SA518391349B1 - Cleaning of water filtration membranes - Google Patents

Abstract

The present invention provides a method for cleaning a water filtration membrane by contacting the membrane with a liquid cleaning composition comprising a DNase. The cleaning process may be Cleaning-In-Place (CIP) or Cleaning-Out-of-Place (COP). In an embodiment, the water flow rate through the membrane (flux) is improved. In another embodiment, the method is preceded or followed by contacting the membrane with a biocidal composition. In another embodiment, the water filtration membrane comprises a biofilm, which preferably includes one or more bacteria selected from the group consisting of Acinetobacter, Bacillus, Comamonas, Escherichia, Pseudomonas, and Sphingomonas species.

Description

CLEANING OF WATER FILTRATION MEMBRANES Full description

Invention background

The present invention relates to cleaning of water filtration membranes using detergent formulations

- (DNase) deoxyribonuclease cleaning compositions membrane filtration Membrane clogging is a problem encountered in membrane filtration processes ae) a major determinant of its practical application in water and wastewater treatment Sele Promise processes 5

and desalination in terms of technology and economics. Membrane occlusion includes inorganic occlusion/exfoliation;

particulate/colloidal embolism” and organic embolism with dead organic matter or microorganisms; which normally

What constitutes a biofilm 10510i. Blockages can be caused by both organic and inorganic components

and Lal) microorganisms simultaneously; These components can interact in terms of mechanism.

0 A common method for cleaning filter membranes is “cleaning-in-place” (010). The typical clean-in-place cycle often involves the use of chemicals of a high pH (for example, “(NaOH) caustic soda solution) and a low pH (for example, (Ji) citric acid). or tetraic acid ¢ (nitric acid, water and biocides for disinfection” for example;

Typical on-site cleaning of membranes used in the dairy industry involves several steps that we include: - pre-washing/rinsing with a stream of clean water; Which can be heated up to 60-80°C and recycled for a period of time. - Circulate the caustic soda solution at a temperature of about 60-580°C for a period of time.

0 - Medium rinse using clean water flow. Rotate the acidic solution for a period of time. Final rinse using a stream of clean water. Final inflation with air.

Similar processes for cleaning membranes exist in other businesses; The individual steps may be applied in a different order depending on the nature of the membrane occlusion. A typical clean-in-place cycle for cleaning membranes used in the enzyme manufacturing industry includes a cola flush and a cold and hot water rinse; and rinse with nitric acid.

The use of harsh chemicals in hal cleaning is undesirable and poses a problem for the environment. In the past years, cleaning methods have been developed in place, including the use of enzymes. International patent application No. 02753/97 relates to a solution containing a protease lipase lug for in-situ cleaning. ang that the solution is effective in cleaning process equipment

process equipment 0 containing milk residue or burnt milk. Compared to a typical clean-in-place procedure; There are many benefits to improved membrane cleaning with deoxyribonuclease enzymes, such as saving energy to pump fluid through less obstructive membranes, and reducing the use of harsh and hazardous chemicals. reducing the cost of acid-base neutralization and post-use discharge; Extended membrane life due to milder cleaning conditions and removal of blockages5 by chemical or physical methods. 686 (Deoxyribonuclease) The term 'DNase' means a polypeptide with deoxyribonuclease activity that catalyzes the hydrolytic cleavage of phosphodiester linkages in the main DNA group; 0 thus hydrolyzes DNA. Examples of enzymes that exhibit β-deoxyribonuclease activity are those covered by the 3.1.11 EC NEC enzyme classes 1 as defined by the recommendations of the International Union of Biochemistry and Molecular Biology (IUBMB) Nomenclature Committee. Biology

The terms “0118565” “DNase enzymes” and “tof expression peptide with deoxyribonuclease activity” are used interchangeably throughout the application. For purposes of the present invention, deoxyribonuclease activity is determined according to the procedure described in Test 1. In one aspect; For deoxyribonuclease of the present invention at least 20 times at least 740 at least 750 at least 760 at least 770 at least 780 at least 0 at least 7295; or at least 72100 deoxyribonuclease activity of a deoxyribonuclease having the amino acid sequence ACID 801100 of Sequence-ID No: 1. The deoxyribonuclease used according to the present invention is a mature polypeptide exhibiting deoxyribonuclease activity” comprising or consisting of an amino acid sequence having at least 780 0 sequence matches with the amino acid sequence indicated as Sequence ID No.: 1 or sequence with ID number: 2. Preferably; the amino acid sequence of deoxyribonuclease has at least 785 Gills sequence; most preferably at least 790 most preferably at least 795 796 7, 798 «299» or more preferably Most preferred 7100 sequence matches the amino acid sequence of Sequence ID#: 1 or Sequence ID#: 2. In one embodiment; The 5 amino acid sequence of the deoxyribonuclease is Sequence ID#:1 or Sequence ID#:2. In the OAT embodiment the deoxyribonuclease is a fungal deoxyribonuclease; A filamentous fugal deoxyribonuclease is preferred more preferably Aspergillus fumigatus and more preferably Aspergillus oryzae deoxyribonuclease. In the AT embodiment the deoxyribonuclease is an Aspergillus oryzae deoxy0' pipenonuclease or a derivative thereof. Still in another embodiment the deoxyribonuclease is a deoxyribonuclease as disclosed die in the international patent application in EP 057883/2015 (now ISPM 155350/2015); which is included for reference under this application.

in one embodiment; number of substitutions amino acid deletions and/or insertions contained in the amino acid sequence from Seq-id:1 or Seq-id:2 up to 0 ex 321 4 5; 6» 7 8; 9 or 10; or up to 5; ex 32d 4; or 5; or up to 2. Amino acid changes can be of a weak nature; are conservative amino acid insertions or substitutions that do not substantially affect protein flexion and/or activity; small deletions; Typically 1-30 amino acids; small amino or carboxyl-terminal extensions; such as a methionine residue at the amino terminus; a tamino—terminal methionine residue; a linker peptide as small as 0-25 residues; or a small extension that facilitates purification by changing the net charge or an “irl 0” function such as a poly-histidine tract, an antigenic epitope, or a .binding domain are examples of conservative substitutions Within the groups of basic amino acids (arginine, lysine, histidine), acidic amino acids (glutamic acid and aspartic acid 5). Polar amino acids (glutamine and asparagine) Hydrophobic amino acids (leucine, isoleucine, valine) aromatic amino acids amino acids (phenylalanine, tryptophan, tyrosine) and small amino acids (glycine, alanine, serine, 0, threonine, and methionine) Aminos that do not generally alter specific activity are known in the art and have been described eg; By 0. Neurath and R.L.

Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are “Thr/Ser (Asp/Glu Val/lle Ala/Ser” Lys/Arg Ala/Pro.Tyr/Phe. Ser/Gly (Ala/Val Serf/Asn (Ala/Thr Ala/Gly Asp/Gly 5 Ala/Glu Leu/Val Leu/lle Asp/Asn 5

Essential amino acids in a polypeptide can be defined according to procedures known in the art;

Jie site-directed mutagenesis or alanine scanning mutagenesis

Cunningham and Wells, 1989, Science ) alanine-scanning mutagenesis.

1081-1085: 244). in the last technique; Alanine mutations are included

5 individuality in each structural unit of the molecule, and mutant molecules are tested

Resulting from deoxyribonuclease activity to identify the amino acid building blocks important for the activity of the molecule.

See also 271:4708-4699, Hilton et al., 1996, J.

Biol.

Chem. It can also

Determine the active site of the deoxyribonuclease or other biological interaction

By physical analysis of structure; As determined by these techniques Jie 0 nuclear magnetic resonance crystallography

crystallography Electron diffraction or photoaffinity labeling

photoaffinity labeling in combination with amino acid mutagenesis of a hypothetical contact site

de Vos et al., 1992, Science See; For example ». 001817/6 contact site

255: 306-312; Smith et al., 1992, J.

Mol.

Biol. 224: 899-904; Wlodaver can also infer amino acid conformity wet al, 1992, FEBS Lett. 309:59-64 5

Primary from alignment with a related polypeptide.

Substitutions and deletions can be made; and/or single or multiple amino acid insertions;

and tested using known methods of mutagenesis; crecombination and/or sale).

distribution.” This is followed by a related check; Jie those revealed by Reidhaar— Olson and Sauer, 1988, Science 241: 53-57: Bowie and Sauer, 1989, 20

Natl.

Acad.

Sci.

USA 86: 2152-2156; .0206 ISPM 22625/95 or

5. Other methods that can be used include error-prone PCR;

Lowman et al., 1991, Biochemistry (lis) phage display lela)

‎U.S.

Patent No. 5,223,409; WO 92/06204 30:10832-10837;

Derbyshire et al., 1986, Gene (region-directed mutagenesis (Ner et al., 1988, DNA 7: 127;145:46). The relationship between two amino acid sequences is described by the variable 'sequence match'. For the purposes of the present invention, the sequence conformity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.

Mol.

Biol. 48: 443-453 5) As implemented in NEEDLE program from the group ((EMBOSS version of BLOSUM62) EMBOSS version 5.0.0 or later is preferred. Variables used include the gap opening sentence 10) gap extension penalty 0.5' and a substitution template EBLOSUMG2 (EMBOSS version of 62/the 8105) The output of Needle named "longest match" (obtained using 10 -nobrief option) is used as a percentage and is calculated as follows: matched residue * 100)/(alignment length - total number of gaps in the alignment). microbial origin Chemically modified or genetically modified mutations are included in the protein It can be a calkaline protease such as serine protease or metalloprotease Serine protease for example can be from the family Jie ST Trypsin 7/0517 or FA 58 Jie Subtilisin 155:0 5001. Metalloprotease enzymes can be es protease for example is thermolysin 0-Mn; Sie family M4 or metalloprotease AT such as those of family M5 1/7 or M8 The term 'substylease 5000018565' refers to a subset of serine proteases according to Siezen et al. , 1991, Protein Engng. 4: 719-737, and Siezen et al., 1997, Protein Science 6: 501-523 The serine proteases are a subset

It is a protease enzyme that contains serine in the active site, which forms a covalent adduct with the substrate. Substilase families can be divided into 6 subfamilies; any; Subtilisin family Thermitase 16111111356 Family Proteinase A Family Lantibiotic peptidase 5 Kexin family Pyrolysin family

Examples of substillases are those derived from bacilli such as Bacillus alkalophilus, amyloliquefaciens, and Bacillus gibsonii. Bacillus subtilis described in US Patent

0 7,262,042 and ISPM 02186/2009; slow cluster subtilisin lentus csubtilisin novo Carlsberg 50011510; Bacillus licheniformis subtilisin BPN subtilisin 309 subtilisin 147 and subtilisin 168 described in IPM 06279/89 and protease BD8 00138 described in IPM 18140/93. Protease enzymes can be

Other useful ones are those described in International Patent 17577/92; 16285/01; 2 and the international patent 16547/02. Examples of trypsin-like proteases are trypsin (Di (from a porcine or bovine dual) and Fusarium protease described in IPC 06270/89; 25583/94 and 5 chymotrypsin proteases are derived from

ala 20 5 described in International Patents 052161/2005 and 052146/2005. An additional preferred protease Ble over the alkaline protease from S. slow-dose is DSM al 5483; as described for example in International Patent 23221/95; Variants of it are described in International Patent 21,760,792, International Patent 23221/95, European Patents 1921147 and 1921148.

Examples of metalloprotease enzymes are neutral metalloproteases as described in IPO 044993/2007 (Genencor Int.) such as those derived from Bacillus amyolaicofaccins. Examples of useful protease enzymes Ble for the variants described are: IPC 19729/92 34946/96 20115/98 20116/98 11768/99” 44452/01” 006602/03 03186/2004” 041979/2004.; 006305/2007 036263/2011 “1: Especially variants with substitutions in one or more of the following positions: 3 4 9 5 27 36 L 68 (T6 7 P 95 96 97 98 99 100 101 102 103” 4 + 106 “118” 120 « 123« 128« 129« 130 160« 167« 170 194 195« 0 199« 205« 206« 217« 218 222« 224« 232« 235« 236« 245« 248« 252 274 using more BPN' Preferably, subtilase variants may include mutations: S3T531;V4I V4 S9R SOR A15T8151 K27R (K27R 360*V68A VOSA N76D760N 78 asr 875, not 97*; RiMS101G V 104 A0/104/A.NO »S 106A ¢S106A RR G118 V oof »61181/H120D 1100 !8;N123S (N123S S128LA8128;B 129q©0129;S0A S130A G160D G167A 2016785 R 170 S 0/2051 0 L217D2170 N218D2180NM222S (M222S A232V/A232K) 235L K235L Q236H “Q236H” Q245R “Q245R” N252K T274 A1274/78 (using BPN numbering) Suitable commercially available protease enzymes include those sold by trade names Alcalase™ “Savinase™ (Ultra Relase™ (Relase™ (Durazym™ (Duralase”™) Liguanase™ (Kannase™ (Polarzyme™ (Primase™ (Ultra Savinase)) 25

518261 Ultra Coronase™ (Coronase™ (Ovozyme™ (Ultra Liquanase™) those sold with the name “(Novozymes A/S) Esperase™ ; Everlase™ (Neutrase™)

Commercial Purafect Purafect™ (Maxapem™ (Maxacal™ (Maxatase™)) “Puramax™ (Purafect OxP™ (Purafect Ox™) (Purafect MA™ (Prime ™)

Opticlean™ (Eraser™ (Excellase™) (FN4™ (FN3™) (FN2™ (Properase™ 5) and BLAP ((Gist-Brocases N.V.) Axapem™ (Danisco/DuPont) Optimase™ (the infiltration shown in Fig. 29 of the patent. American 5,352,604) and its variants (Henkel AG) KAP 4 (Bacillus alkalophilus subtilisin from Kao) lig enzymes can be used, but other types of enzymes; together with inhibitors 0 Protease inhibitors that Jia are reversible inhibitors of the Ole “Jig serine protease activity. Preferably, the protease inhibitor is a subtilisin protease inhibitor (reverse). Jaan can The protease inhibitor is an aldehyde peptide boric acid, tboronic acid, or a derivative thereof.The appropriate boronic acid derivatives are described in International Patent 4,963,655.US Patent 0 INP 12655/95 29223/95 19707/92< 04635/94 4 USP 5,442,100 5,488,157 and 5,472,628 Aldehyde peptides pepti are described de aldehydes appropriate in International Patent 04651/94; 5 13458/968 13459/98 13460/98 13461/96 13461/98 0 13462/98<141736/2007 145963/2007 118375/2009 055052/2010 and 1. alternatively The aldehyde peptide can have the formula as described in International Patent 036153/2011. The aldehyde peptide can be converted to a water—soluble hydrosulfite additive compound by reaction with A sodium bisulfite as

It is described in textbooks; March, J.

Advanced Organic Chemistry, Sie C, 1992 LS, cfourth edition, Wiley-Interscience, US is described in International Patent 004636/2013. lipase/cutinase

5 Suitable lipases and cutinase enzymes include those of bacterial or fungal origin. Chemically modified or transgenic protein mutants are included. Examples include a lipase from Thermomycins 161171007/065” Examples from Thermomycins lanuginosa 1801091705015 (previously called Humicola lanuginosa as described in EPs 258068 and 305216) Cutinase from Sie (Humicola)

0 Humicola Inulin 10501605 as described in IPM 13580/96; Pseudomonas lipase exemplary from LGA Pseudomonas alcaligenes or Pseudomonas pseudoalcaligenes (patent Lng) 2) bulbous Pseudomonas cepacia (EP 331376); Pseudomonas stutzeri (British Patent 1372034); Sparkling fake

S. (Dartois et al., Biochemica et Biophysica Acta 1131: 253-360, 1993) Pseudomonas stearothermophilus (JP 744992/64)

0 Pseudomonas pumilus (IP 16422/91); Streptomyces lipases GDSL (IP6,455,2006); Chitinase from Magnaporthe grisea (IPT 760 + chitinase from Pseudomonas mendocina (USP 5,389,536); lipase from Thermobifida fusca (IPT

‎Ligne (084412/2011 5) a thermophilic lipase Geobacillus stearothermophilus

— 2 1 — 86 | (Pat. 084417/2011) Lipase from Neuron subtilis (Pat. 1 (+)+) Lipase from Streptomyces griseus (Pat. 71 4) and Pristinaespiralis series (Pat. 137147/2012) Other examples are They are lipase variants such as those described in IPC 2 01541/94 EP 260105 407225 EC 5 00292/96 0744/95 2557894 4783/95 22615/95 04079/97 07202/97 600063/7508 /2007 and 109500/2009.Preferred commercially available lipase enzymes include “Ultra™ Lipolase cLipolase™

Novozymes ) Lipoprime™ (Lipoclean™ ¢ Lipolex™ cLecitase™ ¢ Lipex™ 3 0

Genencor ) Lumafast while other commercially available lipase enzymes include Lumafast (A/S sp.) and Bacillus (Gist-Brocades/Genencor Int Inc) Lipomax (Int Inc)

Solvay lipase from Solvay amylase 5 and includes those suitable amylase enzymes of bacterial or fungal origin. Chemically modified or transgenic protein mutants are included. Amylase enzymes include, for example, Jal©-Enzyme Dl 0-8018565 obtained from Bacillus; lie is a special strain of lichenoid bacillus” described in more detail in UK Patent 1,296,839. Examples of suitable amylase enzymes include amylase enzymes with sequence-id:2 in IPL 10603/95 or variants having 790 sequence matches with sequence-id:3 thereof. Preferred variants are described in the international patent 02597/94 «18314/94> 43424/97 and series with ID No.: 4 of 19467/99,; Jie Variables with substitutions in one or more positions of the following: 15« 23 105 106 124 128 133 154 156 78 to 179

— 3 1 — 181 « 188 » 190 197 » 201 202 207 208 209 211 243 264 304 » 305 391 408 » 5 444. Various suitable amylase enzymes include amylase enzymes with Sequence ID No: 6 in ISPM 10355/02 or variants of That has 790 sequence matches with a sequence with ID number: 6. The preferred variants of a sequence with identity number: 6 are those that have omissions in positions 181

1824 and a substitution at position 193. Other suitable amylase enzymes are a hybrid alpha-amylase comprising 1-33 residues of α-amylase derived from amyloliquefaciens shown in sequence ID No. 6 of the patent International 066594/2006

0 and residues 483-36 of lichenoid formulation alpha-amylase indicated in sequence with ID (tad) 4 of ISPM 066594/2006 or variants with 790 identical sequences thereof. Preferred variants of these hybridized alpha-amylases are those with a “deletion or insertion” substitution at one or more of the following positions: >= 48 “T49 49 =” G48 G107 “G107 H156 H156 A181 A181 N 190 190 No al 7 01197 E 201 1201 A 209

5 8209 and Q264. Most of the preferred variants of the hybridized α-amylases include residues 1-33 of the α-amylase derived from bacillus amyloliquefaccins shown in Sequence-ID No. 6 of ISPM 066594/2006 and residues 36-483 of Sequence-id No.: 4 is the one with the substitutions: “M197T

The additional suitable Pld enzymes are amylases with sequence ID: 6 in the international patent 99/19467 or variants thereof that have 790 sequence matches with a sequence with ID No. 6 Preferred variants of a sequence with ID No. 6 are those that have a substitution » deletion or inclusion in

One or more of the following loci: R181 G182 “G182 H183 1183 G4 6184 N195 195L E206 1206 {E212 E212 E216 E216 and K9 269a.” Specifically preferred amylases are those with deletions at positions “G182 R181” or positions H183 and 6184. Additional amylases that can be used are those with Sequence ID: 1; Sequence ID: 3; Sequence ID:: 2 or sequence with identity number: 7 from the international patent 6 or variants thereof have 790 sequence matches with sequence with identity number: 1 sequence with identity number: 2 sequence with identity number: 3 or sequence with identity number: 7. Preferred variants of sequence with identity number: 1 A sequence id#: 2 A sequence id#: 3 or a sequence id#: 7 is one with a 0 substitution; deletion or insertion in one or more of the following positions: 140 ¢181 182 183; 4 195 206 212 243 260 269 304 and 476. More favorably variants are those with deletions at positions 181 and 182 or positions 183 and 184. The most preferred amylase variants are from sequence ID: 1, sequence ID: 2, or sequence ID: 7. those having a deletion in positions 183 and 184 and a replacement in one or more positions 140 195; 5 206 243 260 304 and 476. Other amylase enzymes that can be used are amylases having Sequence ID No.: 2 from ISPM 153815/2008, Sequence ID No.: 10 in ISPM 1, or variants thereof having 790 sequence matches with Sequence ID No: 2 from ISPM 153815 /2008 or 790 sequence matches with a sequence with ID No.: 10 in International Patent 0 66712/01. Preferred variants of a sequence with ID No.: 10 in ISPM 66712/01 are those that have a substitution, deletion or insertion in one or more of the following positions: 176 177; 178 <179 190 201 207 211 and 264. Additional suitable amylase enzymes are lie from amylase enzymes having Sequence-ID:2 of ISPM 061380/2009 or variants having 790 sequence matches with Sequence-ID:2 of 5 thereof. Preferred variants of a sequence with id: 2 are those with a -© and/or

— 5 1 — replace; omission or insertion in one or more of the following positions: Q87 087; Q98 “Q98 S125” “S125 N 128” T7131 131 = “(N128 = 165 1165 K178” K178 R “R180 180 S181” S181 T2 182 G183 M201 201 al “G183 F” F202 202 N 225 1225 S 243 “S243 N 272” (N272 N 282 01282 Y 305 9305 R 309 “R309 D 319 0319 Q320 0320; KER 359 Q359K

K444 4 and J475 6475. in JST form preferably variants from sequence ID: 2 that have the substitution in one or more of the following loci: RQBTE Q8R Q98R S125 A5125/8; N128C © 0128 T131i 11311 T165i 11651 K178L “K178L T182G 11826 al 1l 1012011f

10 202 Y F202Y AR 225 to 225 N 272 E “R” N272E D; E’ ca S243Q Bl Y305R Bl 305 ls “DEAS243Q 309A30; Q0R “Q320R Q359E (Q359E K444E K444E and G475K G475K) and/or delete in position R180 and/or S181 or from 1182 and/or 6183 The most preferred amylase variants from sequence ID: 2 are those with substitutions:

5 41781+11826+73054+64754A+1280L1;'N128C+K178L+T182G+F202Y+Y305R+D319T+G475K ¢€8125A+N128C+K178L+T182G+Y305R+G475C or ACus S1285C+N1285K +T1311+T1651+K178L+T182G+Y305R+G475K Variants are truncated at the ©-ends and optionally also include a substitution at position 243 and/or

0 deletion at position 180 and/or position 181. Other suitable amylase enzymes are alpha-amylases with sequence ID: 12 in ISPM 66712/01 or a variant having at least 790 sequence matches with sequence ID: 2. Preferred amylase variants are those with a “substitution” deletion or insertion at one or more of the following positions of sequence ID No.: 12 in ip. 01/2 6671: “R118 (R28)

R181 ¢N174 6182 D 183 “G184 (D183) G186 6186 W 189 W189 N195 (N195 M202 (M202) Y298 7298 N299 299LAK 302 (K302) S303 “S303 N306 1306 R310” R310 N314 ¢N314 R320 “R320” H324 (H324 E345 E345 Y396 9396R400 “R400 W439) W439 5 R 444 (R444 N 445 445 NO K 446 446 Q 449 “Q449 1 458 (R458) N 471 (N4T1) N 484 [N484] Preferred amylase enzymes in particular include variants with ida of 0183 and 6184 having substitutions R 118 K 41186 N 195 F (N195F Ji 320 K R320K and R 458 K 44586 and variant additionally having substitutions in one or more positions selected from the group: M9 G149 6149 “G182 6186; 0 01202 T 257 1257 Y 295 1295 (N299) M323 E345 (M323) and A339 and the most preferred variant have additional substitutions in all these positions. Other examples are are amylase variants such as those described in IPOs 098531/2011, 001078/2013 and 001087/2013. The commercially available amylase enzymes are “Stainzyme”. Plus™ (Stainzyme™) Termamyl (Termamyl™ (Duramyl™ (Everest™ (Resilience™ (Amplify™ 15))

Rapidase™ (Novozymes A/S) BAN™ 5 Fungamyl™ (Natalase™ ¢Ultra™

Genencor (from Preferenz and 5100 Powerase™ (Purastar™ [Effectenz™] (International Inc. / DuPont) Lyase Enzymes 0 The lyase can be bacilli-derived SU pectoris; specifically Bacillus agaradhirenz 05 or Bacillus lichen planus; or a variant derived thereof; Ole as described in US Patent 6,124,127” IPC 27083/99 07084/99 2 092741/02; 095638/03 Commercially available pectate lyase enzymes are 601810 00 / 86501 p. 4 (Novozymes A/S) Pectaway™

— 7 1 — Cellulase (Sa) Suitable cellulase enzymes are of bacterial or fungal origin. Chemically or genetically modified mutants are included. Suitable cellulase enzymes also include cellulase enzymes of the genus Cgnum 0 ), bacillus; fusiform humicola; pseudo; Thelavia 116187/18; Ole fungal cellulases enzymes produced from fusarium sala

Myceliophthora thermophila Humicola insulinins and Myceliophtora thermophila cOXySporum 5,691,178 5,648,263 “4,435,307 disclosed in US Patents 09259/89. ISPM 5,776,757 Particularly suitable cellulase enzymes are enzymes Cellulase alkaline or neutral 0 has the benefit of color preservation. Examples of “Jie cellulase enzymes” are cellulase enzymes described in EP 257 495 0 < 372 531 0” IN 11262/96; 29397/96; 8 . Other examples are cellulase variants such as those described in IPC 07998/94 EP 315 531 0 US Patents 5,457,046 83 5,763,254 ISPM 2447195 12307/98 and 01544/99. Cellulase™ (Celluzyme™ (Carezyme™) Commercially available cellulase enzymes include 5¢ (Novozymes A/S) Whitezyme™ (Renozyme™ (Endolase™ (Celluclast™) and Genencor (available from Puradax EG 5 < Puradax HA (Puradax «Clazinase™. (Kao Corporation) KAC-500(B)™.

Oxidases <layl/Peroxidases Peroxidase enzymes The appropriate peroxidase enzymes are formed by the f-classification of enzyme 7 . 1 . 1 1 . 1 EC » as a gala by the International Union of Biochemistry and Molecular Biology Nomenclature Committee; or any fragment derived from it that shows peroxidase activity. Suitable peroxidase enzymes include those of plant, bacterial or fungal origin. Chemically modified or transgenic protein mutants are included. Examples of clay peroxidase

Useful include peroxidase enzymes from amassing oS 515M0001100; lia from the gray mold Coprinosis cinerea (European Patent 179.486); and variants thereof, Jie, those described in International Patents 24618/93 10602/95,; and 15257/98. The peroxidase enzymes also include the haloperoxidase enzyme, such as chloroperoxidase, bromoperoxidase,

and compounds that exhibit chloroperoxidase or bromoperoxidase activity. Haloperoxidases are classified according to their specifications of halide ions. Chloroperoxidases (EC. 1.11.1.10) stimulate the formation of hypochlorite from chloride ions.

0 in one embodiment” the haloperoxidase according to the invention is preferably a chloroperoxidase.; Ble is a halo peroxidase from vanadium haloperoxidase, i.e.; .vanadate—containing haloperoxidase In a preferred method according to the present invention is .vanadate-containing haloperoxidase with a .chloride ion source.

5. Je halo peroxidase enzymes have been obtained from many different fungi; Particularly from the group of fungi “dematiaceous hyphomycetes” such as Alternaria gray rot Botrytis caldaromyces 081081101077/065; for example; Caldariomyces fumago Sle “Curvularia Caldariomyces verruculosa and Caldariomyces inaequalis

Halo peroxidase enzymes were also isolated from bacteria such as Pseudomonas; pseudoexamples peruchenia 38m and series; Streptomyces aureofaciens ull. In a preferred embodiment, the haloperoxidase is derived from the genus Curvularia inaequalis or Curvularia verruculosa as Curvularia verruculosa.

CBS Inaqualiz 102.42 as described in ip. 27046/95; or Keofolaria verruculosa CBS 147.63 or Keevularia verruculosa CBS 444.70 as described in International Patent 04102/97; Dendryphiella salina as described in International Patent 79458/01; Drechslera hartlebii as described in International Patent 79459/01; Geniculosporium as described in International Patent 79460/01; or Phaeotrichoconis crotalarie as described in International Patent 79461/01. These include the appropriate oxidase enzymes; Determining which lactase is a component by enzyme classification EC 2 . or any fragment derived thereof which exhibits activity of a recombinant or compound which exhibits similar activity; As 0 catechol oxidase (EC 1.10.3.1) catechol oxidase ©-aminophenol oxidase E-” (EC 1.10.3.4) aminophenol oxidase or (ug ply) bilirubin oxidase (EC 1.3.3.5). 5 Suitable laccase enzymes are enzymes of microbial origin. The enzymes can be derived from plants, bacteria or fungi (including filamentous fungi and yeasts. 5 Suitable examples of fungi include a laccase derived from a strain of Aspergillus fumigatus Gray mold (Aspergillus cCollybia cobrinosis; lie coprenocis mold (sole) Curvularia comatus “Curvularia 5” cCurvularia friesii and “Curvularia plicatilis” Sis cCoriolus CCoriolus hirsutus Curvularia 0 (Japanese Patent 2238885(« 5130) Fomes (gall Lentinus) Myceliophthora eg thermophila Suds fili <Neurospora eg Neurospora crassa crassa (Neurospora) Banaeolus Sie (Panaeolus pseudomonas Papillionacius 0801101782 06115; Sia (Phlebia pseudomonas radiata (IPN 01046/92));

Oe +) 8 Condelleana Rhizoctonia Rhizoctonia solani Yew Sie (Schytalidium cthermophilum or Trametes) Trametes villosa and Trametes versicolor, including suitable examples of bacteria derived from a lacquer strain of Bacillus.

It is preferred for a kerosene derived from Coperinopsis or Micelyophthora; Specifically, LKAZ is derived from gray mold coprenoesis; as disclosed die in International Patent 08325/97; or from Myceleoptora thermophila” as disclosed in International Patent 33836/95. Examples of beneficial perhydrolase enzymes include naturally occurring mycobacterium perhydrolase enzymes or

0 variants of it. A representative enzyme is derived from Mycobacterium smegmatis and its enzyme properties are described; its intention” and its variables; In International Patent 5" 063400/2008. US Patent 0145353/2008; and 0167344/2007. General Description of the Invention

5 The present invention provides a method for cleaning a water filtration membrane by contacting the membrane with a liquid cleaning composition comprising deoxyribonuclease. The cleaning process can be Clean-in-Place or Clean-Out-of-Place (COP) in one embodiment; The rate of water flow through the membrane (reflux) is improved.

0 In another embodiment the pre or post method is by contacting the membrane with a biocidal composition .composition still In an embodiment AT ¢ the water filter membrane includes a biofilm; which includes preferably one or more bacteria selected from the group consisting of the species Acinetobacter

Bacillus, Comamonas, Escherichia ally (30+3) and Sphingomonas- other aspects and embodiments of the invention will be evident from the description and examples. Detailed description:

5 in the context of the present invention; The terms 'water filtration' and 'water treatment' include the filtration and treatment of aqueous solutions Able and aqueous suspensions as well as substantially pure water. It has been shown that extracellular deoxyribonucleic acid (DNA) plays an important role in initiating the binding of biotic and abiotic organisms to membranes. And the acid was released

0 Extracellular deoxyribonucleic acid Wal (eDNA) into the environment from dead cells of the animal; plant or dual bacterium. DNA is sticky and binds to surfaces and other molecules; Thus membrane occlusion is simply triggered by either agglomeration of smaller molecules into larger molecules or initiation of biofilm formation. In addition, microorganisms stick to surfaces; The DNA outside cells also serves as a structural component of a biomembrane. The role of extracellular DNA in biofilms varies from one bacterial species to the next » The extracellular polymers (GAY) Jie proteins and polysaccharides also play important roles; Although it is clear that extracellular DNA ghoul plays a major role in bacterial attachment to surfaces and early biofilm formation.0 Membranes used for water filtration/purification or non-ageous liquids include ultrafiltration (UF) ultrafiltration (MF) microfiltration nanofiltration (NF) nanofiltration reverse osmosis (RO) membranes reverse osmosis When daw membranes, to maintain a certain flow of liquid through the membrane must The operator (530) must apply pressure to the membrane (trans membrane pressure) which leads to increased power consumption. 5 Above a certain pressure the cohesion of the membrane is irreparably damaged.

The use of deoxyribonuclease enzymes to clean water filter membranes can reduce the adhesion of organic material and microorganisms to the membrane; The water/liquid flow through the membrane is improved. Deoxyribonuclease can also be used to clean membranes with conventional chemical cleaning agents, Jie acids, bases, bleaches, and other disinfectants and biocides. The deoxyribonuclease cleaning step can be applied either before or after cleaning procedures using other cleaning chemicals or formulations. Typically used biocides and cleaning chemicals include 2-dibromo-3-nitrilopropionamide (DBNPA) 2,2-dibromo-3-nitrilopropionamide rhodalon sodium hypochlorite hydrogen peroxide (H202) Hydrogen peroxide 0 (HNOs) nitric acid oxalic acid ‎acid An H;_ caustic soda solution; Ethylenediaminetetraacetic acid (01)" _surfactants "surfactants" coagulants antiscalants and dispersants. The membrane cleaning procedure can be either 'clean- In-place” or “clean-out-of-place.” In a clean-in-place procedure 5 the membranes remain attached to the treatment facility while they are subjected to the cleaning formula either in a soak procedure where the membranes are dipped or soaked in the cleaning formula or Flow-through procedure where the cleaning formula is continuously circulated through the membrane In a clean-out-of-place process the membranes are removed from the facility and cleaned in a soak or flow-through procedure 0 Depending on the nature of the blockage; cleaning procedures can be extended to clean-in-place or clean Off-site for a few minutes to several hours, such as overnight A typical membrane cleaning procedure can contain several separate steps by rinsing with water The order of chemical and deoxyribonuclease cleaning steps can vary depending on the nature of the membrane dirt 5 Structure On the characteristics of the deoxyribonucleic enzyme activity Glyase (sha) deoxyribonuclease purification can occur at either low, neutral or high pH and low or high temperature.

preferably The action takes place at a pH of 9-6 and a temperature range of 70-360 degrees

Asi

Sos membrane A biofilm is any group of microorganisms in which cells adhere to each other on a surface; such as a fibrous surface, a metallic surface, or any other solid surface. These adherent cells are often embedded within a self-produced matrix of extracellular polymeric material. (EPS) substance A biofilm is an extracellular polymeric conglomeration generally consisting of extracellular DNA, proteins and polysaccharides Biofilms can form on living or inanimate surfaces A planktonic LIAN growing in a biomembrane is distinguished physiologically from the microbial cells of the same organism, which, on the contrary, are individual cells that can float or swim in a medium. 010 and 11. Bacteria living in a Bale biofilm have significantly different characteristics5 from free-floating bacteria of the same species, as the dense and membrane-protected medium allows them to cooperate and interact in different ways. Vital 20110100658 Cua protect The high-density extracellular matrix and the outer layer of the cells are the inner part of the group. Typical biofilms include one or more bacteria selected from group 0 of the Acinetobacter species; bacilli; Komamonas” and Escherichia; pseudo; Sphingobium 480/01800100145 as one or more selected bacterial species from the group consisting of Acinetobacter calcoaceticus Bacillus amyloliquefaciens SC100 50100; Bacillus amyloliquefaciens SC8 168 Comamonas denitrificans Escherichia coli 5 K-12 Pseudomonas aeruginosa and Sphingomonas mucosissima.

Non-deoxyribonuclease enzyme The non-deoxyribonuclease enzyme optionally combined with the deoxyribonuclease (SUS prion) according to the invention” can be one or more non-deoxyribonuclease enzymes selected from the group consisting of ccarbohydrase has carabinase lull amylase Del cellulase cellulase cutinase ccutinase galactatase amniocentesis; clipase lu! protease and xylanase 10 in general; the properties of the enzyme(s) selected should be compatible with clean-up conditions (ie; best pH; compatibility with other enzymatic and non-enzymatic components” etc.); and the enzyme(s) should be present ) in effective quantities. on cellulose ©10105©.” Many cryohydrase enzymes have been used in cleaning and laundry applications; Jie “lad cellulase pectinase” pectate Ol matanase “arabetase” galactanase and xylanase” (Sang) are all used in liquid formulation composit ion

Mathanase Mathanase can be an alkaline mannanase of the family 5 or 6. It may belong to a wild type of bacillus or Yous specifically Bacillus clausii Chalodurans Lichenoides ; Or Humicola 5 insulins. The appropriate methanase enzymes are also described in IPM 64619/99. The commercially available methanase is (Novozymes A/S) Mannaway™ ga

Perhydrolases and suitable perhydrolases are able to catalyze a super Sle perhydrolysis reaction resulting in the production of a peracid peracid from a carboxylic acid ester (acyl) substrate in the presence of a source of superoxide oxygen Dla) peroxygen (hydrogen peroxide) While many enzymes perform this reaction at low levels, perhydrolase enzymes exhibit a superior hydrolysis ratio: hydrolysis lle often greater than 1. Suitable perhydrolase enzymes can be Plant, bacterial or fungal origin Chemically modified or genetically modified mutants are included in the protein Water filtration membranes A variety of membrane types and designs can be used in treatment processes 05 Water or waste water Types of capillary tube membrane designs include capillary ctube didd hollow chollow fiber multiple tubes 0010-0006 plate-and-frame/flat wafer ¢pleated cartridge filter spiral wound and ceramic Le incl. ceramic disc ceramic disc yum Membranes can be manufactured from one or more of 5 different materials; Ly including, for example, chlorinated polyethylene, sh cpolyacrylonitrile, polysulfone, cpolyethersulfone, polyvinyl alcohol, ¢ polyvinylalcohol, cellulose acetate. Regenerated cellulose Polyvinylidene difluoride Polyethylene difluoride cpolyethlysulphone Polyethylene Unarmed polypropylene Ceramic material Other properties include membranes that can vary based on application ; For example; The pore size of the membrane. Membrane pore size can be larger or smaller Bly on the size of the particles or impurities removed from the water or wastewater. Membrane types » according to the present invention include; those used in ultrafiltration; Microfiltration » Nanofiltration. 5 Membrane Bioreactor Systems

Membrane bioreactor (MBR) systems typically couple two basic processes: biodegradation and membrane separation; In a single process where suspended solids and microorganisms responsible for biodegradation are separated from the water that has been treated by a membrane filtration unit. See, for example, Water Treatment a3 Membrane Processes, McGraw-Hill, 1996, p. 17.2 5 capture the entire biomass within the system; This provides both control over the residence time of microorganisms in the reactor (sludge age); and disinfection of liquid waste. In a typical membrane bioreactor unit; Wastewater effluent is pumped or gravity fed into a cus aeration tank which is brought into contact with biomass which decomposes organic 0 matter in the wastewater. Aeration means such as blowers provide oxygen to the biomass. The resulting mixed liquid is pumped or fed by gravity from the aeration tank to the Cus membrane module mechanically or by gravity through a membrane under pressure or drawn through a membrane under reduced vacuum. in some systems; The aeration tank and membrane tank are the same tank. The effluent is drained from the system while the mixed liquid is returned to the bioreactor. Excess sludge is pumped out to maintain a constant sludge life; The membrane is cleaned regularly by backwashing; chemical washing; Air drying or any combination of these mechanisms. Membrane bioreactor systems have multiple designs. Two of the major membrane bioreactor process designs include immersed/immersed and bypass current. There are also two primary mechanisms of hydraulic 0 operation; It includes pumping and lifting air. These designs and bulky fluid transfer modes are stereotypically referred to as conventional biomass rejection membrane bioreactors. Other designs include: Extraction and diffusion modes that use membranes for purposes (gal) other than separating biomass from treated water. All of these process designs include one or more membrane units incorporating membranes such as those described in aud "Membranes" “Membranes” section mentioned above.

in one of the models; The membranes are in a membrane bioreactor. in another form; The wastewater treatment process takes place in a membrane bioreactor in which a flat-sheet cassette unit or, in one embodiment, a hollow-fiber unit is typically immersed; Wastewater is treated before entering the biofilm reactor. Pretreatment can occur at the wastewater source; or in a pretreatment plant or as part of an overall membrane bioreactor system. This pretreatment can include a Jala bar screen, a grit chamber, or a rotary drum. screen to achieve the removal of coarse solids Other pretreatments may include the removal of substances such as harmful contaminants; oils, fuels, or other toxic substances 0 dlle water processes One or more water treatment processes are described by the present invention. dallas This water includes, but is not limited to reverse osmosis, desalination, purification of drinking water, and wastewater treatment processes It can be water or wastewater hg of the present invention from any source including human waste; 5 Septic tank leakage; septic tank drainage; sewage and wash water drainage (Jie) gray water or shallow water; collected rainwater; groundwater; industrial overflow; seawater (Oeil water) and disposal of human fluid; drainage Highways, storm drains, fresh water malady; slag Processing from product manufacturing industries including electronics » transportation vehicles; cpharmaceuticals; paints; lubricants; plastics; Crude oil, gas, and derived refined products from finished feed and foodstuffs; drinks; textiles; non-textiles; paper and pulp; processed grains; vegetable oils, chemicals, and proteins (eg; enzymes); industrial waste; industrial site wastewater or sewage such as cooling or process water; and agricultural wastewater or sewage. 5 liquid cleaning formula

The liquid detergent composition used Bg of the invention has a form (gale) that is not a solid (or gaseous). The Sl can be pourable, pourable gel, or non-pourable gel. It may be either slanted or structural; slanted is preferred ALL CLEANING FORMULA CAN BE AQUATIC: Typically contains at least 720% by weight and up to 795% water; such as up to 770% water; up to 750% water; up to 740% water; up to to 730 of water, or up to 20 7 of water Other types of liquids may be included, including but not limited to alcatel calkanols amines camines dihydroxyl compounds diols ethers and many Hydroxyl polyols in an aqueous liquid composition The aqueous 0 liquid composition can contain 0-230 of an organic solvent The liquid composition can be aqueous; the water content is less than 710; Jiang Less than 75. The cleaning composition can take the form of a unit dose product The unit dose product is single dose packaging in a non-sealable container for re-use. They are increasingly used in laundry detergents and dishwashing detergents. The detergent dose unit product shall be packaging “J) 5 eg” in a bag made of water soluble film of one wash quantity of detergent. The bags can take any form; shape and material suitable for fixing the formulation, eg without allowing the formulation to be released from the sachet prior to contact with water The sachet shall be made of a water soluble film which encloses an inner volume Said inner volume may be divided into parts of the bag 0 Preferred membranes include polymeric materials materials Preferably polymers formed into a film or wafer Preferred polymers or copolymers or derivatives are polyacrylates 00178015 and water-soluble acrylate copolymers; methyl cellulose and methyl cellulose carboxy methyl cellulose, sodium dextrin, ethyl cellulose 5 cethyl cellulose, chydroxyethyl cellulose, chydroxypropyl methyl cellulose, malt and dextrin 10,000; And polymethacrylate

JS g cmethacrylates Mostly preferred Polyvinyl alcohol copolymers 35M hydroxypropyl cellulose (HPMC) hydroxypropyl methyl cellulose Preferably up to polymer level in the membrane eg puty did alcohol (PVA) polyvinyl alcohol about 760 at least. The average preferred molecular weight is typically around 20,000 to about 150,000. Lind films can be blend compositions containing a hydrodegradable, water-soluble polymer die polyactide and polyvinyl alcohol (known under trade reference M8630 8630 al as sold by Chris Craft In.

Prod.

Of Gary, Ind., US In addition to plasticizers, Jie plasticizers cglycerol ethylene glycerol propylene glycol sorbitol and mixtures thereof. 0 Sachets may include a solid detergent formulation or partial components and/or a liquid cleaning formulation or partial components separated by a water soluble membrane. The liquid components portion may be different in composition from the portions containing solids (see eg » US Patent No. 0011970/2009). The choice of cleaning components can include consideration of the type of membrane to be cleaned; 5 type and/or degree of contamination”; and the temperature at which the cleaning takes place.”; and cleaning product formula. Although the components listed below are categorized by general heading according to a specific function; However, this cannot be construed as a limitation; Because the component can have additional functions as conceived by those skilled in the art. The selection of additional components is within the skill of the experienced; It includes traditional ingredients; Including the typical unrestricted components described below.

Surfactants The detergent composition may include one or more surfactants; which can be anionic, cationic, nonionic, semipolar and/or dipole; or a mixture of that. in a specific embodiment; The composition includes a mixture of one or more nonionic surfactants and one or more anionic surfactants. The surfactant(s) are typically present at a level of approx

1 to 760 by weight» Mos die 71 to about 0 +; or about 73 to about 720 or about 73 to about 7210. The choice of surfactant(s) depends on the desired cleaning application; It includes any conventional surfactant(s) known in the art. (Sag) Use any surfactant known in the art for use in the detergent formulations. When included therein the detergent formulation will typically contain from about 71 to about 0 by weight; such as from about 75 to about S30 including from about 75 to about 715; or about 720 to about 725 of an anionic surfactant. Unbound examples of anionic surfactants include sulfates and sulfonates 0 limitation of alkylbenzene linear sulfonates alkylbenzenesulfonates (as) isomers of linear alkylbenzenesulfonates 0-branched alkylbenzenesulfonates (BABS) alkylbenzenesulfonates phenylalkanesulfonates cphenylalkanesulfonates alpha-olefinsulfonates (AOS) alpha-olefinsulfonates olefin sulfonates calkene sulfonates alkane-3 -diylbis (sulfates) calkane-2,3-diylbis(sulfates) 5-hydroxyalkanesulfonates and disulfonates cdisulfonates alkyl sulfate (AS) sulfates like capers sodium dodecyl sulfate (505); Fatty alcohol sulfates (FAS) fatty alcohol sulfates primary alcohol sulfates (exactly) alcohol ether sulfates (AES) or AEOS or FES Known Also known as alcohol ethoxysulfates or fatty alcohol ether sulfates 0 Secondary alkanesulfonates (SAS) secondary alkanesulfonates Paraffin sulfonates (PS) paraffin sulfonates ester sulfonates Processing csulfonated fatty acid glycerol esters SFMe~ all) alpha-sulfo fatty acid methyl esters or Lo (SES) MES methyl ester sulfonate an alkyl acid—alkyl or alkenylsuccinic dodecenyl/tetradecenyl succinic acid (/015); fatty acid derivatives of amino acids;

diesters and monoesters of sulfo-succinic acid or soaps and combinations thereof. When included in it, the cleaned composition will typically contain from about 70.1 to about 0 by weight of a cationic surfactant. Unrestricted examples of cationic surfactants include alkyl dimethylethanolamine ( (ADMEAQ) alklydimethylethanolamine quat Cetyltrimethylammonium bromide (CTAB) cetyltrimethylammonium bromide gla Jie ga Stearyl ammonium (DSDMAC) dimethyldistearylammonium chloride calkylbenzyldimethylammonium calkyl quaternary ammonium 0 alkoxylated quaternary ammonium compounds and combinations thereof.When included therein the detergent composition will usually contain from about 70.2 to about 0 by weight of a non-ionic surfactant, eg of about 70.5 to about 230; namely from about 71 to about e220 from about 73 to about 710; die from 5 about 73 to about 75; or from about 78 to about 78 S12 Examples of unrestricted non-ionic surfactants include alcohol ethoxylates (AE) or hydrochloric acid (AE) calcohol propoxylates PFA propoxylated fatty alcohols; alkyl esters fatty acid dallas with alkoxylated Jie calkoxylated fatty acid alkyl esters of fatty acid treated with ethoxylated 0 and/or dallas with cpropoxylated alkylphenol ethoxylates (APE) ethoxylates nonylphenol ethoxylates (NPE) nonylphenol ethoxylates; Alkyl Polyglycosides (APG) alkylpolyglycosides calkoxylated amines fatty acid monoethanolamides (FAM) ; SU Ethanolamides (FADA) fatty acid diethanolamides (EFAM) ethoxylated fatty acid monoethanolamides propoxylated fatty acid monoethanolamides

(PFAM); polyhydroxy alkyl fatty acid amides or 17-acyl N-acyl N-alkyl derivatives JSI-N of glucosamine (GA) glucamides or glucamides “((FAGA) fatty acid glucamide) as well as products available under the brand names “TWEEN 5 SPAN” and combinations thereof. When included, the detergent composition will typically contain from about 70.1 to about 720 by weight of a semi-surfactant. Semipolar surfactant. Non-restrictive examples of semipolar surfactants include Jie (AO) amine oxides alkyldimethylamine oxide calkyldimethylamineoxide 71-(cocoalkyl)-17017-di(ise) N-(coco alkyl)-N, N-dimethylamine oxide and oxide ?1-(tallow-alkyl)-11017-bis(2-0-hydroxyethyl)N-(tallow-alkyl)-N,N -bis(2-hydroxyethyl)amine oxide fatty acid alkanolamides, ethoxylated fatty acid alkanolamides, and combinations thereof when included therein The cleaned composition will typically contain from about 70.1 to about 0 by weight of sale zwitterionic surfactant Unlimited examples of zwitterionic surfactants include Colin betaine Alkyldimethylbetaine calkyldimethylbetaine esulfobetaine and combinations thereof. Methods of use 0 as described in the above paragraphs; The present invention provides a method for cleaning a water filtration membrane by contacting the membrane with a liquid detergent composition comprising deoxyribonuclease. preferably The rate of water flow through the membrane (reflux) is improved. The liquid detergent formulation may also include the basic ingredients as described in the above paragraphs. in one embodiment; The cleaning process can be clean-in-place or clean-out-of-place. 5 in an embodiment; Deoxyribonuclease treatment of the water filtration membrane is pre or post by contacting the membrane with a biocidal composition.

in one embodiment; The water filtration membrane includes a biofilm. preferably The biofilm includes one or more bacteria selected from the group consisting of the species Acinetobacter; bacilli; Komamonas” and Escherichia; pseudo; and sphingobium. More preferably the biofilm comprises one or more bacteria selected from the group consisting of Acinetobacter, Bacillus amylolicofaccins 50100 Bacillus amylolicofaccins 50168 Commonas denitrificans Escherichia coli (K-12) Pseudomonas aeruginosa and Sphingobium mucossima; preferably Bacteria Pseudomonas aeruginosa In another embodiment the pH of the cleaning composition is 6-9; preferably a pH of approximately 0 In one embodiment the film is in contact with the cleaning composition at a temperature of 30-100 °C; preferably at a temperature of still in the GAT embodiment the membrane is simultaneously or separately attached to one or more deoxyribonuclease (NDN) enzymes from the group consisting of proteases; Pictate Ol methanase Arabinase Galactanase Xylanan 5 perhydrolase and oxidoreductase The enzyme is preferably a non-deoxyribonuclease Ble than a protease In one embodiment the deoxyribonuclease The RNase used in the detergent formulation is an Aspergillus oryzae deoxyribonuclease or a derivative thereof. preferably A deoxyribonuclease is a polypeptide with deoxyribonuclease activity” comprising or consisting of an amino acid sequence having at least 780 sequence matches with the amino acid sequence indicated as Sequence ID number: 1. More preferably 0 the deoxyribonuclease is a polypeptide with activity A deoxyribonuclease comprising or consisting of an amino acid sequence indicated as sequence ID number: 1. In another embodiment the deoxyribonuclease is a bacillus lichenoid deoxyribonuclease or Fide thereof. preferably A deoxyribonuclease is a polypeptide having deoxyribonuclease activity” comprising or consisting of an amino acid sequence having at least 780 Gills 5 sequences with the amino acid sequence indicated as Sequence ID No: 2.

Test 1: Deoxyribonuclease activity Deoxyribonuclease activity was determined on deoxyribonuclease test agars with methyl green (BD, “Franklin Lakes, NJ, USA”) prepared according to the instruction manual from the supplier. Briefly; 21 aba was dissolved. of deoxyribonuclease test agar in 500 mL of water and was autoclaved 5 sad 15 min at 121 °C The temperature of the autoclaved agar was measured at 48 °C in plas water 20 mL of agar was poured into Petri dishes and allowed to solidify by 0/0 incubation at room temperature On hardened agar plates 5 μl of enzyme solutions were added and deoxyribonuclease activity was observed as colorless areas around the enzyme solutions 10 examples were algal chemicals used as buffer solutions buffers and substrates are commercial products of at least reagent grade 0 The deoxyrdonuclease used in metal 1 was obtained from Aspergillus oryzae and represents the mature polypeptide from a sequence Amino acid shown in the bhui sequence Example 1. Example 1 Treatment of water filter membranes with deoxyribonuclease Bacteria and culture media 0 Seven bacteria commonly found in wastewater and sedimentary water treatment facilities were used to form a multispecies biofilm for this study. The following bacteria were selected: Pseudomonas aeruginosa (ATCC 19429) Acinetobacter calcium acetate (ATCC 23055) 5 (Commonas denitrificans) 700937 (ATCC) Escherichia coli (ATCC 10798) K-12

(NRRL B-59454) Sphingopium mucusissima

Bacillus amyloidofacchinate 50100

Bacillus amyloidofacchinate 50168

Bacteria were cultured on Luria Broth (LB 13; 15.5 g/L; “Beckton Dickenson”).

(NJ Franklin Lakes 5) at 30°C at 200 rpm overnight for membrane tests

implemented bio. All biofilm tests were performed using LB

Calibration curve of a filter plate reflux test

to assess reflux changes; an indicator dye was used; Saad bright “(Clariant Corp, Switzerland) sad 0 The amount of liquid that can pass through the membranes of an EMD 96-eye filter dish

Millipore MultiScreenHTS and a 11018000 curve calibration curve were created for brilliant greens with a focus

70.05 (w/v) in distilled water. A black test dish containing 96 Costar® eyes was also incubated

With a clear flat base with 70.05 amounts of shimmery green pigment ranging from 10-240.

microliters. These size series were run in triplicate and measured on a Biotek Synergy 114 Hybrid (KPR) Reader Kinetic Plate Reader 5 at an absorption of 610 nm. Based on the readings

absorption from each of the corresponding volumes; A standard curve was created in Excel

By plotting the absorbance against the corresponding magnitude of this absorbance reading. He formed the standard curve

This is a linear equation used to evaluate the amount of liquid that can pass through the membranes of a filter dish. 0 Backflow test of a 96-well filter dish

The cultures were grown separately overnight in LB at 30 °C; With shaking at a speed of 200

revolutions per minute. The growth of the culture was measured by the optical density of 600 nm using

Scientific Spectronic 200+ spectrophotometer 1161070. Reduced

All farms to OD 600 600 00 is 0.5. The diluted cultures were added together in equal amounts of 1 mL in a separate tube and gently stirred in a vortex. A filter plate containing 96 samples was also loaded

Type EMD Millipore MultiScreenHTS in mixed cultures. Equal volumes are 100 µL

Distributed in a multi-channel pipette from the mixed culture in columns 1-4 and 9-12. die compare LB medium in equal volumes of 100 µL was added to columns 8-5. Once the dish is ready; A breathable film was applied to the plate. The plate was wrapped in a Jala Tupperware container along with a damp WYPALL towel to maintain 799 RH to ensure that the plate did not dry out. The Tupperware was placed on a vibrator at 200 rpm for 7 days at 30 After incubating the 96-well filter dish for 7 days, the 'breathe easy' membrane in a BSL 2 851,11 hood was removed. The plate was inverted against a WYPALL towel allowing the majority of the fluid in the eyes to be removed. Each eye was washed with 200 μL of phosphate buffer 0. The plate was immediately inverted and the buffer was removed from the eyes of the plate. To ensure that all fluid was removed From the eyes, the inverted plate was gently covered on the towel.From here the following pathways were taken to determine the efficacy of DRNase treatment on blocked membranes: of 1 oa per million in sterile water on dishes tested using treated eyes selected semi-randomly. Sterile water was added to eyes not receiving DRN-treated eyes. Two dishes were incubated, respectively, at . room (22 °C) and 0 °C dl (60 °C (sie) for 1 hour. After incubation, the plates were inverted and the liquid was removed from the eyes. The dishes were rinsed again with phosphate buffer and the liquid was removed. Effect of concentration Enzyme A deoxyribonuclease sample of an ultrafiltrate product was diluted to a final concentration of respectively 0.1 parts per million and 1 oda per million in sterile water on two separate coated plates; Using treated eyes that are chosen semi-randomly. Sterile water was added to the eyes

that did not receive deoxyribonuclease treatment. The two plates were incubated at room temperature (22°C) for 20 minutes. after incubation; The dishes were inverted and the fluid was removed from the eyes. The dishes were rinsed again with phosphate-buffered solution and the liquid was removed. Effect of time A deoxyribonuclease sample of the ultrafiltration yield was diluted to a final concentration of 1 ppm in sterile water on dishes tested using pseudorandomly selected (SA) treated eyes. Sterile water was added to eyes that did not receive Deoxyribonuclease treatment Five plates were incubated at room temperature (22°C) for 0 in the order 0 5 10 20 and 60 minutes After incubation the plates were inverted and the fluid was removed from the eyes The plates were rinsed again with phosphate buffer and the Liquid Once both plates had been rinsed with phosphate buffer, a sample of 200 µL of 70.05 brilliant green indicator dye was added to each eye The filter plate was placed on a Costar® 96 black eye with a flat base Lila and used as a collection plate. This mixture was placed in an Eppendorf 5810 Centrifuge 5 with an empty balance.The Baal centrifuge was set for 1 minute at 150 relative centrifugal force at room temperature (22°C). centrifuge; the collection dish has been removed from the centrifuge The amount of indicator dye that passed through the membranes into each of the corresponding eyes was measured on a Biotek Synergy 114 Hybrid Kinetic Reader Plate Reader at an absorption of 610 nm. using the equation formed from the calibration curve 0 (described above); An amount can be calculated from each eye of the collection dish. The percentage of regurgitation was calculated by dividing the eyes that had been treated with their non-obstructed comparison samples. This is done using untreated dX blocked eyes to show reflux comparisons between DRNase-treated and untreated membranes. 5 Backflow test of a filter plate of 96 eyes pre-treated with deoxyribonuclease

The aforementioned seven microorganisms were grown separately overnight in LB at 30 °C; With shaking at a speed of 200 revolutions per minute. The growth of the culture was measured by optical density at 0 nm using a Thermo Scientific Spectronic +200 spectrophotometer and each culture was diluted to 600 OD of 0.5. The diluted cultures were added together in equal amounts of 1 mL in a separate tube and gently stirred in a vortex. A filter dish containing 96 EMD Millipore MultiScreenHTS eyes mixed cultures along with deoxyribonuclease ultrafiltrate of three species (Aspergillus oryzae and Bacillus subtilis) were loaded into the selected eyes. Treated eyes were randomly selected while sterile water was added to eyes that did not receive deoxyribonuclease treatment. Once the dish is ready; A 'breathable 0 easy' membrane was placed on the plate. The dish was daly wrapped in a Tupperware container along with a damp WYPALL towel to maintain the 799 RH to ensure that the dish never dried out. The Tupperware was placed on a vibrator at 0 rpm for 7 days at 30° Agia after incubating the 96-well filter dish for 7 days; The 'breathe easy' membrane in the lid [851,1] was removed. The plate was inverted against a CWYPALL towel allowing removal of all fluid 5 in the eyes. The eyes were washed with 200 μL of a phosphate-buffered solution. The plate was immediately inverted and the The buffer solution from the eyes of the plate.To ensure that all the liquid is removed from the eyes,the inverted plate was gently covered on the towel.Once the filter dishes had been rinsed with the phosphate buffer solution,200 μL of 70.05 brilliant green indicator dye was added to each eye.The filter dish was placed on the Costar® 96 sample 0. Moisturizer with flat, clear base 60W test plate as collection plate This mixture was placed in an Eppendorf 5810 centrifuge with an empty balance The centrifuge was set for one minute at a speed of 150 centrifugal force at 150 °C. room temperature (22 °C).After centrifugation, the collection plate AY was removed from the centrifuge and the amount of indicator dye that passed through the membranes to the collection plate was measured on a Biotek Synergy 114 Hybrid Reader Kinetic Plate Reader 5 at an uptake of . 610 nm The amount that passed through each eye was also calculated n using a calibration test (described above). By dividing the eyes processed by comparison samples

— 9 3 — Unblocked E. Reflux percentage is configured. This was done using untreated blocked eyes as well; To show reflux comparisons between membranes treated with deoxyribonuclease and those not. Results Effect of temperature Temperature showed little effect on the effectiveness of deoxyribonuclease, where deoxyribonuclease treatments given at room temperature showed very different Jia to no reflux f compared to deoxyribonuclease treatments given at elevated temperatures. Reversion of bio-clogged 0 membranes treated with DRN-Jin was greater than 788 compared to non-clogged control samples (see Table 1). Effect of enzyme concentration when comparing DRNase concentrations of 1 ppm and 0.1 ppm; There was little to no difference between doses 5. Effect of time At each of the time points (5 min; 10 min; 20 min; 60 min); both deoxyRNase and RNase treatments showed a significant increase in reflux compared to eyes treated with water. However, little improvement in regurgitation was seen after application of treatment longer than 20 minutes.At this time point, the RNase-treated membranes had reached 790 regurgitations to those un-fouled membranes (see Table 1). Conclusion Percentage of flux by volume that passed through the membranes of a 96-well filter dish sealed by a polyspecies biofilm pretreated with deoxyribonuclease (DNA) of Aspergillus oryzae ultrafiltration at concentrations of 1 sha per million. Treated eyes twitched

— 0 4 — Using ultrafiltration yields a significant increase in reflux compared to those without processing. Refluxes in the obstructed membranes were restored to as many as 788 non-occlusive membrane refluxes. Table 1. Overview of test results for a 96-well filter dish backflow. Each site located in the middle of the father Amount of reflux/amount of comparison sample (reflux ratio) Optimum temperature 1 ppm | 155.88 µl/169.05 µl (791.7) untreated 2 µl/167.69_µl (772.0) rt 1 ppm | en in | 150.52 μl /158.59 μl per million 20 min (794.9) untreated 106.3 als Se /162.0 μl (765.6) room temperature 1 ppm | 101.08 ils Kia (168.84 μl min (59.9)

4-1 0

Untreated 30.8 1693/5558.30 μl (12.3)

room temperature 1 ppm | 136.12 µL/170.74 µL minutes (179.7)

Untreated 45.25 ss Kae 169.20 μl (126.7)

room temperature 1 ppm | 152.76 µL/159.26 µL 0 min (195.9)

Untreated 2 µL/155.17 µL (153.4)

room temperature 1 3a per million | 155.04 Soe 55 (174.03 μl 60 min) (789.6)

Untreated 105.52 fs Sue (172.95 µl (161.0)

Claims (6)

— 2 4 — Elements of protection 1. Method for cleaning a Cus ewater filtration membrane The water filtration membrane includes a biological cbiofilm The method (i) involves contacting the membrane with a liquid cleaning composition containing deoxyribonuclease (DNase) deoxyribonuclease and one or more selected non-deoxyribonuclease enzymes from arabinose arabinose cryohydrase carbohydrase cellulase cutinase s galactanase lipase ble 018002008 covidoreductase pectinase 0600856 pectate lyase perhydrolase ¢<perhydrolase protease and xylanase ¢xylanase or (ii) contact membrane membrane with liquid cleaning composition includes 0 Separate Jug cdeoxyribonuclease. Membrane contact with one or more non-deoxyribonuclease enzymes selected from the camylase arabinose arabinose carbohydrate ccarbohydrase C cellulase cutinase ccutinase galactanase cgalactanase lipase clipase manase cmannase oxidoreductase covidoreductase pectinase 6000m pectate lyase ¢pectate lyase perhydrolase ¢perhydrolase protease 5 and zeplanase xylanase 2. the method according to claim 1; Where the cleaning process is Cleaning- (CIP) In-Place or Cleaning-Out-of-Place (COP). 3. The method according to claim 1; Where the rate of water flow through the membrane (flux) is improved 4. the method according to claim 1; preceded or followed by contact of the membrane with a biological killing composition -biocidal composition 5. The method in accordance with claim 1; The biofilm includes one or more bacteria selected from the group consisting of species of Acinetobacter, Bacillus, Comamonas, Escherichia, Pseudomonas, and Sphingomonas. . Sphingomonas 5 6.. the method in accordance with claim 1; Where the biological membrane includes one or more bacteria selected from the group consisting of Calcoaceticus 011010700127 Bacillus amyloliquefaciens SC10 SC100 Amyloliquefaciens 58 50168 Bacillus amyloliquefaciens Escherichia coli Comamonas denitrificans 0 Escherichia coli K-12 K-12 Pseudomonas aeruginosa and Sphingomonas mucosissima. 7. Method Wy [Cus] The cleaning composition has a pH of 9-6. 8. The method in accordance with claim 1; Where the membrane is in contact with the cleaning composition at a temperature between 30-100 C. 9. The method according to claim ol where deoxyribonuclease is 0 Aspergillus oryzae deoxyribonuclease or a derivative thereof. 0. The method according to claim 1; Where deoxyribonuclease is a polypeptide with a cdeoxyribonuclease activity that includes or consists of an amino acid sequence that is at least 780 percent identical to the 10 5 amino acid sequence shown In a sequence with identity number: 1. 1. The method according to Claim 1 where deoxyribonuclease is a polypeptide that includes deoxyribonuclease activity and which includes or consists of an amino acid sequence indicated by sequence ID No.: 1. 12. The method according to claim 1 where deoxyribonuclease is a deoxyribonuclease 060771000016886 of Bacillus licheniformis or a derivative thereof. 3. The method according to Claim 1 where the deoxyribonuclease is a polypeptide that includes deoxyribonuclease activity and which includes or consists of an amino acid sequence that is at least 780 percent identical to the amino acid sequence amino acid shown in sequence with ID number: 2. 4. The method according to claim 1; Where the biological membrane includes (A) biofilm aeruginosa Pseudomonas aeruginosa 15 5. Method according to claim 1” wherein the pH of the cleaning composition is approximately neutral. 6. Method [yaaa] 12d Protection 1 Cua Membrane is in contact with the cleaning composition at a temperature between 50-80"C. Sued Authority for Intellectual Property RE .¥ + \ A 0 § 8 Ss o + < M SNE A J > E K J TE I UN BE Ca a a a ww > Ld Ed H Ed - 2 Ld, provided that the annual financial consideration is paid for the patent and that it is not null and void for violating any of the provisions of the patent system, layout designs of integrated circuits, plant varieties and industrial designs, or its implementing regulations. Ad Issued by + bb 0.b The Saudi Authority for Intellectual Property > > > This is PO Box 1011 .| for ria 1*1 v= ; Kingdom | Arabic | For Saudi Arabia, SAIP@SAIP.GOV.SA