WO2019185610A1 - Procédé de réduction de l'hydrophobie du sol - Google Patents

Procédé de réduction de l'hydrophobie du sol Download PDF

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WO2019185610A1
WO2019185610A1 PCT/EP2019/057544 EP2019057544W WO2019185610A1 WO 2019185610 A1 WO2019185610 A1 WO 2019185610A1 EP 2019057544 W EP2019057544 W EP 2019057544W WO 2019185610 A1 WO2019185610 A1 WO 2019185610A1
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Prior art keywords
surfactant
soil
acid sequence
seq
lipase
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PCT/EP2019/057544
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English (en)
Inventor
Alexander Wissemeier
David Edward Mainwaring
Pandiyan Murugaraj
Rohan DAVIES
Wolfgang Weigelt
Uwe Thiel
Kai-Uwe Baldenius
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Basf Se
Grains Research And Development Corporation (Grdc)
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Publication of WO2019185610A1 publication Critical patent/WO2019185610A1/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
    • 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

Definitions

  • the present invention relates to a method of reducing soil water repellency and/or for increasing water holding capacity using a lipase and at least one surfactant.
  • 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 combination of a specific lipase and a surfactant leads to a reduced soil water repellency and an increased water holding capacity of soils.
  • the present invention relates to the use of
  • the at least one surfactant may be selected from anionic, cationic, nonionic and amphoteric surfactants, block polymers, polyelectrolytes, and mixtures thereof.
  • the at least one surfactant is selected from the group consisting of a block polymer (P) comprising at least one polyethyleneoxide moiety and at least one polypropyleneox- ide moiety, an alcohol alkoxylate (E) and/or an alcohol ethoxylate (L) of the general formula (XIII)
  • R 3 -0-(C 2 H 4 0)s-H (XIII) wherein R 3 is linear or branched Cs to C20 alkyl, and
  • s has a value of from 1 to 40.
  • composition comprising a block polymer (P) comprising at least one polyethyleneoxide moiety and at least one polypropyleneoxide moiety, and an alcohol alkoxylate (E); and
  • R 3 -0-(C 2 H 4 0)s-H (XIII) wherein R 3 is linear or branched Cs to C20 alkyl, and
  • s has a value of from 1 to 40
  • the surfactant may be a mixture of ethylenoxide-propyleneoxide block copolymer, 2-propylhep- tanol alkoxylate and a triblock polymer of ethylenoxide-propyleneoxide-ethylenoxide.
  • the surfactant may comprise sodium-di-ethyl-hexyl-sulfosucccinate and an iso C13-alcohol eth- oxylate.
  • polypeptide or an enzymatically active fragment thereof is 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.
  • the surfactant is applied in a concentration of 0.02 % to 4 % (v/v), preferably of 0.04 % to 3% (v/v), more preferably of 0.06% to 2% (v/v) and most preferably of 0.08 % to 1 .0 % (v/v).
  • 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 soil may be non-wetting soil.
  • the present invention further relates to a method for reducing soil water repellency and/or for enhancing water holding capacity of soils comprising applying (a) an isolated polypeptide having lipase activity and being selected from the group consisting of:
  • the polypeptide and the at least one surfactant may be applied to the area of groundcover sim- ultaneously.
  • the at least one surfactant may be selected from anionic, cationic, nonionic and amphoteric surfactants, block polymers, polyelectrolytes, and mixtures thereof.
  • the at least one surfactant is selected from the group consisting of a block polymer (P) comprising at least one polyethyleneoxide moiety and at least one polypropyleneox- ide moiety, an alcohol alkoxylate (E) and an alcohol ethoxylate (L) of the general formula (XIII)
  • R 3 -0-(C 2 H 4 0)s-H (XIII) wherein R 3 is linear or branched Cs to C20 alkyl, and
  • s has a value of from 1 to 40.
  • composition comprising a block polymer (P) comprising at least one polyethyleneoxide moiety and at least one polypropyleneoxide moiety, and an alcohol alkoxylate (E); and
  • R 3 -0-(C 2 H 4 0)s-H (XIII) wherein R 3 is linear or branched Cs to C20 alkyl, and
  • s has a value of from 1 to 40
  • the surfactant may be a mixture of ethylenoxide-propyleneoxide block copolymer, 2-propylhep- tanol alkoxylate and a triblock polymer of ethylenoxide-propyleneoxide-ethylenoxide.
  • the surfactant may comprise sodium-di-ethyl-hexyl-sulfosucccinate, a polymeric alcohol ethox- ylate and bis(2-ethylhexyl)maleate.
  • polypeptide or an enzymatically active fragment thereof is 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, more preferably of between 0.6 kg to 20 kg, most preferably of between 1 kg to 5 kg.
  • the surfactant is applied in a concentration of 0.02 % to 4 % (v/v), preferably of 0.04 % to 3% (v/v), more preferably of 0.06% to 2% (v/v) and most preferably of 0.08 % to 1.0 % (v/v).
  • the present invention also relates to a composition
  • a composition comprising:
  • the at least one surfactant is selected from the group consisting of a block polymer (P) comprising at least one polyethyleneoxide moiety and at least one polypropyleneoxide moiety, an alcohol alkoxylate (E) and/or an alcohol ethoxylate (L) of the general formula (XIII)
  • R 3 -0-(C 2 H 4 0) s -H (XIII) wherein R 3 is linear or branched Cs to C20 alkyl, and
  • s has a value of from 1 to 40.
  • Figure 1 Comparison of Day 7 infiltration rate (upper graph) and water retention (lower graph) in soil columns treated with surfactant only and surfactant+lipase (after a cumulative rain of 1 1 mm from two rain events)
  • Figure 2 Comparison of Day 105 infiltration rate (upper graph) and water retention (lower graph) in soil columns treated with surfactant only and surfactant+lipase (after a cumulative rain of 500 mm from five rain events)
  • 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.
  • 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 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 5% or 10%, preferably by at least 15% or 20%, more preferably by at least 25% or 30% and most preferably by at least 40% or 50%. 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 5% or 10%, preferably by at least 15% or 20%, more preferably by at least 25% or 30% and most preferably by at least 40% or 50%.
  • water holding capacity refers to the amount of water that a given amount of soil can hold. This has an impact on the water supply of any crops planted on the soil, but also on the retention 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 applying 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 is increased by at least 3% or 5%, preferably by at least 10% or 15%, more preferably by at least 20% or 25% and most preferably by at least 30%.
  • 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%, preferably by at least 10% or 15%, more preferably by at least 20% or 25% and most preferably by at least 30% 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 is increased.
  • the soil water repellency is re-umbled by at least 5% and the water holding capacity is increased by at least 3%, preferably the soil water repellency is reduced by at least 20% and the water holding capacity is increased by at least 10%, more preferably the soil water repellency is reduced by at least 30% and the water holding capacity is increased by at least 20% and most preferably the soil water repellency is reduced by at least 50% and the water holding capacity is increased by at least 30%.
  • 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 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. Pref- erably, 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 mix, and soils irrigated with treated wastewater.
  • the soil is agricultural land which means that it is used for planting crops.
  • the agricultural land has not been treated by tillage (so-called no-till agriculture).
  • the agricultural land comprises 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.
  • the total area of agricultural 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°.
  • 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.
  • the enzyme used in the method of the present invention is 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 having an amino acid sequence with at least 70% sequence identity to the amino acid sequence according to SEQ ID No. 1 or an enzymatically active fragment thereof;
  • an "enzymatically active fragment" of the lipase having the amino acid sequence according to SEQ ID No. 1 is understood to refer to a smaller part of the lipase which consists of a contigu- ous amino acid sequence found in SEQ ID No. 1 and which has lipase activity.
  • the fragment of the lipase according to SEQ ID No. 1 comprises at least amino acid residues 87 to 285 of SEQ ID No.1 , more preferably it comprises at least amino acid residues 61 to 291 of SEQ ID No.1 , even more preferably it comprises at least amino acid residues 41 to 301 of SEQ ID No. 1 and most preferably it comprises at least amino acid residues 11 to 311 of SEQ ID No. 1 .
  • a "fragment" of the nucleic acid sequence according to SEQ ID No. 2 is understood to refer to a smaller part of this nucleic acid sequence which consists of a contiguous nucleotide sequence found in SEQ ID No. 2 and which encodes a protein having lipase activity.
  • the frag- ment of the nucleic acid sequence according to SEQ ID No. 2 encodes at least amino acid resi- dues 87 to 285 of SEQ ID No.1 , more preferably it encodes at least amino acid residues 61 to 291 of SEQ ID No.1 , even more preferably it encodes at least amino acid residues 41 to 301 of SEQ ID No. 1 and most preferably it encodes at least amino acid residues 11 to 311 of SEQ ID No. 1.
  • 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 acid serine on position 87 of SEQ ID No. 1 , the amino acid aspartate on position 241 of SEQ ID No.
  • 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.
  • variants have lipase activity, i.e. the ability to cleave a suitable lipid substrate.
  • the variants of the lipase according to SEQ ID No. 1 have essentially the same lipase activity as the lipase 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 lipase characterized by the sequence according to SEQ ID No. 1.
  • the activity of the variants can be compared with the activity of the lipase according to SEQ ID No. 1 by incubating the variant and the wild-type lipase according to SEQ ID No. 1 with a suita- ble substrate under suitable conditions and detecting the amount of the cleavage products of the variant and the wild-type lipase.
  • the lipase according to SEQ ID No. 1 or an enzymatically active variant thereof as described herein is preferably recombinantly 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 dis closure.
  • expression systems include but are not limited to: Aspergillus niger, Asper gillus oryzae, Hansenula polymorpha, Thermomyces lanuginosus, fusarium oxysporum, Fusarium heterosporum, Escherichia coH, Bacillus, preferably Bacillus subti/is, or Bacillus Hchen- iformis, Pseudomonas, preferably Pseudomonas Huorescens, Pichia pastoris (also known as Ko- magataella phaffii), Myceliopthora thermophile (C1), Schizosaccharomyces pom be, Burkholderia glumae, Burkholderia plantarii, Trichoderma, preferably Trichoderma reesei and Saccharomyces, preferably Saccharomyces cerevisiae.
  • the lipase according to SEQ ID No. 1 or an enzymatically active variant thereof as described herein is produced using one of the ex- pression systems listed above.
  • Suitable vectors for expressing the lipase and cultivation condi- tions for producing the lipase are known to the skilled person.
  • the lipase according to SEQ ID No. 1 or an enzymatically active variant thereof as described herein may be isolated from the host microorganism by well-known methods including centrifu- gation and filtration which remove 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 ex- change chromatography, hydrophobic interaction chromatography, mixed mode chromatog- raphy or hydroxyapatite chromatography.
  • 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 according to SEQ ID No. 1 or a variant thereof as described above may be used in combination with another enzyme which may be useful in reducing soil water repellency 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 according to SEQ ID No. 1 or a variant thereof is used in combination with a chitinase, 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 Aspergillus nigerand the protease may be from Aspergillus oryzae.
  • the lipase is combined with at least one surfac- tant.
  • “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 interface. According to its ionic charge, a surfactant is called non-ionic, anionic, cationic, or amphoteric. 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.
  • Non-ionic surfactants may be compounds of the general formulae (la) and (lb):
  • R 1 is selected from C 1 -C 23 alkyl and C 2 -C 23 alkenyl, wherein alkyl and/or alkenyl are linear or branched; examples are n-CzHis, n-CgH-ig, n-CnH23, n-Ci3H27, n-CisH3i, n-Ci7H35, i-CgH-ig, i- C12H25.
  • R 2 is selected from H, C1-C20 alkyl and C2-C20 alkenyl, wherein alkyl and/or alkenyl are linear or branched.
  • R 3 and R 4 each independently selected from C1-C16 alkyl, wherein alkyl is linear or branched; examples are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, 1 ,2-dimethylpropyl, isoamyl, n-hexyl, isohexyl, sec-hexyl, n- heptyl, n-octyl, 2-ethylhexyl, n-nonyl, n-decyl, isodecyl.
  • R 5 is selected from H and C1-C18 alkyl, wherein alkyl is linear or branched.
  • n is in the range of zero to 200, preferably 1-80, more preferably 3-20; n and o, each inde- pendently in the range of zero to 100; n preferably is in the range of 1 to 10, more preferably 1 to 6; o preferably is in the range of 1 to 50, more preferably 4 to 25.
  • the sum of m, n and o is at least one, preferably the sum of m, n and o is in the range of 5 to 100, more preferably in the range of from 9 to 50.
  • non-ionic surfactants of the general formula (I) may be of any structure, is it block or ran- dom structure, and is not limited to the displayed sequence of formula (I).
  • Non-ionic surfactants may further be compounds of the general formula (II), which might be called alkyl-polyglycosides (APG):
  • R 1 is selected from C 1 -C 17 alkyl and C 2 -C 17 alkenyl, wherein alkyl and/or alkenyl are linear or branched; examples are n-C7Hi 5 , n-CgHig, n-CnH23, n-Ci3H27, n-CisH3i, n-C ⁇ Hss, i-CgHig, i- C12H25.
  • R 2 is selected from H, C1-C17 alkyl and C2-C17 alkenyl, wherein alkyl and/or alkenyl are linear or branched.
  • G 1 is selected from monosaccharides with 4 to 6 carbon atoms, such as glucose and xylose.
  • the integer w of the general formula (II) is in the range of from 1.1 to 4, w being an average number.
  • Non-ionic surfactants may further be compounds of general formula (III):
  • AO is selected from ethylene oxide (EO), propylene oxide (PO), butylene oxide (BO), and mix- tures thereof.
  • R 6 is selected from C 5 -C 17 alkyl and C 5 -C 17 alkenyl, wherein alkyl and/or alkenyl are linear or branched.
  • R 7 is selected from H, Ci-Cie-alkyl, wherein alkyl is linear or branched.
  • Non-ionic surfactants may further be selected from sorbitan esters and/or ethoxylated or propoxylated sorbitan esters.
  • Non-limiting examples are products sold under the trade names SPAN and TWEEN.
  • Non-ionic surfactants may further be selected from alkoxylated mono- or di-alkylamines, fatty acid monoethanolamides (FAMA), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxy alkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamides, GA, or fatty acid glucamide, FAGA), and combinations thereof.
  • FAMA fatty acid monoethanolamides
  • FADA fatty acid diethanolamides
  • EFAM ethoxylated fatty acid monoethanolamides
  • PFAM propoxylated fatty acid monoethanolamides
  • polyhydroxy alkyl fatty acid amides or N-acyl N-alkyl derivatives of glucosamine (glucamides, GA, or fatty acid glucamide, FAGA), and combinations thereof.
  • Mixtures of two or more different non-ionic surfactants may also be used in the present inven- tion.
  • Amphoteric surfactants are those, depending on pH, which can be either cationic, zwitterionic or anionic. Surfactants may be compounds comprising amphoteric structures of general formula (IV), which might be called modified amino acids (proteinogenic as well as non-proteinogenic):
  • R 8 is selected from H, C1-C4 alkyl, C2-C4 alkenyl, wherein alkyl and/or are linear or branched.
  • R 9 is selected from C1-C22- alkyl, C2-C22- alkenyl, C10-C22 alkylcarbonyl, and C10-C22 alkenylcar- bonyl.
  • R 10 is selected from H, methyl, -(CH 2 ) 3 NHC(NH)NH2, -CH 2 C(0)NH 2 , -CH 2 C(0)0H, - (CH 2 ) 2 C(0)NH 2 , -(CH 2 ) 2 C(0)0H, (imidazole-4-yl)-methyl, -CH(CH 3 )C 2 H 5 , -CH 2 CH(CH 3 ) 2 , - (CH 2 ) 4 NH 2 , benzyl, hydroxymethyl, -CH(OH)CH 3 , (indole-3-yl)-methyl, (4-hydroxy-phenyl)-me- thyl, isopropyl, -(CH2)2SCH 3 , and -CH2SH.
  • R x is selected from H and Ci-C4-alkyl.
  • Surfactants may further be compounds comprising amphoteric structures of general formulae (Va), (Vb), or (Vc), which might be called betaines and/or sulfobetaines:
  • R 11 is selected from linear or branched C7-C22 alkyl and linear or branched C7-C22 alkenyl.
  • R 12 are each independently selected from linear C1-C4 alkyl.
  • R 13 is selected from C1-C5 alkyl and hydroxy C1-C5 alkyl; for example 2-hydroxypropyl.
  • A- is selected from carboxylate and sulfonate.
  • Surfactants may further be compounds comprising amphoteric structures of general formula (VI), which might be called alkyl-amphocarboxylates:
  • R 11 is selected from C 7 -C 22 alkyl and C 7 -C 22 alkenyl, wherein alkyl and/or alkenyl are linear or branched, preferably linear.
  • R 14 is selected from -CH 2 C(0)0-IVI + , -CH 2 CH 2 C(0)0-M + and -CH 2 CH(0H)CH 2 S0 3 -M + .
  • R 15 is selected from H and -CH 2 C(0)O
  • the integer r in general formula (VI) is in the range of 2 to 6.
  • Non-limiting examples of further suitable alkyl-amphocarboxylates include sodium cocoampho- acetate, sodium lauroamphoacetate, sodium capryloamphoacetate, disodium cocoamphodiace- tate, disodium lauroamphodiacetate, disodium caprylamphodiacetate, disodium capryloam- phodiacetate, disodium cocoamphodipropionate, disodium lauroamphodipropionate, disodium caprylamphodipropionate, and disodium capryloamphodipropionate.
  • Surfactants may further be compounds comprising amphoteric structures of general formula (VII), which might be called amine oxides (AO):
  • R 16 is selected from Cs-C-is linear or branched alkyl, hydroxy Cs-C-is alkyl, acylamidopropoyl and C8-C18 alkyl phenyl group; wherein alkyl and/or alkenyl are linear or branched.
  • R 17 is selected from C 2 -C 3 alkylene, hydroxy C 2 -C 3 alkylene, and mixtures thereof.
  • each residue can be independently selected from C1-C3 alkyl and hydroxy C1-C3;
  • R 15 groups can be attached to each other, e.g., through an oxygen or nitrogen atom, to form a ring structure.
  • integer x in general formula (VII) is in the range of 0 to 5, preferably from 0 to 3, most pref- erably 0.
  • Non-limiting examples of further suitable amine oxides include C10-C18 alkyl dimethyl amine ox- ides and Ce-Ci 8 alkoxy ethyl dihydroxyethyl amine oxides.
  • Examples of such materials include dimethyloctyl amine oxide, diethyldecyl amine oxide, bis-(2-hydroxyethyl)dodecyl amine oxide, dimethyldodecylamine oxide, dipropyltetradecyl amine oxide, methylethylhexadecyl amine ox ide, dodecylamidopropyl dimethyl amine oxide, cetyl dimethyl amine oxide, stearyl dimethyl amine oxide, tallow dimethyl amine oxide and dimethyl-2-hydroxyoctadecyl amine oxide.
  • a further example of a suitable amine oxide is cocamidylpropyl dimethylaminoxide, sometimes also called cocamidopropylamine oxide.
  • Mixtures of two or more different amphoteric surfactants may also be used in the present inven- tion.
  • 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.
  • Anionic surfactants may be compounds of general formula (VIII), which might be called (fatty) alcohol/alkyl (ethoxy/ether) sulfates [(F)A(E)S] when A- is SO 3 , (fatty) alcohol/alkyl (eth- oxy/ether) carboxylate [(F)A(E)C] when A- is -RCOO:
  • R 1 is selected from Ci-C23-alkyl (such as 1 -, 2-, 3-, 4- Ci-C23-alkyl) and C2-C23-alkenyl, wherein alkyl and/or alkenyl are linear or branched, and wherein 2-, 3-, or 4-alkyl; examples are n-CzHis, n-CgH-ig, n-CnH23, n-Ci3H27, n-CisH3i, n-C- ⁇ Ftas, 1-C9H19, i-Ci2H25.
  • R 2 is selected from H, Ci-C2o-alkyl and C2-C2o-alkenyl, wherein alkyl and/or alkenyl are linear or branched.
  • R 3 and R 4 each independently selected from Ci-Ci 6 -alkyl, wherein alkyl is linear or branched; examples are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, 1 ,2-dimethylpropyl, isoamyl, n-hexyl, isohexyl, sec-hexyl, n- heptyl, n-octyl, 2-ethylhexyl, n-nonyl, n-decyl, isodecyl.
  • A- is selected from -RCOO , -SO 3 and RSO 3 , wherein R is selected from linear or branched C 1 - Ce-alkyl, and C 1 -C 4 hydroxyalkyl, wherein alkyl is.
  • M + is selected from H and salt forming cations.
  • Salt forming cations may be monovalent or multi- valent; hence M + equals 1/v M v+ . Examples include but are not limited to sodium, potassium, magnesium, calcium, ammonium, and the ammonium salt of mono-, di, and triethanolamine.
  • m is in the range of zero to 200, preferably 1 -80, more preferably 3-20; n and o, each inde- pendently in the range of zero to 100; n preferably is in the range of 1 to 10, more preferably 1 to 6; o preferably is in the range of 1 to 50, more preferably 4 to 25.
  • the sum of m, n and o is at least one, preferably the sum of m, n and o is in the range of 5 to 100, more preferably in the range of from 9 to 50.
  • Anionic surfactants of the general formula (VIII) may be of any structure, block copolymers or random copolymers.
  • anionic surfactants include salts (M + ) of C12-C18 sulfo fatty acid alkyl esters (such as C12-C18 sulfo fatty acid methyl esters), Cio-Cis-alkylarylsulfonic acids (such as n-Cio- Cis-alkylbenzene sulfonic acids) and C10-C18 alkyl alkoxy carboxylates.
  • M + in all cases is selected from salt forming cations.
  • Salt forming cations may be monovalent or multivalent; hence M + equals 1/v M v+ . Examples include but are not limited to sodium, potas- sium, magnesium, calcium, ammonium, and the ammonium salt of mono-, di, and triethanola- mine.
  • Non-limiting examples of further suitable anionic surfactants include branched alkylbenzenesul- fonates (BABS), phenylalkanesulfonates, alpha-olefinsulfonates (AOS), olefin sulfonates, al- kene sulfonates, alkane-2, 3-diylbis(sulfates), hydroxyalkanesulfonates and disulfonates, sec- ondary alkanesulfonates (SAS), paraffin sulfonates (PS), sulfonated fatty acid glycerol esters, alkyl- or alkenylsuccinic acid, fatty acid derivatives of amino acids, diesters and monoesters of sulfo-succinic acid.
  • BABS branched alkylbenzenesul- fonates
  • AOS alpha-olefinsulfonates
  • olefin sulfonates
  • Anionic surfactants may be compounds of general formula (IX), which might be called N-acyl amino acid surfactants:
  • R 19 is selected from linear or branched C6-C 2 2-alkyl and linear or branched C6-C2 2 -alkenyl such as oleyl.
  • R 20 is selected from H and Ci-C4-alkyl.
  • R 21 is selected from H, methyl, -(CH 2 ) 3 NHC(NH)NH2, -CH 2 C(0)NH 2 , -CH 2 C(0)OH, - (CH 2 ) 2 C(0)NH 2 , -(CH 2 ) 2 C(0)0H, (imidazole-4-yl)-methyl, -CH(CH 3 )C 2 H 5 , -CH 2 CH(CH 3 ) 2 , - (CH 2 )4NH 2 , benzyl, hydroxymethyl, -CH(OH)CH 3 , (indole-3-yl)-methyl, (4-hydroxy-phenyl)-me- thyl, isopropyl, -(CH 2 ) 2 SCH 3 , and -CH2SH.
  • R 22 is selected from -COOX and -CH 2 S0 3 X, wherein X is selected from Li + , Na + and K + .
  • Non-limiting examples of suitable N-acyl amino acid surfactants are the mono- and di-carbox- ylate salts (e.g., sodium, potassium, ammonium and ammonium salt of mono-, di, and triethano- lamine) of N-acylated glutamic acid, for example, sodium cocoyl glutamate, sodium lauroyl glu tamate, sodium myristoyl glutamate, sodium palmitoyl glutamate, sodium stearoyl glutamate, disodium cocoyl glutamate, disodium stearoyl glutamate, potassium cocoyl glutamate, potas- sium lauroyl glutamate, and potassium myristoyl glutamate; the carboxylate salts (e.g., sodium, potassium, ammonium and ammonium salt of mono-, di, and triethanolamine) of N-acylated ala- nine, for example, sodium cocoyl alaninate, and triethanolamine lauroyl alaninate; the carbox
  • Anionic surfactants may further be selected from the group of soaps.
  • Suitable are salts (M + ) of saturated and unsaturated C 12 -C 18 fatty acids, such as lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, (hydrated) erucic acid.
  • M + is selected from salt forming cat- ions. Salt forming cations may be monovalent or multivalent; hence M + equals 1/v M v+ .
  • Exam- pies include but are not limited to sodium, potassium, magnesium, calcium, ammonium, and the ammonium salt of mono-, di, and triethanolamine.
  • suitable soaps include soap mixtures derived from natural fatty acids such as tallow, coconut oil, palm kernel oil, laurel oil, olive oil, or canola oil.
  • Such soap mixtures comprise soaps of lauric acid and/or myristic acid and/or palmitic acid and/or stearic acid and/or oleic acid and/or linoleic acid in different amounts, depending on the natural fatty ac- ids from which the soaps are derived.
  • anionic surfactants include salts (M + ) of sulfates, sul- fonates or carboxylates derived from natural fatty acids such as tallow, coconut oil, palm kernel oil, laurel oil, olive oil, or canola oil.
  • Such anionic surfactants comprise sulfates, sulfonates or carboxylates of lauric acid and/or myristic acid and/or palmitic acid and/or stearic acid and/or oleic acid and/or linoleic acid in different amounts, depending on the natural fatty acids from which the soaps are derived.
  • Mixtures of two or more different anionic surfactants may also be used in the present invention.
  • Mixtures of non-ionic and/or amphoteric and/or anionic surfactants may also be used in the pre- sent invention.
  • 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 ter- tiary amines.
  • Cationic surfactants may be compounds of the general formula (X) which might be called qua- ternary ammonium compounds (quats):
  • R 23 is selected from H, C 1 -C 4 alkyl (such as methyl) and C 2 -C 4 alkenyl, wherein alkyl and/or alkenyl is linear or branched.
  • R 24 is selected from C 1 -C 4 alkyl (such as methyl), C 2 -C 4 alkenyl and C 1 -C 4 hydroxyalkyl (such as hydroxyethyl), wherein alkyl and/or alkenyl is linear or branched.
  • R 25 is selected from C 1 -C 22 alkyl (such as methyl, C 18 alkyl), C 2 -C 4 alkenyl, C 12 -C 22 alkylcarbon- yloxymethyl and C 12 -C 22 alkylcarbonyloxyethyl (such as C 16 -C 18 alkylcarbonyloxyethyl), wherein alkyl and/or alkenyl is linear or branched.
  • R 26 is selected from C12-C1 8 alkyl, C2-C4 alkenyl, C12-C22 alkylcarbonyloxymethyl, C12-C22 alkyl- carbonyloxyethyl and 3-(Ci2-C22 alkylcarbonyloxy)-2(Ci2-C22 alkylcarbonyloxy)-propyl.
  • X is selected from halogenid, such as Ch or Br.
  • Non-limiting examples of further cationic surfactants include, amines such as primary, second- ary and tertiary monoamines with C18 alkyl or alkenyl chains, ethoxylated alkylamines, alkox- ylates of ethylenediamine, imidazoles (such as 1-(2-hydroxyethyl)-2-imidazoline, 2-alkyl-1-(2- hydroxyethyl)-2-imidazoline, and the like), quaternary ammonium salts like alkylquaternary am- monium chloride surfactants such as n-alkyl(Ci2-Ci8)dimethylbenzyl ammonium chloride, n- tetradecyldimethylbenzylammonium chloride monohydrate, and a naphthylene-substituted qua- ternary ammonium chloride such as dimethyl-1 -naphthylmethylammonium chloride.
  • amines such as primary
  • Particularly suitable cationic surfactants that may be:
  • ester quats in particular quaternary esterified mono-, di- and trialkanolamines which are esterified with C8-C22-carboxylic acids;
  • imidazoline quats in particular 1-alkylimidazolinium salts of formulae XI or XII
  • R 27 is selected from Ci-C25-alkyl and C2-C25-alkenyl
  • R 28 is selected from Ci-C 4 -alkyl and hydroxy-Ci-C4-alkyl
  • R 29 is selected from Ci-C4-alkyl, hydroxy-Ci -C h alky I and a R*-(CO)-R 30 -(CH2)r radical, wherein R* is selected from Ci-C2i-alkyl and C2-C2i-alkenyl; R 30 is selected from-O- and -NH-; j is 2 or 3.
  • 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).
  • the surfactant comprises a block polymer (P) comprising at least one polyethyleneoxide moiety and at least one polypropyleneoxide moiety, an alcohol alkoxylate (E) and/or an alcohol ethoxylate (L) of the general formula (XIII)
  • R 3 -0-(C 2 H 4 0)s-H (XIII) wherein R 3 is linear or branched Cs to C20 alkyl, and
  • s has a value of from 1 to 40.
  • the block polymer (P) is preferably a triblock polymer comprising one polyethyleneoxide moiety and two polypropyleneoxide moieties, or two polyethyleneoxide moieties and one polypropylene- oxide moiety.
  • the block polymer (P) is a pentablock polymer comprising two polyethyleneoxide moieties and three polypropyleneoxide moieties, or three polyethyleneoxide moieties and two polypropyleneoxide moieties.
  • the block polymer (P) comprises
  • the block polymer (P) is or comprises a polyethyleneox- ide polypropyleneoxide polyethyleneoxide (EO-PO-EO) triblock polymer.
  • the polypropyleneox- ide moiety in the EO-PO-EO triblock polymer may have a molar mass of 250 to 5000 g/mol, preferably from 400 to 3000 g/mol, and in particular from 600 to 1500 g/mol.
  • the EO-PO-EO triblock polymer may contain 3 to 90 mol%, preferably 25 to 85 mol%, and in particular 50 to 80 mol% of the polypropyleneoxide moiety.
  • the block polymer (P) is or comprises a polyethyleneox- ide polypropyleneoxide polyethyleneoxide polypropyleneoxide polyethyleneoxide (EO-PO-EO- PO-EO) pentablock polymer.
  • the polypropyleneoxide moiety in the EO-PO-EO-PO-EO pen- tablock polymer may have a molar mass of 500 to 5000 g/mol, preferably from 750 to 3500 g/mol, and in particular from 1000 to 2500 g/mol.
  • the EO-PO-EO triblock polymer may contain 3 to 90 mol%, preferably 25 to 80 mol%, and in particular 50 to 70 mol% of the polypropylene- oxide moiety.
  • the alcohol alkoxylate (E) is selected in particular among alcohol alkoxylates of the formula (XIV)
  • R is branched Cs-Cso-alkyl
  • n, p independently of one another are an integer from 2 to 16, preferably 2, 3, 4 or 5;
  • x+y+z have a value of 1 to 100.
  • m is 2
  • x is from 5.0 to 5.5
  • n 3
  • y is from 4.5 to 5.0
  • p 2
  • z is from 2 to 2.5.
  • Preferred substances are EO/PO block alcohol alkoxylates in which the EO:PO ratio is 1 : 1 to 4:1 , in particular 1.3:1 to 3:1.
  • the degree of ethoxylation is, as a rule, 1 to 20, preferably 2 to 15, in particular 4 to 10
  • the degree of propoxylation is, as a rule, 1 to 20, preferably 1 to 8, in particular 2 to 6.
  • the total degree of alkoxylation, i.e. the total of EO and PO units, is, as a rule, 2 to 40, preferably 3.to 25, in particular 6 to 15.
  • these alcohol alkoxylates take the form of the EO type, with the EO block being bonded terminally, however.
  • Preferred PO/EO block alcohol alkoxylates are those in which the PO:EO ratio is 1 :10 to 3:1 , in particular 1 :6 to 1.5:1.
  • the degree of ethoxylation is, as a rule, 1 to 20, preferably 2 to 15, in particular 4 to 10
  • the degree of propoxylation is, as a rule, 0.5 to 10, preferably 0.5 to 8, in particular 1 to 6.
  • the total degree of alkoxylation i.e. the total of EO and PO units given as an average number, is, as a rule, 1.5 to 30, preferably 2.5 to 21 , in particular 5 to 16.
  • the alcohol alkoxylates (E) are based on primary, alpha-branched alcohols of the general formula (XV) in which
  • R 1 , R 2 independently of one another are hydrogen or Ci-C26-alkyl.
  • R 1 and R 2 independently of one another are Ci-C 6 -alkyl, in particular C2-C 4 -alkyl, for example C3-alkyl.
  • alcohol alkoxylates which are based on 2-propylheptanol.
  • These include, in particular, alcohol alkoxylates of the formula (XIV) in which R is a 2-propylheptyl radi cal, i.e. R 1 and R 2 in formula (XV) are in each case n-propyl.
  • Such alcohols are also referred to as Guerbet alcohols. They can be obtained for example by dimerization of corresponding primary alcohols (for example R 1 ⁇ 2 CH2CH20H) at elevated tem- perature, for example 180 to 300°C, in the presence of an alkaline condensing agent such as potassium hydroxide.
  • corresponding primary alcohols for example R 1 ⁇ 2 CH2CH20H
  • an alkaline condensing agent such as potassium hydroxide
  • Alkoxylates which are employed for the purposes of this preferred embodiment, which is based on Guebert alcohols, are mainly alkoxylates of the EO type. Particularly preferred are ethox- ylates with a degree of ethoxylation of 1 to 50, preferably 2 to 20, in particular approximately 3 to 10. The correspondingly ethoxylated 2-propylheptanols may be mentioned especially among these.
  • the alcohol alkoxylates to be used are based on C13-oxo alcohols.
  • C13-oxo alcohol refers to an alcohol mixture whose main component is formed by at least one branched C13-alcohol (isotridecanol).
  • C13-alcohols include, in par- ticular, tetramethylnonanols, for example 2,4,6,8-tetramethyM -nonanol or 3,4,6,8-tetramethyM - nonanol and furthermore ethyldimethylnonanols such as 5-ethyl-4,7-dimethyl-1 -nonanol.
  • Suitable C13-alcohol mixtures can generally be obtained by hydrogenation of hydroformylated trimeric butane, as described in US2005/0170968. In particular, it is possible to proceed as fol lows:
  • the C12-olefin fraction is hydroformylated by reacton with carbon monoxide and hydrogen in the presence of a suitable catalyst
  • Advantageous C13-alcohol mixtures are essentially free from halogens, i.e. they contain less than 3 ppm by weight, in particular less than 1 ppm by weight, of halogen, in particular chlorine.
  • the alcohol alkoxylates (E) may have an average molecular weight of preferably at least 200 g/mol, more preferably at least 300 g/mol, most preferably at least 400 g/mol, particularly prefer- ably at least 500 g/mol, particularly more preferably at least 600 g/mol, particularly most prefera- bly at least 650 g/mol.
  • the alcohol alkoxylates (E) may have an average molecular weight of preferably up to 10000 g/mol, more preferably up to 5000 g/mol, most preferably up to 3000 g/mol, particularly preferably up to 2000 g/mol, particularly more preferably up to 1500 g/mol, particularly most preferably up to 1200 g/mol, particularly up to 1000 g/mol, for example up to 850 g/mol.
  • the alcohol alkoxylates (E) may have an average molecular weight of from 300 to 2000 g/mol, preferably from 400 to 1000 g/mol, and particularly from 650 to 850 g/mol.
  • R 3 -0-(C 2 H 4 0)s-H (XIII) wherein R 3 is linear or branched Cs to C20 alkyl, and
  • s has a value of from 1 to 40
  • R 3 is preferably branched C10 to C15 alkyl, more preferably branched C12 to C14 alkyl, most preferably isotridecyl.
  • s has a value of from 1 to 40, preferably 2 to 30, more preferably 3 to 20, most preferably 4 to 15, particularly preferably 5 to 10, particularly preferably 7 to 9, for example 8.
  • Suitable C13-alcohol mixtures for example isotridecanol mixtures, as precursor for alcohol eth- oxylate (L), can generally be obtained by hydrogenation of hydroformylated trimeric butene. In particular, it is possible to proceed as follows:
  • the C12-olefin fraction is hydroformylated by reacton with carbon monoxide and hydrogen in the presence of a suitable catalyst
  • C13-alcohol mixtures for example isotridecanol mixtures, as precursor for alcohol ethoxylate (L)
  • the degree of branching is defined as the number of methyl groups in one molecule of the alcohol minus 1.
  • the mean degree of branching is the statistical mean of the degrees of branching of the molecules of a sample. The mean number of methyl groups in the molecules of a sample can be determined readily by 'H-NMR spectroscopy.
  • the signal area corresponding to the methyl protons in the 'H-NMR spectrum of a sample is di- vided by three and then divided by the signal area of the methylene protons if the CH n — OH group divided by two.
  • the alcohol ethoxylate (L) may have an average molecular weight of preferably at least 200 g/mol, more preferably at least 300 g/mol, most preferably at least 350 g/mol, particularly prefer- ably at least 400 g/mol, particularly more preferably at least 450 g/mol, particularly most prefera- bly at least 500 g/mol.
  • the alcohol alkoxylates (E) may have an average molecular weight of preferably up to 2000 g/mol, more preferably up to 1500 g/mol, most preferably up to 1000 g/mol, particularly preferably up to 800 g/mol, particularly more preferably up to 700 g/mol, par- ticularly most preferably up to 650 g/mol, particularly up to 600 g/mol.
  • the alcohol ethoxylate (L) may have an average molecular weight of from 200 to 2000 g/mol, preferably from 400 to 1000 g/mol, and particularly from 500 to 600 g/mol.
  • the surfactant is a composition (A) comprising a block polymer (P) comprising at least one polyethyleneoxide moiety and at least one polypropyleneoxide moiety, and an alcohol alkoxylate (E) as discussed above.
  • the block polymer (P) is contained in an amount of from 4 wt.-% to 96 wt.-%, more preferably from 8 wt.-% to 92 wt.-%, most preferably from 12 wt.-% to 88 wt.-%, particularly preferably from 16 w.-% to 84 wt.-%, particularly more preferably from 20 wt.-% to 80 wt.-%, particularly even more preferably from 24 wt.-% to 76 wt.-%, particularly most prefera- bly from 28 wt.-% to 72 wt.-%, for example preferably from 32 wt.-% to 68 wt.-%, for example more preferably from 36 wt.-% to 64 wt.-%, for example even more preferably from 40 wt.-% to 60 wt.-%, for example most preferably from 44 wt.-% to 56 wt.-
  • the alcohol alkoxylate (E) is contained in an amount of from 4 wt.-% to 96 wt.-%, more preferably from 8 wt.-% to 92 wt.-%, most preferably from 12 wt.-% to 88 wt- %, particularly preferably from 16 w.-% to 84 wt.-%, particularly more preferably from 20 wt.-% to 80 wt.-%, particularly even more preferably from 24 wt.-% to 76 wt.-%, particularly most pref- erably from 28 wt.-% to 72 wt.-%, for example preferably from 32 wt.-% to 68 wt.-%, for example more preferably from 36 wt.-% to 64 wt.-%, for example even more preferably from 40 wt.-% to 60 wt.-%, for example most preferably from 44 wt.-% to 56 wt
  • the surfactant is a composition (B) comprising an alcohol eth- oxylate (L) of the general formula (XIII)
  • R 3 is linear or branched Cs to C20 alkyl
  • s has a value of from 1 to 40
  • the alcohol ethoxylate (L) is contained in an amount of from 4 wt.-% to 96 wt.-%, more preferably from 8 wt.-% to 92 wt.-%, most preferably from 12 wt.-% to 88 wt- %, particularly preferably from 16 w.-% to 84 wt.-%, particularly more preferably from 20 wt.-% to 80 wt.-%, particularly even more preferably from 24 wt.-% to 76 wt.-%, particularly most pref- erably from 28 wt.-% to 72 wt.-%, for example preferably from 32 wt.-% to 68 wt.-%, for example more preferably from 36 wt.-% to 64 wt.-%, for example even more preferably from 40 wt.-% to 60 wt.-%, for example most preferably from 44 wt.-% to 56 w
  • the surfactant comprises sodium-di-ethyl-hexyl-sulfosucccinate and an iso C13-alcohol ethoxylate.
  • the surfactant comprises a mixture of ethylenoxide-propyleneoxide block copolymer, 2-propylheptanol and a triblock polymer of ethylenoxide-propyleneoxide-eth- ylenoxide.
  • the surfactant comprises a mixture of equal volumes of (a) ethylenoxide- propyleneoxide block copolymer; and (b) 2-propylheptanol and a triblock polymer of ethylenox- ide-propyleneoxide-ethylenoxide.
  • auxiliaries such as solvents or liquid carriers, solid carriers, surfactants, adjuvants, dispersants, emulsifiers, wetters, adjuvants, solubilizers, penetration en- hancers, protective colloids, adhesion agents, thickeners, humectants, compatibilizers, bacteri- cides, anti-freezing agents, anti-foaming agents, colorants, tackifiers, binders, preservatives, an- tioxidants, and odorants may be used in addition to the lipase according to SEQ ID No. 1 or a variant thereof as defined herein and to the at least one surfactant.
  • surfactants such as solvents or liquid carriers, solid carriers, surfactants, adjuvants, dispersants, emulsifiers, wetters, adjuvants, solubilizers, penetration en- hancers, protective colloids, adhesion agents, thickeners, humectants, compatibilizer
  • 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.
  • 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 according to SEQ ID No. 1 or a variant thereof as defined herein and to the at least one surfactant.
  • 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
  • the polypeptide having lipase activity may be applied to the groundcover in a concentration of between 0.001 kg to 60 kg of polypeptide per hectare, preferably of between 0.016 kg to 10 kg per hectare, more preferably of between 0.06 kg to 2 kg per hectare, most preferably of be- tween 0.1 kg to 0.5 kg per hectare.
  • the polypeptide having lipase activity may be applied to the groundcover in a concentration of 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 0.001 % to 5% or 3% (w/v), preferably of 0.002% to 2% or 1 % (w/v), more preferably of 0.003% to 0.08 or 0.06%
  • w/v w/v
  • concentration of the lipase is 0.006% (w/v).
  • the lipase is dissolved in water in a concentration of 0.01 % to 50% or 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 % (w/v).
  • concentration of the lipase is 0.06% (w/v).
  • the lipase is dissolved in water in a concentration of 0.001 % to 0.01 % (w/v), preferably of 0.002% to 0.008% (w/v), more preferably of 0.003% to 0.007% (w/v) and most preferably of 0.006% (w/v).
  • the lipase is dissolved in water in a concentration of 0.1 % to 5% (w/v), preferably of 0.5% to 3% (w/v), more preferably of 0.8% to 2% (w/v) and most preferably of 1 % (w/v).
  • large volume ap- plications 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 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% (w/v) 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% (w/v) 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 may be supplied as a concentrated suspension and diluted to the concentrations in- dicated above before the application to soil.
  • the lipase may also be supplied in powder form and either dissolved before the application to soil or applied to the soil in powder form. If the li- pase is applied to the soil in powder form, it is washed into the soil by rain or irrigation water with a volume of 0.5 L to 15 L, preferably with a volume of 1 L to 10 L per square meter, more preferably with a volume of 4 to 8 L per square meter.
  • the surfactant is used in a concentration of 0.02 % to 4 % (v/v), (v/w), (w/w) or (w/v), preferably of 0.04 % to 3% (v/v), (v/w), (w/w) or (w/v), more preferably of 0.06% to 2% (v/v), (v/w), (w/w) or (w/v) and most preferably of 0.08 % to 1.0 % (v/v), (v/w), (w/w) or (w/v).
  • the surfactant is used in a concentration of 0.02 % to 4 % (v/v), preferably of 0.04 % to 3% (v/v), more preferably of 0.06% to 2% (v/v) and most preferably of 0.08 % to 1.0 % (v/v).
  • the lipase is used in a concentration of 0.001 % to 5% or 3% (w/v)and the surfactant is used in a concentration of 0.02 % to 4 % (v/v).
  • the lipase is used in a concentration of 0.002% to 2% or 1 % (w/v) and the surfactant is used in a concentration of 0.04 % to 3% (v/v).
  • the lipase is used in a concentration of 0.003% to 0.08 or 0.06% (w/v) and the surfactant is used in a concentration of 0.06% to 2% (v/v) and most preferably the lipase is used in a concentration of 0.005% to 0.01 % (w/v) and the surfactant is used in a con- centration of 0.08 % to 1.0 % (v/v).
  • the lipase is used in a concentration of 0.01 % to 50% or 30% (w/v) and the surfactant is used in a concentration of 0.02 % to 4 % (v/v).
  • the lipase is used in a concentration of 0.02% to 20% or 10% (w/v) and the surfactant is used in a concentration of 0.04 % to 3% (v/v).
  • the lipase is used in a concentration of 0.03% to 0.8 or 0.6% (w/v) and the surfactant is used in a concentration of 0.06% to 2% (v/v) and most prefera- bly the lipase is used in a concentration of 0.05% to 0.1 % (w/v) and the surfactant is used in a concentration of 0.08 % to 1.0 % (v/v).
  • the lipase is used in a concentration of 0.006% (w/v) and the surfactant is used in a concentration of 0.52 % (v/v). In one embodiment the lipase according to SEQ ID No.
  • the surfactant comprising sodium-di-ethyl- hexyl-sulfosucccinate and an iso C13-alcohol ethoxylate is used in a concentration of 0.52 % (v/v).
  • the lipase is used in a concentration of 0.06% (w/v) and the surfactant is used in a concentration of 0.52 % (v/v). In one embodiment the lipase according to SEQ ID No.
  • the surfactant comprising sodium-di-ethyl-hexyl- sulfosucccinate and an iso C13-alcohol ethoxylate is used in a concentration of 0.52 % (v/v).
  • the lipase is used in a concentration of 0.0015% (w/v) and the surfactant is used in a concentration of 0.04 % (v/v).
  • the lipase according to SEQ ID No. 1 is used in a concentration of 0.006% (w/v) and the surfactant comprising a mixture of eth- ylenoxide-propyleneoxide block copolymer, 2-propylheptanol alkoxylate and a triblock polymer of ethylenoxide-propyleneoxide-ethylenoxide is used in a concentration of 0.04 % (v/v).
  • the lipase is used in a concentration of 0.015% (w/v) and the surfactant is used in a concentration of 0.04 % (v/v). In one embodiment the lipase according to SEQ ID No.
  • the surfactant comprising a mixture of eth- ylenoxide-propyleneoxide block copolymer, 2-propylheptanol alkoxylate and a triblock polymer of ethylenoxide-propyleneoxide-ethylenoxide is used in a concentration of 0.04 % (v/v).
  • the lipase according to SEQ ID No. 1 or an en- zymatically active variant thereof and the at least one surfactant is applied to an area of ground- cover.
  • 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.
  • groundcover is potting mix. Potting mix, which is also re- ferred to potting soil, is typically a blend of ingredients that is used to grow plants, preferably, the potting mix comprises a combination of peat moss, vermiculite, coir fiber, perlite, pine bark, sand, compost, and further ingredients. It may also contain native soil.
  • applying includes any activity by which the lipase and the surfactant come into con- tact with the area of groundcover.
  • the lipase and the surfactant can be dissolved in water and applied as a solution.
  • a granulate can be prepared from the combination of lipase and surfactant and the granulate is then applied to the groundcover in solid form which is dis solved when water is applied to the soil.
  • the lipase and the surfactant are applied in irrigation water.
  • the lipase and the surfactant are applied extensively and non-directional to turfgrass soil.
  • the lipase and the surfactant are applied extensively and non-directional to turfgrass soil in irrigation water. In one embodiment the lipase and the surfactant are applied di- rectional to turfgrass soil using a watering can.
  • the lipase and the surfactant are applied to turfgrass soil as part of a top- dressing which comprises particles to which the lipase is coupled.
  • Topdressing refers to a mate- rial 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 and the surfactant serves to prepare a non- wetting soil for the seeding of crops.
  • the lipase and the surfactant are ap- plied selectively to those locations were 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-se- lectively to the entire application site (so-called blanket application).
  • the lipase and the surfactant are applied simultaneously, for example dis solved in the same solution such as irrigation water.
  • the surfactant is applied to the soil before the lipase is applied.
  • the surfactant may be applied not later than two days, preferably not later than one day, more preferably not later than 18 hours and most pref- erably not later than 12 hours before the lipase is applied.
  • Example 1 Determination of water droplet penetration time of soil treated with lipase and sur- factant
  • Soil cores with a diameter of 7.4 cm and about 6.5 cm in depth were removed from a golf course near Limburgerhof showing Localized Dry Spot symptoms with a soil auger.
  • the soil cores were transferred to a growth room having a temperature of constantly 20 °C and placed in 1 liter beakers for further treatments.
  • the soil cores were treated with 300 mL of one of the following solutions for 24 h:
  • the soil cores were placed in plastic pots with holes at the bottom for 20 days in the growth chamber with no further treatment or irrigation. Then the cores were halved with a knife and at 2 cm below the soil surface the water droplet penetration test was performed by determining the time required for water droplets with a volume of 10 pi to penetrate into the sample soil layer.
  • Example 2 Determination of water holding capacity of soil treated with lipase and surfactant
  • Soil cores with a diameter of 7.4 cm and about 6.5 cm in depth were removed from a golf course near Limburgerhof showing Localized Dry Spot symptoms with a soil auger.
  • the soil cores were transferred to a growth room having a temperature of constantly 20 °C for further treatments.
  • Each soil core was treated by irrigation with 25 mL solution (equivalent to about 6 mm irrigation) of one of the following compositions:
  • Example 3 Long term efficacy of surfactant+lipase soil treatment compared to surfactant only treatment:
  • a surfactant and surfactant + lipase pre-treatment protocol was used to mimic agricultural prac- tice where, in the field, banded surfactant treatments infiltrate the soil surface and dry prior to subsequent rainfall events. Pre-treatment was carried out within the packed soil column, which remained undisturbed prior to water infiltration measurements. Kojonup, a Western Australia wheat growing soil with extreme water repellence (MED >4) was used for long-term efficacy studies.
  • MED >4 extreme water repellence
  • Soil columns consisted of glass tubes with a diameter of 10 mm with soil particle diameters of ⁇ 420 pm packed to a consistent density with ⁇ 0.05 variation. All soils were pre-dried at 40 °C prior to packing. Infiltration measurements were carried out on soil columns pre-treated with so- lutions of surfactant + lipase (T2) which was then compared to surfactant only treated soil (T 1 ).
  • Pre-treatment with surfactant only (T1 ) utilized a surfactant blend of (1 :1) EO(18)/PO(29)-block copolymer and 2-propylheptanol alkoxylate + 5.2EO + 4.7PO + 2.3EO applied at a rate of 300 pL per column of 0.4 g/L aqueous surfactant solution that corresponded to 180 pg/cm 2 on the column, which is equivalent to an agricultural practice of 2 L/ha banded application if about 10% of the field is treated.
  • the surfactant + lipase pre-treatment (T2) was carried out similarly with the surfactant blend (T1 ) as described above together with the addition of 0.015% w/v lipase en- zyme. All columns were then dried at 40 °C. Table 3: Details of water application stages to mimic rainfall events, infiltration measurements and drying periods over 105 days
  • the wetting front movement indicates how deep the water has infiltrated and the quantity of water infiltrated provides information on how much of the water is retained in the pores of the soil bed in the packed column.
  • Infiltration distance and water retention were determined as a function of time. When the infiltration over 10 cm depth was complete and drainage starts, the application of wa- ter was continued at a constant hydraulic head to simulate a continuous rainfall event. Infiltration rates measured for all soil samples were replicated in independent duplicate runs.
  • Figs. 1 and 2 compare the efficiency of lipase enzyme pre-treatment to surfactant only pre-treat- ment in terms of (a) infiltration rate and (b) water retention in the upper soil zone for time peri- ods up to 105 days.
  • Fig. 1 provides the early water infiltration rate and water retention 7 days after a simulated cumulative rain of 1 1 mm from two rain events (Table 3). At day 7, the infiltra tion rate is marginally higher when lipase was used initially compared to only surfactant. The water retention after 7 days is significantly improved (20 %) by initial pre-treatment with lipase enzyme. By comparison, over the longer term (day 105, Fig. 2), both the infiltration rate and the water retention is significantly improved by initial pre-treatment with lipase enzyme.
  • the at least one surfactant is selected from anionic, cationic, nonionic and amphoteric surfactants, block polymers, polyelectrolytes, and mixtures thereof.
  • R 3 -0-(C 2 H 4 0)s-H (XIII) wherein R 3 is linear or branched Cs to C20 alkyl, and
  • s has a value of from 1 to 40.
  • composition comprising a block polymer (P) comprising at least one polyethyleneoxide moiety and at least one polypropyleneoxide moiety, and an alcohol alkoxylate (E); and
  • R 3 -0-(C 2 H 4 0)s-H (XIII) wherein R 3 is linear or branched Cs to C20 alkyl, and
  • s has a value of from 1 to 40
  • polypeptide or an enzy- matically active fragment thereof is 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.
  • the soil is selected from ag- ricultural land, pasture, coastal dune sands, forest, shrub lands, parks, turfgrass soils, no-till ag- riculture, and soils irrigated with treated wastewater.
  • a method for reducing soil water repellency and/or for enhancing water holding capacity of soils comprising applying
  • polypeptide and the at least one surfactant are applied to the area of groundcover simultaneously.
  • the at least one surfactant is selected from anionic, cationic, nonionic and amphoteric surfactants, block polymers, polyelectrolytes, and mixtures thereof.
  • the at least one surfactant is selected from the group consisting of a block polymer (P) comprising at least one polyethylene- oxide moiety and at least one polypropyleneoxide moiety, an alcohol alkoxylate (E) and an alcohol ethoxylate (L) of the general formula (XIII)
  • R 3 -0-(C 2 H 4 0)s-H (XIII) wherein R 3 is linear or branched Cs to C20 alkyl, and
  • s has a value of from 1 to 40.
  • composition comprising a block polymer (P) comprising at least one polyethyleneoxide moiety and at least one polypropyleneoxide moiety, and an alcohol alkoxylate (E); and
  • R 3 -0-(C 2 H 4 0)s-H (XIII) wherein R 3 is linear or branched Cs to C20 alkyl, and
  • s has a value of from 1 to 40
  • the surfactant is a mixture of ethylenoxide-propyleneoxide block copolymer, 2-propylheptanol alkoxylate and a triblock polymer of ethylenoxide-propyleneoxide-ethylenoxide.
  • the surfactant comprises sodium-di-ethyl-hexyl- sulfosucccinate, a polymeric alcohol ethoxylate and bis(2-ethylhexyl)maleate.
  • a composition comprising:
  • the at least one surfactant is selected from the group consisting of a block polymer (P) comprising at least one polyethyleneoxide moiety and at least one polypropyleneoxide moiety, an alcohol alkoxylate (E) and/or an alcohol ethoxylate (L) of the general formula (XIII)
  • R 3 -0-(C 2 H 4 0) S -H (XIII) wherein R 3 is linear or branched Cs to C20 alkyl, and
  • s has a value of from 1 to 40.

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  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Life Sciences & Earth Sciences (AREA)
  • Soil Sciences (AREA)
  • Materials Engineering (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

La présente invention concerne un procédé de réduction de l'hydrophobie du sol et/ou d'augmentation de la capacité de rétention d'eau à l'aide d'une lipase et d'au moins un tensioactif.
PCT/EP2019/057544 2018-03-26 2019-03-26 Procédé de réduction de l'hydrophobie du sol WO2019185610A1 (fr)

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Cited By (3)

* 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
KR102570654B1 (ko) * 2023-04-17 2023-08-25 주식회사 다봄씨엔에스 양계장 배설물에서 발생된 암모니아를 제올라이트로 흡착하여 토양개량에 활용하는 방법
WO2024121058A1 (fr) 2022-12-05 2024-06-13 Novozymes A/S Composition comprenant une lipase et un peptide

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Cited By (3)

* 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
KR102570654B1 (ko) * 2023-04-17 2023-08-25 주식회사 다봄씨엔에스 양계장 배설물에서 발생된 암모니아를 제올라이트로 흡착하여 토양개량에 활용하는 방법

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