WO2001023534A1 - A method for treating biofilm and other slime products with endo-beta-1,2-galactanase - Google Patents

Abstract

The invention describes a method for degrading polysaccharides by using an enzyme preparation comprising endo-β-1,2-galactanase activity. In the process industry and in the paper machine environment in particular, microbes form biofilms on surfaces, consisting of microbes, fibres, and polysaccharide that protects the microbe cells, increases the viscosity of the biofilm, and protects the biofilm against drying. By treating biofilms and other slime deposits in the process industry with the enzyme preparation according to the invention, the problems caused by biofilms to the process industry can be reduced. Endo-β-1,2-galactanase can also be used in the preparation of various mono- or oligosaccharides.

Description

A method for treating biofilm and other slime products with endo-Beta-1,2 -galactanase

The object of the present invention is a method according to the preamble of claim 1 for degrading microbial polysaccharides.

The invention also relates to a method according to the preamble of claim 11 for manufacturing monosaccharides or oligosaccharides.

Another object of the invention is a method according to the preamble of claim 16 for treating slime deposits containing microbial polysaccharides.

Another object of the invention is an enzyme preparation according to the preamble of claim 31.

The formation of deposits or biofilms caused by microbes on various surface materials is a common problem in the processing industry. The common tendency of bacteria to grow in biofilms, attached to the surfaces, leads to the formation of slime deposits in a paper machine environment in particular. As biofilms enable the growth of microbes in oligo- trophic stream flows, paper machines offer excellent conditions for the growth of microbes.

In addition to wood fibre and inorganic material, biofilms generally consist of mixed populations of microbes, polymers produced by microbes. Microbial polysaccharides constitute an essential component of biofilms. The polysaccharides produced by the microbes contained in the biofilms bind the microbe cells to the surfaces and to the insoluble substances precipitated on the biofilm, and a matrix is formed, consisting of microbes, precipitated ingredients (fibres, among others) and polysaccharides, protecting the microbe cells, stabilizing the biofilm, increasing its viscosity, and protecting the biofilm against drying. Fibrous carbohydrates often form the main fraction of the carbo- hydrates of the biofilm. The increased amounts of sugars typical of the heteropoly- saccharides of the bacteria indicate the presence of bacterial polysaccharides in the slime deposits.

Bacteria are continuously carried to the paper machine environment by raw material (water, fibres, especially mechanical pulp and recycled fibre) and the chemicals used in paper manufacture. The paper machine environment is convenient for the growth of bacteria because of its suitable temperature (30-50°C), its pH (4-10), and because there are nutrients present, which flow into the paper machine along with pulp and the paper chemicals. This is why there are always microbes in paper machines. According to the research by Vaisanen et al. (1998), the most common microbes at the wet end of the paper machine included Bacillus coagulans and other Bacillus species, Burkholderia cepacia, Ralstonia pickettii. The paper chemicals contained the Aureobacterium, B. cereus, B. licheniformis, B. sphaericus, Bordetella, Hydrogenophaga, Klebsiella pneumoniae, Pantoea agglomer- ans, Pseudomonas stuzeri, Staphylococcus species. The most common bacteria in the pink slimes, which accumulated in the area of the wire and the printing sector, included the Deinococcus, Aureobacterium and Brevibacterium species. It was discovered that 91 of the 131 strains that were examined degraded raw material in papermaking. Coloured slimes were formed by the strains of the Deinococcus, Acinetobacter, and Methylobacterium (pink), Aureobacterium, Pantoea and Ralstonia (yellowish) and Microbulbifer genera (brown).

In the process industry, biofilms cause various kinds of disadvantages, e.g., a decrease in flow rates, blocking of tube systems, corrosion, and an increase in the consumption of energy. In paper machines, microbial deposits can also cause holes in the paper, breaks in the paper web, or it can reduce the removal of water. Reducing the use of water in paper manufacture has increased the nutrient contents of the circulation water and, thus, the prerequisites for the formation of biofilm have increased. The bacteria of the biofilms are more resistant to the anti-microbial substances generally used in paper machines than free- living cells. The slime deposits act as continuous sources of infections, transferring contaminating organisms to the circulation water and even to the final product. Furthermore, many microorganisms found in biofilms decompose paper materials, such as cellulose fibres, starch, casein, and resin adhesives or they can cause deterioration of the product quality by generating odours, tastes, undesirable colouration, and microbiological contamination. Attempts have been made to solve the problem with slime in paper machines by using various substances poisonous to microbes, i.e., biocides, together with dispersing agents, and by seeking a suitable combination of biocides for each machine. However, the use of many biocides will be considerably restricted in the near future because of their toxicity, and so an alternative solution should be found for the slime and microbe problems of paper machines.

Proposals have been made to use enzymes that decompose biofilms to replace or increase the effect of conventional biocides. In some cases, levanase enzyme has been found to be effective in controlling the deposits. However, its impact is limited to fructose polymer levan, which is mostly a very minor component in the biofilms of paper machines.

Attempts have also been made to solve the problem with slime in paper machines by using commercial enzymes in compositions that have been optimised by using biofilm tests for each case separately. However, the enzymatic methods used today have not been able to solve the slime problem of paper machines in a satisfactory manner.

Ratto et al. (1998a and 1998b) have analysed the carbohydrate compositions and the pro- tein and ash contents of paper machine slime deposits. The samples contained microbiological and chemical deposits containing microbes and microbial polysaccharides in addition to pulp fibres and inorganic compounds. Carbohydrates, cellulose, and xylan derived from wood were the main components of the carbohydrates of the deposits. Increased amounts of arabinose, galactose, mannose, rhamnose or fructose indicated the presence of bacterial polysaccharides in the deposits.

Buchert et al. (1998) and Ratto et al. (1998a) have isolated samples of paper machine slime deposits, and isolated from them microbe strains that produce large quantities of slime. In that case, they discovered that the Klebsiella pneumoniae species was common. They found that the Klebsiella species produced heteropolysaccharides containing various amounts of neutral sugars and uronic acids, such as galactose, glucose, mannose, and galacturonic acid. The isolated bacterial polysaccharides were used to find, in soil and compost samples, microbe strains that are capable of degrading polysaccharides. The growth medium of a mixed culture that was enriched by using the polysaccharide of a K. pneumoniae strain was active against the polysaccharides produced by some K pneumoniae strains. In the biofilm tests, a cell-free enzyme preparation, which had been prepared from the cells of the mixed culture by sonication, inhibited the formation of a matrix containing cell aggregates on steel plates.

The publications mentioned above describe neither the purification nor the characterisation of the polysaccharides produced by the microbes that are isolated from paper machines. Neither do the above-mentioned publications describe the isolation nor the characterisation of the enzyme(s) produced by the mixed microbe cultures isolated from the soil and compost samples. They do not describe the functional mode of the enzyme(s).

Pellikka (1997) has described an enzyme that was able to degrade the polysaccharide pro- duced by the Klebsiella pneumoniae VTT E-97860 strain. The enzyme had been isolated from the growth medium of the mixed culture by using the above-mentioned polysaccharide as the only source of carbon. Proposals were made to use the enzyme for eliminating slime deposits from industrial water but, nonetheless, the operating mechanism of the enzyme had not been clarified at all, and there was no explanation as to the kind of chemi- cal bonds the enzyme influences or which polysaccharide bonds the enzyme breaks and, consequently, the enzyme could not be named.

Patent publication EP-A-896998 suggests that an enzyme that degrades saccharide gum be used in detergents. β-l,2-galactanase is mentioned as one of the enzymes that degrade saccharide gum. However, the publication does not mention any commercial β-1 ,2- galactanase, it neither describes the purification of this enzyme nor says from where the enzyme could be isolated. Patent publication WO-A-9826807 proposes the use of galactanases when treating biofilms, and US-A-5985593 suggests treating microbe samples containing mycobacteria with galactanes, among others, but the publication does not mention β-1 ,2-galactanase, nor does it describe the isolation of galactanases or suggest any commercial galactanase. Consequently, publications according to the prior art do not describe an effective solution for the slime problem in paper machines or other process industry, nor is there a commercial product available, which could be used to solve the slime problem.

The purpose of the present invention is to eliminate the disadvantages of the prior art and to provide a new method for degrading polysaccharides. The purpose of the invention is especially to present an enzyme that has endo-β-l,2-galactanase activity. As far as we know, such enzyme activity has not been described before.

To be more specific, the object of the invention is a method for degrading microbial polysaccharides by using an enzyme preparation characterized by what is stated in the characterizing part of claim 1.

According to the invention, an enzyme preparation comprising endo-β-l,2-galactanase enzyme can be used to degrade polysaccharides that have two monosaccharide units with a β-1, 2 bond between them. One of the monosaccharides is galactose and the other one galactose, galacturonic acid, glucuronic acid, mannose, glucose, arabinose, xylose, rham- nose or some other monosaccharide. According to a preferred embodiment of the invention, the enzyme is used to degrade polysaccharides that have a β-1, 2 bond between ga- lactose and galacturonic acid.

The enzyme preparation according to the invention can be used to prepare various mono- or oligosaccharides. To be more specific, the invention relates to a method for preparing mono- or oligosaccharides according to the characterizing part of claim 11. The method according to the invention can be used to manufacture mono- or oligosaccharides by enzymatically hydrolysing microbial polysaccharides. Certain oligosaccharides can possibly be used as probiotics, for example.

An especially significant application comprises the use of the enzyme preparation according to the invention in degrading slime deposits in various environments. To be more specific, the object of the invention is a method for treating slime deposits containing microbial polysaccharides in accordance with the characterizing part of claim 16. The enzyme preparation comprising endo-β-l,2-galactanase can be used to decompose biofilms and other slime deposits found in the process industry. Bacterial polysaccharides occur not only in paper machine environments but also in other process industries, such as the food processing industry. For example, in clean water and sewage piping, there are slime deposits containing bacterial polysaccharides. It is especially preferable to use the enzyme preparation comprising endo-β-l,2-galactanase for solving the problem with slime in paper machines.

The enzyme preparation, which can be used, for example, in the process industry or in the manufacture of mono- or oligosaccharides, is characterized by what is stated in the characterizing part of claim 31.

It is preferable to use the enzyme preparation according to the invention together with enzyme preparations comprising other enzyme activities. Such enzyme activities include, for example, protease, β-glucanase, mannanase, chitinase, lysozyme, hemicellulase, pectinase, lipase, and levanase. Enzyme preparations comprising these substances are commercially available. A suitable combination of enzymes is selected in accordance with the application.

The invention is useful in many respects in the process industry in particular. Flow rates can be increased, the risk of blocking the piping is decreased, there is less corrosion, and the consumption of energy can be reduced. Reducing the slime in paper machines reduces breaks in the paper web and improves the removal of water. There is less deterioration of paper because of perforation, and the transfer of microbes along with the end product to the end user or the consumer is decreased.

Endo-β-l,2-galactanase could be used for cleansing the clean water and sewage pipes, possibly together with other enzyme activities and/or chemicals.

As endo-β-galactanase enzyme has not been described before, the use of endo-β galac- tanase enzyme according to the invention in the manufacture of mono- and oligosaccha- rides provides a new method for manufacturing certain oligosaccharides or sugars.

Deposition Data

In accordance with the Budapest Agreement, Klebsiella pneumoniae VTT-E-97860 was recorded in the DSMZ Deutsche Sammlung von Mikroorganismen recording plant according to the Budapest Agreement on 28 September 1999, at the address of Maschero- derweg 1 b, D-38124 Braunschweig, Germany, and it received the classification number DSM 13058.

The invention is described in detail with the aid of the following detailed description and with reference to some experimental examples.

The term "polysaccharide" refers to large molecules formed by several (more than 20) large units formed by monosaccharide molecules. Oligosaccharides refer to chains formed by a few (2 to 20) sugar units. The polysaccharide according to this invention specifically refers to a polysaccharide containing two monosaccharides and a β-1, 2 bond between them. One of the polysaccharides is galactose and the other one is galactose, galacturonic acid, glucuronic acid, mannose, glucose, arabinose, xylose, rhamnose or some other mono- saccharide. According to a preferred embodiment of the invention, the polysaccharide in this invention refers to a polysaccharide containing a β-1, 2 bond between galactose and galacturonic acid.

The enzyme activity described in this invention is enriched from soil and compost samples by using a polysaccharide that consists of oligosaccharide units, which contain a galacturonic acid, two mannoses and a galactose and, as a side chain, a mannose, with which a pyruvic acid in acetal form is bound, and where there is a β-1, 2 bond between galactose and galacturonic acid. Thus the structure of the polysaccharide is as follows: -2)-α-D-GalpA-(l→3)-α-D-Manp-(l→2)-α-D-Manp-(l→3)-β-D-Galp-(l→

/ α-D-Manp-(l→4) 4 6 \ /

C

/ \

H₃C COOH

The polysaccharide is produced by the Klebsiella pneumoniae VTT-E-97860 strain, which was deposited in the DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen collection of strains on 28 September 1999 with the classification number DSM 13058. The polysaccharide can be produced with the aid of the K. pneumoniae strain, as described in Example 1. Other Klebsiella pneumoniae strains can also produce a corresponding poly- saccharide. Dutton and Parolis (1986), for example, have described a polysaccharide of a similar structure as a Klebsiella serotype 3.

However, the invention is not limited to the polysaccharide produced by the above- mentioned Klebsiella pneumoniae strain or Klebsiella pneumoniae species and to its use in enriching microorganisms that produce endo-β-1 ,2-galactanase, but microorgamsms that produce endo-β-l,2-galactanase can also be enriched by using polysaccharides derived from other microbes, as long as the polysaccharides have a structure that has two mono- saccharides with a β-1, 2 bonding between them. One of the monosaccharides is galactose and the other one galactose, galacturonic acid, glucuronic acid, mannose, glucose, arabinose, xylose, rhamnose or some other monosaccharide, preferably by using a polysaccharide, where the β-1, 2 bonding is between galactose and galactoronic acid. Correspondingly, the enzyme according to the invention is able to degrade polysaccharides, which have the structure mentioned above, in various environments. According to the invention, polysaccharide can be isolated from bacterial strains, which have been isolated from paper machine slime deposits and which produce large quantities of slime. According to a preferred embodiment of the invention, the polysaccharide is isolated from the growth media of Klebsiella pneumoniae strains, as in this invention we stated that these bacteria were common among the bacteria that produce large quantities of slime.

In the research behind the invention, the ability of the strains to produce polysaccharides was examined by plating. In this case, we discovered that, in a medium containing saccha- rose, which is commonly used in screening bacteria that produce slime, producers of levan, a polymer mainly consisting of fructose, could be isolated. To avoid the formation of levan in the following experiments, we selected glucose as the source of carbon in the screening medium. Furthermore, we produced polysaccharides by using strains selected by preliminary screening, growing as slimy colonies on the plates, by cultivation in shake flasks in liquid cultures. We followed the polysaccharide yield in the liquid cultures by measuring the viscosity of the medium or by precipitating the formed polysaccharide from the growth medium.

The strains that produced polysaccharides, chosen for further testing, were identified. It turned out that many producers of polysaccharides isolated from the paper machine slime samples represented bacterial species that had not been described before. In screening, we found several Klebsiella pneumoniae strains that produce polysaccharides.

Polysaccharides were produced in shake flask cultivation in various media. The source of carbon, and the nitrogen content of the medium in particular had an impact on the formation of polysaccharides. When saccharose was used as the source of carbon, several strains that were examined produced levan. To isolate others than the levanase enzyme, we selected a nitrogen-restricted glucose medium for a future production medium, the polysaccharides produced on which being various heteropolysaccharides. The polysaccharides were isolated from the growth media by precipitating with ethanol. The precipitated polysaccharides were dissolved in water and treated with protease (Neutrase, Novo) to remove any protein residue, and reprecipitated. After precipitation, some samples contained a good deal of phosphates. To remove salts, the samples were dialyzed. Finally, the polysaccharides were freeze-dried.

For further research, we selected a strain of the Klebsiella pneumoniae species that produces large quantities of slime (VTT-E-97860). The polysaccharide was isolated from the K pneumoniae growth medium by concentration, ultrafiltration, and precipitation, as described in Example 1. To precipitate any impurities, the concentrated polysaccharide solution was heat treated, so that impurities were precipitated and the viscosity of the solution was decreased. The decrease in viscosity enabled further concentration, after which the polysaccharide was precipitated with ethanol.

The structure of the polysaccharide was defined by the NMR technique. On the basis of the spectra of the enzyme-hydrolysed samples, the structure of the polysaccharide could be determined. As mentioned above, a capsular polysaccharide of a similar structure had been described before as a K pneumoniae serotype K3 (Dutton and Parolis, 1986). An enzyme that degrades this polysaccharide has not been characterized earlier.

Enzyme production experiments were used to find and isolate, from soil and compost samples, enzymes that degrade slime polysaccharides, and to examine the ability of both pure strains and mixed cultures to degrade polysaccharides. The screening of enzyme- producing strains was carried out in the form of micro titre plate or test tube cultivation in a nutrient medium, where the polysaccharide to be examined was the only source of carbon. The ability of the screened strains to degrade polysaccharide was studied, for example, by following the turbidity of the growth medium and changes in its viscosity.

For the screening, various bacteria were first selected from the applicant's own culture collection, the bacteria having been isolated from paper machine slimes, paper machines or mills, and bacteria, which were known to produce various enzymes that degrade polysaccharides. In screening, several bacteria were found, which were able to use as the only source of carbon the fructose polymers (levan) produced by the B. licheniformis and P. βuorsecens strains in a saccharose medium. Hydrolysis tests were used to ensure that the growth media of these strains contained enzyme activity that would hydrolyse the above- mentioned levan polysaccharides and commercial levan, producing fructose as a hydrolysis product. None of the bacteria selected for this screening was capable of degrading the VTT-E-84211 and the T-E-97860 polysaccharides of the K. pneumoniae strains.

As the collection strains examined did not produce the enzyme that degrades Klebsiella polysaccharides, screening of strains that produce the enzyme was continued by enriching from factory and natural samples mixed cultures, which were able to use the isolated heteropolysaccharides as the only source of carbon. The capability of the cultures to degrade polysaccharide was followed on the basis of changes in the viscosity in a liquid medium. This screening method proved to be effective.

Mixed cultures that degrade heteropolysaccharides were isolated from soil and compost samples. As in the media containing other carbohydrates the cultures quickly lost their desired property for an unknown reason, possibly because of changes in the proportions of species in the culture, these cultures were maintained in media, where the bacterial poly- saccharide was the only source of carbon. In several cases, the enzyme was not secreted into the growth medium but had attached to the surface of a cell, from where it was released by slight sonication. Enzyme activity was determined by a viscosimetric method, and the activity unit (AU) was defined as enzyme concentration, which affected a lcP/min decrease in viscosity.

Thus the present invention also relates to a method for isolating enzyme-producing microorganisms, the enzymes being capable of degrading the selected, slime-producing polysaccharides. The method comprises the following steps: soil and compost samples are collected from various environments, - the samples are suspended in water or a buffer solution, such as a saline solution, a growth medium is inoculated by the solution or directly by the soil or compost sample, the growth medium containing, as the only source of carbon, polysaccharide that contains a β-1 , 2 bond between two monosaccharides, so that one of the mono- saccharides is galactose, and the other one galactose, galacturonic acid, glucuronic acid, mannose, glucose, arabinose, xylose, rhamnose or another monosaccharide, the medium is incubated in suitable conditions and the culture filtrate is measured for viscosity, for further testing, a culture is selected, in which the viscosity is decreased; optionally a culture that exhibits the most intense and rapid decrease in viscosity, - cells are separated and the culture is again grown in suitable conditions in a medium that contains the selected polysaccharide, and optionally, a sample is taken from the growth medium and the cells are separated and suspended in a buffer solution or a growth medium, the cells are sonicated and separated, the filtrate is recovered, and optionally, - the sample of the growth medium or the filtrate is brought into contact with the selected polysaccharide and it is established whether or not the enzyme is capable of degrading the selected polysaccharide,

- the cells of the culture are recovered; optionally from a culture, the medium of which or the solution obtained from the sonication has been found to be capable of degrading the selected polysaccharide, and the cells of the culture are grown in a medium that contains the selected polysaccharide.

The isolation method described in this invention can be repeated, even though the basis consists of soil or compost samples. Although the soil samples were different, and the compost samples originated from compost samples that contained different raw material and were at different stages, more than 80% of the samples contained microbes that were capable of degrading polysaccharide, when the polysaccharide in question was used as the only source of carbon in their medium. The capability of the mixed culture to degrade polysaccharide remained from one generation to another, when the culture was continuously grown in a medium that contained the selected polysaccharide as the only source of carbon.

The growth medium of the microbe culture isolated according to the invention or a cell extract that is obtained, for example, by sonicating the cells and recovering the cell-free solution, contains 0.02-0.03 mg/ml of endo-β-l,2-galactanase enzyme, calculated as protein on the basis of observations made in connection with the invention, or 50-200 AU/ml, calculated as enzyme activity. For manufacturing an industrially usable enzyme preparation, it is preferable to concentrate the growth medium or the solution obtained by sonication, and/or the enzyme must be purified in one or more protein purification steps, so that the enzyme preparation contains more than 0.1 mg/ml of endo-β-l,2-galactanase enzyme, calculated as protein, preferably 0.250 mg/ml, and most preferably 0.5 mg/ml. When calculated as enzyme activity, the enzyme activity is more than 200 AU/ml, preferably more than 500 AU/ml, and most preferably over 1000 AU/ml.

However, the present invention is not limited to the enzyme isolation method described here but the enzyme can be isolated or produced by other methods as well. The enzyme according to the invention can be produced by microorganisms, to which an enzyme- coding gene has been transferred or which endogenously produce the enzyme, but the production capacity of which has possibly been improved by mutation or by the methods of gene technology.

A gene that encodes endo-β-l,2-galactanase can be isolated from a microbial culture, which contains cells of one or more different microbe species, and transferred to a suitable host microorganism, preferably under effective regulation areas. The producer organism is preferably one that is capable of secreting the enzyme outside the cell, so that the growth medium can be used as an enzyme preparation as such, or by slightly processing, for example, by concentrating, adding substances that increase the stability, and buffers, etc.

The term "enzyme preparation" refers to any preparation that contains at least one enzyme. Thus, the enzyme preparation can be a growth medium or a cell extract containing one or more enzymes, an isolated enzyme or a mixture formed by one or more enzymes. In addition to enzyme activity, the enzyme preparation preferably contains additives, which are commonly used in enzyme preparations that are intended for the process industry of the application in question. Such additives include buffers and stabilizers, for example. In paper machine applications, these additives are preferably agents that are used in the applications of the chemical pulp or pulp industry. If the enzyme preparation is intended for another processing industry or for treating clean water or sewage piping, the additives are selected so that they suit the purpose of use in question. To characterize the enzyme that hydrolyses the polysaccharide produced by the K. pneumoniae VTT-E-97860 strain, the enzyme was purified chromatographically, as described in Example 2. Purification consisted of two chromatographic steps based on hydrophobic interactions, followed by anion exchange chromatography and gel filtration as the final step of purification.

The purified enzyme was used to hydrolyse the polysaccharide produced by the K. pneumoniae VTT-E-97860 strain, which was used as a substrate, and the NMR method was used to determine, which bond of the polysaccharide the enzyme broke. It was discovered that the enzyme broke the β-1 ,2 bond between galactose and galacturonic acid.

When the mode of action the enzyme is known, various enzyme combinations specific for various purposes of use can be planned.

The enzyme according to the invention is preferably endo-β- 1 ,2-galactanase, the molecular weight of which is 110 - 130 kDa. The pH optimum of the enzyme according to the invention is within 5-6.5, preferably about 5.5. The pH stability of the enzyme at 40°C is within 5 - 7, preferably within 5.4 - 6.0.

The amount of enzyme that is needed when hydrolysing the β-1 ,2 bonds between the galactose and the galacturonic acid found in polysaccharides, or the corresponding bonds between galactose and galactose, galacturonic acid, glucuronic acid, mannose, glucose, arabinose, xylose or rhamnose or another monosaccharide, is 50-500000 AU/g, preferably 500-50000 AU/g, most preferably 1000 - 10000 AU/g of polysaccharide.

The polysaccharides are hydrolysed at 20 - 60°C or 20 - 70°C, preferably at 35 - 55°C, most preferably at 35 - 50°C. The pH of the hydrolysing solution is 4 - 8, or 4 - 9, preferably the pH is 5 - 7, and more preferably the pH is 5.5 - 6.0. The hydrolysing time can be 5 minutes to 10 days, depending on the application and the amount of enzyme used. When preparing mono- or oligosaccharides, the hydrolysing time is preferably less than 1 day or a few tens of minutes, 5 min to 1 day, preferably 20 min to 5 hours, most preferably 30 min to 2 hours.

The enzyme according to the present invention is suitable for degrading biofilms and other slime deposits found in various environments. The enzyme preparation, which possibly contains suitable additives, such as buffers, needed in the environment in question, is then brought into contact with the slime deposit in conditions that are suitable for the enzyme to act. The enzyme treatment is continued until an essential portion of the polysaccharide degrades under the effect of the enzyme. In this case, the essential portion means that at least 50% of the β-1, 2 bonds contained by the slime deposit degrade, preferably more than 80%, and most preferably more than 90% of the β-1 ,2 bonds degrade.

In the method according to the invention, the slime deposit is brought into contact with an enzyme preparation containing endo-β- 1 ,2-galactanase alone or together with other enzyme activities. The treating temperature is 20 - 60°C or 20 - 70°C, preferably 35 - 55°C, and most preferably 35 - 50°C. The treatment is carried out at a pH of 4 - 8 or 4 - 9, preferably at a pH of 5 - 7, most preferably at a pH of 5.5 - 6.0. The enzyme preparation is added into the circulation water, particularly in paper machines. A suitable amount of enzyme in this case is 5 - 10000 AU/litre of circulation water, preferably 20 - 50000 AU/litre, most preferably 200 - 2000 AU/litre of circulation water. As the treatment is not distinctly stopped at a certain point in time, the processing time varies from a few hours to several days.

The treatment with endo β-l,2-galactanase can be carried out separately or simultaneously with other enzyme treatments, or before such treatment. It is especially preferable to com- bine the treatment with endo-β- 1 ,2-galactanase with a treatment with protease, β-glucanase, mannanase, chitinase, levanase, hemicellulase, lipase, pectinase, or lysozyme. When endo-β- 1 ,2-galactanase is used simultaneously with the above-mentioned enzymes, it is preferable to use an enzyme preparation, to which endo-β- 1 ,2-galactanase has been added or which has been produced by a strain, which is genetically modified to produce large amounts of the enzyme in question in order for the enzyme preparation to have substantially high endo-β-l,2-galactanase activity. The enzyme treatment according to the invention can also be combined with one or more chemical treatments, or their combinations. Such treatments can be, for example, treatments with a dispersing agent or a biocide, so that the biocide is selected from among biocides, where the active ingredient is, for example: glutaraldehyde, DBNPA, MBT, chloro- 2-methylisothiazoline-3-one, methyl-4-isothiazoline-3-one, Bronopol, dazomete, sodium hypochlorite, peracetic acid or BCDMH (bromochlorodimethyl hydantoin).

Examples

Example 1 Isolation of polysaccharide

The microbe strains that produce slime polysaccharides were derived from various culture collections or from slime samples of paper machines. The capability of the strains to produce polysaccharides was examined by plating. At an early stage of the study, it was discovered that in a medium containing saccharose, mainly producers of levan could be iso- lated; therefore, glucose was selected for a future source of carbon in the screening medium. The strains, which had been selected by preliminary screening and which grew in the form of slimy colonies on the plates, were further used to produce polysaccharides by cultivation on a shaker in liquid cultures. The production of polysaccharide in the liquid cultures was followed by measuring the viscosity of the medium or by precipitating the formed polysaccharide from the growth medium.

The polysaccharide-producing strains that were selected for further testing were identified (API, FAME, ribotyping, 16S rDNA sequencing). It turned out that many producers of polysaccharide isolated from the paper machine slime samples represented bacterial spe- cies that had not been described earlier. In screening, several Klebsiella pneumoniae strains that produced polysaccharides were found. When comparing with one another the K. pneumoniae strains isolated from different paper machines, it was stated, on the basis of ribotyping, that their ribotypes were different.

Polysaccharides were produced by cultivation on a shaker in various media. The source of carbon and the nitrogen content of the medium in particular had an impact on the formation of polysaccharides. In order to isolate other than the levanase enzyme, we selected a nitrogen-restricted glucose medium for a future production medium, the polysaccharides produced on which being various heteropolysaccharides. The polysaccharides were isolated from the growth media by precipitating with ethanol. The precipitated polysaccharides were dissolved in water and treated with protease (Neutrase, Novo) to remove possible protein residue, and reprecipitated. After precipitation, some samples contained a good deal of phosphates. To remove salts, the samples were dialyzed. Finally, the polysaccharides were freeze-dried.

The polysaccharide yields in liquid cultures were fairly low, mostly less than lg/1.

The total carbohydrate content of the polysaccharide preparations was determined by the phenol sulphuric acid method. The composition of monosaccharides and uronic acids of the polysaccharides was defined by HPLC after acid hydrolysis. When hydrolysing the polysaccharides, it was stated that the optimal conditions for hydrolysis should be estab- lished for each polysaccharide: a suitable acid concentration and hydrolysing time. The acid hydrolysis was mainly carried out by using 0.5N - 2N sulphuric acid at 105°C. The hydrolysing time was 2 to 5h. To define the fructose content, the polysaccharides were hydrolysed with 2N TFA at 20°C for lh.

Testing was continued on the Klebsiella pneumoniae VTT-E-97860 strain, which produced very slimy colonies on the culture plates. For extracellular polysaccharide production, the strain was grown in liquid medium in shake flasks and a laboratory fermenter. The polysaccharide yield in shake flasks (measured as total carbohydrates in the freeze-dried polysaccharide preparation) was about lg/1 of the growth medium. The polysaccharide was composed of mannose, galactose, and galacturonic acid. It also contained some glucose and methyl glucuronic acid (Table 1). Table 1. Carbohydrate yield and the sugar composition of the K. pneumoniae polysaccharide produced in shake flasks.

Yield Man Gal Glc GalA MeGluA

( i^"1) (%) (%) (%) (%) (%)

0.95 62.6 23.5 0.9 12.10 0.9

The Klebsiella pneumoniae VTT-E-97860 strain was used to produce polysaccharide in a 51 Erlenmeyer flask that contained 1 litre of a culture medium. The medium contained 20g r¹ glucose, 0.5g l^"1 yeast extract (Difco), 0.6g l^"1 (NH₄)₂SO₄ (Riedel-de Haen), 3.18g l^"1 KH₂PO₄ (Merck), 5.2g \^Λ K₂HPO₄ (Merck), 0.3g l^"1 MgSO₄x7H₂O (Merck), 0.05g r'CaCfe (Merck) and 1ml per litre of a nutrient solution (0.2g l^"1 ZnSO₄x7H₂O (Merck), 0.2g l^"1 CuSO₄x5H₂O (Merck), 0.2g l^"1 MnSO₄xH₂O (Merck), 0.2g l^'1 CoCl₂x6H₂O (Merck), 0.2g l^"1 FeSO x7H₂O (Merck). After 5 days of cultivation on a rotary shaker (120 rev min^"1) at +30°C, NaCl (0.9%)) was added to the growth medium to assist separation of the partially cell bound polysaccharide. The medium was homogenized with a mixer (Ystral). The cells were separated by centrifugation. The polysaccharide was precipitated from the supernatant with 3vol ethanol at +4°C overnight. The polysaccharide was separated by centrifu- gation and dissolved in water. To remove protein impurities, the dissolved polysaccharide solution was treated with protease (Neutrase, Novo) at +37°C for lh. The polysaccharide was precipitated with ethanol, dissolved in water, dialyzed (MWCO 12-14 kDa, Medicell, London, UK) against water and freeze-dried.

For a larger scale polysaccharide production, fermentations were carried out in a Chemap CF 2000 laboratory fermenter with a working volume of 10 litres, and in a pilot scale fer- menter with a working volume of 1200 litre. The fermentation conditions were: temperature 30°C, pO₂ >10% (Chemap) or pO₂ >30% (pilot), pO₂ was controlled by agitation, pH 7-8.5 (controlled by an automatic addition of NaOH). The growth medium contained 30g/l of glucose, 0.5g/l of yeast extract and the same salts as the Erlenmeyer flask cultivation. The amount of the polysaccharide produced was calculated from the amount of mannose, analysed in the centrifuged culture broth by HPLC after acidic hydrolysis (2 N H SO , 105°C, 2h). The cells were separated by centrifugation, the broth was concentrated by ultra filtration, and the polysaccharide was precipitated from the supernatant with ethanol. Gel electrophoresis

The proteins in the samples were characterized by SDS electrophoresis by 10% polyacry- lamide gel according to Laemmli (1970), using the Ready Gel Cell system (Bio-Rad Labo- ratories, Hercules, CA, USA), and following the manufacturer's instructions. The calibration mixture 7707S (Biolabs, NE, USA) was used as the standard.

Chemical analyses

The protein concentration was determined by a spectrophotometer by measuring the ad- sorption of the samples at a wavelength of 280nm. The concentration was obtained assuming that the absorbance value of 1 unit was equal to 1 mg/ml of protein concentration.

The total carbohydrate content of the polysaccharide preparations was determined by the phenol sulphuric acid method (Dubois et al. 1956). To determine the composition of the monosaccharides and uronic acids, the isolated polysaccharide was hydrolysed with 2N H₂SO₄ at 105°C for 2h. The hydrosylate was analysed by HPLC (Dionex).

Structure of polysaccharide

For a structural analysis and hydrolysis experiments, the polysaccharide was purified with an anion exchange resin (DEAE Sepharose FF, Pharmacia-LKB, Uppsala, Sweden). The resin was equilibrated with a buffer, the pH of which was 7 and the conductivity 190 μS/cm. The polysaccharide was mixed with the resin and shaken for lh at +20°C. The resin was separated by centrifugation.

The structure of the polysaccharide was determined by using 2D NMR after the enzyme treatment. The polysaccharide was treated with a purified enzyme endo-β- 1 ,2-galactanase for 24h (a dosage of 280 mg/g of polysaccharide), after which the mixture was boiled (5 min) and freeze-dried. The freeze-dried hydrosylate was dissolved in deuterium oxide (D2O, 99.8 atom%, Fluka) and the solution was clarified by centrifugation. The sample was measured for the ID IH and 13C NMR spectra and the 2D COSY, relayCOSY, TOCSY, HMQC, and HSQC spectra at 50°C. The NMR analysis showed that the polysaccharide was composed of five sugar units, the structure of which was as follows:

→2)- -D-GalpA-(l→3)-α-D-Manp-(l→2)-α-D-Manp-(l→3)-β-D-Galp-(l→

/ α-D-Manp-(l→4)

4 6

C

/ \ H₃C COOH

Example 2

Screening and isolation of enzyme Mixed cultures from soil and compost samples were screened for a capability to degrade the polysaccharide isolated in Example 1. To enrich the cultures that degrade polysaccharide, the samples were inoculated into a culture medium containing 5g l^"1 of the polysaccharide of K. pneumoniae and 6.7g l^"1 of Yeast nitrogen base (Difco) in 50 mM potassium phosphate buffer, pH 7. After 12 days of incubation at +30°C on a rotary shaker (120 rev. min^"1), 750μl of the cultures were reinoculated into 15ml of fresh medium and incubated for 3 days. The capability of the culture to degrade polysaccharide was followed by viscosity measurements (Brookfield DV II).

When screening from the soil and compost samples microbes that produce polysaccharide- degrading enzyme activity, by incubating the samples in a medium containing the K. pneumoniae polysaccharide as the only source of carbon, as described above, we discovered polysaccharide-degrading activity in 6 of the 7 cultures that were cultivated in the polysaccharide medium (Table 3). The mixed culture No. 3 isolated from soil was the most effective in degrading polysaccharide during the second incubation. This culture was selected for enzyme production. The mixed culture was kept in the growth medium at +4°C until the production of the enzyme. Table 2

Sample I Incub. 5 d I Incub. 12 d II Incub. 1 d II Incub. 3 d

No. Origin Viscosity (cP)

Control 5.2 6.3 4.4 4.6

1 Compost, straw 5.2 2.7

2 Compost, paper 1,2 1.3 3.7 2.5

3 Soil 5.0 1.1 1.7 1.0

4 Compost 1.1 1,2 4.1 1.3

5 Sand and leaves 5.2 1,2 2.1 1,2

6 Soil and leaves 5.0 5.3

7 Compost and leaves 5.2 1.3 2.4 1,2

Correspondingly, when using other than the K. pneumoniae polysaccharide described above, we discovered that, generally, it was possible to isolate from the soil or compost samples the cultures that degrade each microbial polysaccharide by using this method.

For enzyme production, the mixed culture No. 3 (VTT-E-97916) was incubated in shake flasks containing 500ml of polysaccharide medium at +30°C for 3-5 days. The cells were separated from the growth medium by centrifugation at +4°C. The cells were suspended in 50ml of 50mM kalium phosphate buffer, pH 7. The suspension was sonicated to release the cell bound enzyme into a liquid phase. The cells were removed from the enzyme solution.

The following purification steps were carried out at +4°C. Purification was started by a hydrophobic interaction chromatography (HIC). The conductivity of the enzyme solution was adjusted to 106 mS/cm and the pH to 6. The solution was applied to a butyl sepharose column (Butyl Sepharose FF, Pharmacia-LKB, 16mm x 80mm), which had been equilibrated to the same conductivity (106 mS/cm) and pH value (pH 6) with 0.8M of ammonium sulphate in 20mM kalium phosphate buffer, pH 6. After feeding the sample, the column was washed with the above-mentioned equilibrating buffer and then eluted with a decreasing linear gradient of ammonium sulphate (80ml) from 0.8M to 0M in 20mM kalium phosphate buffer, pH 6. The flow rate was 1.5ml min^"1 and fractions of 9ml were collected during the entire run and assayed for the ability to degrade the K. pneumoniae VTT-E-97860 polysaccharide. The active fractions were pooled and purification by HIC was repeated. The conductivity and the pH of the sample were adjusted to correspond to the equilibration buffer (1.0M ammonium sulphate in 20mM kalium phosphate, pH 6). The sample (93ml) was applied to the same column, which previously had been equilibrated with 1.0M of ammonium sulphate in 20mM kalium phosphate, pH 6. After feeding the sample, the column was washed with the equilibrating buffer and then eluted with the decreasing linear ammonium sulphate gradient (80ml) from 1M in 20mM kalium phosphate buffer (pH 6) to 0M in 2mM kalium phosphate buffer (pH 6). During the gradient, the enzyme eluted in one peak, and the active fractions were pooled.

The obtained fraction (36ml) containing the enzyme was buffered in 20mM kalium phos- phate, pH 6.2, by gel filtration (Sephadex G-25 coarse, Pharmacia LKB, 50 x 150mm).

The enzyme was further purified by anion exchange (AE) chromatography (DEAE Sepharose FF, Pharmacia-LKB, 10mm x 110mm) in a column. The enzyme was bound to resin in 20mM kalium phosphate, pH 6.2, at a flow rate of 0.8ml min^"1. The enzyme was eluted from the column with an increasing linear gradient of sodium chloride from 0 to 0.15M, during which action the enzyme was eluted in one peak. The most active fractions (a 3ml) were pooled (12ml, AE pool I), and three fractions eluting before and after collecting the pool I were combined to form a second sample (18ml, AE pool II). Both above-mentioned samples were concentrated by ultra filtration (Amicon, Amicon Diaflo, PM10 membrane, Beverly, MA, USA), the AE pool I was concentrated 8-fold and the AE pool II was con- centrated 22-fold.

The last purification step, the gel filtration, was performed at room temperature. The pool was applied to the column of Sephracryl S-300 HR (Pharmacia-LKB), equilibrated with 0.1M of sodium chloride in 50mM kalium phosphate buffer, pH 6. The flow rate was 0.6ml min^"1 and fractions of 2.9ml were collected. The enzyme eluting from the DEAE pool I was divided into two different pools (G pool I and G pool II) and the enzyme eluting from the DEAE pool II was combined to form one pool (G pool III). The total activity of the cell-free enzyme preparation, which was obtained from cells grown in 500ml of a culture medium, was 14 874AU. The first purification step (HIC 1) resulted in a 25-fold increase in the specific activity of the enzyme with a yield of 74%. In this case, the enzyme did not bind properly to the resin and, consequently, elution was not optimal. The active fractions were pooled and purification was repeated (HIC2). This time, the enzyme was bound and it eluted in a single peak, resulting in the removal of about 50% of the protein impurities and about a 10% loss of the total activity. The active fractions that eluted from the anion exchange chromatographic columns were divided into two pools. Pool 1 contained the four most active fractions and pool 2 three fractions, which had eluted before, and three fractions, which had eluted after the most active fractions. After concentration by ultra filtration, the purification coefficients were 229 (pool 1) and 1 11 (pool 2). In the AE, 15% of the proteins and nearly 50% of the total activity were recovered. In SDS-PAGE, both pools produced one clearly dark band, but many significantly lighter bands were also observed. Both pools were further purified by gel filtration. In gel filtration, some impurities were removed but a great deal of activity was lost and the purification coefficient decreased. After gel filtration, the pool 1 was divided into two separate pools. After gel filtration of pool 2, the active fractions were collected into one pool. The concentration of the pool resulted in a further loss of activity. In the SDS-PAGE, one very fine band was observed.

Table 3. Purification of the enzyme that degrades the K pneumoniae VTT-E-97860 polysaccharide

Purification step Volume Total protein Total activity Specific activity Yield Purification (ml) (mg) (AU) (AU/ mg protein) (%) Coefficient

Cell-free extract 37 444 14874 34 1 fflC l 95 13 10944 853 74 25

HIC 2 38 6 9302 1466 63 44

AE Pool I 12,5 0,65 4200 6462 28 193

Pool II 18 0,63 1166 1851 8 55

Pool 1 cone. 1,4 0,39 3024 7687 20 229

Pool II cone. 0,8 0,38 1402 3720 9 1 11

GF Pool I 16 0,34 307 914 2,1 27 Pool II 1 1 0,22 111 504 0,7 15

Pool III 14 0,31 134 436 0,9 13

Pool I cone. 1 0,05 58 1252 0,4 37

Pool II cone. 1,05 67 0,5

Pool III cone. 1 0,06 41 638 0,3 19

The molecular weight of the protein was 110-130 kDa, determined from the mobility in SDS-PAGE and the gel filtration column. According to the MALDI mass spectropho- tometer, the molecular weight was about 117-120 kDa.

Enzyme activity assay

The enzyme activity was determined as the ability of the enzyme to decrease the viscosity of the K. pneumoniae VTT-E-97860 polysaccharide in a 0.5% polysaccharide substrate solution made in 50mM kalium phosphate buffer (pH 7) at +30°C. In the assay, 50μl of enzyme was added to 450μl of substrate. The decrease in the viscosity was followed by a viscometer (Brookfield LVTDV-II CP, MA, USA) for 4 min. The viscosity was measured within 60-180 s after adding the enzyme. The sample was diluted so that the viscosity decreased at a velocity of 0.007- 0.0035 cP/s during the measurement. The activity unit (AU) was determined as an amount of enzyme activity that in the measuring conditions reduced the viscosity by lcP in 1 minute.

Determination of pH optimum

The pH optimum was determined by hydrolysing the 0.5% polysaccharide substrate in lOOmM acetic acid or phosphate buffer, pH 3.5-8.4, with an enzyme that was purified by anion exchange chromatography. 50μl of the sample was added into 1ml of substrate. To determine reference values, the test was also performed using an inactivated enzyme. All the samples were incubated for 15 min and boiling the samples for 8 min stopped the enzymatic reaction. The viscosity was determined using the Brookfield viscometer. The activity at certain pH values was defined as the difference between the viscosity of the sample and that of the reference sample. The pH optimum of the enzyme was about 5.5; below 5, the activity of the enzyme decreased rather quickly.

pH and thermal stability The pH and the thermal stability of non-purified and purified enzyme were determined within the pH of 4.0-8.8. The pH of the enzyme solution was adjusted by using 25%) acetic acid or 1M NaOH. The enzyme was kept at 40°C or 50°C (the purified only) for lh. 50μl of enzyme were added into 1ml of 0.5% polysaccharide substrate in 200mM phosphate buffer, pH 7. The reference sample was hydrolysed with an untreated enzyme and, in the reference samples the enzyme was replaced with a buffer. The samples were incubated for 25 min. Boiling of the samples for 8 min stopped the reaction. Measurement of the activity was based on the determination of viscosity, as described above.

The pH stability of the non-purified enzyme (more than 80% of the activity left) was within 5.8 - 7 at 40°C and that of the purified enzyme within 5.4 - 6.0. When incubated at 50°C for lh, the purified enzyme lost its activity.

Hydrolysis experiments

To determine the bond that the enzyme hydrolysed, 2ml of purified polysaccharide, which contained 1.66mg of carbohydrate in 1ml, was hydrolysed with 200μl of enzyme (280 AU ml^"1) for 48h at +30°C. The enzyme was obtained from the preparation purified by ion exchange chromatography. The hydrosylate was freeze-dried and the structure of the formed oligomers was analysed by 2D NMR.

In accordance with the NMR analysis performed on the hydrolysed polysaccharide, the enzyme broke the β-D-Galp-(l→2) bond. Example 3

Hydrolysis experiments

The capability of the commercial mixed enzymes Econase CE (Primalco) and Pectinex Ultra SPL (Novo) to degrade the levan polysaccharides produced by the saccharose medium of the B. licheniformis STFI-79133 and B. licheniformis E-91446 strains and the heteropolysaccharide produced by the K. pneumoniae VTT-E-97860 strain was examined by hydrolysis experiments. The enzyme preparations examined did not contain any activity that would hydrolyse these polysaccharides.

The study also examined the effect of various enzyme preparations (Econase CE, Primalco; Pecti-nex Ultra SPL, Novo; Neutrase, Novo; Aspergillus foetidus hemicellulase and Rhodotermus β-(l→3)-glucanase) on commercial microbial polysaccharides (welane, gellane, xanthane, alginate, curdlane). Curdlane, which is a linear β-(l— >3)-glucane, was effectively hydrolysed with the Econase CE, Pectinex, Ultra SPL and β-(l — >3)-glucanase enzymes. In the enzyme preparations used, no other activities were found that hydrolyse the microbial polysaccharides that were examined.

The effect of the enzyme that degrades the polysaccharide produced by K. pneumoniae on the formation of K. pneumoniae biofilm and its attachment was examined on a laboratory scale by growing microbes in different nutrient solutions. The formation of biofilm was followed both by traditional cultivation and epifluorescence microscopy.

By following the growth of the Klebsiella pneumoniae VTT-E-97860 strain and the pH level of the nutrient solution in preliminary tests, two different nutrient solutions were selected as the growth medium: a nutrient solution consisting of peptone and 0.2M kalium phosphate buffer and a nutrient solution consisting of glucose and 0.2M kalium phosphate buffer. Biofilm was cultured in both nutrient solutions both with an enzyme addition (sterile filtrated enzyme solution 460AU/200ml of the nutrient solution) and without the enzyme addition. At two days intervals, 100ml of the old nutrient solution was removed from the incubator and a corresponding amount of fresh nutrient solution was added, as well as 460 AU of the enzyme. The conditions and the formation of biofilm were followed by making culture, microscopic, and pH determinations on Klebsiella pneumoniae biofilm cultures of 2, 4, 6, 8, 10, and 14 days of age.

According to the cultivation results, after 10 days of growing, the enzyme seemed to have an effect that decreased the growth of K. pneumoniae biofilm. A similar result was obtained by a previous series of tests that were conducted by using a non-sterile enzyme, even though, on the basis of numeric microscopic results, no coπesponding effect that decreased biofilm could clearly be observed. However, when examined with a microscope, the microbes of the biofilm that was grown in the enzyme-containing nutrient solution more than 10 days seemed essentially more evenly distributed than cells that had grown in the enzyme-free nutrient solution and that formed aggregates. Similar differences were obtained by different types of nutrient solutions between cultivations carried out with the enzyme and without the enzyme.

Furthermore, we examined the effect of the enzyme on the formation of Klebsiella pneumoniae biofilm on a pilot scale. Pilot tests were carried out in a test hall by cultivation equipment of a total volume of 87 litres. The formation of biofilm was examined by growing the Klebsiella pneumoniae microbe in a pure nutrient solution and in a nutrient solution, to which the enzyme that degrades the polysaccharide of Klebsiella pneumoniae, produced in the project, was added in a similar way as in the laboratory tests (a dosage of 2.2 AU/ml).

In the test, the K pneumoniae VTT-E-97860 strain was grown in a nutrient solution containing phosphate buffer (0.16M, pH 7), glucose (2%), yeast extract (0.05%), and salts. A reference run contained 75 litres of the culture solution and an enzyme run contained 37 litres. On the basis of visual inspection, we could state that the enzyme-containing run had less slime than the reference run. In the reference run, the cover of the container, the walls above the fluid level, and the set of sample grids were covered by a substantial amount of slime. In the enzyme run, the amount of slime accumulated on the walls was considerably smaller than and the composition of the slime was different (dryer) from the slime in the reference run. In the laboratory tests, the effect of the enzyme on the formation of K. pneumoniae biofilm could be observed after 10 days of cultivation. According to the cultivation results, the enzyme reduced the growth of K. pneumoniae biofilm and, when examined with a microscope, we could see that the use of the enzyme degraded the microbe aggregates and thus changed the structure of the biofilm.

According to the tests on the pilot scale, the addition of enzyme reduced the formation of slime and changed its composition.

References

Buchert et al. (1998) Enzymes for the improvement of paper machine runnability. 7th Int. Confe. Biotechnology in the pulp and paper industry, Vancouverm 16-19. June 1998, A225-A228.

Dubois, M et al. (1956) Colorimerric method for determination of sugars and related sub- stances. Anal Chem 28: 350-356.

Dutton GGS, Parolis H (1986) The use of bacteriphage depolymerization in the structural investigation of the capsular polysaccharide from klebsiella serotype K3. Carbohydr Res 149: 411-423.

Laemmli, UK (1970) Cleavage of structural proteins during the assembly of he head bacte- riophage T4. Nature (London) 227: 680-685.

Pellikka T (1997) Mikrobipolysakkaridia hajottavan entsyymin tuotto, puhdistus ja karak- terisointi (Production, Purification, and Characterization of Microbial Polysaccharide- degrading Enzyme.) Thesis. The Department of Biochemistry and Biotechnology, the University of Kuopio. 68 p. Ratto et al. (1998a) Effect of Enzymes degrading bacterial polysaccharides on biofilm formation. COST Action El, Paper Recyclability. Improvement of recyclability and the recycling paper industry of the future. Ed. by A. Blanco et al. Las Palmas de Gran Canaria, 24-26 November 1998. Posteri.

Ratto et al. (1998b) Bacterial polysaccharides in paper machine slime deposits. Med. Fac. Landbouww. Univ. Gent. 63/4a: 1183-1186.

Claims

CLAIMS:

1. A method for degrading microbial polysaccharides, characterized in that the polysaccharide is brought into contact with an enzyme preparation comprising more than 200 AU/ml of endo-β- 1 ,2-galactanase.

2. A method according to claim 1, characterized in that the enzyme preparation comprises more than 500 AU/ml of endo-β- 1 ,2-galactanase.

3. A method according to claim 1 or 2, characterized in that in the polysaccharide, there is a β-1, 2 bond between galactose and the second monosaccharide, so that the second monosaccharide is galactose, galacturonic acid, glucuronic acid, mannose, glucose, arabi- nose, xylose, or rhamnose, and that endo-β- 1 ,2-galactanase is able to degrade this bond.

4. A method according to any one of claims 1 to 3, characterized in that in the polysaccharide, there is a β-1, 2 bond between galactose and galacturonic acid, and that endo-β- 1 ,2-galactanase is able to degrade this bond.

5. A method according to any one of claims 1 to 4, characterized in that the molecular weight of the endo-β- 1 ,2-galactanase enzyme is 110-130 kDa.

6. A method according to any one of claims 1 to 5, characterized in that the pH optimum of the endo-β- 1,2-galactanase enzyme is within 5-6.5.

7. A method according to any one of claims 1 to 6, characterized in that the enzyme treatment is carried out at a temperature of 20 - 60°C, preferably at 35 - 55°C, and most preferably at 35 - 50°C.

8. A method according to any one of claims 1 to 7, characterized in that the enzyme treatment is carried out at a pH of 4 - 8, preferably at a pH of 5 - 7, and most preferably at a pH of 5.5 - 6.0.

9. A method according to any one of claims 1 to 8, characterized that the treating time is from 5 min. to 10 days, preferably from 20 min. to 1 day, and most preferably from 30 min. to 2h.

10. A method according to any one of claims 1 to 9, characterized in that the amount of enzyme used is 50 - 500000 AU/g, preferably 500 - 50000 AU/g, and most preferably 1000 - 10000 AU/g of polysaccharide.

11. A method for preparing monosaccharides of oligosaccharides, characterized in that the microbial polysaccharide is brought into contact with an enzyme preparation comprising endo-β- 1 ,2-galactanase.

12. A method according to claim 11, characterized in that, in the polysaccharide, there is a β-1, 2 bond between two monosaccharides, of which the one is galactose and the other one galactose, galacturonic acid, glucuronic acid, arabinose, xylose, mannose, glucose or rhamnose.

13. A method according to claim 11 or 12, characterized in that the enzyme preparation comprises more than 200 AU/ml, preferably more than 500 AU/ml of endo-β- 1,2- galactanase.

14. A method according to any one of claims 11 to 13, characterized in that the molecular weight of the endo-β- 1 ,2-galactanase enzyme is 110 - 130 kDa.

15. A method according to any one of claims 11 to 14, characterized in that the pH optimum of the endo-β- 1 ,2-galactanase enzyme is within 5 - 6.5.

16. A method for treating slime deposits containing microbial polysaccharides, characterized in that the slime deposit is brought into contact with an enzyme preparation comprising more than 200 AU/ml of endo-β- 1 ,2-galactanase.

17. A method according to claim 16, characterized in that the enzyme preparation comprises more than 500 AU/ml of endo-β- 1 ,2-galactanase.

18. A method according to claim 16 or 17, characterized in that in the polysaccharide, there is a β-1, 2 bond between galactose and the second monosaccharide, so that the second monosaccharide is galactose, galacturonic acid, glucuronic acid, arabinose, xylose, mannose, glucose or rhamnose, and that endo-β- 1 ,2-galactanase is able to degrade this bond.

19. A method according to any one of claims 16 to 18, characterized in that, in the polysaccharide, there is a β-1 ,2 bond between galactose and galacturonic acid, and that endo-β- 1 ,2-galactanase is able to degrade this bond.

20. A method according to any one of claims 16 to 19, characterized in that the molecular weight of the endo-β-l,2-galactanase enzyme is 110 - 130 kDa.

21. A method according to any one of claims 16 to 20, characterized in that the pH optimum of the endo-β- 1 ,2-galactanase enzyme is within 5 - 6.5.

22. A method according to any one of claims 16 to 21, characterized in that the enzyme treatment is carried out at a temperature of 20 - 60°C, preferably at 35 - 55°C, most preferably at 35 - 50°C.

23. A method according to any one of claims 16 to 22, characterized in that the enzyme treatment is carried out at a pH of 4 - 8, preferably at a pH of 5 -7, most preferably at a pH of 5.5 - 6.

24. A method according to any one of claims 16 to 23, characterized in that the treating time is from 5 min. to 10 days, preferably from 20 min. to 1 day, most preferably

25. A method according to any one of claims 16 to 24, characterized in that a slime deposit is treated with an enzyme preparation, and before or after or simultaneously with at least one of the following enzymes: protease, β-glucanase, mannanase, chitinase, levanase, hemicellulase, pectinase, lipase or lysozyme.

26. A method according to any one of claims 16 to 25, characterized in that the slime deposit is treated with an enzyme preparation, and before and after or simultaneously with at least one biocide, the active ingredient of which is selected from among the following: glutaraldehyde, DBNPA, MBT, chloro-2-methylisothiazoline-3-one, methyl-4- isothiazoline-3-one, Bronopol, dazomet, sodium hypochloride, peracetic acid or BCDMH (bromochlorodimethyl-hydantoin).

27. A method according to any one of claims 16 to 26, characterized in that the method is used to degrade slime deposits in the process industry.

28. A method according to any one of claims 16 to 27, characterized in that the method is used to degrade slime deposits in paper machines.

29. A method according to claim 28, characterized in that the enzyme preparation is dosed into the circulation water of the paper machine.

30. A method according to claim 28 or 29, c h a r a c t e r i z e d in that the amount of the dosed enzyme is 5 - 10000 AU/litre, preferably 20 - 5000 AU/litre, most preferably 200 - 2000 AU/litre of circulation water.

31. An enzyme preparation, characterized in that it comprises more than 200 AU/ml, preferably more than 500 AU/ml of endo-β- 1 ,2-galactanase.

32. An enzyme preparation according to claim 31, characterized in also comprising additives, such as stabilizing agents and, optionally, buffers.

33. An enzyme preparation according to claim 31 or 32, characterized in that it is intended to be used for degrading microbial polysaccharides in the paper machine environment and, in addition to endo-β- 1 ,2-galactanase, comprising stabilizing agents that are suitable for the chemical pulp or pulp industry and, optionally, any of the following: buffers, one or more chemicals, such as a dispersing agent, a biocide or one or more other enzymes.

34. An enzyme preparation according to any one of claims 31 to 33, characterized in that the molecular weight of the enzyme is 110 - 130 kDa, and the pH optimum within 5

-6.5.