493-3.
  • the“P-containing fertilizer” is any fertilizer providing any form of the chemical element phosphorus (P) or containing any chemical compounds incorporating the chemical ele- ment phosphorus (P), including but not limited to phosphate-containing fertilizers or fertilizers containing P.
  • the P-containing fertilizer is selected from the group consisting of a NPK fertilizer, a NP fertilizer, a PK fertilizer, or a P fertilizer.
  • the P-containing fertilizer is a NPK fertilizer.
  • combinations of these fertilizers may be used as ad- ditional P-containing fertilizer.
  • P fertilizers, K fertilizers, and N fertilizers are straight fertilizers, i.e. fertilizers that contain only one of the nutritive elements P, K, and N. It is to be understood, however, that these fertilizers may additionally comprise at least one additional nutritive element selected from S, Ca, Mg, Fe, Mn, Cu, Zn, Mo, B, Ni, and Cl.
  • NPK fertilizers, NP fertilizers, and PK fertilizers are multinutrient fertilizers, i.e. fertilizers that comprise combinations of the nutritive elements P, K, and N as indicated by the terms“NPK”, “NP”, and“PK”. It is to be understood, however, that these fertilizers may additionally comprise at least one additional nutritive element selected from S, Ca, Mg, Fe, Mn, Cu, Zn, Mo, B, Ni and Cl.
  • the NPK fertilizers, NP fertilizers, and PK fertilizers may be provided as complex fertilizers or bulk-blend or blended fertilizers.
  • complex fertilizer refers to a compound fertilizer formed by mixing ingredients that react chemically. In bulk-blend or blended fertilizers, two or more granular fertilizers of similar size are mixed to form a compound fertilizer.
  • Dual element fertilizers are preferably dual element fertilizers with Ca, Mg, Fe, Mn, Zn or Ni which may be applied as soluble salts of chloride, sulfate, nitrate or in chelated form (e.g.
  • Single or dual element fertilizers of Mo are available as salts of molybdate, B as boric acid or borates.
  • Ammonium (NH 4 + ) compounds present in nitrogen-containing fertilizers are converted by soil microorganisms to nitrates (NO 3 ) in a relatively short time in a process known as nitrification.
  • the nitrification process typically leads to nitrogen leakage and environmental pollution.
  • approximately 50% of the applied nitrogen fertilizers are lost during the year following fertilizer addition (see Nelson and Huber; Nitrification inhibitors for corn pro- duction (2001), National Corn Handbook, Iowa State University).
  • nitrification inhibitors mostly together with fertilizers, can be used.
  • Suitable nitrification inhibitors include linoleic acid, alpha-linolenic acid, methyl p-coumarate, methyl feru- late, methyl 3-(4-hydroxyphenyl) propionate (MHPP), Karanjin, brachialacton, p-benzoquinone sorgoleone, 2-chloro-6-(trichloromethyl)-pyridine (nitrapyrin or N-serve), dicyandiamide (DCD, DIDIN), 3,4-dimethyl pyrazole phosphate (DMPP, ENTEC), 4-amino-1 ,2,4-triazole hydrochloride (ATC), 1-amido-2 -thiourea (ASU), 2-amino-4-chloro-6-methylpyrimidine (AM), 2-mercapto-ben- zothiazole (MBT), 5-ethoxy-3-trichloro
  • Pesticides are substances which aim to control pests and include herbicides, insecticides, ne- maticides, mollluscicides, piscicides, avicdes, rodenticides, bactericides, insect repellents, ani- mal repellents, antimicrobials and fungicides.
  • the water-repellent soil is a fairy ring caused by basidiomycetes
  • a fungicide is used in combination with the lipase. More preferably, the fungizide is a fungizide which can be ap- plied through the soil.
  • the present invention also relates to the use of (a) an isolated polypeptide having lipase activity as disclosed herein; and (b) at least one fungicide for the treatment of at least one fairy ring.
  • the present invention also relates to a method for treating at least one fairy ring comprising ap- plying (a) an isolated polypeptide having lipase activity as disclosed herein; (b) at least one fun- gicide to an area of groundcover.
  • Suitable fungicides include strobilurins, carboximides, oxidizing agents, polyoxins and sterol de- methylation inhibitors. Specific examples include, but are not limited to, Azoxystrobin, Triflox- istrobin, Picoxystrobin, Pyraclostrobin, Sedaxane, Penthiopyrad, Penflufe, Fluopyram, Fluxapy- roxad, Boscalid, Oxathiapiprolin, Metalaxyl, Metalaxyl-M, Ethaboxam, DMM, Cyproconazole, Difenoconazole, Prothioconazole, Flutriafol, Thiabendazole, Ipconazole, Tebuconazole, Triad- imenol, Prochloraz, Fluquinconazole, TTZ, Fludioxinil, Carboxin, Silthiofarm, Ziram, Thiram, Carbendazim, TPM, Valifenalate, Oxa
  • fungicides which are marketed for the treatment of fairy rings and which may be used in combination with the lipase include azoxystrobin (marketed under the name Heritage ® ), flutolanil (marketed under the name Prostar ® ), hydrogen dioxide (marketed under the name Zerotol ® ), polyoxin D (marketed under the name Endorse ® ), triadimefon (marketed un- der the name Bayleton ® FLO), pyraclostrobin (marketed under the name Insignia ® ), a mixture of pyraclostrobin and triticonazole (marketed under the name Pillar ® G Intrinsic), a mixture of azoxystrobin and propiconazole (marketed under the name Headway ® ) and a mixture of triad- imefon and trifloxystrobin (marketed under the name Tartan ® Stressgard ® ).
  • azoxystrobin marketed under the name Heritage ®
  • flutolanil marketed under the name Prostar ®
  • Insecticides which may be used in combination with the lipase include, but are not limited to, Fipronil, BAS 450, IL-39, Clothianidin, Thiamethoxam, Acetamiprid, Dinotefuran, Imidaclophd, Thiaclophd, Sulfoxaflor, Methiocarb, Tefluthrin, Bifenthrin, Cypermethrin, Alphacypermethrin, Spinosad, Cyazypyr, Rynaxapyr, Thiodicarb, Triflumezopyrim (Mesoionic), Acephate, Chlorpyri- phos, Flupyradifurone, Tetraniliprole, IL-101 , S-1587, Oxazosulfyl, Cyclaniliprole and Fluxamet- amide.
  • an insecticide selected from the group consisting of Fipronil, BAS 450, IL-39, Clothianidin, Thiamethoxam, Imidacloprid, Thiacloprid, Sulfoxaflor, Tefluthrin, Spinosad, Cyazypyr, Rynaxapyr, Triflumezopyrim (Mesoionic), Acephate and Chlorpyriphos is used in combination with the lipase.
  • an insecticide selected from the group consisting of Fipronil, BAS 450, IL-39, Clothianidin, Thiamethoxam, Imidacloprid, Thiacloprid, Sulfoxaflor, Tefluthrin, Spinosad, Cyazypyr, Rynaxapyr, Triflumezopyrim (Mesoionic), Acephate and Chlorpyriphos is used in combination with the lipase.
  • Nematicides which may be used in combination with the lipase include, but are not limited to, Thiodicarb, Abamectin, Tioxazafen, Bacillus firmus and Pasteuria nishizawae.
  • the polypeptide having lipase activity may be applied to the groundcover in a concentration of between 0.01 kg to 600 kg of polypeptide per hectare, preferably of between 0.16 kg to 100 kg per hectare, more preferably of between 0.6 kg to 20 kg per hectare, most preferably of be- tween 1 kg to 5 kg per hectare.
  • the lipase is dissolved in water in a concentration of 0.01 % to 30% w/v, preferably of 0.02% to 20% or 10% w/v, more preferably of 0.03% to 0.8 or 0.6% w/v, even more preferably of 0.04% to 0.4% or 0.3% w/v and most preferably of 0.05% to 0.1 %.
  • concentration of the lipase is 0.06% w/v.
  • the lipase is dissolved in water in a concentration of 0.01 % to 0.1 % w/v, preferably of 0.02% to 0.08% w/v, more preferably of 0.03% to 0.07% and most preferably of 0.06% (w/v).
  • the lipase is dissolved in water in a concentration of 1 % to 50% w/v, preferably of 5% to 30% w/v, more preferably of 8% to 20% and most preferably of 10% (w/v).
  • the lipase is dissolved in a great volume of water for application to a great area of soil.
  • the lipase is dissolved in a small volume of water for application to a small area.
  • the lipase is applied to an area of groundcover.
  • groundcover includes, but is not limited to, soil, natural soil, potting soil, sand, silt, clay, turfgrasses and other plants and forms of vegetation used to cover and pro- tect the soil, as well as composites of organic materials that form within or as part of such groundcovers, such as thatch and mat layers, and also includes potting mixes.
  • groundcover is soil, more preferably, groundcover is water-repellent or non-wetting soil. In another preferred embodiment, groundcover is potting mix.
  • Potting mix which is also re- ferred to potting soil, is typically a soilless blend of ingredients that is used to grow plants, pref- erably, the potting mix comprises a combination of peat moss, vermiculite, coir fiber, perlite, pine bark, sand, compost, and further ingredients.
  • the potting mix may also comprise native soil.
  • applying includes any activity by which the lipase come into contact with the area of groundcover.
  • the lipase can be dissolved in water and applied as a solution.
  • a granulate can be prepared from the lipase and the granulate is then applied to the groundcover in solid form which is dissolved when water is applied to the soil.
  • the lipase is applied in irrigation water. In one embodiment the lipase is ap- plied extensively and non-directional to turfgrass soil. In one embodiment the lipase is applied extensively and non-directional to turfgrass soil in irrigation water. In one embodiment the lipase is applied directional to turfgrass soil using a watering can.
  • the lipase is applied to turfgrass soil as part of a topdressing which corn- prises particles to which the lipase is coupled.
  • Topdressing refers to a material applied to the top of a ground covering, usually in order to obtain a desirable effect on the groundcover, and includes sand or other particulate material.
  • the application of the lipase serves to prepare a non-wetting soil for the seeding of crops.
  • the lipase is applied selectively to those locations of the non-wetting soil where the seeds will be placed, e.g. the seeding row, so that they can prepare the soil for seed uptake.
  • the lipase is applied non-selectively to the en- tire application site (so-called blanket application).
  • Non-naturally occurring variant lipase enzymes were created using rational design single site mutagenesis and multisite mutagenesis.
  • the variant lipase enzymes include single point amino acid modifications, insertions, or deletions compared to a parent enzyme (LIP062, which is the amino acid sequence of SEQ ID 1 , and is encoded by nucleic acid sequence of SEQ ID NO:2) at 18 different amino acid residue positions: 23, 33, 82, 83, 84, 85, 160, 199, 254, 255, 256, 258, 263, 264, 265, 268, 308, 31 1 , or any combination thereof, wherein the variant lipase enzymes has lipase activity.
  • LIP062 which is the amino acid sequence of SEQ ID 1 , and is encoded by nucleic acid sequence of SEQ ID NO:2
  • LIP062 which is the amino acid sequence of SEQ ID 1 , and is encoded by nucleic acid sequence of SEQ ID NO:2
  • LIP062 which is the amino acid
  • Variant lipase enzymes were also created with various combinations of the single point modifica- tions of the parent enzyme (LIP062), wherein the variant lipase enzymes have lipase activity.
  • LIP062 parent enzyme
  • the single point modifications and various combinations of single point modifications are listed in Table: 1.
  • the variant lipase enzymes were obtained by constructing expression plasmids containing the encoding polynucleotide sequences, transforming plasmids into Pichia pastor/s ( Komagataella phaffit) and growing the resulting expression strains in the following way.
  • Fresh Pichia pastoris cells of the expression strains were obtained by spreading the glycerol stocks of sequence-con- firmed strains onto Yeast extract Peptone Dextrose (YPD) agar plates containing Zeocin. After 2 days, starter seed cultures of the production strains were inoculated into 100 ml.
  • BMGY Buffered Glycerol complex Medium
  • the cell-free supernatants were ultrafiltered using a lab-scale tangential flow filtration (TFF) system with a molecular weight cut-off of 5 kD (Spectrum Labs). Samples were first concentrated 10-20X and then buffer-exchanged 5X into 50 mM HEPES pH 7.5. The resultant retentate was centrifuged at 27000xg for 1 hour, and then sterile filtered through 0.2 pm filters to remove any production organisms or particulate matter. Total protein content of the final samples was determined using the Braford assay. Lipases were lyophilized to form pow- der.
  • TMF lab-scale tangential flow filtration
  • the activity of the variant lipase enzymes was determined using natural substrates in solution. Natural lipid substrates were prepared at 5 mM final concentration in 0.25 % sodium deoxycholate by sonication. Substrate (15 pL) was mixed with 30 uL fluorescein (0.25 pg/mL in 10 mM CaCI2) and 10 pL recovered lipase ( ⁇ 1 -2 pg/mL) pre-diluted in 5 mM Hepes pH 7.5. Products of lipid hydrolysis were monitored by the drop in fluorescence due to pH change (485 nm/525 nm for excitation/emission), recorded kinetically every 30 seconds for 10 min at 26°C . Activity on a log scale was proportional with the fluorescence change per min. The results are shown below in Table 2, expressed as percentage of parent (LIP062) fluorescence change at same protein con- centration.

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Abstract

La présente invention concerne un procédé pour améliorer la disponibilité de l'eau dans le sol à l'aide d'une lipase.
PCT/EP2019/057395 2018-03-26 2019-03-25 Procédé de réduction de la répulsion d'eau du sol WO2019185519A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023116569A1 (fr) 2021-12-21 2023-06-29 Novozymes A/S Composition comprenant une lipase et un renforçateur
WO2024121058A1 (fr) 2022-12-05 2024-06-13 Novozymes A/S Composition comprenant une lipase et un peptide

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013181240A2 (fr) 2012-05-30 2013-12-05 University Of Georgia Research Foundation, Inc. Procédés et compositions réduisant le caractère hydrofuge des sols
WO2014181099A1 (fr) * 2013-05-08 2014-11-13 Croda International Plc Traitement de sol

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013181240A2 (fr) 2012-05-30 2013-12-05 University Of Georgia Research Foundation, Inc. Procédés et compositions réduisant le caractère hydrofuge des sols
WO2014181099A1 (fr) * 2013-05-08 2014-11-13 Croda International Plc Traitement de sol

Non-Patent Citations (28)

* Cited by examiner, † Cited by third party
Title
"Current Protocols in Molecular Biology", 1985, JOHN WILEY & SONS
"Essential Molecular Biology: A Practical Approach", IRL PRESS AT OXFORD UNIVERSITY PRESS
"Nucleic Acids Hybridization: A Practical Approach", 1991, IRL PRESS AT OXFORD UNIVERSITY PRESS
ATANASSOVA; DOERR, EUROP. J. OF SOIL SCI., vol. 61, 2010, pages 298 - 313
BACHMANN, J.; ELLIES, A.; HARTGE, K.H.: "Development and application of a new sessile drop contact angle method to assess soil water repellency", J. OF HYDROLOGY, vol. 231, 2000, pages 66 - 75, XP085048042, DOI: doi:10.1016/S0022-1694(00)00184-0
BARTON; COLMER, AGRIC. WATER MANAGEMENT, vol. 99, 2011, pages 1 - 7
CREIGHTON: "Proteins", 1984, W.H. FREEMAN AND COMPANY
DA-VIES ET AL., CROP UPDATES 2012, 2012, pages 358 - 362
DEBANO ET AL., J. HYDROL., vol. 231, 2000, pages 4 - 32
DEKKER ET AL., AUST. J. OF SOIL RES., vol. 43, 2005, pages 403 - 441
DEKKER ET AL., SOIL SCI., vol. 163, 1998, pages 780 - 796
DOERR ET AL., EARTH-SCI. REVIEWS, vol. 51, 2000, pages 33 - 65
DOERR, S.H.: "Earth Surf. Landforms", vol. 23, 1998, article "On standardizing the ''water drop penetration time and the ''molarity of an ethanol droplet'' technique to classify soil hydrophobicity: A case study using medium textured soils", pages: 663 - 668
KISS; SIMIHAIAN: "Improving Efficiency of Urea Fertlizers by Inhibition of Soil Urease Activity", 2002, SPRINGER
LUSHENG ZENG ET AL: "Evaluation of Direct Application of Enzymes to Remediate Soil Water Repellency", LIFE SCIENCE WEEKLY, vol. 49, 15 July 2014 (2014-07-15), pages 3060 - 666, XP055491518, ISSN: 1552-2466 *
MCCUTCHEON: "Detergents and Emulsifiers", 2016
MCCUTCHEON: "Functional Materials", 2016, MC PUBLISHING CO
MOORE ET AL., J. HYDROL. HYDROCHEM., vol. 58, no. 3, 2010, pages 142 - 148
MULLER; DEUER, AGRIC., ECOSYSTEMS AND ENVIRON., vol. 144, 2011, pages 208 - 221
NEEDLEMAN; WUNSCH, J. MOL. BIOL., vol. 48, 1970, pages 443 - 453
NEEDLEMAN; WUNSCH, J. MOL. BIOL., vol. 48, 1979, pages 443 - 453
NELSON; HUBER: "National Corn Handbook", 2001, IOWA STATE UNIVERSITY, article "Nitrification inhibitors for corn production"
ROPER, BIOLOGIA, vol. 61, no. 19, 2006, pages S358 - S362
SAMBROOK; RUSSELL: "Molecular Cloning: A Laboratory Manual", 2001, COLD SPRING HARBOUR LABORATORY PRESS
TILLMAN ET AL., AUSTR. J. SOIL RES., vol. 27, 1999, pages 637 - 644
UPADHYAY, IND. J. BIOTECHNOL., vol. 11, 2012, pages 381 - 388
WOCHE ET AL., EUR. J. SOIL SCI., vol. 56, 2005, pages 239 - 251
ZENG, HORTSCIENCE, vol. 49, no. 5, 2014, pages 662 - 666

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023116569A1 (fr) 2021-12-21 2023-06-29 Novozymes A/S Composition comprenant une lipase et un renforçateur
WO2024121058A1 (fr) 2022-12-05 2024-06-13 Novozymes A/S Composition comprenant une lipase et un peptide

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