DK202100639A1 - Method for controlling slime in a pulp or paper making process - Google Patents

Abstract

The present invention pertains to the field of pulp or paper making. More specifically the present invention relates to a method of preventing a build-up of slime or removing slime from a surface contacted with water from a pulp or paper making process. The present invention can control slime in an efficient and environmentally friendly way.

Description

DK 2021 00639 A1

METHOD FOR CONTROLLING SLIME IN A PULP OR PAPER MAKING PROCESS

REFERENCE TO SEQUENCE LISTING This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.

FIELD OF THE INVENTION The present invention pertains to the field of pulp or paper making. More specifically the present invention relates to a method of preventing a build-up of slime or removing slime from a surface contacted with water from a pulp or paper making process.

BACKGROUND OF THE INVENTION Most modern paper mills are operating a warm and closed loop water system under neutral or alkaline conditions which provide a good environment for the growth of microorganisms. In pulp mills, the pH and temperature conditions in the process water (white water) circuit of the pulp drying machines are beneficial for the growth of microorganisms. The microbes in the system or process show slime build-up, i.e. surface-attached, growth, and free-swimming, i.e. planktonic, — growth. Slime can develop on the surfaces of a process equipment and can rip off from the surfaces. It can reduce water flow; block devices such as filters, wires, or nozzles; deteriorate the final product quality, e.g. by causing holes or colored spots in the final product; or increase downtime due to the need for cleaning or due to breaks in the process. The slime is difficult to remove from the surfaces of the process equipment and often require the use of very strong chemicals. Controlling slime-forming microorganisms by applying toxic biocides is becoming increasingly unacceptable due to environmental concerns and safety. For example, biocides constitute toxicants in the system, and pollution problems are ever present. Planktonic microbes may be efficiently controlled by the biocides; however, the use of biocides has not solved all slime problems in paper or board industry, since microorganisms growing in slime are generally more resistant to biocides than the planktonic microbes. In addition, the efficacy of the toxicants is minimized by the slime itself, since the extracellular polysaccharide matrix embedding the microorganisms hinders penetration of the chemicals. Biocides may induce bacterial sporulation and after the treatment of process waters with biocides, a large number of spores may exist in a final product. 1

DK 2021 00639 A1 There is a need in the paper industry to control slime deposits in an efficient and environmentally friendly way.

SUMMARY OF THE INVENTION The present invention provides a method of preventing a build-up of slime or removing slime from a surface contacted with water from a pulp or paper making process, comprising contacting said water with a DNase. In one embodiment, the method is an efficient and environmentally friendly way to prevent a build-up of slime or remove slime from a surface contacted with water. The treatment of water from a pulp or paper making process by contacting it with a DNase can efficiently prevent a build-up of slime or remove slime from a surface contacted with the water. The treatment can further reduce downtime by avoiding the need for cleaning or breaks in the pulp or paper making process; reduce spots or holes in a final product; reduce spores in a final product, reduce blocking of devices such as filters, wires, or nozzles, or partly or totally replace biocides. The treatment is efficient and environmentally friendly.

The present invention also relates to a method of manufacturing pulp or paper, comprising subjecting water from pulp or paper making process to a DNase. In an embodiment, the method of present invention prevents the build-up of slime or removes slime from a surface contacted with water from a pulp or paper making process. In another embodiment, the method of the present invention controls or reduces odour from a pulp or paper making process.

The present invention further relates to use of a DNase in preventing the build-up of slime or removing slime from a surface contacted with water from a pulp or paper making process.

The present invention further relates to a composition for preventing a build-up of slime or removing slime from a surface contacted with water from a pulp or paper making process, comprising a DNase and an additional enzyme; a DNase and a surfactant, or a DNase and an additional enzyme, and a surfactant.

Proteases and polysaccharide degrading enzymes have been described in the literature for slime control in papermaking. In a recent review on the control of microbiological problems in papermaking, it discloses the use of several enzyme classes (Pratima Bajpai, Pulp and Paper Industry: Microbiological Issues in Papermaking Chapter 8.4, 2015 Elsevier Inc, ISBN: 978-0- 2

DK 2021 00639 A1 12-803409-5). The industrial benchmark in use as an enzymatic green technology for microbial control in papermaking is based on protease enzymes which prevent bacteria from attaching to a surface and thus preventing slime build-up (Martin Hubbe and Scott Rosencrance (eds.), Advances in Papermaking Wet End Chemistry Application Technologies, Chapter 10.3, 2018 TAPPI PRESS, ISBN: 978-1-59510-260-7). Our invention based on the use of a DNase enzyme has a completely different mode of action from the use of a protease and it was found to have a highly superior effect in the control of slime when compared to the commercial benchmark protease. At the same protein dosage, the prevention effect of the DNase is improved by at least 10%, for example, about 10-5000%, preferably 15-3000%, more preferably 20-2000% compared to the one achieved by the best-in-class protease.

DETAILED DESCRIPTION OF THE INVENTION In one aspect, the present invention provides a method of preventing a build-up of slime or removing slime from a surface contacted with water from a pulp or paper making process, comprising contacting said water with a DNase. In one embodiment, the present invention provides a method of preventing a build-up of slime from a surface contacted with water from a pulp or paper making process, comprising contacting said water with a DNase. In another embodiment, the present invention provides a method of removing slime from a surface contacted with water from a pulp or paper making process, comprising contacting said water with a DNase.

Microorganisms such as, e.g., bacterium, mycoplasma (bacteria without a cell wall) and certain fungi, secrete a polymeric conglomeration of biopolymers, generally composed of extracellular nucleic acids, proteins, and polysaccharides, that form a matrix of extracellular polymeric substance (EPS). The EPS matrix embeds the cells causing the cells to adhere to each other as well as to any living (biotic) or non-living (abiotic) surface to form a sessile community of microorganisms referred to as a biofilm, slime layer, or slime, or a deposit of microbial origin. A slime colony can also form on solid substrates submerged in or exposed to an aqueous solution, or form as floating mats on liquid surfaces. Primarily, the microorganisms involved in slime formation are different species of spore-forming and nonspore-forming bacteria, particularly capsulated forms of bacteria which secrete gelatinous substances that envelop or encase the cells. Slime forming microorganisms also include filamentous bacteria, filamentous fungi of the mold type, yeasts, and yeast-like organisms. The pulp or paper making processes contain warm waters (e.g. 45-60 degrees C) that are rich in biodegradable nutrients and have a beneficial pH (e.g. pH 4-9) thus providing a good environment for the growth of microorganisms.

3

DK 2021 00639 A1 By contacting water from a pulp or paper making process with a DNase, the present invention provides an efficient and environmentally friendly way to prevent a build-up of slime or remove slime from a surface contacted with the water. The slime mainly comprises a matrix of extracellular polymeric substance (EPS) and slime forming microorganisms.

According to the present invention, the term “DNase” or “deoxyribonuclease” means a polypeptide with DNase activity that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA. Examples of enzymes exhibiting DNase activity are those covered by enzyme classes EC 3.1.11 to EC 3.1.31, as defined in the recommendations of the Nomenclature Committee of the International Union of Biochemistry — and Molecular Biology (IUBMB). In one aspect, the DNase of the present invention has at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the DNase activity of the DNase having the amino acid sequence of SEQ ID NO: 1, the mature polypeptide of SEQ ID NO: 2, the mature polypeptide of SEQ ID NO: 3, or the mature polypeptide of SEQ ID NO: 4. The DNase used according to the present invention is a mature polypeptide exhibiting DNase activity, which comprises or consists of an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence shown as SEQ ID NO: 1, the mature polypeptide of SEQ ID NO: 2, the mature polypeptide of SEQ ID NO: 3, or the mature polypeptide of SEQ ID NO: 4. The relatedness between two amino acid sequences is described by the parameter “sequence identity”. For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al, 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of > BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the —nobrief option) is used as the percent identity and is calculated as follows: (Identical Residues x 100)/(Length of Alignment — Total Number of Gaps in Alignment). 4

DK 2021 00639 A1 In an embodiment, the amino acid sequence of the DNase is SEQ ID NO: 1, the mature polypeptide of SEQ ID NO: 2, the mature polypeptide of SEQ ID NO: 3, or the mature polypeptide of SEQ ID NO: 4. In another embodiment, the DNase is a fungal DNase, preferably a Bacillus DNase, a Morchella DNase, a Urnula DNase or Neosartorya DNase; more preferably Bacillus cibi DNase, Morchella costata DNase or Neosartorya massa DNase.

In another embodiment, the DNase is a Bacillus cibi DNase, a derivative or a variant thereof.

In another embodiment, the DNase is Morchella costata DNase, a derivative or a variant thereof.

In another embodiment, the DNase is Urnula DNase, a derivative or a variant thereof.

In another embodiment, the DNase is Neosartorya massa DNase, a derivative or a variant thereof.

In yet another embodiment, the DNase is a DNase as disclosed in International patent application no.

WO 2019/081724, which is hereby incorporated by reference.

In an embodiment, the number of amino acid substitutions, deletions and/or insertions introduced into the amino acid sequence of SEQ ID NO: 1, the mature polypeptide of SEQ ID NO: 2, the mature polypeptide of SEQ ID NO: 3, or the mature polypeptide of SEQ ID NO: 4 is upto 20, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; or up to 10, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; or up to 5. The amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H.

Neurath and R.L.

Hill, 1979, In, The Proteins, Academic Press, New York.

Common substitutions are Ala/Ser, Val/lle, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/lle, Leu/Val, Ala/Glu, and Asp/Gly.

Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and 5

DK 2021 00639 A1 Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for DNase activity to identify amino acid residues that are critical to the activity of the molecule.

See also, Hilton et al., 1996, J.

Biol.

Chem. 271: 4699-4708. The active site of the DNase or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids.

See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J.

Mol.

Biol. 224: 899- 904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide.

Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc.

Natl.

Acad.

Sci.

USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al, 1991, Biochemistry 30: 10832-10837; U.S.

Patent No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al, 1988, DNA 7: 127). In a preferred embodiment, the DNase is a DNase variant, which compared to a DNase with SEQ ID NO: 1, comprises two or more substitutions selected from the group consisting of: T11, TIL, T1V, S13Y, T22P, S25P, S27L, S39P, S42G, S42A, S42T, S57W, S57Y, S57F, S59V, S591, S59L, V76L, V76l, Q109R, S116D, S116E, T127V, T1271, T127L, S144P, A147H, S167L, S1671, S167V, G175D and G175E, wherein the positions correspond to the positions of SEQ ID NO: 1 (numbering according to SEQ ID NO: 1) wherein the variant has a sequence identity to the polypeptide shown in SEQ ID NO: 1 of at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% and wherein the variant has DNase activity.

In a more preferred embodiment, the DNase variant is a variant comprising one or more of the substitution sets selected from the group consisting of: T11+S13Y, T11+T22P, T11+S25P, T11+S27L, T11+S39P, T11+S42G, T11+S42A, T11+SA42T, m— T1+S57W, T11+S57Y, T11+S57F, T11+S59V, T11+S59I, T11+S59L, T11+V76L, T11+V76I, T11+Q109R, T11+S116D, T1+S116E, T11+T127V, T11+T1271, T11+T127L, T11+S144P, T11+A147H, T11+S167L, T11+S1671, T11+S167V, T1+G175D, T1+G175E, T1L+S13Y, T1L+T22P, T1L+S25P, T1L+S27L, T1L+S39P, T1L+S42G, T11+S42A, T1L+S42T, T1L+S57W, T1L+S57Y, T1L+S57F, T1L+S59V, T1L+S59I, TIL+S59L, T1L+V76L, T1L+V76I, TIL+Q109R, 6

DK 2021 00639 A1 T1L+S116D, T1L+S116E, T1L+T127V, T1L+T1271, T1L+T127L, T1L+S144P, T1L+A147H, T1L+S167L, T1L+S1671, T1L+S167V, T1L+G175D, T1L+G175E, T1V+S13Y, T1V+T22P, T1V+S25P, T1V+S27L, T1V+S39P, T1V+S42G, T1V+S42A, T1V+S42T, T1V+S57W, T1V+S57Y, T1V+S57F, T1V+S59V, T1V+S59I, T1V+S59L, T1V+V76L, T1V+V76I, T1V+Q109R, T1V+S116D, T1V+S116E, T1V+T127V, T1V+T1271, TIV+T127L, T1V+S144P, T1V+A147H, T1V+S167L, T1V+S1671, T1V+S167V, T1V+G175D, T1V+G175E, S13Y+T22P, S13Y+S25P, S13Y+S27L, S13Y+S39P, S13Y+S42G, S13Y+S42A, S13Y+SA42T, S13Y+S57W, S13Y+S57Y, S13Y+S57F, S13Y+S59V, S13Y+S591, S13Y+S59L, S13Y+V76L, S13Y+V76l, S13Y+Q109R, S13Y+S116D, S13Y+S116E, S13Y+T127V, S13Y+T1271, S13Y+T127L, S13Y+S144P, S13Y+A147H, S13Y+S167L, S13Y+S1671, S13Y+S167V, S13Y+G175D, S13Y+G175E, T22P+S25P, T22P+S27L, T22P+S39P, T22P+S42G, T22P+S42A, T22P+S42T, T22P+S57W, T22P+S57Y, T22P+S57F, T22P+S59V, T22P+S59I, T22P+S59L, T22P+V76L, T22P+V761, T22P+Q109R, T22P+S116D, T22P+S116E, T22P+T127V, T22P+T127I, T22P+T127L, T22P+S144P, T22P+A147H, T22P+S167L, T22P+S1671, T22P+S167V, T22P+G175D, T22P+G175E, S25P+S27L, S25P+S39P, S25P+S42G, S25P+S42A, S25P+SA42T, S25P+S57W, S25P+S57Y, S25P+S57F, S25P+S59V, S25P+S59I, S25P+S59L, S25P+V76L, S25P+V761, S25P+Q109R, S25P+S116D, S25P+S116E, S25P+T127V, S25P+T1271, S25P+T127L, S25P+S144P, S25P+A147H, S25P+S167L, S25P+S167I, S25P+S167V, S25P+G175D, S25P+G175E, S27l+S39P, S27L+S42G, S27L+S42A, > S27L+SA2T, S27L+S57W, S27L+S57Y, S27L+S57F, S27L+S59V, S27L+S59I, S27L+S59L, S271+V76L, S27L+V76I, S27L+Q109R, S271+S116D, S27L+S116E, S27L+T127V, S271+T1271, S27L+T127L, S271+S144P, S27L+A147H, S27L+S167L, S27L+S16T71, S271+S167V, S27L+G175D, S27L+G175E, S39P+S42G, S39P+S42A, S39P+S42T, S39P+S57W, S39P+S57Y, S39P+S57F, S39P+S59V, S39P+S59|, S39P+S59L, S39P+V76L, S39P+V76l, S39P+Q109R, S39P+S116D, S39P+S116E, S39P+T127V, S39P+T1271, S39P+T127L, S39P+S144P, S39P+A147H, S39P+S167L, S39P+S1671, S39P+S167V, S39P+G175D, S39P+G175E, S42G+S57W, S42G+S57Y, S42G+S57F, S42G+S59V, S42G+S591, S42G+S59L, S42G+V76L, S42G+V761, S42G+Q109R, S42G+S116D, S42G+S116E, S42G+T127V, S42G+T1271, S42G+T127L, S42G+S144P, S42G+A147H, >S42G+S167L, S42G+S1671, S42G+S167V, S42G+G175D, S42G+G175E, S42A+S57W, S42A+S57Y, S42A+S57F, S42A+S59V, S42A+S591, S42A+S59L, S42A+V76L, S42A+V76l, S42A+Q109R, S42A+S116D, S42A+S116E, S42A+T127V, S42A+T1271, S42A+T127L, S42A+S144P, S42A+A147H, S42A+S167L, S42A+S1671, S42A+S167V, S42A+G175D, S42A+G175E, S42T+S57W, S42T+S57Y, S42T+S57F, S42T+S59V, S42T+S59|, SA2T+S59L, S42T+V76L, S42T+V76I, S42T+Q109R, S42T+S116D, S42T+S116E, S42T+T127V, 7

DK 2021 00639 A1 S42T+T1271, S42T+T127L, S42T+S144P, S42T+A147H, S42T+S167L, S42T+S1671, S42T+S167V, S42T+G175D, S42T+G175E, S57W+S59V, S57W+S59I, S57W+S59L, S57W+V76L, S57W+V761, S57W+Q109R, S57W+S116D, S57W+S116E, S57W+T127V, S57W+T1271, S57W+T127L, S57W+S144P, S57W+A147H, S57W+S167L, S57W+S1671, S57W+S167V, S57W+G175D, S57W+G175E, S57Y+S59V, S57Y+S591, S57Y+S59L, S57Y+V76L, S57Y+V76I, S57Y+Q109R, S57Y+S116D, S57Y+S116E, S57Y+T127V, S57Y+T1271, S57Y+T127L, S57Y+S144P, S57Y+A147H, S57Y+S167L, S57Y+S167I, S57Y+S167V, S57Y+G175D, S57Y+G175E, S57F+S59V, S57F+S59I, S57F+S59L, S57F+V76L, S57F+V76l, S57F+Q109R, S57F+S116D, S57F+S116E, S57F+T127V, S57F+T1271, S57F+T127L, S57F+S144P, S57F+A147H, S57F+S167L, S57F+S16T1, S57F+S167V, S57F+G175D, S57F+G175E, S59V+V76L, S59V+V76I, S59V+Q109R, S59V+S116D, S59V+S116E, S59V+T127V, S59V+T1271, S59V+T127L, S59V+S144P, S59V+A147H, S59V+S167L, S59V+S1671, S59V+S167V, S59V+G175D, S59V+G175E, S591+V76L, S591+V76I, S59+Q109R, S591+S116D, S59+S116E, S59+T127V, S59+T1271, S59I1+T127L, S59+S144P, S59+A147H, S59+S167L, S59+S1671, S591+S167V, S591+G175D, BS59+G175E, S59L+V76L, S59L+V76I, S59L+Q109R, S59L+S116D, S59L+S116E, S59L+T127V, S591+T1271, S59L+T127L, S59L+S144P, S59L+A147H, S59L+S167L, S591+S1671, S59L+S167V, S59L+G175D, S59L+G175E, V76L+Q109R, V76L+S116D, V76L+S116E, V76L+T127V, V76L+T1271, V76L+T127L, V76L+S144P, > V76L+A147H, V76L+S167L, V76L+S1671, V76L+S167V, V76L+G175D, V76L+G175E, V761+Q109R, V76+S116D, V76+S116E, V761+T127V, V761+T1271, — V76I+T127L, V761+S144P, V76I+A147H, V76+S167L, V76+S1671, V761+S167V, V761+G175D, V761+G175E, Q109R+S116D, Q109R+S116E, Q109R+T127V, Q109R+T1271, Q109R+T127L, Q109R+S144P, Q109R+A147H, Q109R+S167L, Q109R+S1671, Q109R+S167V, Q109R+G175D, Q109R+G175E, S116D+T127V, S116D+T1271, S116D+T127L, S116D+S144P, S116D+A147H, S116D+S167L, S116D+S1671, S116D+S167V, S116D+G175D, S116D+G175E, S116E+T127V, S116E+T1271, S116E+T127L, S116E+S144P, S116E+A147H, S116E+S167L, S116E+S1671, S116E+S167V, S116E+G175D, S116E+G175E, T127V+S144P, T127V+A147H, T127V+S167L, T127V+S1671, T127V+S167V, T127V+G175D, m— T127V+G175E, T1271+S144P, T127I+A147H, T1271+S167L, T1271+S1671, T1271+S167V, T1271+G175D, T1271+G175E, T127L+S144P, T127L+A147H, T127L+S167L, T127L+S1671, T127L+S167V, T127L+G175D, T127L+G175E, S144P+A147H, S144P+S167L, S144P+S1671, S144P+S167V, S144P+G175D, S144P+G175E, A147H+S167L, A147H+S16T1, A147H+S167V, A147H+G175D, A147H+G175E, S167L+G175D, S167L+G175E, —S$1671+G175D, S1671+G175E, S167V+G175D and S167V+G175E.

8

DK 2021 00639 A1 In a further embodiment, the DNase variant is selected from the group consisting of: i.

T1+S13Y+T22P+S25P+S27L+S39P+S42G+S57W+S59V+V76L+S144P+A147H+S167 L+G175D; i.

T1+S13Y+T22P+S27L+S42G+S57W+S59V+V76L+Q109R+S116D+T127V+S144P+A1 47H+S167L+G175D; iii.

T1+S13Y+T22P+S25P+S27L+S39P+S42G+S57W+S59V+V76L+Q109R+S116D+T127 V+S8144P+A147H+S167L+G175D; iv.

T11+S13Y+T22P+S25P+S27L+S39P+S42G+S57W+S59V+V76L+T77Y+Q109R+S116D +T127V+S144P+A147H+S167L+G175D; v.

T11+T22P+D561+S57W+V76L+Q109R+S116D+A147H+G162S+S167L+G175N+N178; vi.

T11+S13Y+T22P+S27L+S39P+S42G+D561+S57W+S59V+V76L+Q109R+S116D+T127 V+S144P+A147H+S167L+G175D; vii.

T11+T22P+S25P+S27L+S42G+D561+S57Y+S59V+V76L+T77Y+Q109R+S116D+T127V +S8144P+A147H+Q166D+S167L+G175D+S181L; viii.

T11+S13Y+T22P+S25P+S27L+S39P+S42G+D561+S57W+S59V+V76L+Q109R+S116D +T127V+S144P+A147H+S167L+G175D; ix.

T11+S13Y+T22P+S25P+S27L+S39P+S42G+D561+S57W+S59V+V76L+Q109R+S116D +T127V+S144P+A147H+Q166D+S167L+G175D; x.

T11+S13Y+T22P+S27R+S39P+S42G+D561+S57W+S59V+V76L+Q109R+S116D+T127 V+S144P+A147H+S167L+G175D; xi.

T11+S13Y+T22P+S27L+S39P+S42G+D561+S57W+S59V+T65L+V76L+Q109R+S116D +T127V+S144P+A147H+S167L+G175D; xii.

T11+S13Y+T22P+S27L+L33K+S39P+S42G+D561+S57W+S59V+T65V+V76L+Q109R+ S116D+T127V+S144P+A147H+S167L+G175D; xiii.

T11+S13Y+T22P+S25P+S27R+S39P+S42G+S57W+S59V+S66W+V76L+T77Y+Q109R +S8116D+T127V+S144P+A147H+S167L+G175D; xiv.

T11+S13Y+T22P+S25P+S27L+L33K+S39P+S42G+S57W+S59V+T65V+V76L+T77Y+Q 109R+S116D+T127V+S144P+A147H+S167L+G175D; xv.

T11+S13Y+T22P+S25P+S27L+L33K+S39P+S42G+S57W+S59V+S66Y+V76L+T77Y+Q 109R+S116D+T127V+S144P+A147H+S167L+G175D; xvi.

T11+S13Y+T22P+S25P+S27L+S39P+S42G+S57W+S59V+T65V+S66Y+V76L+T77Y+Q 109R+S116D+T127V+S144P+A147H+S167L+G175D; xvii. — T11+S13Y+T22P+S27L+S39P+S42G+D56L+S57W+S59V+T65V+V76L+Q109R+S1 16D+T127V+S144P+A147H+G162D+S1671L+G175D; xviii. — T11+S13Y+T22P+S27R+S39P+S42G+D56L+S57W+S59V+T65V+V76L+Q109R+S1 9

DK 2021 00639 A1 16D+T127V+S144P+A147H+S167L+G175D; xix. T11+S13Y+T22P+S27R+S39P+S42G+D56L+S57W+S59V+T65V+V76L+Q109R+S116 D+T127V+S144P+A147H+G162D+S167L+G175D; xx. T11+S13Y+T22P+S27K+S39P+S42G+D561+S57W+S59V+T65V+V76L+S106L+Q109R +S8116D+T127V+S144P+A147H+S167L+G175D; xxi. T11+S13Y+T22P+S27K+S39P+S42G+D561+S57W+S59V+T65V+V76L+Q109R+S116D +T127V+S130A+S144P+A147H+S167L+G175D; xxii. — T11+S13Y+T22P+S27L+S39P+S42G+D56L+S57W+S59V+T65L+V76L+Q109R+S1 16D+T127V+S130A+S144P+A147H+S167L+G175D; and xxiii. — T11+S13Y+T22P+S27L+S39P+S42G+D561+S57W+S59V+T65V+V76L+Q109R+S11 6D+T127V+S144P+A147H+G162D+S167L+G175. In a further preferred embodiment, the DNase variant is selected from the group consisting of: a. T11 +T22P +S57W +V76L +A147H +S167L, b. T1I +T22P +S57W +V76L +A147H +G175D, c. T1l +T22P +S57W +V76L +S167L+G175D, d. T11 +T22P +S57W +A147H +S167L+G175D, e. T1l +T22P +V76L +A147H +S167L+G175D, f. T11 +S57W +V76L +A147H +S167L+G175D, g. T22P +S57W +V76L +A147H +S167L+G175D, and h. T11 +T22P +S57W +V76L +A147H +S167L+G175D. The DNase is added in an amount effective to preventing a build-up of slime or removing slime from a surface contacted with water from a pulp or paper making process. In a preferred embodiment, the DNase is added in an amount of 0.001-1000 mg enzyme protein/L, preferably

0.005 -500 mg enzyme protein/L, more preferably 0.01 mg -100 mg enzyme protein/L, such as,

0.05 mg- 50 mg enzyme protein/L, or 0.1 - 10 mg enzyme protein/L. The DNase treatment may be used to control (i.e., reduce or prevent) build-up of slime or remove slime from a surface contacted with water from a pulp or paper making process in any desired environment. In one embodiment, the surface is a solid substrate submerged in or exposed to an aqueous solution, or forms as floating mats on liquid surfaces. In preferred embodiment, the surface is solid surface, for example, a plastic surface or a metal surface. The solid surface can come from a manufacturing equipment, such as surfaces of the pulpers, headbox, machine frame, foils, suction boxes, white water tanks, clarifiers and pipes. The DNase treatment may be used to control (i.e., reduce or prevent) a build-up of slime or 10

DK 2021 00639 A1 remove slime from a surface contacted with water from a pulp or paper making process. In the present context, the term “water” comprises, but not limited to: 1) cleaning water used to clean a surface in paper-making; 2) process water added as a raw material to the pulp or paper making process; 3) intermediate process water products resulting from any step of the process for manufacturing the paper material; 4) waste water as an output or by-product of the process; 5) water mist in the air, generated by clearing water, process water or waste water at a certain humidity and temperature. In an embodiment, the water is cleaning water, process water, wastewater, and/or water mist in the air. In a particular embodiment, the water is, has been, is being, or is intended for being circulated (re-circulated), i.e., re-used in another step of the process. In a preferred embodiment, the water is process water from recycled tissue production. In a preferred embodiment, the water is process water from liquid packaging board production. In a preferred embodiment, the water is process water from recycled packaging board process. The term “water” in turn means any aqueous medium, solution, suspension, e.g., ordinary tap water, and tap water in admixture with various additives and adjuvants commonly used in pulp or paper making processes. In a particular embodiment the process water has a low content of solid (dry) matter, e.g., below 20%, 18%, 16%, 14%, 12%, 10%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or below 1% dry matter. The water may vary in properties such as pH, conductivity, redox potential and/or ATP. In a preferred embodiment, the water has pH from 4 to 10, conductivity from 100 uS/cm to 12000 uS/cm, redox potential from -500 mV to 1500 mV and cellular ATP from 0.1 ng/ml to 1000 ng/ml. In a more preferred embodiment, the water has pH from 5 to 9, conductivity from 1000 uS/cm to 8000 uS/cm, redox potential from -300 mV to 500 mV and cellular ATP from 1 ng/ml to 500 ng/ml. In the most preferred embodiment, the water has pH from 6.1 to 7.6, conductivity from 1772 uS/cm to 5620 uS/cm, redox potential from -110 mV to 210 mV and cellular ATP from 4.2 ng/ml to 114 ng/ml.

In one embodiment, the pulp or paper making process of the present invention can be carried out separately in a pulp making mill and paper making mill. In a preferred embodiment, the pulp or paper making process is a paper making process which can be carried out in a paper making mill. In another embodiment, the pulp or paper making process is a pulp and paper making process which can be carried out in an integrated paper mill. The process of papermaking starts with the stock preparation, where a suspension of fibers and water is prepared and pumped to the paper machine. This slurry consists of approximately 89.5% water and approximately 0.5% pulp fiber and flows until the “slice” or headbox opening where the fibrous mixture pours onto a traveling wire mesh in the Fourdrinier process, or onto a rotating cylinder in the cylinder. As the wire moves along the machine path, water drains through the mesh while fibers align in the direction of the wire. After the web forms on the wire, the paper machine needs to remove 11

DK 2021 00639 A1 additional water. It starts with vacuum boxes located under the wire which aid in this drainage, then followed by the pressing and drying section where additional dewatering occurs. As the paper enters the press section, it undergoes compression between two rotating rolls to squeeze out more water and then the paper web continues through the steam-heated dryers to lose more moisture. Depending on the paper grade being produced, it will sometimes undergo a sizing or coating process in a second dry-end operation before entering the calendaring stacks as part of the finishing operation. At the end of the paper machine, the paper continues onto a reel for winding to the desired roll diameter. The machine tender cuts the paper at this diameter and immediately starts a new reel. The process is now complete for example in grades of paper used in the manufacture of corrugated paperboard. However, for papers used for other purposes, finishing and converting operations will now occur, typically off-line from the paper machine (Pratima Bajpai, Pulp and Paper Industry: Microbiological Issues in Papermaking, Chapter 2.1, 2015 Elsevier Inc, ISBN: 978-0-12-803409-5).

In one embodiment, fibrous material is turned into pulp and bleached to create one or more layers of board or packaging material, which can be optionally coated for a better surface and/or improved appearance. Board or packaging material is produced on paper machines that can handle higher grammage and several plies.

The temperature and pH for the DNase treatment in the pulp or paper making process is not critical, provided that the temperature and pH is suitable for the enzymatic activity of the DNase.

Generally, the temperature and pH will depend on the system, composition or process which is being treated. Suitable temperature and pH conditions include 5°C to 120°C and pH 1 to 12, however, ambient temperatures and pH conditions are preferred. For paper production processes, the temperature and pH will generally be 15°C to 65°C, for example, 45°C to 60°C and pH 3 to 10, for example, pH 4 to 9.

The treatment time will vary depending on, among other things, the extent of the slime problem and the type and amount of the DNase employed. The DNase may also be used in a preventive manner, such that, the treatment time is continuous or carried out a set point in the process.

In a preferred embodiment, the DNase is used to treat water in a pulp or paper making process for manufacturing paper or packaging material. The term “paper or packaging material” refers to paper or packaging material which can be made out of pulp. In an embodiment, the paper and packaging material is selected from the group consisting of printing and writing paper, tissue and towel, newsprint, carton board, containerboard and packaging papers.

12

DK 2021 00639 A1 The term “pulp” means any pulp which can be used for the production of a paper and packaging material.

Pulp is a lignocellulosic fibrous material prepared by chemically or mechanically separating cellulose fibers from wood, fiber crops or waste paper.

For example, the pulp can be supplied as a virgin pulp, or can be derived from a recycled source, or can be supplied as a combination of a virgin pulp and a recycled pulp.

The pulp may be a wood pulp, a non-wood pulp or a pulp made from waste paper.

A wood pulp may be made from softwood such as pine, redwood, fir, spruce, cedar and hemlock or from hardwood such as maple, alder, birch, hickory, beech, aspen, acacia and eucalyptus.

A non-wood pulp may be made, e.g., from flax, hemp, bagasse, bamboo, cotton or kenaf.

A waste paper pulp may be made by re-pulping waste paper such as newspaper, mixed office waste, computer print-out, white ledger, magazines, milk cartons, paper cups etc.

In other preferred embodiments, the DNase is added in combination (such as, for example, sequentially or simultaneously) with an additional enzyme and/or a surfactant.

Any enzyme having lipase, cutinase, protease, pectinase, laccase, peroxidase, cellulase, glucanase, xylanase, mannanase, lysozyme, amylase, glucoamylase, galactanase, and/or levanase activity can be used as additional enzymes in the present invention.

Below some non- limiting examples are listed of such additional enzymes.

The enzymes written in capitals are commercial enzymes available from Novozymes A/S, Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark.

The activity of any of those additional enzymes can be analyzed using any method known in the art for the enzyme in question, including the methods mentioned in the references cited.

An example of a lipase is the RESINASE A2X lipase.

Examples of cutinases are those derived from Humicola insolens (US 5,827,719); from a strain of Fusarium, e.g.

F. roseum culmorum, or particularly F. solani pisi (WO 90/09446; WO > 94/14964, WO 94/03578). The cutinase may also be derived from a strain of Rhizoctonia, e.g.

R. solani, or a strain of Alternaria, e.g.

A. brassicicola (WO 94/03578), or variants thereof such as those described in WO 00/34450, or WO 01/92502. Examples of proteases are the ALCALASE, ESPERASE, SAVINASE, NEUTRASE and DURAZYM proteases.

Other proteases are derived from Nocardiopsis, Aspergillus, Rhizopus, Bacillus alcalophilus, B. cereus, B. natto, B. vulgatus, B. mycoide, and subtilisins from Bacillus, especially proteases from the species Nocardiopsis sp. and Nocardiopsis dassonvillei such as 13

DK 2021 00639 A1 those disclosed in WO 88/03947, and mutants thereof, e.g. those disclosed in WO 91/00345 and EP 415296. Specific examples of pectinase that can be used are pectinase AEI, Pectinex 3X, Pectinex 5X and Ultrazyme 100. Examples of peroxidases and laccases are disclosed in EP 730641; WO 01/98469; EP 719337; EP 765394: EP 767836; EP 763115; and EP 788547. Examples of cellulases are disclosed in co-pending application US application US 60/941,251, which is hereby incorporated by reference.

In an embodiment the cellulase preparation also comprises a cellulase enzymes preparation, preferably the one derived from Trichoderma reesei.

Examples of endoglucanases are the NOVOZYM 613, 342, and 476, and NOVOZYM 51081 enzyme products.

An example of a xylanase is the PULPZYME HC hemicellulase.

Examples of mannanases are the Trichoderma reesei endo-beta-mannanases described in — Ståhlbrand et al, J.

Biotechnol. 29 (1993), 229-242. Examples of amylases are the BAN, AQUAZYM, TERMAMYL, and AQUAZYM Ultra amylases.

An Example of glucoamylase is SPIRIZYME PLUS.

Examples of galactanase are from Aspergillus, Humicola, Meripilus, Myceliophthora, or Thermomyces. > Examples of levanases are from Rhodotorula sp.

Surfactants can in one embodiment include poly(alkylene glycol)-based surfactants, ethoxylated dialkylphenols, ethoxylated dialkylphenols, ethoxylated alcohols and/or silicone based surfactants.

Examples of poly(alkylene glycol)-based surfactant are poly(ethylene glycol) alkyl ester, poly(ethylene glycol) alkyl ether, ethylene oxide/propylene oxide homo- and copolymers, or poly(ethylene oxide- co-propylene oxide) alkyl esters or ethers.

Other examples include ethoxylated derivatives of primary alcohols, such as dodecanol, secondary alcohois, 14

DK 2021 00639 A1 poly[propylene oxide], derivatives thereof, tridecylalcohol ethoxylated phosphate ester, and the like.

Specific presently preferred anionic surfactant materials useful in the practice of the invention comprise sodium alpha-sulfo methyl laurate, (which may include some alpha-sulfo ethyl laurate) for example as commercially available under the trade name ALPHA-STEP™-ML40; sodium xylene sulfonate, for example as commercially available under the trade name STEPANATE™- X; triethanolammonium lauryl sulfate, for example as commercially available under the trade name STEPANOL'M-WAT; diosodium lauryl sulfosuccinate, for example as commercially available under the trade name STEPAN™-Mild SL3; further blends of various anionic surfactants may also be utilized, for example a 50%-50% or a 25%-75% blend of the aforesaid ALPHA-STEP™ and STEPANATE™ materials, or a 20%-80% blend of the aforesaid ALPHA- STEP™ and STEPANOL™ materials (All of the aforesaid commercially available materials may be obtained from Stepan Company, Northfield, III). Specific presently preferred nonionic surfactant materials useful in the practice of the invention comprise cocodiethanolamide, such as commercially available under trade name NINOL™- 11CM; alkyl polyoxyalkylene glycol ethers, such as relatively high molecular weight butyl ethylenoxide-propylenoxide block copolymers commercially available under the trade name TOXIMUL™-8320 from the Stepan Company. Additional alkyl polyoxyalkylene glycol ethers may be selected, for example, as disclosed in U.S. Pat. No. 3,078,315. Blends of the various nonionic surfactants may also be utilized, for example a 50%-50% or a 25%-75% blend of the aforesaid NINOL™ and TOXIMUL™ materials. Specific presently preferred anionic/nonionic surfactant blends useful in the practice of the invention include various mixtures of the above materials, for example a 50%-50% blends of the aforesaid ALPHA-STEP™ and NINOL™ materials or a 25%-75% blend of the aforesaid STEPANATE™ and TOXIMUL™ materials. Preferably, the various anionic, nonionic and anionic/nonionic surfactant blends utilized in the practice of the invention have a solids or actives content up to about 100% by weight and preferably have an active content ranging from about 10% to about 80%. Of course, other blends or other solids (active) content may also be utilized and these anionic surfactants, nonionic surfactants, and mixtures thereof may also be utilized with known pulping chemicals such as, for example, anthraquinone and derivatives thereof and/or other typical paper chemicals, such as caustics, defoamers and the like. 15

DK 2021 00639 A1 The method of the present invention is an efficient and environmentally friendly way to prevent a build-up of slime or remove slime from a surface contacted with water. In a preferred embodiment, the method of the present invention can further reduce downtime by avoiding the need of cleaning or breaks in the pulp or paper making process; reduce spots or holes in a final product; reduce spores in a final product; or reduce blocking of devices such as filters or wires or nozzles, or partly or totally replace biocides. In another preferred embodiment, the method of the present invention can reduce downtime by avoiding the need of cleaning or breaks in the pulp or paper making process. Cleaning stops or breaks and the corresponding downtime are the most common runnability problems in a pulp or paper making mill. By reducing cleaning time and the amount of breaks the method of the present invention will increase production. In another preferred embodiment, the method of the present invention can reduce spots or holes in a final product. Quality of paper or paperboard is affected by sheet defects from microbiological deposition. By controlling the slime, the method of the present invention effectively reduces spots or holes in a final product. In another preferred embodiment, the method of the present invention can reduce blocking of devices such as filters or wires or nozzles. Slime can block devices such as filters or wires or nozzles. By controlling slime, the method of the present invention effectively reduces blocking of devices such as filter or wires or nozzles. In another preferred embodiment, the method of the present invention allows a partial or total reduction on the use of conventional biocides in use. The method of present invention provides a greener alternative to toxic biocides which are needed by the pulp and paper industry.

It was found that the method of the present invention has a highly superior effect in the control of slime when compared to the commercial benchmark protease. At the same protein dosage, the prevention effect of the DNase is improved by about 10-5000%, preferably 15-3000%, more preferably 20-2000% compared to the one achieved by the best-in-class protease.

In another aspect, the present invention relates to a method of preventing a build-up of slime or removing slime from a surface contacted with water from a pulp or paper making process, comprising the steps of (a) preparing a composition comprising DNase; and (b) adding the composition to the water from a pulp or paper making process.

In another aspect, the present invention provides a method of manufacturing pulp or paper, comprising subjecting water from pulp or paper manufacturing process to a DNase. In an embodiment, the method of present invention prevents the build-up of slime or removes slime from a surface contacted with water from a pulp or paper making process. In another 16

DK 2021 00639 A1 embodiment, the method of the present invention reduces odour from a pulp or paper making process.

In another aspect, the present invention provides use of DNase in preventing the build-up of slime or removing slime from a surface contacted with water from a pulp or paper manufacturing process.

In a preferred embodiment, the water is cleaning water, process water, wastewater, and/or water mist in the air. In another preferred embodiment, the DNase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1, the mature polypeptide of SEQ ID NO: 2, the mature polypeptide of SEQ ID NO: 3, or the mature polypeptide of SEQ ID NO: 4.

In another aspect, the present invention relates to a composition for preventing a build-up of slime or removing slime from a surface contacted with water from a pulp or paper making process, comprising a DNase and an additional enzyme; a DNase and a surfactant; or a DNase, an additional enzyme and a surfactant.

Any enzyme having lipase, cutinase, protease, pectinase, laccase, peroxidase, cellulase, glucanase, xylanase, mannanase, lysozyme, amylase, glucoamylase, galactanase, and/or levanase activities can be used as additional enzymes in the composition of the invention.

Various anionic, nonionic and anionic/nonionic surfactant can be used as the surfactant in the composition of the invention.

The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description.

Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.

17

DK 2021 00639 A1

EXAMPLES Materials and methods Chemicals used as buffers and substrates were commercial products of at least reagent grade. The process waters from the industrial papermaking process were sampled in the water circulation loop of the paper machine. They were stored in a refrigerated room at ca. 5°C and used as described in the examples. Specific enzymes used in the examples: DNase-1 DNase variant, | DNase variant of SEQ ID NO: 1 herein with prepared according to | substitutions T1l + S13Y + T22P + S27L + WO 2019/081724 L33K + S39P + S42G + D561 + S57W + S59V + T65V + V76L + Q109R + S116D + T127V + S144P + A147H + S167L + G175D DNase-2 DNase derived from | DNase shown in SEQ ID NO: 2 herein Morchella costata, prepared according to SEQ ID NO: 11 of WO2018177203 DNase-3 DNase derived from | DNase shown in SEQ ID NO: 3 herein Urnula sp-56769, prepared according to SEQ ID NO: 5 of WO2018177203 DNase-4 DNase from | DNase shown in SEQ ID NO: 4 herein Neosartorya massa SEQ ID NO: 184 of WO2017059802 Protease a Bacillus — clausii | SEQ ID NO: 5 herein protease, prepared according to SEQ ID NO: 1 in WO 2011/036263 18

DK 2021 00639 A1 Process water samples used in the examples: Process Conductivity Redox potential | Cellular ATP ” Origin pH øn fm Recycled PW1 packaging board 3120 -40 229 production Recycled PW2 packaging board | 7.2 4460 517 production 4 Cellular ATP, Adenosine Triphosphate, was measured with LuminUltra test kit QuenchGone21 Industrial (QG211TM), EXAMPLE 1 Measurement of slime prevention effect by DNase using process water from recycled packaging board process A sample of process water, PW1, from the paper machine water loop from an industrial production of recycled packaging board was used as microbiol inoculum for the slime cultivation experiments in a micro-titer plate (MTP; 96 wells; Thermo Scientific Nunc Edge microwell 96F well plate, clear, with lid, Sterile). This process water was mixed with a buffer (800 mM MES pH

6.8) in 85:15 volume proportion, and 130 ul. was added to each MTP well followed by the addition of 20 pL of diluted enzyme or sterilized RO water (control — without enzyme). The MTP plate was incubated at 40°C for 96 hours in an incubator (Heraeus B 6120). Each column of the MTP plate corresponds to a different treatment (control vs. enzyme) done in six wells. The enzymes were diluted to target concentration in the final volume (150 ul) in 20 mM sterilized RO water. After the incubation time, the solution was discarded from the MTP plates and the wells were gently washed with 300 ul of 0.9% NaCl solution in one step. After discarding the washing solution the slime was fixated at 60°C for 30 min in an benchtop orbital shaker (Thermo Scientific, MaxQ 4450) and was allowed to cool before 150 ul of 0.095% crystal violet (CAS No. 548-62-9) solution was added to the wells and left for 15 mins to stain the slime that was formed. The crystal violet solution was then discarded and 300 pL of 0.9% NaCl solution was gently added to the wells in two consecutive steps while discarding the washing solution after each washing step. Finally, 150 ul of 40% acetic acid was added and let it to react for 20 min. 19

DK 2021 00639 A1 The amount of color released from the slime was measured by the Absorbance (ABS) at 600 nm in a spectrophotometer (SpectraMax plus 384) and was used to quantify the amount of slime that was produced on the plastic surface. Average of 6 ABS measurements of all samples (outliers excluded according to the Median Absolute Deviation method) was used to calculate the resulting % of slime reduction of each enzyme treatment in relation to the control according to the below formula. The Blank was measured as being the ABS of nutrient medium without process water. If more than one control was present in the MTP (i.e. more than one column for the same sample), the average of the corresponding number of wells was calculated. . . i Control ” stank) - (AB rrestment = ABS + . Slime reduction (36) = ABS (pam gen ABSpiank). » 100% It is seen in Table 1 that the DNase-1 achieves the best slime prevention effect ranging from 35% to 76% versus the commercial benchmark protease ranging from 0 to 59% with the same enzyme protein dosage range applied. A clear dosage response is observed for both treatments, where the DNase-1 outperforms the commercial benchmark protease at all enzyme protein concentrations, showing improvements versus the protease from 1149% to 30% depending on the actual enzyme protein dosage.

Table 1 Treatment Enzyme ABS at | Slime Relative improvement in protein (EP) | 600 nm | reduction slime reduction by DNase dosage (mg versus protease at same Coa PP føl LA osm Jo |- Poe [move wm Foe | æjfm | Poems [® pose ox [- Poems (9 øje |- Oe |B Jose jen | (æt j® Jem {me {me

DK 2021 00639 A1 EXAMPLE 2 Measurement of slime prevention effect by DNase using process water from recycled packaging board process A sample of process water, PW1, from the paper machine water loop from an industrial production of recycled packaging board was used as microbiol inoculum for the slime cultivation experiments in a micro-titer plate as described in Example 1. The effect of alternative DNases was tested.

Incubation, measurement of absorbance and calculation of slime reduction was performed as — described in Example 1. Three different DNases had slime prevention properties as seen in Table 2. DNases-2, DNase-3 and DNases-4 had a slightly lower performance than the protease.

Table 2 Treatment Enzyme protein | ABS at 600 nm Slime reduction (EP) dosage (mg EP/L)

21

Claims (16)

DK 2021 00639 A1 CLAIMS

1. A method of preventing a build-up of slime or removing slime from a surface contacted with water from a pulp or paper making process, comprising contacting said water with a DNase.

2. The method according to claim 1, wherein the DNase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1, the mature polypeptide of SEQ ID NO: 2, the mature polypeptide of SEQ ID NO: 3, or the mature polypeptide of SEQ ID NO: 4.

3. The method according to claim 1 or 2, wherein the DNase is a DNase variant, which compared to a DNase with SEQ ID NO: 1, comprises two or more substitutions selected from the group consisting of: T11, TIL, T1V, S13Y, T22P, S25P, S27L, S39P, S42G, S42A, S42T, S57W, S57Y, S57F, S59V, S591, S59L, V76L, V76I, Q109R, S116D, S116E, T127V, T1271, T127L, S144P, A147H, S167L, S1671, S167V, G175D and G175E, wherein the positions correspond to the positions of SEQ ID NO: 1 (numbering according to SEQ ID NO: 1) wherein the variant has a sequence identity to the polypeptide shown in SEQ ID NO: 1 of at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% and wherein the variant has DNase activity.

4. The method according to claim 3, wherein the DNase variant is selected from the group consisting of: I. — T11+S13Y+T22P+S25P+S27L+S39P+S42G+S57W+S59V+V76L+S144P+A147H +S8167L+G175D;

1. — T11+S13Y+T22P+S27L+S42G+S57W+S59V+V76L+Q109R+S116D+T127V+S14 4P+A147H+S167L+G175D; MM. — T11+S13Y+T22P+S25P+S27L+S39P+S42G+S57W+S59V+V76L+Q109R+S116D +T127V+S144P+A147H+S167L+G175D; IV. — T11+S13Y+T22P+S25P+S27L+S39P+S42G+S57W+S59V+V76L+T77Y+Q109R+ S116D+T127V+S144P+A147H+S167L+G175D; 22

DK 2021 00639 A1 V. — T11+T22P+D561+S57W+V76L+Q109R+S116D+A147H+G162S8+S1671+G175N+ N178; VI. — T11+S813Y+T22P+S27L+S39P+S42G+D561+S57W+S59V+V76L+Q109R+S116D +T127V+S144P+A147H+S1671+G175D; VII. — T11+T22P+S25P+S271+S42G+D561+S57Y+S59V+V76L+T77Y+Q109R+S116D+ T127V+S144P+A147H+Q166D+S1671+G175D+S181L; VII. — T11+S13Y+T22P+S25P+S27L+S39P+S42G+D56/+S57W+S59V+V76L+Q109R+ S116D+T127V+S144P+A147H+S167L+G175D; IK. — T11+S13Y+T22P+S25P+S27L+S39P+S42G+D561+S57W+S59V+V76L+Q109R+ S$116D+T127V+S144P+A147H+Q166D+S167L+G175D; X. — T11+S13Y+T22P+S27R+S39P+S42G+D56+S57W+S59V+V76L+Q109R+S116D +T127V+S144P+A147H+S1671+G175D; Xl. — T11+S13Y+T22P+S27L+S39P+S42G+D56+S57W+S59V+T65L+V76L+Q109R+ S116D+T127V+S144P+A147H+S167L+G175D; XI. — T11+S13Y+T22P+S27L+L33K+S39P+S42G+D56/+S57W+S59V+T65V+V76L+Q1 09R+S116D+T127V+S144P+A147H+S167L+G175D; XII. — T11+S13Y+T22P+S25P+S27R+S39P+S42G+S57W+S59V+S66W+V76L+T77Y+ Q109R+S116D+T127V+S144P+A147H+S167L+G175D; XIV. — T11+S13Y+T22P+S25P+S27L+L33K+S39P+S42G+S57W+S59V+T65V+V76L+T 77Y+Q109R+S116D+T127V+S144P+A147H+S167L+G175D; XV. — T11+S13Y+T22P+S25P+S27L+L33K+S39P+S842G+S57W+S59V+S66Y+V76L+T 77Y+Q109R+S116D+T127V+S144P+A147H+S167L+G175D; XVI. — T11+S13Y+T22P+S25P+S271+S39P+S42G+S57W+S59V+T65V+S66Y+V76L+T 77Y+Q109R+S116D+T127V+S144P+A147H+S167L+G175D; XVII. — T11+S13Y+T22P+S27L+S39P+S42G+D56L+S57W+S59V+T65V+V76L+Q109R+ S$116D+T127V+S144P+A147H+G162D+S167L+G175D; XVII. — T11+S13Y+T22P+S27R+S39P+S42G+D56L+S57W+S59V+T65V+V76L+Q109R+ S116D+T127V+S144P+A147H+S167L+G175D; XIX. — T11+S13Y+T22P+S27R+S39P+S42G+D56L+S57W+S59V+T65V+V76L+Q109R+ S$116D+T127V+S144P+A147H+G162D+S167L+G175D; XX. — T11+S13Y+T22P+S27K+S39P+S42G+D561+S57W+S59V+T65V+V76L+S106L+ Q109R+S116D+T127V+S144P+A147H+S167L+G175D; XXI. — T11+S13Y+T22P+S27K+S39P+S42G+D561+S57W+S59V+T65V+V76L+Q109R+ S$116D+T127V+S130A+S144P+A147H+S167L+G175D; XXII. — T11+S13Y+T22P+S27L+S39P+S42G+D56L+S57W+S59V+T65L+V76L+Q109R+ 23

DK 2021 00639 A1 S116D+T127V+S130A+S144P+A147H+S167L+G175D; and XXII. — T11+S813Y+T22P+S27L+S39P+S42G+D561+S57W+S59V+T65V+V76L+Q109R+ S116D+T127V+S144P+A147H+G162D+S167L+G175.

5. The method according to any of claims 1-4, wherein the DNase is added in an amount of

0.001-1000 mg enzyme protein/L, preferably 0.005 -500 mg enzyme protein/L, more preferably 0.01 mg -100 mg enzyme protein/L, such as, 0.05 mg - 50 mg enzyme protein/L, or 0.1 - 10 mg enzyme protein/L.

6. The method according to any of claims 1-5, wherein the water is cleaning water, process water, wastewater, and/or water mist in the air; preferably, the water has pH from 4 to 10, conductivity from 100 uS/cm to 12000 uS/cm, redox potential from -500 mV to 1500 mV and cellular ATP from 0.1 ng/ml to 1000 ng/ml; more preferably, the water has pH from 5 to 9, conductivity from 1000 uS/cm to 8000 uS/cm, redox potential from -300 mV to 500 mV and cellular ATP from 1 ng/ml to 500 ng/ml; most preferably, the water has pH from 6.1 to 7.6, conductivity from 1772 uS/cm to 5620 uS/cm, redox potential from -110 mV to 210 mV and cellular ATP from 4.2 ng/ml to 114 ng/ml.

7. The method according to any of claims 1-6, wherein the surface is a plastic surface or a metal surface.

8. The method according to any of claims 1-7, wherein the surface is the surface from a manufacturing equipment, such as surfaces of the pulpers, headbox, machine frame, foils, suction boxes, white water tanks, clarifiers and pipes.

9. The method according to any of claims 1-8, wherein the pulp or paper making process is a process for manufacturing paper or packaging material, preferably the paper or packaging material is selected from the group consisting of printing and writing paper, tissue and towel, newsprint, carton board, containerboard and packaging papers.

10. The method according to any of claims 1-9, further comprising contacting said water with lipase, cutinase, protease, pectinase, laccase, peroxidase, cellulase, glucanase, xylanase, mannanase, lysozyme, amylase, glucoamylase, galactanase, and/or levanase.

11. The method of any of claims 1-11, wherein the method is an efficient and 24

DK 2021 00639 A1 environmentally friendly way to prevent a build-up of slime or remove slime from a surface contacted with water, preferably the method reduces downtime by avoiding the need for cleaning or breaks in the pulp or paper making process; reduces spots or holes in a final product; reduces blocking of filters, wires or nozzles, or partly or totally replaces biocides.

12. A method of preventing a build-up of slime or removing slime from a surface contacted with water from a pulp or paper making process, comprising the steps of (a) preparing a composition comprising DNase; and (b) adding the composition to the water from a pulp or paper making process.

13. A method of manufacturing pulp or paper, comprising subjecting water from pulp or paper making process to a DNase.

14. Use of DNase in preventing the build-up of slime or removing slime from a surface contacted with water from a pulp or paper making process.

15. The use according to claim 15, wherein the DNase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1, the mature polypeptide of SEQ ID NO: 2, the mature polypeptide of SEQ ID NO: 3, or the mature polypeptide of SEQ ID NO: 4.

16. A composition for preventing a build-up of slime or removing slime from a surface contacted with water from a pulp or paper making process, comprising DNase and an additional enzyme; DNase and a surfactant; or DNase, an additional enzyme and a surfactant.

DK 2021 00639 A1

SEQUENCE LISTING <110> Novozymes A/S <120> METHOD FOR CONTROLLING SLIME IN A PULP OR PAPER MAKING PROCESS <130> 15325-DK-NP <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 182 <212> PRT <213> Bacillus cibi <400> 1 Thr Pro Pro Gly Thr Pro Ser Lys Ser Ala Ala Gln Ser Gln Leu Asn 1510 15 Ala Leu Thr Val Lys Thr Glu Gly Ser Met Ser Gly Tyr Ser Arg Asp Leu Phe Pro His Trp Ile Ser Gln Gly Ser Gly Cys Asp Thr Arg Gln 40 45 Val Val Leu Lys Arg Asp Ala Asp Ser Tyr Ser Gly Asn Cys Pro Val 50 55 60 Thr Ser Gly Ser Trp Tyr Ser Tyr Tyr Asp Gly Val Thr Phe Thr Asn 65 70 75 80 Pro Ser Asp Leu Asp Ile Asp His Ile Val Pro Leu Ala Glu Ala Trp 85 90 95 Arg Ser Gly Ala Ser Ser Trp Thr Thr Ser Lys Arg Gln Asp Phe Ala 190 105 110 Asn Asp Leu Ser Gly Pro Gln Leu Ile Ala Val Ser Ala Ser Thr Asn 115 120 125 Arg Ser Lys Gly Asp Gln Asp Pro Ser Thr Trp Gln Pro Pro Arg Ser 130 135 140 Gly Ala Ala Cys Gly Tyr Ser Lys Trp Trp Ile Ser Thr Lys Tyr Lys 145 150 155 160 Trp Gly Leu Ser Leu Gln Ser Ser Glu Lys Thr Ala Leu Gln Gly Met 165 170 175 Leu Asn Ser Cys Ser Tyr 180 <210> 2 <211> 203 <212> PRT <213> Morchella costata <400> 2 Met Lys Leu Thr Ala Val Ala Leu Phe Phe Thr Thr Ala Leu Ala Ala 1510 15 Pro Thr Leu Glu Lys Arg Thr Pro Pro Asn Ile Pro Thr Ala Ala Ser 20 25 30 Ala Asn Thr Met Leu Ala Ala Leu Thr Thr Arg Thr Thr Asp Ala Thr 35 40 45 Gly Tyr Ser Arg Asp Leu Phe Pro His Trp Ile Thr Gln Ser Gly Ser 50 55 60 Cys Asn Thr Arg Glu Val Val Leu Ala Arg Asp Gly Ser Asn Val Val 65 70 75 80 Gln Ala Ser Asp Cys Ser Ala Ser Ser Gly Thr Trp Phe Ser Pro Tyr 85 90 95 Asp Gly Ala Thr Trp Thr Ala Ala Ser Asp Leu Asp Ile Asp His Val 190 105 110 Val Pro Leu Ser Asp Ala Trp Lys Ser Gly Ala Asn Thr Trp Thr Thr 115 120 125 Ala Gly Arg Gln Ala Phe Ala Asn Asp Leu Thr Asn Pro Gln Leu Ile 130 135 140 Ala Val Thr Asp Asn Val Asn Gln Ala Lys Gly Asp Lys Ser Pro Asp 145 150 155 160 Ala Trp Lys Pro Pro Leu Thr Ser Tyr Tyr Cys Thr Tyr Ala Arg Met 165 170 175 Trp Val Lys Val Lys Ser Val Tyr Ser Leu Ser Val Thr Ser Ala Glu 180 185 190

DK 2021 00639 A1 Arg Ser Ala Leu Thr Ser Met Leu Asn Thr Cys 195 200 <210> 3 <211> 213 <212> PRT <213> Urnula sp <400> 3 Met Lys Phe Ser Thr Val Leu Pro Leu Val Phe Thr Leu Val Ala Gly 1510 15 Ala Pro Ala Pro Thr Pro Ile Val Asp Gln Ala Ala Ile Glu Lys Arg

Leu Pro Thr Gly Ile Pro Thr Ala Ala Thr Ala Lys Thr Leu Leu Ala 40 45 Gly Leu Gly Thr Arg Thr Thr Asp Ala Thr Gly Tyr Asp Arg Asp Leu 50 55 60 Phe Pro His Trp Ile Thr Ile Ser Gly Asn Cys Asn Thr Arg Glu Thr 65 70 75 80 Val Leu Asn Arg Asp Gly Thr Asn Leu Asp Ile Gly Thr Asp Cys Tyr 85 90 95 Pro Asp Ser Gly Thr Trp Val Ser Pro Tyr Asp Gly Ala Thr Trp Thr 190 105 110 Ala Ala Ser Asp Val Asp Ile Asp His Val Val Pro Leu Ser Glu Ala 115 120 125 Trp Lys Ala Gly Ala Asn Ala Trp Thr Thr Ala Gln Arg Gln Ala Phe 130 135 140 Ala Asn Asp Leu Thr Asn Pro Gln Leu Val Ala Val Thr Asp Asn Val 145 150 155 160 Asn Ser Ala Lys Gly Asp Lys Thr Pro Asp Leu Trp Lys Pro Pro Leu 165 170 175 Thr Ser Phe His Cys Thr Tyr Ala Arg Met Tyr Val Lys Val Lys Ser 180 185 190 Val Tyr Ser Leu Thr Val Lys Ser Ala Glu Arg Thr Ala Leu Thr Ser 195 200 205 Met Leu Ala Thr Cys 210 <210> 4 <211> 207 <212> PRT <213> Neosartorya massa <400> 4 Met Thr Arg Leu Leu Leu Ala Ala Leu Leu Gly Thr Ser Leu Val Thr 1510 15 Ala Ile Pro Ala Pro Val Ala Leu Pro Thr Pro Pro Gly Ile Pro Ser 20 25 30 Ala Ala Thr Ala Glu Ser Glu Leu Ala Ala Leu Thr Val Ala Ala Gin 35 40 45 Gly Ser Ser Ser Gly Tyr Ser Arg Asp Leu Phe Pro His Trp Ile Ser 50 55 60 Gln Gly Gly Ser Cys Asn Thr Arg Glu Val Val Leu Ala Arg Asp Gly 65 70 75 80 Ser Gly Val Val Lys Asp Ser Asn Cys Tyr Pro Thr Ser Gly Ser Trp 85 90 95 Tyr Ser Pro Tyr Asp Gly Ala Thr Trp Thr Gln Ala Ser Asp Val Asp 190 105 110 Ile Asp His Val Val Pro Leu Ala Asn Ala Trp Arg Ser Gly Ala Ser 115 120 125 Lys Trp Thr Thr Ser Gln Arg Gln Ala Phe Ala Asn Asp Leu Thr Asn 130 135 140 Pro Gln Leu Met Ala Val Thr Asp Asn Val Asn Gln Ala Lys Gly Asp 145 150 155 160 Asp Gly Pro Glu Ala Trp Lys Pro Pro Leu Thr Ser Tyr Tyr Cys Thr 165 170 175 Tyr Ala Lys Met Trp Val Arg Val Lys Tyr Val Tyr Asp Leu Thr Ile 180 185 190

DK 2021 00639 A1 Thr Ser Ala Glu Lys Ser Ala Leu Val Ser Met Leu Asp Thr Cys 195 200 205 <210> 5 <211> 269 <212> PRT <213> Bacillus clausii <400> 5 Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala 1510 15 His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp

Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser 40 45 Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr 50 55 60 His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu 65 70 75 80 Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala 85 90 95 Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala 100 105 110 Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser 115 120 125 Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly 130 135 140 Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser 145 150 155 160 Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln 165 170 175 Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile 180 185 190 Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr Tyr 195 200 205 Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala 210 215 220 Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile 225 230 235 240 Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu 245 250 255 Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg 260 265