FI109921B - Method and enzyme preparation for process industry - Google Patents

Description

109921

The present invention relates to a process for the degradation of microbial polysaccharides according to the preamble of claim 1.

The invention also relates to a process for the preparation of monosaccharides or oligosaccharides according to the preamble of claim 13.

10

The invention further relates to a method for treating mucus collections containing microbial polysaccharides according to the preamble of claim 17.

The invention also relates to an enzyme preparation for use in the degradation of microbial polysaccharides 15 in the process industry as defined in the preamble of claim 33 for use in the preparation of mono- or oligosaccharides according to the preamble of claim 35.

The formation of microbial deposits or biofilms on various surface materials is a common problem in the process industry. The general tendency of bacteria to grow on biofilms adheres to surfaces, leading to the formation of mucus deposits, · | · 'Especially in a paper machine environment. Paper machines provide excellent growth conditions • · · • · ·. microbes, because biofilms allow microbes to grow in the low-flux streams found there.

• · · 25. : 1. Biofilms generally consist of mixed populations of microbes, polymers produced by microbes, in addition to wood fiber and inorganic material. Microbial polysaccharides are an essential part of the biofilms of sleep. Polysaccharides produced by microbes contained in biofilms • '1': binding microbial cells on surfaces and insoluble matter precipitated on biofilm and. 1 · ·, 30 consists of microbes, precipitated materials (including fibers) and polysaccharides ....: a matrix which in turn protects microbial cells, stabilizes biofilm, increases its viscosity and protects biofilm from drying out. Fiber carbohydrates often form the main fraction of biofilm carbohydrates. Increased amounts of sugars typical of bacterial heteropolysaccharides indicate the presence of bacterial polysaccharides in mucus deposits.

5 The paper machine environment is constantly carrying bacteria along with raw materials (water, fibers, especially mechanical pulp and recycled fiber) and chemicals used in papermaking. The paper machine environment is favorable due to the temperature suitable for microbial growth (30-50 ° C) and pH (4-10) and the presence of nutrients flowing into the paper machine along with pulp and paper chemicals. Therefore, paper machines 10 always have microbes. Väisänen et al. (1998) found that Bacillus coagulans and other BacillusAapt, Burkholderia cepacia, Ralstonia pickettii were the most common microbes in the wet end of a papermaking machine. Paper chemicals included Aureobacterium, B. cereus, B. licheniformis, B. sphaericus, Bordetella, Hydrogenophaga, Klebsiella pneumoniae,

Pantoea agglomerans, Pseudomonas stuzeri, StaphylococcusAajeja. Of the pink limes that accumulated in the wire region and in the printing sector, the most common bacteria were Deinococcus, Aureobacterium, and BrevibacteriumAajit. 91 of the 131 strains tested were found to degrade raw materials for paper making. Colored mucus consisted of Deinococcus, Acinetobacter, and Methylobacterium (pink), Aureobacterium, Pantoea, and Ralston (yellowish) and Microbulbiferxxx (brown).

·. ·. 20 • · '/ Biofilms cause a variety of disadvantages in the process industry. flow rates • · ·. reduced pipeline clogging, corrosion and increased energy consumption.

• · ·. ·. : On paper machines, microbial seams can also cause paper punctures, web breaks. ·: *. or impair drainage. Reducing the use of water in papermaking has increased. ': *. 25 nutrient levels in circulating water and thus the conditions for biofilm formation have increased. Biofilm bacteria are more resistant to commonly used paper machines: '· antimicrobial agents than free cells. Mucus deposits act as a continuous source of infection, transferring contaminating organisms to the recirculated water and even to the final product. ·. ·. Many microorganisms also found in biofilms decompose. · · ·. 30 paper materials such as cellulose fibers, starch, casein and glue can. causes degradation of the product by producing odors, flavors, undesirable discoloration and microbiological contamination.

On paper machines, an attempt has been made to solve the air problem by using various substances toxic to microbes 109921 3, or biocides, together with dispersants, and by finding a suitable biocide combination for each machine. However, many biocides will be severely restricted in the near future due to their toxicity, so there should be some alternative solution to the mucus and microbial problems in paper machines.

5

Biofilm-degrading enzymes have been proposed to replace or enhance the effect of conventional biocides. In some cases, levanase enzyme has been found to act to control deposits. However, its effect is limited to the fructose polymer levan, which is usually a very minor component in paper machine biofilms.

10

Attempts have also been made to solve the Airborne Problem of Paper Machines using commercially available enzymes in various combinations optimized on a case by case basis. However, the enzymatic methods currently in use have not satisfactorily solved the Airborne Problem of paper machines.

Rättö et al. (1998) have analyzed the carbohydrate compositions and protein and ash contents of mucus in a paper machine. Samples contained microbiological and chemical clusters containing microbes and microbial polysaccharides in addition to pulp fibers and inorganic compounds. Wood-derived carbohydrates, cellulose and xylan, were. 20 major components of carbohydrates. Increased levels of arabinose, galactose, • «! iI; * mannose, rhamnose or fructose depicted the presence of bacterial polysaccharides in ·· _ collections.

• * * • · *, ·: ·. Buchert et al. (1998) and Rättö et al. (1998) have isolated samples from a paper machine ♦ 25 collection and isolated from them mucus-producing microbial strains. This is the case

Klebsiella pneumoniae is common. Klebsiella species were found to produce hetero-: polysaccharides with varying amounts of neutral sugars and uronic acids such as · '. galactose, glucose, mannose and galacturonic acid. Isolated bacterial polysaccharides, *, *, were used to locate soil and microbial strains capable of breaking down polysaccharides. · ·. 30 compost samples. A polysaccharide-enriched mixed culture of a strain of K. pneumoniae was active against poly- * t-M saccharides produced by some strains of K. pneumoniae. In biofilm experiments, a cell-free enzyme preparation prepared from mixed culture cells by sonication inhibits the formation of a matrix containing cellular aggregates in steel plates.

4, 109921

The above publications do not describe purification or characterization of polysaccharides produced by microbes isolated from paper machines. The isolation of enzymes produced or produced by microbial cultures isolated from soil and compost samples and Karak-5 terisation are also not described in the above publications. The mode of action of the enzyme or enzymes is not described. Prior art publications do not describe a well-functioning solution to the Air Problem in paper machines or other process industries and there is no commercially available product that could be used to solve the Air Problem.

It is an object of the present invention to overcome the disadvantages of the prior art and to provide a novel method for the degradation of polysaccharides. In particular, it is an object of the invention to provide an enzyme having endo-β-1,2-galactanase activity. To our knowledge, such enzyme activity has not been described previously.

More particularly, the invention relates to a process for the degradation of microbial polysaccharides by means of an enzyme preparation, characterized in what is stated in the characterizing part of claim 1.

According to the invention, an enzyme preparation 20 containing the endo-P1, 2-galactanase enzyme can be used to cleave polysaccharides having two monosaccharide units with a p1, 2 bond, one of the monosaccharides being galactose and the other being: galactose, galacturonic acid, , glucose, arabinose,. “Xylose or rhamnose. According to a preferred embodiment of the invention, the enzyme: is used to cleave the polysaccharides in which the β-1,2 bond is between galactose and galactose · · · · · · · · · · · · · U ur.

• · · * · * * »»

The enzyme preparation of the invention can be used to prepare various mono- or oligo- saccharides. More specifically, the invention thus relates to a process for the preparation of mono- or oligosaccharides according to the characterizing part of claim 13: 30. The process of the invention can be used to prepare "'': mono- or oligosaccharides by enzymatic hydrolysis of microbial polysaccharides.

. '. : Certain oligosaccharides may be used, for example, as probiotics.

A particularly important application is the use of an enzyme preparation according to the invention

S

More particularly, the present invention relates to a process for treating mucus collections containing microbial polysaccharides according to the characterizing part of claim 17. The enzyme preparation of Εηάο-β-1,2-5 galactanase can be used to degrade other biofilms of the process industry. Mucus containing bacterial polysaccharides is also found, for example, in clean water and sewage pipelines, and it is particularly advantageous to use an enzyme preparation containing endo-β-1,2-10 galactanase to solve the air problem on paper machines.

The enzyme preparation used in the process industry is characterized in what is defined in the characterizing part of claim 33 and the enzyme preparation used in the preparation of mono- or oligosaccharides 15 is characterized in what is defined in the characterizing part of claim 35.

The enzyme preparation of the invention is preferably used in combination with enzyme preparations containing other enzyme activities. Such enzyme activities include, for example, protease, β-glucanase, mannanase, chitinase, lysozyme, hemicellulase,. : ': pectinase, lipase and levanase containing enzyme preparations commercially available: * ·': available. The appropriate enzyme combination is selected according to the application.

: *: The invention is useful in many respects, especially in the process industry. Flow rates can be increased, the risk of pipeline clogging is reduced, corrosion occurs less, and energy consumption can be reduced. On paper machines, slime reduction reduces web breaks and improves drainage. Paper pollution due to perforation is reduced and microbial migration with the end product to the end user or ... · consumer is reduced.

: 30 "'" Endo-β-1,2-galactanase could be used to keep clean water and sewage pipelines »· 109921 6 L possibly in combination with other enzyme activities and / or chemicals.

Since the enzyme endo-β-galactanase has not been described previously, the use of the enzyme endo-β-galactanase according to the invention in the preparation of mono- and oligosaccharides provides a novel route for the preparation of certain oligosaccharides or sugars.

Deposit information 10 Klebsiella pneumoniae VTT-E-97860 was deposited with the Budapest Depository, DSMZ Deutsche Sammlung von Mikroorganismen, Mascheroderweg 1b, D-38124 Braunschweig, Germany, on September 28, 1999 under the Budapest Treaty and received the deposit number DSM 13058.

The invention will be explained in more detail by the following detailed description and by reference to experimental examples.

The term "polysaccharide" refers to large molecules formed by a plurality (more than 20) units of monosaccharide molecules. By oligosaccharides is meant. . 20 chains of a few (2-20) sugar units. In particular, the polysaccharide of the present invention refers to a polysaccharide containing two <$ · ί ·. # Monosaccharides and a P-1,2 bond between them, one of the monosaccharides being · · ·. ·,: Galactose and other galactose, galacturonic acid, glucuronic acid, mannose, glucose,

AA

. : ·. arabinose, xylose or rhamnose. According to a preferred embodiment of the invention

• A A

The term polysaccharide as used herein refers to a polysaccharide containing a P-1,2 bond

A

between galactose and galacturonic acid.

A ·

AA

• ”]: The enzyme activity described in this invention is enriched in soil and compost samples. y. by means of a polysaccharide consisting of oligosaccharide units containing galactic acid. 30 uronic acid, two mannose and galactose, and a side chain of mannose containing. *. - pyruvic acid in acetal form, bound with galactose and galactose,

»AA

, · · ·. there is a β-1,2 bond between uronic acid. The structure of the polysaccharide is thus as follows:

• A

I 1G9921 j 7 -2) -aD-GalpA- (1 -> 3) -cc-D-Manp- (1 -> 2) -aD-Manp- (1 -> 3) -pD-Galp- (1 - »/ AD-Manp- (1-4) 4 6 5 \ / c / \ h3c cooh 10 Polysaccharide is produced by strain Klebsiella pneumoniae VTT-E-97860 deposited in DSMZ strain collection of Microorganism und Zellkulturen on 28 September 1999, Accession No. DSM 13058. The polysaccharide can be produced by the K. pneumoniae strain as described in Example 1. Other Klebsiella pneumoniae karmas are capable of producing the corresponding polysaccharide, for example Dutton and Parolis (1986) have described a similar polysaccharide with Klebsiella.

However, the invention is not limited to the polysaccharide produced by the aforementioned Klebsiella pneumonie karma, or -Klebsiella pneumoniae child, and its use for endo-β-1,2-galactanase. . 20 microorganisms, but endo-β-1,2-galactanase-producing microorganisms can be enriched with polysaccharides derived from other microbes. The polysaccharides are structured so that they have two mono-. a saccharide and a β-1,2 bond between them, one of the monosaccharides being galactose and the other galactose, galacturonic acid, glucuronic acid, mannose, glucose, arabinose, xylose or rhamnose, preferably by means of a polysaccharide wherein β-1,2- the bond is between galactose and galcturonic acid. Correspondingly, the enzyme according to the invention is capable of degradation. and polysaccharides in various environments having said structure.

»: Y: According to the invention, the polysaccharide can be isolated from the mucus collections of the paper machine from isolated mucus-producing bacterial strains. According to a preferred embodiment of the invention, the polysaccharide is isolated from growth media of Klebsiella pneumoniae because:. · · ·, These bacteria were found to be widespread among mucus producing bacteria.

109921 8

In the studies underlying the invention, the ability of the strains to produce polysaccharides was examined by plate spreading. It was then found that the sucrose-containing medium commonly used to screen for mucus-producing bacteria isolated the levan producers of the mainly fructose polymer. Glucose was chosen as the carbon source for the screening medium in the future to prevent levan formation. Polysaccharides were further produced by shaking cultures in solution cultures at selected strains growing at initial screening, with slime colonies. The production of polysaccharide in solution cultures was monitored by measuring the viscosity of the medium or precipitating the polysaccharide formed from the growth medium.

10

Polysaccharide production strains selected for further testing were identified. It turned out that many polysaccharide producers isolated from paper machine samples previously represented! non-native bacterial species. Several strains of Klebsiella pneumoniae producing polysaccharides were found in the screening. When K. pneumoniae strains isolated from different paper machines were compared on the basis of ribotyping on each of them, each strain examined had a different skin type.

Polysaccharides were produced in agitator cultures on various media. In particular, the carbon source and nitrogen content of the substrate had an effect on the formation of polysaccharides. When sucrose was used as the 20 carbon source, many strains studied produced levan. In order to isolate non-* · # # levanase enzyme, the production medium was further selected as the nitrogen-restricted glu- «1 • ·! .. co-medium on which the polysaccharides produced were various heteropolysaccharides. The polysaccharides were isolated from the growth solutions by ethanol precipitation. The precipitated polysaccharides were dissolved in t · '; · In water and treated with protease (Neutrase, Novo) to remove any traces of protein. ·: ·. 25 sex and was precipitated again. After precipitation, some of the samples were rich in phosphates. Samples were dialyzed to remove salts. Finally, the polysaccharides were freeze-dried.

• · «·« • ·. 1 1 1. A mucus-producing strain of Klebsiella pneumoniae (VTT-E-97860) was selected for further studies. Polysaccharide concentrations of 1 · ', 30 clubs were isolated from K. pneumoniae growth medium by ultrafiltration and precipitation as described in Example 1. To precipitate the impurities, the concentrated polysaccharide solution was subjected to a heat treatment to impurity the impurities. precipitated and the solution viscosity decreased. The decrease in viscosity allowed for further concentration, after which the polysaccharide was precipitated with ethanol.

! 109921 9

The structure of the polysaccharide was determined by NMR. Based on the spectra of enzymatically hydrolyzed samples, the structure of the polysaccharide could be determined. As noted above, a capsular polysaccharide of similar structure has previously been described by K. pneumoniae serotype K3 (Dutton and Parolis, 1986). However, the presence of this polysaccharide in biofilms of the process industry has not been previously reported and the enzyme which degrades this polysaccharide has not been isolated or characterized.

To determine and isolate mucus polysaccharide-degrading enzymes from soil and compost samples, the ability of both pure strains and mixed cultures to degrade poly-10 saccharides was investigated in enzyme production assays. Screening of enzyme-producing strains was performed as microtiter plate or test tube cultures on medium in which the polysaccharide to be studied was the sole carbon source. The ability of the strains to be screened to degrade the polysaccharide was investigated e.g. by monitoring the turbidity of the culture medium and changes in its viscosity.

15

Initially, a variety of bacteria isolated from papermaking machinery, paper machines or mills, as well as bacteria known to produce various polysaccharide-degrading enzymes, were selected for screening. During the screening, several bacteria were found that were able to use the fructose polymers produced on sucrose medium by strains of B. licheniformis and P. fluorescens as the sole carbon source. Hydrolysis experiments confirmed that the growth solutions of these strains contained enzyme activity which hydrolyzed, e.m. levan polysaccharides and commercial levan to produce fructose as a hydrolysis product.

! * *. None of the bacteria selected for this screening were able to degrade the polysaccharide of K. pneumoniae strain VTT-E-84211 and VTT-E-97860.

• »· 25 • · ·

Since K7eZwze // a polysaccharides were not produced by the collections examined, ·. enzyme, screening of enzyme producing strains was continued by enriching the factories. * '·. mixed samples of natural samples which were able to use isolated heteropolysaccharides as the sole carbon source. The ability of the cultures to degrade the polysaccharide was monitored t! * T ..! - 30 on a liquid medium based on changes in viscosity. This screening method proved to work.

»| *

• I

'' * Mixed cultures of heteropolysaccharides were isolated from soil and compost samples.

These cultures were maintained on media in which bacterial polysaccharide was the sole source of carbon 10 10 9921, since media containing other carbohydrates rapidly lost their desired property for a more unknown reason, possibly due to a change in culture species. In many cases, the enzyme was not secreted into the growth medium but was attached to the cell surface, from which it was detached by gentle sonication. Enzyme activity 5 was determined by a viscometric method, and the activity unit (AY) was defined as the enzyme concentration that caused a decrease in viscosity of 1 cP / min.

Thus, the present invention also relates to a method for isolating enzyme-producing microorganisms capable of degrading selected mucus-producing polysaccharides. The method comprises the steps of: - collecting soil and compost samples from a variety of media, - suspending samples in a buffer solution such as physiological saline, - inoculating with a solution or directly with a soil or compost sample a culture solution containing a selected polysaccharide containing two monosaccharides, pl, wherein one of the monosaccharides is galactose and the other galactose, galacturonic acid, glucuronic acid, mannose, glucose, arabinose, xylose or rhamnose, - incubate the medium under suitable conditions and measure the viscosity of the culture filtrate, - select for further experiments, 20 cultures with the fastest and highest viscosity decreases,. ·. separating the cells and re - culturing the culture under appropriate conditions on medium which:. *: ·. contains the selected polysaccharide, - collecting the culture medium or separating the cells from the culture medium, suspending them in a buffer or medium, sonicating the cells, separating the cells and collecting the filtrate, · T: - contacting the culture solution with the polysaccharide to be examined enzyme to degrade the selected polysaccharide, ': ·': - the cells of the culture are harvested, optionally from the culture medium or '. ,, · sonication solution has been found to be capable of degrading the selected polysaccharide and; . 30 - grow cells on medium containing the selected polysaccharide.

• · ·, · | j The method of isolation described in this invention is reproducible even if starting from • M »» ....: soil or compost samples. Although soil samples were different and compost samples were derived from compost samples containing different raw materials and at different stages, more than 80% of the 109921 11 samples contained microbes that were able to degrade the polysaccharide when used. polysaccharide as their sole carbon source in their substrate. The ability of the mixed culture to degrade the polysaccharide was maintained from one generation to the next through continuous culture on a medium which! contained the selected polysaccharide as the sole carbon source.

! I 5

The culture solution or cell extract of the microbial culture isolated according to the invention obtained, for example, by sonication of the cells and recovery of the cell-free solution contains the enzyme endo-β-1,2-galactanase based on the observations made in the invention as calculated as 0.02-0.03 mg / ml protein or enzyme activity. calculated from 50 to 200 AU / ml. For the preparation of an industrially usable enzyme preparation, it is preferable to concentrate and / or purify the enzyme preparation by means of one or more protein purification steps so that the enzyme is present in an amount of endo-β-1,2-galactanase per ml of protein , preferably 0.250 mg / ml, most preferably 0.5 mg / ml. Calculated as enzyme activity, the enzyme activity is greater than 15,200 AY / ml, preferably greater than 500 AY / ml, most preferably greater than 1,000 AY / ml.

However, the present invention is not limited to the enzyme isolation method disclosed herein, but the enzyme may be isolated or obtained by other methods. The enzyme according to the invention can be produced by microorganisms into which the gene encoding the enzyme 20 has been introduced or which naturally produce the enzyme but which are * * ;;; · Productivity may have been improved by mutation or genetic engineering.

A microbe from a culture containing cells of one or more microbial species can isolate a gene encoding endo-β-1,2-galactanase and transfer it to a suitable host microorganism. · · T *: t to 25 organisms, preferably under effective control regions.The producer organism is * s # • 1 (preferably such that it is capable of secreting the enzyme outside the cell, min.): The culture medium can be used as such or only to a minor extent. · Modified by measures such as concentration, addition of stability enhancing agents and buffers, etc.

• I · 30

By the term "enzyme preparation" is meant any preparation containing at least '; '·' One enzyme. The enzyme preparation may thus be a culture solution or a cell extract containing one or more enzymes, an isolated enzyme or a mixture of one or more enzymes. In addition to enzyme activity, the enzyme preparation preferably contains 1G9921 12! auxiliaries commonly used in enzyme preparations for use in the process industry. Such adjuvants include, for example, buffers and stabilizers. In a paper machine application, these excipients are preferably substances used in pulp or pulp industry applications. If the enzyme preparation is intended for other process industry or for the treatment of clean water or sewage pipelines, the excipients are selected for the intended use.

To characterize the polysaccharide hydrolyzing enzyme produced by K. pneumoniae strain VTT-E-97860, the enzyme was purified by chromatography as described in Example 10 2. Purification consisted of two chromatographic steps based on hydrophobic interactions followed by final anion exchange chromatography and purification.

The purified enzyme was used to hydrolyze the polysaccharide produced by K. pneumoniae strain VTT-E-15 97860 and used as a substrate to determine which linkage the enzyme cleaved from the polysaccharide. The enzyme was found to cleave the β-1,2 bond between galactose and galacturonic acid.

When the mode of action of an enzyme is known, different combinations of enzymes may be designed specifically for a variety of uses.

«· •. The enzyme according to the invention is preferably an endo-β-1,2-galactanase having a molecular weight of 11. · 'Weight is 110-130 kDa. The pH optimum of the enzyme of the invention is in the range 5-6.5, * ': preferably the pH optimum is about 5.5. The pH stability of the enzyme at 40 ° C is between pH 5 and 7,; Preferably between 5.4 and 6.0.

. . Amount of enzyme required to hydrolyze galactose in polysaccharides • · · • · ♦ *. / and β-1,2-bonds between galacturonic acid or the like between galactose and galactose, galact uronic acid, glucuronic acid, mannose, glucose, arabinose, xylose or rhamnose, · 50 · 30, preferably 500- 50,000 AY / g, most preferably 1000-10,000 AY / g polysaccharide.

The hydrolysis of the polysaccharides is carried out at 20-60 ° C, preferably 35-55 ° C, most preferably 35-50 ° C. The pH of the hydrolysis solution is 4-8, preferably pH 5-7, 109921 13 most preferably pH 5.5-6.0. The hydrolysis time may be from 5 minutes to 10 days, depending on the application used and the amount of enzyme used. In the preparation of mono- or oligosaccharides, the hydrolysis 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.

5

The enzyme of the present invention is suitable for degradation of biofilms and other mucus deposits in various environments. The enzyme preparation, which may contain suitable excipients, such as buffers, required in the environment, is then contacted with the mucus under conditions suitable for the enzyme to function.

10

In the method of the invention, the mucus is contacted with an enzyme preparation containing endo-β-1,2-galactanase, alone or in combination with other enzyme activities. The treatment temperature is 20-60 ° C, preferably 35-55 ° C, most preferably 35-50 ° C. The treatment is carried out at pH 4-8, preferably at pH 5-7, most preferably at pH 5.5-6.0. The enzyme preparation is added to the circulation water, especially on paper machines. A suitable amount of enzyme is then 5 to 10,000 AY / liter of circulating water, preferably 20 to 50,000 AY / liter, most preferably 200 to 2,000 AY / liter of circulating water. Because processing is not clearly terminated at a specific time, processing time varies from a few hours to several days.

The Endo-β-1,2-galactanase treatment may be performed separately or concurrently with, or prior to, other enzymatic treatments. It is particularly advantageous to combine endo-β-1,2-galactanase treatment with protease, β-glucanase, mannanase, chitinase, leva-1 ”. nasal, hemicellulase or lysozyme treatment. When endo-β-1,2-galactanase is used concurrently with the above-mentioned enzymes, it is preferable to use an enzyme preparation with the addition of endo-β-1,2-galactanase. galactanase or produced by a strain * that is genetically modified to produce large quantities of the coenzyme to make the enzyme ready. ' # | the tea would have substantially high endo-β-1,2-galactanase activity.

• · • ·, f ·. The enzyme treatment according to the invention can also be incorporated into one or more keramins. 30 for all processing. This may be, for example, a biocide treatment wherein the biocide is selected from: • 1 I · 1 · I · · · · 109921 14: glutaraldehyde, DBNPA, MBT, chloro-2-methylisothiazolin-3-one, methyl-4-isothiazolin 3-One, Bronopol, Dazometh, Sodium Hypochlorite, Peracetic Acid BCDMH (Bromochlorodimethylhydantoin).

5 Examples Example 1 Isolation of polysaccharide 10

Microbial strains producing mucus polysaccharides were derived from different strain collections or mucus samples from a paper machine. The ability of the strains to produce polysaccharides was examined by plate decomposition. In the early stages of the investigation it was found that the sucrose-containing medium was used to isolate mainly the levan producers, so that in the future glucose was chosen as the carbon source for the screening medium. Polysaccharides were further produced by shaking cultures in solution cultures at selected strains growing at initial screening in plate-like slime colonies. Production of polysaccharide in solution cultures was monitored by measuring the viscosity of the medium or precipitating the polysaccharide formed from the growth medium.

Polysaccharide production strains selected for further experiments were identified (API, FAME, ribotyping, '; 16S rDNA sequencing). Many poly-1: ·: saccharide producers isolated from paper machine samples were found to represent bacterial species not previously described. Screening found. "': Multiple polysaccharide producing strains of Klebsiella pneumoniae. Comparing different paper: * ·: strains of K. pneumoniae isolated from machines with basic ribotype::: 25 showed that they were of different ribotype .

I »·

Polysaccharides were produced in agitator cultures on various media. Especially the chassis. The carbon source and the nitrogen content had an effect on the formation of polysaccharides.

In order to isolate the non-levanase enzyme, the following medium was selected as the production medium:::: 30 glucose-restricted medium with polysaccharides of various heteropoly-: saccharides. The polysaccharides were isolated from the growth solutions by ethanol precipitation. Precipitated. ·. ·. polysaccharides were dissolved in water and treated with protease (Neutrase, Novo) to remove any traces of protein. "*. After precipitation, some of the samples 109921 15 were rich in phosphates. To remove salts, the samples were lyophilized. Finally, the polysaccharides were lyophilized.

Polysaccharide yields in solution cultures were quite low, most often below 1 g / 1 5 i | The total carbohydrate content of the polysaccharide preparations was determined by the phenolic sulfuric acid method. The monosaccharide and uronic acid composition of the polysaccharides was determined by HPLC after acid hydrolysis. When hydrolyzing the polysaccharides, it was found that the optimum hydrolysis conditions should be determined for each polysaccharide: a suitable acid concentration and a hydrolysis time. Acid hydrolysis was generally performed with 0.5N to 2N sulfuric acid at 105 ° C. The hydrolysis time was 2-5 h. To determine the fructose content, the polysaccharides were hydrolyzed with 2N TFA at 20 ° C for 1 h.

Experiments were continued on Klebsiella pneumoniae strain VTT-E-97860, which produced highly li-15 colonies on growth plates. To produce extracellular polysaccharides, the strain was grown on liquid medium in shake flasks and in a laboratory fermenter. The yield of polysaccharide in the shake flasks (measured as the total carbohydrate content of the freeze-dried polysaccharide preparation) was about 1 g / L of culture medium. The polysaccharide consisted of mannose, galactose and galacturonic acid. It also contained some glucose and methyl glucuronic acid (Table 1).

• · · · · / ”Table 1. Carbohydrate yield and sugar composition of K. pneumoniae polysaccharide in shake flask cultivation.

· ". * ·: Saanto Man Gal Glc GalA MeGluA

v: (g r1) (%) (%) (%) (%) (%) '* 0.95 62.6 23.5 0.9 12.10 0.9. · · ·. 25 Klebsiella pneumoniae strain VTT-E-97860 produced 5 l of polysaccharide. '. In an Erlenmeyer flask containing 11 media. The medium contained 20 g Γ1 glucose, 0.5 g λ! Γ1 yeast extract (Difco), 0.6 g Γ1 (NH4) 2 SO4 (Riedel-de Haen), 3.18 g Γ1 KH2PO4 (Merck), 5.2 g F1 K2HPO4 (Merck), 0.3 g F1 MgSO4x7H2O (Merck), 0.05 g CaCl2; ; (Merck) and 1ml / L nutrient solution (0.2g F1 ZnSO4x7H2O (Merck), 0.2g F1CuSO4x5H2O * 30 (Merck), 0.2g F1MnSO4xH2O (Merck), 0.2g F1 CoCl2x6H2O (Merck), 0.2g F1

FeSO 4 x 7H 2 O (Merck). The culture was grown for 5 days in a shaker (120 rpm) at 109921 at 16 + 30 ° C, after which NaCl (0.9%) was added to the culture medium to facilitate the removal of the partially bound polysaccharide. The medium was homogenized with a mixer (Ystral). The cells were separated by centrifugation. The polysaccharide was precipitated from supernatant 3 butter. ethanol at + 4 ° C overnight. The polysaccharide was separated by centrifugation and dissolved in water.

To remove protein impurities, soluble polysaccharide was treated with protease (Neutrase, Novo) at + 37 ° C for 1 hour. The polysaccharide was precipitated with ethanol, dissolved in water, dialyzed (MWCO 12-14 kDa, Medicell, London, UK) against water and freeze-dried.

To produce the larger scale polysaccharide, fermentations were made in a Chemap 10 CF 2000 laboratory fermenter with a 10 L growth volume and a pilot-size fermenter (1200 L growth volume). The fermentation conditions were: temperature 30 ° C, pO 2> 10% (Chemap) or pC> 2> 30% (pilot), pC> 2 adjusted with stirring, pH 7-8.5 (adjusted by automatic addition of NaOH). The medium contained 30 g / l glucose, 0.5 g / l yeast extract and the same salts as in Erlenmeyer flask cultivation. The amount of polysaccharide produced in the load was chained from the amount of mannose analyzed from the centrifuged growth broth by HPLC after acid hydrolysis (2N H2SO4, 105 ° C, 2h). The cells were separated by centrifugation, the broth was concentrated by ultrafiltration, and the polysaccharide was precipitated from the supernatant with ethanol.

20 Gel Electrophoresis •. ' '· The proteins in the samples were characterized by SDS electrophoresis on a 10% polyacrylic • ...: amide gel according to Laemmlin (1970) using the Ready Gel Cell system (Bio-Rad

:. ' i Laboratories, Hercules, CA, USA) and following the manufacturer's instructions. Calibration Alloy 7707S

'25 (Biolabs, NE, USA) was used as a standard.

•

Chemical analysis *; · Protein concentration was determined by spectrophotometry by measuring the adsorption of the samples:; 30 at 280 nm. The concentration was obtained on the assumption that the absorbance value of '1 unit' corresponded to a protein concentration of 1 mg / ml.

The total carbohydrate content of the polysaccharide preparations was determined by the phenol-sulfuric acid method (Dubois et al. 1956). Determination of the monosaccharide-uronic acid composition 109921 µl of the isolated polysaccharide was hydrolyzed with 2NtSO 4 at 105 ° C for 2h. The hydrolyzate was analyzed by HPLC (Dionex).

Structure of the polysaccharide

For structural analysis and hydrolysis experiments of the polysaccharide, the polysaccharide was purified with an anion exchange resin (DEAE Sepharose FF, Pharmacia-LKB, Uppsala, Sweden). The resin was equilibrated with a buffer of pH 7 and a conductivity of 190 pS / cm. The polysaccharide was mixed with the resin and shaken for 1 h at + 20 ° C. The resin was separated by centrifugation.

10

The structure of the polysaccharide was determined by 2D NMR after enzyme treatment. The polysaccharide was treated with the purified enzyme endo-β-1,2-galactanase for 24 h (dose 280 mg / g polysaccharide), after which the mixture was boiled (5 min) and lyophilized. The freeze-dried hydrolyzate was dissolved in deuterium oxide (D20, 99.8 Atom%, Fluka) and the solution clarified by centrifugation. The sample was measured with 1 H and 13 C NMR spectra and 2D COSY, relayCOSY, TOCSY, HMQC and HSQC spectra at 50 ° C.

NMR analysis showed that the polysaccharide consisted of five sugar units having the following structure: 20. Y: -> 2) -aD-GalpA- (1 -> 3) -aD-Manp- (1 -> 2) - <xD-Manp- (1 -> 3) -pD-Galp- (1 -> / aD-Manp- (l-> 4): .Y 4 6

Y * 25 W

: c Λ h3c cooh • · t · · • »109921 18

Example 2 ί

Enzyme Screening and Isolation Mixed cultures capable of disrupting the polysaccharide isolated in Example 1 were screened from soil and compost samples. To enrich the polysaccharide-degrading cultures, the samples were inoculated into a medium containing 5 g pne1 K. pneumoniae polysaccharide and 6.7 g Γ1 Yeast nitrogen base (Difco) in 50 mM potassium phosphate buffer, pH 7. After 12 days incubation at +30 ° C in a mixer (120 rpm) min'1) 750 μΐ cultures were inoculated into 15 ml of fresh medium and incubated for 3 days. The ability of the culture to degrade the polysaccharide was monitored by viscosity measurements (Brookfield DVII).

By screening microbes for polysaccharide-degrading enzyme activity from soil and compost samples by incubating samples on a medium containing K. pneumoniae polysaccharide 15 as the sole carbon collar as described above, polysaccharide-degrading activity in 6 mixed cultures was observed. Ground-isolated mixed culture number 3 was most effective in breaking down the polysaccharide during the second incubation. This culture was selected for enzyme production.). The mixed culture was stored in the culture medium at + 4 ° C until the enzyme was produced.

20, · - Table 2.

• .Sample I Incub. 5 d I Incub. 12 d II Incub. 1 d II Incub. 3 d '···' No Origin Viscosity (cP)

Check ^ 2 63 4Λ 4 ~ 6 «tl l" '1 Compost, straw 5.2 2.7' 2 Compost, paper 1.2 1.3 3.7 2.5 3 Country 5.0 1.1 1.7 1.0! .. * 4 Compost 1.1 1.2 4.1 1.3 '·' 5 Santa and leaves 5.2 1.2 2.1 1.2 6 Earth and leaves 5.0 5.3 ·; · '7 Compost and leaves 5.2 1.3 2.4 1.2 • * * 109921 19

Similarly, when using a K. pneumoniae polysaccharide other than that described above, it was generally found that cultures that degrade each microbial polysaccharide could be isolated from soil or compost samples by this method.

To produce the enzyme, mixed culture # 3 (VTT-E-97916) was incubated in shake flasks containing 500 ml of polysaccharide medium at + 30 ° C for 3-5 days. Cells were separated from the medium by centrifugation at + 4 ° C. The cells were suspended in 50 of 50 mM potassium phosphate buffer, pH 7. The suspension was sonicated to release the bound enzyme into the liquid phase. The cells were removed from the enzyme solution.

10

The following purification steps were performed at + 4 ° C. Purification was started by hydrophobic interaction chromatography (HIC). The conductivity of the enzyme solution was adjusted to 106 mS / cm and pH 6. The solution was applied to a butyl sepharose column (Butyl Sepharose FF, Pharmacia-LKB, 16 mm x 80 mm column) equilibrated to 15 with the same conductivity (106 mS / cm) and pH 6 with 0.8 M ammonium sulfate in 20 mM potassium phosphate buffer, pH 6. After feeding the sample, the column was washed with em and then the column was eluted with a decreasing linear gradient of ammonium sulfate (80 mL) from 0.8 M to 0 M in 20 mM potassium phosphate buffer, pH 6. The flow rate was 1.5 mL min -1 and 9 mL was collected over the entire run: n 20 fractions from which the polysaccharide degradation ability of K. pneumoniae VTT-E-97860 was measured.

..:: The active fractions were pooled and further purified by a second HIC purification step.

'. * The conductivity and pH of the sample were adjusted to equilibration buffer (1.0 M ammonium sulfate in 20 mM potassium phosphate, pH 6). A sample (93 mL) was applied to the same column previously equilibrated with 1.0 M ammonium sulfate in 20 mM potassium phosphate, pH 6. After sample loading, the column was washed with equilibration buffer and then eluted with a decreasing linear ammonium sulfate gradient (80 mL). 20 of M. , mM in potassium phosphate buffer (pH 6) to 0 M in 2 mM potassium phosphate buffer (pH 6).

!, 'The enzyme eluted as a single peak during the gradient and the active fractions were pooled.

'; ; ' The resulting enzyme-containing fraction (36 mL) was buffered in 20 mM potassium phosphate, pH 6.2; Gel filtration (Sephadex G-25 coarse, Pharmacia LKB, 50 x 150 mm). Enzyme 'V was further purified by anion exchange chromatography (AE) (DEAE Sepharose FF,

'' Pharmacia-LKB, 10 mm x 110 mm) column. The enzyme was bound to the resin at 20 mM

potassium phosphate, pH 6.2, at a flow rate of 0.8 mL min -1. The enzyme was eluted from column 109921 with a linear gradient from 0 to 0.15 M sodium chloride, during which the enzyme eluted as a single peak. The most active fractions (? 3 mL) were pooled (12 mL, AE fraction I) and the three fractions eluting before and after fraction I collection were combined into another sample (18 mL, AE fraction II). Both e.m. samples were concentrated by 5 ultrafiltration (Amicon, Amicon Diaflo, PM 10 membrane, Beverly, MA, USA), AE fraction I was concentrated 8-fold and AE fraction II was concentrated 22-fold.

The final purification step, gel filtration, was carried out at room temperature. The fraction was applied to a Sephracryl S-300 HR column (Pharmacia-LKB) equilibrated with 0.1 M sodium chloride in 50 mM potassium phosphate buffer, pH 6. The flow rate was 0.6 mL min -1 and 2.9 mL fractions were collected. The enzyme eluting from DEAE fraction II was divided into two different fractions (G fraction I and G fraction II) and the enzyme eluting from DEAE fraction II was combined into one fraction (G fraction III).

The total activity of the cell-free enzyme preparation obtained from 500 ml medium grown cells was 14 874 AU. The first purification step (HIC 1) resulted in a 25-fold increase in specific enzyme activity in a yield of 74%. The enzyme was then not properly bound to the resin and therefore elution was not optimal. The active fractions were collected and purified again (HIC2). This time, the enzyme bound and eluted as a single peak, resulting in the removal of about 50% of the protein impurities and loss of about 10% of total V activity. Active fractions eluting! * ··: Anion exchange chromatography columns were divided into two fractions. Fraction 1 contained four. * '': Most active fractions and fraction 2 three fractions eluted: '> · before and three fractions eluted after most active fractions ::: 25. After concentration by ultrafiltration, the purification factors were 229: (fraction 1) and 111 (fraction 2). In AE, 15% of the protein and almost 50% of the total activity was recovered. In SDS-PAGE, both fractions clearly produced an ambush. · Zone i, but several significantly weaker zones were also observed.

• »

Both fractions were further purified by gel filtration. In the gel filtration, some impurities were removed but at the same time much activity was lost and the purification factor :: decreased. Fraction 1 was divided into two separate fractions after gel filtration. Fraction 2; '; *; after gel filtration, the active fractions were collected into a single fraction. Further activity was lost in the concentration of fraction: ''. One thin »1 * band was detected on SDS-PAGE.

j 109921 21

Table 3. Purification of K. pneumoniae VTT-E-97860 polysaccharide-degrading enzyme. Purification step Volume. Total Protein Total Active Spes. activity Yield Purification (ml) (mg) (AU) (AU / mg prot.) (%)

Cell Free Extract 37 444 14874 34 1 HIC 1 95 13 10944 853 74 25 HIC 2 38 6 9302 1466 63 44 AE Fractionate 12.5 0.65 4200 6462 28 193

Fraction 18 0.63 1166 1851 8 55

Fraction I Cons. 1.4 0.39 3024 7687 20 229

Fraction II Cons. 0.8 0.38 1402 3720 9 111 GF Fraction 16 0.34 307 914 2.1 27

Fractionates! 11 0.22 111 504 0.7 15

Fractionation 14 0.31 134 436 0.9 13

Fraction I Cons. 1 0.05 58 1252 0.4 37

Fraction II Cons. 1.05 67 0.5

Fraction III Cons. 1 0.06 41 638 0.3 19

The protein had a molecular weight of 110-130 kDa as determined by mobility on SDS-PAGE and 5 gel filtration columns. The molecular weight of the MALDI mass spectrophotometer was about 117-120 kDa.

. . Enzyme Activity Assay • · ·, 10 Enzyme activity was measured as the ability of an enzyme to reduce K. pneumoniae VTT-E-97860 • »

viscosity of polysaccharide in 50 mM potassium phosphate buffer, polysaccharide substrate, pH

. y. 7 in 0.5% solution at + 30 ° C. In the assay, 50 μΐ of enzyme was added to 450 µL of substrate. The decrease in viscosity was monitored with a viscometer (Brookfield LVTDV-II CP, MA, USA) for 4 min. Viscosity was measured between 60-180 s after enzyme addition. The sample was diluted so that the viscosity decreased during measurement *, .. at a rate of 0.007 to 0.0035 cP / s. The activity unit (AU) was determined to be; V j is the amount of enzyme activity which, under the conditions of measurement, decreased the viscosity by 1 cP for 1 minute.

Determination of pH Optimal pH The pH optimum was determined by hydrolyzing 0.5% polysaccharide substrate in 100 mM acetic acid or phosphate buffer pH 3.5-8.4 with anion purified by 5 chromatography. 50 μΐ of the sample was added to 1 ml of substrate. To determine reference values, the experiment was also performed with an inactivated enzyme. All samples were incubated for 15 min and the enzymatic reaction was terminated by boiling the samples for 8 min. Viscosity was measured with a Brookfield viscometer. Activity at certain pH values was determined as the difference in viscosity between sample and control.

10

The enzyme had an optimum pH of about 5.5, and below pH 5 the enzyme activity decreased rapidly.

pH and thermal stability

pH and thermal stability of the purified and unpurified enzyme were determined as pH

between 4.0 and 8.8. The pH of the enzyme solution was adjusted with 25% acetic acid or 1M NaOH.

The enzyme was kept at 40 ° C or 50 ° C (purified only) for 1 hour. 50 μΐ of enzyme was added to 1 ml of 0.5% polysaccharide substrate in 200 mM phosphate buffer, pH 7.

The control sample was hydrolyzed with untreated enzyme and the control enzyme was replaced with buffer. Samples were incubated for 25 min. The reaction was quenched by boiling. * ': samples for 8 min. The activity measurement was based on the determination of the viscosity as described above.

»« · • · · *; The pH stability (more than 80% of the activity remaining) of the crude enzyme at 40 ° C was between 5.8 and 7 and the purified enzyme between 5.4 and 6.0. Purified enzyme. lost activity when incubated for 1 hour at 50 ° C.

> · • i 'I' Hydrolysis experiments • · · 30 «· *; For determination of enzyme hydrolysed linkage, 2 ml of purified polysaccharide which:. *. · Containing 1.66 mg of carbohydrate in 1 ml was hydrolyzed with 200 μΐ enzyme (280 AU ml'1) for 48 'to 1 h at +30 ° C. The enzyme was obtained from a preparation purified by ion exchange chromatography.

109921 23

The hydrolyzate was freeze-dried and the structure of the oligomers formed analyzed by 2D NMR.

i!

According to NMR analysis of the hydrolyzed polysaccharide, the enzyme cleaved the β-5 D-Galp (1-2) bond.

Example 3

Hydrolysis tests

The ability of commercial blend enzymes Econase CE (Primalco) and Pectinex Ultra SPL (Novo) to degrade levan polysaccharides produced on sucrose medium of B. licheniformis STFI-79133 and B. licheniformis strains and AT. pneumoniae The heteropolysaccharide produced by strain VTT-E-97860 was resolved by hydrolysis experiments. The enzyme preparations examined did not contain hydrolyzing activity of these polysaccharides.

The study also investigated the effect of various enzyme preparations (Econase CE, Primalco; Pectinex Ultra SPL, Novo; Neutrase, Novo; Aspergillus foetidus hemicellulase and Rhodotermus β- (1-3) glucanase) on commercial microbial polysaccharides (welan, gellan, curdlan was). Kurdlan, a linear β- (1-3) -glucan, hydro-; efficiently lysed with Econase CE, Pectinex, Ultra SPL, and β- (1-3) glucanase enzyme; / us. No other hydrolysing activities of the microbial polysaccharides studied were found *

t I

'*'. used enzyme preparations.

The effect of the K. pneumoniae-produced polysaccharide-degrading enzyme produced on the project on the formation and adhesion of K. pneumoniae biofilm was investigated on a laboratory scale by growing microbes in various nutrient media. Biofilm • ·, ·· · formation was monitored by both conventional culture and epifluorescence microscopy.

The growth medium was selected for growth of Klebsiella pneumoniae VTT-E-97860 and: pH of 30 medium by following two different media in preliminary experiments: Y; Peptone and 0.2 M Potassium Phosphate Buffer and glucose and 0.2 M Potassium Phosphate Buffer Nutrition. Every two days, 100 ml of the old medium was removed from the growth medium and an equivalent amount of fresh medium and 460 AY enzyme were added, conditions, and biofilm formation were monitored by culture, microscopy and pH for 2, 4, 6, 8, 10 and 14 days. of 5 Klebsiella / wewmomae biofilm cultures.

According to the culture results, after 10 days of cultivation, the enzyme appeared to act to inhibit the growth of K. pneumoniae biofilm. A similar result was obtained in a previous experiment with a non-sterile enzyme, although the corresponding biofilm-reducing effect was not clearly detectable by numerical microscopy results. However, the microbes of the biofilm grown in the enzyme-containing medium for more than 10 days, when viewed under the microscope, appeared to be more evenly distributed than the cells grown in the non-enzymatic medium which formed the aggregates. Different types of nutrient solutions showed parallel differences between enzyme-15 and non-enzyme cultures.

In addition, the effect of the enzyme on the formation of Klebsiella pneumoniae biofilm on a pilot scale was investigated. Pilot experiments were performed in a test hall with a total volume of 87 l of culture equipment. Biofilm formation was investigated by growing Klebsiella pneumoniae -20 microbes in nutrient medium alone and in medium supplemented with K. .

In the experiment, K. pneumoniae strain VTT-E-97860 was grown in a medium containing: T; 25 phosphate buffer (0.16 M, pE17), glucose (2%), yeast extract (0.05%) and salts. The growth medium was 75 l in reference run and 37 in enzyme run 1. Visual inspection showed that the run containing the enzyme was less than the reference run.

• *! * I In the reference run, the tank lid, the walls above the liquid surface, and the sample tray were • i * *.,. · Abundant mucus. The amount of mucus accumulated on the walls of the enzyme run was significantly: V: 30 lower and the composition of the mucus was different (drier) than that of the reference run.

• · »* · '1 * In laboratory experiments, the effect of the enzyme on the formation of K. pneumoniae biofilm was i i ·'; ] · 'Observed after 10 days of rearing. According to the results of the study, the enzyme

! I

'1 ·' reduced the growth of K. pneumoniae biofilm, and the use of the enzyme was seen under the microscope to degrade microbial aggregates and thereby alter the structure of the biofilm.

25 109921

According to pilot scale experiments, the addition of enzyme reduced mucus formation and altered its composition.

5 References

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

10

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

Dutton GGS, Parolis H (1986) Depolymerization of the use ofbacteriphage in structural 15 assays for capsular polysaccharide from Klebsiella serotype K3. Carbohydr Res I 149: 411–423.

Laemmli, UK (1970) Cleavage of structural proteins during assembly of head bacteriophage T4. Nature (London) 227: 680-685 20

Ratto et al. (1998) Effect of Enzymes on Degrading Bacterial Polysaccharides on Biofilm • · ,, 1 · Formation. COST Action El, Paper Recyclability. Improvement of Recyclability and the Future of the Recycling Paper Industry. Ed. By A. Blanco et al. Las Palmas de Gran Canaria,

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* ,,. · 24-26 November 1998. Poster • · • 1 · • «· • Λ *« · * 1 1 * · »* it1 t t t> t tl») 1 $ f · III • · I I ·.

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Claims (36)

1. Process digestion of microbipolysaccharides, characterized by the polysaccharide being contacted with an enzyme preparation containing over 200 AY / ml of endo-β-1,2-galactanase. 2. A process according to claim 1, characterized in that the avat enzyme preparation contains more than 500 AY / ml of endo-β-1,2-galactanase. 10 3. A process according to claim 1 or 2, characterized in that in the polysaccharide between the galactose and a second monosaccharide there is a p1, 2-bond, the second monosaccharide being either galactose, galacturonic acid, glucuronic acid, mannose, glucose, arabinose, xylose or rhamnose, and that the endo-β1, 2-galactanase enhances disintegration of this binding. 15 4. A process according to any one of claims 1-3, characterized in that there is a β-1,2-bond between the galactose and galacturonic acid in the polysaccharide, and that the endo-β-1,2-galactanase enhances this binding. 20 Process according to any of claims 1-4, characterized in that the molecular weight of the endo-β-1,2-galactanase enzyme is up to 110-130 kDa. • · • · »» • · · » Process according to any one of claims 1-5, characterized in that the pH optimum of the galactanase enzyme of the endo-β-1,2-β is in the range 5-6,5. Process according to any one of claims 1-6, characterized in that the enzyme treatment is carried out at a temperature of 20 - 60 ° C, preferably 35 - 55 ° C, in particular: 35 - 50 ° C. • ·. *. 30 Method according to any one of claims 1-7, characterized in that ', ···. the enzyme treatment is carried out at a pH of 4-8, preferably at a pH of 5-7, especially •, at a pH of 5.5-6.0. 109921 32 9. The method according to claims 1-8, characterized by reduced processing time up to 5 minutes. - 10 days, preferably 20 minutes. - 1 day, especially 30 min. - 2 hours. 5 Process according to any of claims 1-9, characterized by the amount of enzyme used up to 50 - 500,000 AY / g, preferably 500 - 50,000 AY / g, in particular 1000 - 10,000 AY / g polysaccharide. 10 11. A method according to any of claims 1-10, characterized in that the polysaccharide is produced by a bacterium. Method according to any one of claims 1-11, characterized in that the polysaccharide is produced by the bacterium Klebsiella pneumoniae. 15 A process for the preparation of monosaccharides or oligosaccharides, characterized in that a polysaccharide containing a P1-2 bond between two monosaccharides, one of which is galactose and the other of either galactose, galacturonic acid, glucuronic acid, arabinose, xylose, mannose, glucose or rhamnose, is contacted with an enzyme preparation containing endo-β-1,2-galactanase. .. j j Process according to claim 13, characterized in that the enzyme preparation :: contains endo-β-1,2-galactanase in an amount of over 200, preferably above 500 AY / ml. »·“ · 25 15. A process according to claim 14 or 15, characterized in that the molecular weight of the endo-β-1,2-galactanase enzyme is up to 110-130 kDa. 16. A process according to any of claims 13-15, characterized in that the pH optimum of the endo-β- • 1,2-galactanase enzyme is in the range 5-6.5. 30 Method for treating mucus assemblies containing microbipolysaccharides, characterized in that the mucus assembly is contacted with an enzyme preparation containing '200' / ml of endo-β-1,2-galactanase. »» »» · 109921 33 Method according to claim 17, characterized in that the avat enzyme preparation contains over 500 AY / ml endo-β-1,2-galactanase. 5 Process according to Claim 17 or 18, characterized in that there is a β-1,2-bond in the polysaccharide between the galactose and a second monosaccharide, the second monosaccharide being either galactose, galacturonic acid, ginkuronic acid, arabinose, xylose, mannose, glucose or rhamnose and the endo-β-1,2-galactanase enhances cleavage of this binding. 10 20. A process according to any one of claims 17-19, characterized in that there is a β-1,2-bond between the galactose and galacturonic acid in the polysaccharide and that the endo-β-1,2-galactanase enhances this binding up. 15 Method according to any of claims 17-20, characterized in that the molecular weight of the endo-β-1,2-galactanase enzyme is up to 110-130 kDa. Method according to any of claims 17-21, characterized in that the pH optimum of the endo-β-1,2-galactanase enzyme is in the range 5-6.5. 20 The method according to any of claims 17-22, characterized in that:; : The enzyme treatment is carried out at a temperature of 20 - 60 ° C, preferably 35 - 55 ° C, in the '1' especially 35 - 50 ° C. I · * · · '25 Process according to any of claims 17-23, characterized in that: # The enzyme treatment is carried out at a pH of 4-8, preferably at a pH of 5-7, especially at a pH of 5.5-6. A method according to any of claims 17-24, characterized in that the treatment time is up to 5 minutes. - 10 days, preferably 20 minutes. - 1 day, in particular •: 30 min. - 2 hours. 109921: 34 i ! The method according to claims 17-25, characterized in that the mucus collection contains polysaccharide produced by the bacterium Klebsiella pneumoniae. Process according to any one of claims 17-26, characterized in that the mucus collection 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. Process according to any one of claims 17-27, characterized in that the mucus collection is treated with an enzyme preparation and before or after or simultaneously with at least one biocide selected from the group: glutaraldehyde, DBNPA, MBT, chloro-2-methyl-isothiazoline-3 one, methyl 4-isothiazolin-3-one, Bronopol, dazomet, sodium hypochlorite, peracetic acid BCDMH (bromochlorodimethylhydantoin). 15 Process according to any of claims 17-28, characterized in that the method is used for the decomposition of mucus assemblies in the process industry. 30. A method according to any of claims 17-29, characterized in that the method is used for decomposing mucus assemblies in paper machines. 20 Process according to claim 30, characterized in that the enzyme preparation. '. is served in the circulation water in a paper machine. i · i • »» Method according to Claim 30 or 31, characterized in that the amount of enzyme: · · 25 is intended to be dispensed at 5-10,000 AY / liter, preferably 20-5,000 AY / liter, in particular 200 - 2000 AY / liter. circulation water. 33. Enzyme preparation for decomposition of microbipolysaccharides in the process industry, "" characterized by containing endo-β-1,2-galactanase in an amount of over 200 ... · 30 AY / ml, preferably over 500 AY / ml, and that the preparation further contains auxiliaries, such as stabilizers and possibly buffers. 109921 35 34. Enzyme preparation according to claim 33, characterized in that it is intended for use in cleaving microbipolysaccharides in a paper machine environment and that, in addition to endo-β-1,2-galactanase, contains stabilizers suitable for cellulose and | pulp industry and selectively any of the following: buffers, one or more chemicals, such as biocide, and one or more other enzymes. 35. Enzyme preparation for use in the preparation of mono- or oligosaccharides, characterized in that it contains endo-β-1,2-galactanase in an amount above 200 AY / ml, preferably above 500 AY / ml, and the preparation further contains auxiliary agents. , Such as stabilizers and selectable buffers. Enzyme preparation according to any of claims 33-35, characterized in that the molecular weight of the enzyme is 110 to 130 kDa and the pH optimum is between 5-6.5. • I * * · ·> · · Let I »* *> ·