The present invention relates to the control of extractives during paper manufacture. In particular the invention concerns a process for enzymatically hydrolyzing steryl esters in pulp suspensions or in the process waters. This invention provides also a novel enzyme composition capable of hydrolyzing the ester linkages in steryl esters.

The extractives of wood are a very heterogenous group of compound. The extractives are partially disperse in process waters during pulping, bleaching and paper manufacture. A part of the lipophilic extractives are in the form of colloidal droplets, which can further form aggregates and which, when adhering to surfaces on paper machines can cause breakages of the paper web in the paper manufacture. The extractives can also cause sticky spots in the paper, resulting in impaire paper product quality. Sticky spots in the paper give problems with the runnability of the paper machine and in further coating and printing operations.

In food packaging materials the extractives can cause odour and taste problems. When water circulations are closed the problems caused by the extractives become even more serious. This all results in increased production costs and lower product quality.

The amount and composition of wood extractives vary greatly. The amount of extractives in spruce wood is about half of that in pine and birch. About 30 % of the extractives of softwoods are triglycerides, 20 % are steryl esters and 25 % resin acids (Sjöström, 1981).

The composition of birch extractives is totally different from the composition of the extractives of softwood. There are no resin acids in birch, and the relative amounts of neutral components and fatty acid esters are in birch clearly more abundant than in pine or spruce.

The amount of extractives in softwood pulp cooked by the alkaline kraft method is usually less than 0.1 %. The high pH of the cooking process dissolves and disperses the extractives, which are liberated to the washing water in washing stage. Triglycerides and part of the steryl esters contained in the extractives are hydrolyzed at alkaline cooking conditions. The amount of extractives of birch and aspen kraft pulp is usually 0.3 to 0.8 %. The reason for this high extractive content is the poor dissolution and dispersion of the

birch extractives during the cooking process. The steryl esters that remain unhydrolyzed are particularly difficult to remove from the pulp. Some steryl esters, such as esters of 4,4-dimethyl sterols present especially in hardwoods, are very stable and resist alkaline hydrolysis even in kraft coking, and thus their relative amount increases in the extractives remaining in kraft pulp.

In mechanical pulping of softwood, most of the extractives are disperse as colloidal droplets and released into the process water because of the strong mechanical shear forces and the high temperature, but the extractives are not chemically altered. Also during dithionite and peroxide bleaching of mechanical pulps the triglycerides and steryl esters remain nearly unchanged.

Traditionally the control of the extractives during paper manufacture is carried out by ageing of chips prior to pulping. Alternatively, selective microbial treatments have been used (CartapipTM, Cariant Corp. USA) or enzyme treatments (e. g. Resinase (D, Novo Nordisk A/S) or addition of various chemicals (e. g. fine talc, cationic polymers). Resinase treatment results in hydrolysis of triglycerides present in the extractives (Mustranta et al, 1998). The pitch control with resinase has resulted in better and steady operations and improved product quality (Fujita et al. 1992). The problem of the resinase treatment is its limitation to triglycerides, which account only for about 30-40 % of the extractives.

Resinase lipases hydrolyze triglycerides by liberating fatty acids and glycerol.

In order to avoid pitch problems lipase treatment has been suggested in various patent publications: WO 91/07542 describes lipase hydrolysis of resin simultaneously with peroxy bleaching, US 5,338,403 describes lipase hydrolysis of resin simultaneously with reductive bleaching of pulp. However, both of these publications describe the use of ResinaseX, which according to Mustranta et al. (1998) does not hydrolyse steryl esters.

US 5,176,796 describes acylglycerol lipase treatment of paper-stock slurry or white water in a process for mechanical pulp, US 5,256,252 suggests the addition of lipase and cationic polymer for diminishing pitch deposits in a pulp and papermaking process and WO 92/13130 suggests the addition of thermostable lipase to white-water or to pulp.

Breuil et al. (1997) have described the treatment of aspen wood chips by the commercial fungus product CartapipTM (Clariant Corp. USA). The fungus removed triglycerides very

efficiently. However, CartapipTm did not effectively remove the steryl esters and waxes.

Leone et al. (1998) have also disclosed the screening of fungi capable of growing on aspen steryl esters/waxes. The ability of fungi to consume steryl esters/waxes was assessed in the presence of other carbon sources including glucose and triglycerides. The best consumers of steryl esters/waxes were Aspergillus luchuensis, Phanerochaete chrysosporium and Cunninghamella elegans. However, Leone et al. did not isolate or purify any enzymes, which would be responsible of the steryl ester and/or wax consumption.

International Patent Application WO 94/23052 describes the isolation and purification of a cholesterol esterase/lipase enzyme from Pseudomonas fragi. The same enzyme component responsible for cholesterol esterase activity is according to the publication also responsible for lipase activity. It is suggested that the enzyme may be useful for the treatment of eggs, in processes for hydrolysis of resin in pulp, and in processes for hydrolysis or synthesis of sterols or lanolin or in a cleaning composition.

Although certain microorganism strains have been shown to be capable of metabolizing steryl esters (Leone et al, 1998), an efficient enzymatic method for hydrolysis of steryl esters has not been disclosed previously for the control of extractives during paper manufacture. The prior art suggests the treatment of wood logs or chips by microorganisms or alternatively the treatment of pulp with lipase such as resinase.

Because in the first case those treatments have been applied to whole wood or wood chips, the extractives and pitch is removed only partially and the processes are difficult to control. In the latter case the lipase treatment has only acted on triglycerides.

In WO 94/23052, the treatment of resin-containing papermaking pulp by using Pseudomonas fragi cholesterol esterase/lipase enzyme has been suggested. However, said document does not describe the conditions under which pulp should be treated, neither does it describe any results achieved with the enzyme treatment applied on pulp.

Surprisingly, it has now been found that resin and pitch problems can effectively be controlled during paper manufacture by a new enzyme treatment process. The process waters originating from mechanical, chemimechanical or chemical pulping processes or

circulating in these processes or the circulated process waters originating from bleaching or from paper or board machine can be treated by new enzyme preparations capable of degrading the steryl ester linkages in steryl esters or by a combine treatment with a steryl esterase and a lipase.

According to another embodiment of this invention mechanical, chemimechanical or chemical pulps (pulp suspensions or slurries) can be treated by the enzymatic method of this invention. The enzyme compositions of this invention can be added to the pulp suspensions in connection with mechanical, chemimechanical or chemical pulping or before, during or after bleaching or at any stage of the paper manufacturing process before the paper machine.

The process waters and/or pulp suspensions are preferably treated in the first stages of the manufacture of wood-containing paper and board i. e. the process waters circulated in mechanical or chemi-mechanical pulping processes and/or pulp suspensions from these processes. It is advantageous to treat the process waters and/or pulp suspensions from mechanical or chemimechanical pulping, because the steryl esters remain nearly unchanged in the mentioned processes. The hydrophilicity of steryl esters is increased and thus their tendency to form aggregates and adhere to machine parts during the subsequent processing steps of the paper or board manufacturing process is decreased.

Advantageously also pulp suspensions and/or process waters from chemical pulping are treated with the enzyme preparations of this invention, because especially hardwood comprises very stable steryl esterases which resist alkaline hydrolysis in kraft coking. It is also advantageous to treat sulphite pulp made from hardwood or softwood and/or process waters originating from these processes by the enzymatic method of this invention.

According to one further embodiment the enzyme treatment can be carried out at the head box, where the different components of paper (or board) are mixed together.

Wood extractives from various pulping processes are disperse as colloidal droplets to the process waters. Especially, if the process conditions (pH, temperature, amount of calcium) are changed the colloidal droplets form aggregates, which adhere to the surfaces

on paper machines. When fixed to pulp the aggregates are less harmful than when present in the process waters. This is due to the potential adhesion of the aggregates to machine or wire parts. Accordingly it is most advantageous to treat process waters by the enzymatic method of this invention.

The consistency of pulp is usually 5 % to 35 %, or typically 10 % to 20 %. The consistency of the pulp suspension to be treated by the method of this invention is preferably 5 % or less, more preferably 3 %, or less, still more preferably 2 % or less and most preferably 1 % or less. For example, the consistency of the pulp suspension at the head box of a paper machine is about 1 %. In process waters the consistency is typically 0%.

This invention provides also new enzyme preparations capable of efficiently degrading the steryl ester linkages in steryl esters of wood extractives. These enzyme preparations are derived preferably from strains belonging to Trichoderma, Paecilomyces, Rhizomucor, Candida, Bacillus or Pseudomonas, most preferably from Trichoderma, Paecilomyces, Rhizomucor, Candida or Pseudomonas. The strains belong preferably to the species Trichoderma reesei, Trichoderma longibrachiatum, Trichoderma citrinoviride, Paecilomyces variotii, Rhizomucor miehei, Candida tropicalis, Bacillus pumilus, Bacillus subtilislmacerans, Bacillus subtils or Pseudomonas stutzeri. Thus by growing these strains on a suitable medium it is possible to produce an enzyme preparation capable of hydrolyzing the ester linkages of steryl esters. A still more effective enzyme preparation can be achieved by concentrating and/or purifying the culture medium of the production strain.

According to the present invention it is also possible to isolate the gene or genes encoding an enzyme or enzymes capable of degrading the steryl ester linkages in steryl esters of wood extractives. The gene is then transfered to a suitable host, preferably under the regulation of a strong promoter, making capable of an efficient production of the desired enzyme product. This invention thus comprises also genetical constructions containing DNA sequences encoding steryl esterases, vectors containing these genetical constructions and hosts expressing the genetical constructions and producing steryl esterases.

More specifically, the process of the invention is characterized by what is stated in the characterizing part of claim 1 and 28.

The enzyme preparation is, again, characterized by what is stated in the characterizing part of claim 17 and 23.

The invention provides considerable advantages. The paper machine runnability is improved due to less breakages of the paper web and decrease in the frequency of cleaning the machine, which also result in better runnability of the paper machine. There are less sticky spots and yellowing in the paper, which result in better paper product quality. Furthermore there are less problems during coating and printing. The risk that the paper breaks for example during printing is diminished. There are also clear savings in chemical costs, because less fixatives or other paper chemicals are needed. Due to the increased hydrophilicity of extractives the deresination efficiency is also improved during the pulp washing. The treatment of pulp slurry before bleaching results in better bleachability of the pulp.

Next the invention will be examine more closely with the aid of a detailed description and a number of working examples.

In the attache drawings Figure 1 shows the pH optimum of steryl esterase produced by Rhizomucor miehei Figure 2 shows the pH stability of steryl esterase produced by Rhizomucor miehei Deposits The strains were deposited according to the Budapest Treaty on December 16,1998 at the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1 b, D-38124 Braunschweig, Deutschland, and assigne as follows: Paecilomyces variotii VTT D-76048, assigne as DSM 12587 Trichodennza citrinoviride VTT D-9869, assigne as DSM 12589 Rhizomucor miehei VTT D-82193, assigne as DSM 12588

Futhermore the following strains were deposited according to the Budapest Treaty on March 8,2000 at the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1 b, D-38124 Braunschweig, Deutschland, and assigne as follows: Candida tropicalis VTT-C-84159, assigne as Pseudomonas stutzeri VTT-E-97825, assigne as "Mechanical pulping"stands for manufacturing processes with which the raw material for paper manufacture is defiberized. Mechanical pulp is mainly manufactured by the grinding and refining methods, in which the raw material is subjected to periodical pressure impulses. Due to the friction heat, the structure of the wood is softened and its structure loosened, leading finally to separation of the fibres. By"TMP"is meant thermomechanical pulp, by"GW"is meant groundwood pulp, by"PGW"is meant pressurized groundwood pulp, by"RMP"is meant refiner mechanical pulp, by"PRMP" pressurized refiner mechanical pulp and by"CTMP"chemithermomechanical pulp. The mechanical pulps are mostly prepared from softwood, but also from aspen wood.

"Chemical pulping"stands for manufacturing processes with which substantial amounts of the lignin and hemicellulose components of wood are removed by alkaline cooking methods."Kraft pulping"is used synonymously with"sulphate cooking"and it designates the cooking method in which sodium sulphide and sodium hydroxide are used as principal cooking chemicals. Mechanical pulping methods save lignin and carbohydrates which results in higher yield compare to chemical pulping."Sulphite pulp"is made from softwood or from harwood by"sulphite coking".

"Bleaching"traditionally denoted processes with which the residual lignin of cellulose pulps is solubilized by using chlorine or chlorine dioxide. Presently the pulps are frequently also bleached by oxygen gas, hydrogen peroxide, ozone or by combine sequences including these substances as well as the above-mentioned traditional bleachingchemicals..

Mechanical pulps are bleached under rather mild conditions using different chemicals such as peroxide or dithionite.

In this invention"process waters"are the inner circulation waters of mechanical or chemimechanical pulping process. The process water typically comprises the"brown water"received from the dewatering process of grinded pulp. The process water in this invention thus, e. g. means the water received by processes which enhance the dry matter content of the raw material, such as squeezing or filtering. It is also possible to treat by the methods of this invention the brown water or washing water from mechanical or chemimechanical pulping processes circulated to paper or cardboard machine.

In addition to the process waters of mechanical or chemimechanical pulps, the method of the invention is also suitable for the treatment of the process waters of other cellulosic pulps, i. e. chemical pulps. Thus according to one preferred embodiment of our invention the problems caused by extractives and pitch in paper manufacturing process can be decreased by hydrolyzing enzymatically steryl ester groups in process waters of various pulping processes.

By process waters are furthermore meant waters which are circulated from the bleaching process or from the paper or board machine totally or partially counterwise to the pulping process or washing waters resulting from the mentioned processes. By process waters are meant thus any process waters such as white water or any recycle and reused waters in the pulping processes.

According to another preferred embodiment of this invention various pulps or pulp suspensions, can be treated with the process of this invention. The processes of this invention can be applied on pulp suspensions from mechanical, chemimechanical and chemical pulping processes.

According to one further embodiment of this invention the enzymatic treatment can be carried out at the head box of the paper machine, where the components of paper or board are mixed together.

"Paper machine"denotes in this context also board and cardboard machines.

It is most advantageous to use the treatment method of this invention for treating the pulp

suspensions and/or process waters from mechanical pulping processes, because these pulp suspensions or process waters contain the highest amount of steryl esters. It is advantageous also to treat the pulp suspensions and/or process waters from chemical pulping of hardwood such as birch, aspen and eucalyptus, because only part of the steryl esters contained in hardwood are hydrolyzed (i. e. ester linkage cleaved) during the kraft cooking process. It is also advantageous to treat pulp suspensions and/or process waters from sulphite pulp made from hardwood or from softwood.

According to the present invention, at least a part of the steryl ester linkages of steryl esters are hydrolyzed resulting in free fatty acids and sterols, which are less prone to form deposits than their esters. This hydrolysis is carried out by using steryl esterases originating from micro-organisms capable of efficiently producing steryl esterase activity into their culture medium.

The treatment method of this invention is able to hydrolyze 20 %, preferably 50 %, most preferably 90 % of the steryl esters of the process waters or pulp suspensions. This results in savings of chemical costs and reduction in the frequency of cleaning machines.

Furthermore this results in improved washing (deresination efficiency) of the pulp. The amount of hydrolyzed steryl esters is calculated as described in Example 4.

According to one preferred embodiment of the invention, the enzyme preparation used comprises the cultivation liquid or medium of a steryl esterase producing microorganism. Preferably, such a cultivation liquid or medium or filtrate or extract is concentrated before use. According to another preferred embodiment, the enzyme preparation comprises a purifie enzyme, isolated from a cultivation liquid or medium.

The term"enzyme preparation"denotes any product which contains at least one enzyme.

Thus, such an enzyme preparation may be a culture liquor or filtrate containing one or more enzymes, an isolated enzyme or a mixture of one or more enzymes. In addition to the enzymatic activity such a preparation preferably contains adjuvants which commonly are used in enzyme preparations intended for application in the pulp and paper industry.

Such adjuvants are typically comprise of, for instance, buffering agents and stabilizing agents.

The enzyme preparation useful for treatment of process waters and/or pulp suspensions comprises an essential steryl esterase enzyme activity and may contain also another enzyme activity, preferably lipase activity, lignin degrading activity, hemicellulase or pectinase activity.

Suitable microorganism strains which are capable of producing steryl esterases can be isolated from organic matter from a pulp mill or from the vicinity thereof or from any other potential source containing material with this sterol structure. Alternatively culture collection strains can be screened for their ability to use steryl esters as their carbon source.

Especially suitable in this invention are trains, isolated from pulp mill sites, which are capable of degrading steryl esters typically found in the pitch of wood such as sitosteryl ester.

The present invention also discloses a method of isolating microbial strains capable of producing steryl esterases. In summary it comprises the steps of : -collecting samples of microorganism-containing organic matter from a pulp mill or the vicinity thereof or any other potential source containing material with this carbohydrate structure, -suspending the samples in a suitable liquid or buffer, e. g. in a physiological salt solution, inoculating media containing as a carbon source steryl esters with aliquots of the diluted suspension or directly the material collecte as described before, incubating the inoculated medium in a suitable container, e. g. plate, shake flask or fermentor, in conditions which favour microbial growth, preferably until more than appr. 50 % of the carbon source containing steryl esters is consume, collecting the microbial cells from the cultivation medium, -optionally repeating the cultivation, and -optionally purifying the colonies or storing the possible mixed microbial population using standard microbiological methods.

The growth medium contains steryl esters as carbon source together with suitable nitrogen sources. The method is illustrated in more detail in Example 1.

Steryl esterases can be produced with a microbial strain or mixed microbial populations isolated according to the above method by cultivating the microorganisms on growth media containing steryl esters.

Steryl esterase production can be induced by substances containing steryl ester linkages such as DCS water (water containing dissolve and colloidal substances =DCS preferably from e. g. mechanical pulp), soya bean meal, cholesterol oleate or a synthetic steryl ester (sitosterol ester).

New microorganism strains capable of hydrolyzing the ester linkages in steryl esters can be screened for by using the following method steps: -cultivating different micro-organism strains on a screening medium containing steryl esters as an inducor; -separating the culture filtrate and using it as an enzyme source in the hydrolysis of a substance containing steryl ester linkages; -carrying out the hydrolysis in suitable conditions; -analyzing the steryl esters formed from the rection mixture by using thin layer chromatography or other suitable method; and -choosing the best strains for further studies.

The invention is not, however, limited to the indicated origins of the enzyme or to the isolation method, and the enzyme can also be obtained by other methods.

Thus, it is possible to produce the steryl esterase enzyme by microorganisms, which have been mutated or genetically constructed to produce the desired enzyme, or by other production host trains, to which the gene encoding this enzyme has been transfered.

Suitable recombinant DNA methods for constructing steryl esterage producing recombinant strains are known to a person skilled in the art and are described in Sambrook et al (1989) and Glick and Pasternak (1998). Similar methods as are used for constructing recombinant strains producing lipase can be used in constructing strains of this invention

and are described for example in WO 92/05249. Suitable production hosts for producing recombinant steryl esterases are for example Aspergillus and Trichoderma systems especially for genes originating from fungal sources and Bacillus for genes originating from bacterial sources. The screening of steryl esterase producing strains can be done by testing the capability of the strains to hydrolyse cholesterol oleate or some synthetic steryl ester. Cholesterol oleate comprises chemically similar linkages as steryl ester comprising substrates.

The steryl esterase preparation can be derived from a microorganism strain selected from the group consisting of microorganisms of the genera Trichoderma, Paecilomyces, Rhizomucor, Candida, Bacillus and Pseudomonas, preferably from Trichoderma, Paecilomyces, Rhizomucor, Candida and Pseudon7onas. More preferably the strains belong to the species Trichoderma reesei, Trichoderma longibrachiatum, Trichoderma citrinoviride, Paecilomyces variotii, Rhizomucor miehei, Candida tropicalis, Bacillus pumilus, Bacillus subtilis/macerans, Bacillus subtils or Pseudomonas stutzeri, most preferably to strains belonging to Paecilomyces variotii, Trichoderma citrinoviride, Rhizomucor miehei, Candida tropicalis or Pseudomonas stutzeri. Examples of preferred steryl esterase producing strains are listed in Tables 1 to 7. By mutants and variants thereof are meant natural, mutated or genetically engineered derivatives of the strains still possessing an essential steryl esterase producing capability. All these microorganisms alone or as a combination can be used to produce steryl esterases, which are able to hydrolyze the steryl ester linkage in steryl esters.

According to a preferred embodiment, the enzyme preparations are prepared by cultivating on a suitable cultivation medium comprising steryl esters any of the above-mentioned steryl esterase producing microorganisms.

Esterases are enzymes, which catalyze the hydrolysis of ester linkages. Esterases can be devided according to their substrate specificity to several classes of enzymes. Lipases (EC 3.1.1.3) belong to esterases and they hydrolyze ester linkages in triglycerides. Sterol esterase (EC 3.1.1.13) hydrolyzes steryl esters.

The present enzyme preparations typically contain suitable adjuvants such as buffering agents or stabilizers, conventionally used in enzyme preparations intended for use on pulp

andpaper.

The steryl esterase treatment can be conducted separately, simultaneously with another enzymatic treatment, or before such a treatment. It is particularly preferred to combine the steryl esterase treatment with a lipase treatment. When the steryl esterase treatment is carried out simultaneously with a lipase treatment it is preferred to use an enzyme preparation to which the steryl esterase has been added, or which has been produced by a strain genetically improved to produce high steryl esterase activity, in order to obtain a preparation with an essentially high steryl esterase activity. According to the invention, the steryl esterase treatment can also be combine with treatments with other enzymes like hemicellulases, pectinases and/or lignin modifying (oxidative enzyme) activities.

Furthermore the steryl esterase treatment can be carried out with at least one paper making chemical such as alum or fixatives.

The process is not limited to a certain wood raw material, but it can be applied generally to both soft and hardwood species, such as species of the order of Pinacae (e. g. the families of Picea and Pinus), Salicaceae (e. g. the family of Populus) and the species in the family of Betula and species of Eucalyptus.

The amount of enzyme needed for hydrolyzing a substantial amount of the steryl esters contained in the process waters or in pulp suspensions is, when calculated as steryl esterase activity, 1-1000 nkat/g, preferably 20-500 nkat/g pulp or extractives or 1-4000 nkat/g steryl esters, preferably 200-1000 nkat g steryl esters. It is possible with these amount of enzymes to hydrolyze 20 %, preferably 50 %, most preferably 90 % of the steryl esters contained in the process waters or in the pulp suspensions of paper manufacturing process. When the steryl esterase activity is calculated as steryl esterase activity per liter of process waters, the necessary enzyme activity varies from 1-1000 nkat/l, preferably 20- 500 nkat/l of process water.

Lipase can originate from any microorganism capable of producing this enzyme, such as from the genus Aspergillus or Pseudomonas. Hemicellulose hydrolyzing enzymes can originate from any microorganism capable of producing these enzymes, such as from filamentous fungi Trichoderma, Aspergillus, Penicillium, Paecilomyces, Sclerotium,

Sporotrichum, Thielavia, Polyporus, Tyromyces or from bacteria, such as Bacillus or Streptomyces. The invention is not, however, limited to the indicated origins of the enzyme nor to the isolation method, and the enzyme can also be obtained by other methods. Lignin modifying enzymes, such as laccase can originate from the genus Trametes, for example from Trametes versicolor or Trametes hirsuta or from some other white rot fungi.

According to the process of this invention a commercial lipase preparation, such as Resinase 0 Novo Nordisk A/S) or laccase preparation such as preparation from Trametes villosa (Novo Nordisk A/S) can be used.

The process water and/or pulp suspension is treated by using steryl esterases with or without lipases and possible with other enzymes in 20-90 °C, preferable 30-60 °C, 10 min to 24 hours, preferable 0,5- 2 hours. The treatment can be performed in pH 3 to 9, preferably in pH 4 to 8 most preferably 6 to 8. Hence, when treating alkaline pulps the pH adjustment may be needed.

As is clear to a person skilled in the art, the enzyme preparations of this invention should not contain any substantial amount of cellulase activities. Cellulase negative strains not capable of producing one or more cellulases, can be prepared by mutation or genetical engineering methods as described for example in US 5,298,405 or alternatively cellulase activity can be removed by well known protein purification methods.

The following non-limiting examples illustrate the invention: Example 1 Isolation of potentially steryl esterase producing fungi from a chip pile Wood chips from a chip pile (mixed pine/spruce) of Finnish paper mill were incubated in 0.9 % NaCl for 2 h and plated on agar containing DCS-fraction (medium containing dissolve and colloidal substances (DCS) see Example 2) from TMP pulp. Sporulating fungi were further purifie by plating spore suspensions on DCS-agar. The isolates were cultivated further on liquid medium containing DCS-fraction as carbon source and Yeast Nitrogen Source as nitrogen source. The culture filtrates were screened for steryl esterase activity by using cholesterol oleate as substrate.

The composition of DCS-agar was: 270 ml DCS-fraction from TMP 30 ml Yeast Nitrogen Base (Difco) 0.33 g saponin 6gagas 7.5 ml 0.4 % chloramphenicol 7.5 ml 0.4 % chlorotetracycline By this method was isolated a new Trichoderma strain identifie as Trichoderma citrinoviride (VTT D-98697) which was deposited at the DSMZ and assigne as DSM 12589.

Example 2 Screening of microorganisms capable of hydrolyzing the ester linkages in steryl esters Different micro-organisms were cultivated on the screening medium containing dissolve and colloidal substances (DCS) from TMP pulp as carbon source and Yeast Nitrogen Base (Difoo) as nitrogen source. DCS fraction was prepared from the TMP pulp by diluting it to 10% consistency with distille water whereafter the suspension was agitated at 60 °C for 3 hours at 150 rpm. The suspension was centrifuged at 500 g for 30 min and the DCS fraction (supernatant) was separated. After the cultivation the culture filtrate was separated and used as an enzyme source. The sterol esterase activity was tested using cholesterol oleate as substrate. The rection mixture contained 1 ml of the culture filtrate and 0.5 mg of cholesterol oleate. The mixture was incubated at 40°C for 18h, whereafter the rection mixture was evaporated, dissolve in hexane and analyzed by thin layer chromatography. The eluent contains petroleum ether-diethylether-acetic acid (50: 50: 2) and the visualization of substances was carried out by rection with sulfuric acid. The hydrolysis of the cholesterol oleate is indicated as +/-in Table 1.

Table 1. Microorganism Hydrolysis of cholesterol oleate Trichoderma longibrachiatum + VTT D-74075 Candida tropicalis + VTT C-84159 Candida tropicalis + VTT C-84160 Comamonas testosteroni VTT E-84214 Comamonas testosteroni VTT E-86249 Galactomyces geotrichum VTT D84228 Bacillus pumilus + NS-034 Bacillus subtilislmacerans + NS-066 Bacillus subtils + NS-075 Bacillus circulans Nus-119 Phanerochaete chrysosporium VTT D-84237 Phanerochaete chrysosporium VTT D-85242 Paecilomyces variotii +++ VTT D-76048 Rhizomucor miehei ++ VTT D-82193 Rhizomucor pusillus VTT D-70016 Trichoderma citrinoviride +++ VTT D-98697

According to Table 1 Paecilomyces variotii, Rhizomucor miehei and Trichoderma citrinoviride strains were able to produce the highest steryl esterase activity.

Example 3 Production of enzyme preparations having steryl esterase activity The most potential strains producing steryl esterase activity were further cultivated at different pu-values using different inducers (DCS water, soy bean meal or cholesterol oleate) in the cultivation media. After the cultivation (+30 °C, 7 d) the culture filtrate was separated by centrifugation, whereafter the sterol esterase and lipase activities were measured.

The sterol esterase activity was measured by spectrophotometer. The rection mixture contained 3 ml of 0,7 M phosphate buffer (pH 7,5), 100 pl of cholesterol oleate solution, 20 pl of H202 solution, 20 Ill of catalase solution, 20 p1 of cholesterol oxidase and 50 Ill of sample solution (culture filtrate). The increase of absorbance at 240 nm was a measurement of the cholesterol esterase activity. One unit of esterase activity (nlcat) was destine as the amount of enzyme that liberated 1 mol cholesterol.

Production of sterol esterase with Paecilomyces variotii, Rhizomucor miehei and Trichoderma citrinoviride at different conditions (media, pH) is shown in Table 2.

Table 2. Strain Inducer Cultivation Steryl esterase Lipase activity H activit (nkat/ml (nkat/ml) Paecilomyces DCS-water 6.40 0.34 58 variotii DCS-water + 5.80 0.30 26 starch Soya 5.54 1.14 67 Cholesterol 5.59 0.43 33 oleate Rhizomucor DCS-water 6.66 0.24 32 miehei DCS-water + 5.80 0.31 25 starch Soya 5.54 0.38 280 Trichoderma DCS-water 6.40 0.03 43 citrinoviride DCS-water + 5.80 0.23 0 starch Soya 5.54 0.30 42 Lecithin 5.54 0.23 0.2 Cholesterol 5.59 0.21 18 oleate I

By selecting a proper inducer the steryl esterase levels in the culture filtrate can be enhanced. In the studied cases soya bean meal was the most efficient inducor.

Example 4 Treating TMP process waters with an enzyme preparation comprising steryl esterase activity.

DCS fraction was isolated from spruce TMP. DCS fraction containing extractives was treated with sterol esterase (I Ox concentrated culture filtrate) produced in Example 3. The analysis of the extractives was carried out by gas chromatography after extraction with ether (Örså and Holmbom, 1994) The results are shown in Table 3. In the DCS fraction the the steryl ester content was 45.6 mg/l. After treatment with P. variotii, R. miehei and T. citrinoviride enzymes, the steryl ester content was reduced even by 52 and 38 %. Table 3. Hydrolysis of sterol esters and triglycerides present in DCS-fraction of TMP pulp by different fungi Sterol esters mg/l Triglyserides mg/l Reference 45.6 63.1 Paecilomyces 40.6 43,3 variotii Rhizomucor miehei 21.8 3.4 Trichoderma 28.3 30.5 citrinoviride

Example 4 Properties of the enzymes having steryl esterase activity.

The characteristics of the sterol esterase produced by Rhizomucor miehei were determined.

The enzyme was found to have pH optimum in the range of 6-8 (see Figure 1). The sterol esterase was stable at pu-values of 4-8 (see Figure 2).

Example 5 The screening of new microbial strains producing steryl esterases was repeated.

Microorganisms from the VTT culture collection or other culture collections and some strains isolated from spruce were cultivated in shake flasks. The cultivations were carried out with media containing spruce extractives (10 % DCS water + Yeast Nitrogen Base solution) or soya meal as inducers. Trichoderma strains were also cultivated on media containing soya, solka floc and spent grain.

A soy bean meal medium contained: Soy meal 30 g I-' Dissolve starch 10 g I-, Peptone 2 g 1-1

(NH4) 2SO4 1 g l-1 gl-1KH2PO410 MgSO4#7H2OMgSO4#7H2O1 g l-1 pH was adjusted to 4.5-5.5 for fungi and to pH 6,5 for bacteria.

Distiller's spent grain-Solca floc-soy bean meal medium contained: Soy bean meal 30 g l-' SF (Solka floc) 20 g l-1 Distiller s spent 10 g l-1 grain KH2PO4 15 1-1 l-1(NH4)2SO45 Both of the media were prepared in distille water. Distiller's spent grain medium was used only for Trichoderma and the pH was adjusted to 5,5.

After one week cultivation the culture filtrates were separated and the effect on cholesterol oleate, synthetic steryl ester (sitosterol ester) and triolein (triglyceride) was investigated by TLC (thin layer chromatography) analysis. Soya meal which is known to contain steryl esters seemed to be a good inducer of esterases. New potential strains, Candida tropicalis (VTT-C-84159), Pseudomonas stutzeri VTT-E-97825 and Trichodejrna reesei, were found to produce steryl esterase on soya meal media.

Table 4. The most potential microorganisms producing steryl esterase. Hydrolysisresults(TLC)StrainMedium CholesterolTrioleinSyntheticsteryl ester oleate Soya++++++Pseudomonasstutzeri VTT-E-97825 VTT-Soya+++++Candidatropicalis C-84159 Trichoderma reesei Soya + solka floc + ++ +++ ++ VTT-D-74083, QM 6a spent grain (ATCC 13631) Trichoderma reesei VTT-Soya ++ ++ ++ D-74070, QM 9136 (ATCC 26920) Trichoderma reesei VTT-Soya ++ ++ ++ D-86271, Rut C-30 (ATCC 56765) Rhizomucor miehei VTT-Soya ++ +++ ++ D-82193, DSM 12588

The new potential sources of steryl esterases were tested on the steryl esters and triglycerides of spruce. I % DCS water was treated with different culture filtrates (40°C, pH 7,20 h) after which the samples were analyse by GC. T. reesei QM 6a (ATCC 13631) was the most effective strain to hydrolyse steryl esters (59 %) and almost 90 % of triglycerides were also hydrolysed. Candida tropicalis (VTT-C-84159), which was isolated from a Finnish paper mill, showed also very promising results (Table 5).

Table 5. Treating of 1% DCS water with different culture filtrates (2.5 ml/10 ml). Enzyme Culture Substrate Steryl esters (mg/1) Triglycerides (mg/1) medium 0 h 20 Hydro-0 h 20 h Hydro- h lysed lysed Trichodenma neesei Soya + 1% DCS 12.4 5.1 59% 20.0 2.2 89% QM 6a (ATCC solka floc water 13631) + spent grain Trichoderma reesei Soya 1% DCS 12.6 6.0 52 % 20.5 3.1 85% QM 9136 (ATCC water 26920) Trichoderma neesei Soya 1% DCS 12.5 6.7 46 % 20.6 2.1 90 % Rut C-30 (ATCC water 56765) Pseudomonas Soya 1% DCS 12.2 8.8 28% 21.0 18.6 11 % stutzeri(VTT-E-water 97825) Pseudomonas DCS water 1% DCS 12.2 7.8 36 % 19.7 8.2 58% stutzeri(VTT-E- water 97825) Candida tropicalis Soya 1% DCS 12.2 7.0 43 % 20.2 13 94 % (VTT-C-84159) water

Example 6 Hydrolysis of steryl esters by partially purifie steryl esterase The steryl esterases of R. rniehei was partially purifie with hydrophobic interaction chromatography (HIC). To investigate the effect of steryl esterase on the spruce extractives, 1% DCS fraction was treated with dosage 4000 nkat of esterase/g of steryl esters (40°C, pH 5,20 h) after which the samples were analyse by GC (Table 6). Purifie R. miehei esterase hydrolyse over 50 % of steryl esters, but only 70 % of triglycerides were hydrolysed, which indicates that it is advantageous to combine steryl esterase treatment with lipase treatment.

Table 6. Treating of DCS waters with R. miehei esterase. Enzyme Source Substrate Steryl esters Triglycerides % hydrolyzed % hydrolyzed R. miehei Culture filtrate I % DCS water 42 63 R. miehei Purifie 1% DCS water 52 76 R. miehei Purifie 0,5% DCS 53 70 water

Example 7 Table 7. Side-activities of steryl esterase culture filtrates SAMPLE Mannanase Xylanase Beta-glucanase HEC nkat/ml nkat/ml nkat/ml nkat/ml Commercial lipases (1%) 1 Resinase#, Novo #1, 6 4,8 1,3 0 2 Candida rugosa (lipase B2), #0,7 #1, 4 0 0 Biocatalysts = 70 nkat/g 3 Pseudomonas sp., Amano #2,4 0 #0, 8 = 80 0 nkat/g 4 Geotrichum candidum, 0 0 1,1 = 110 nkat/g 0 Biocatalysts Growthmedia 5 T. reesei (D-74083) distiller's 66,3 3081 388 104,1 spent grain QM 6a 6 T. reesei QM 6a 12,5 40,1 103,6 7,0 (ATCC13631) soya 7 T. reesei QM 9136 (ATCC-3,2 3,5 21, 1 #3, 3 26920) distiller's spent grain 8 T. reesei QM 9136 soya 0 3,6 18,7 0 9 T. reesei Rut C-30 (ATCC 37,5 1716 570,4 98,6 56765) soya 10 P. stutzeri (E-97825) soya 4,7 21,9 11,3 6,0 I I R. miehei (D-82193) soija 43,6 4,0 2775-3, 2 HIC-purified growth niedia 12 HIC-T. reesei QM 6a 68,1 Not done Not done Not done (ATCC13631) distiller's spent grain 13 HIC-T. reesei QM 9136 5,3 0 45,1 0 (ATCC 26920) distiller's spent grain 14 HIC-T reesei Rut C-30 19,7 ~ 1,8 160,4 18,9 soya(ATCC56765) 15 HIC-P. stutzeri (E-97825) #4,2 0 4,0 0 soya 16 HIC-P. stutzeri (E#97825) #1,0 0 #0, 6 0 DCS-water

The side-acitivities were measured by methods described in Ståhlbrand et al (1993) (mannanase), Bailey et al. (1992) (xylanase), Zurbriggen et al. (1990) (beta-glucanase) and IUPAC (endoglucanase, HEC). Only T. reesei QM 6a (ATCC13631) and T. reesei Rut C- 30 (ATCC 56765) produced a substantial amount of cellulase into the culture medium. T. reesei QM 9136 (ATCC 26920) is a cellulase negative mutant strain, which produces no or only a hardly mesurable amount of cellulase measured as HEC activity.

Literature references: Bailey, M. J., Biely, P. and Poutanen, K. (1992) Interlaboratory testing ofmethods for assay of xylanase activity. J. Biotechnol. 23: 257-270.

Breuil, C., Leone, R., Peng, J. and Serreqi, A. (1997) Some fungi can degrade aspen steryl esters and waxes. Proc. 9th Int. Symp. Wood Pulp Chem., Vol 1, pp 60: 1-3.

Fujita et al. (1992) Paperrnakers Conference, TAPPI Proceedings 1992, pp 73-79.

Glick, B. R. and Pasternak, J. J. (1998) Molecular biotechnology: Principles and applications of recombinant DNA. 2nd Edition, ASM Press, Washington, D. C.

IUPAC (International Union of Pure and Applied Chemistry) (1987) Measurement of cellulase activities. Pure and Appl. Chem. 59: 257-268.

Leone, R., Serreqi, A. and Breuil, C. (1998). The degradation of aspen steryl esters and waxes by wood-inhabiting fungi. Proc. 7th Int. conf. on Biotechnology in the Pulp and Paper Industry. Vol B. pp. B: 1 1-14.

Mustranta, A., Spetz, P., Ekman, R., Luukko, K. and Buchert, J. (1998) Enzymatic modification of dissolve and colloidal substances in process waters of mechanical pulping. Proc. EUCEPA 1998 Symp. Chemistry in Papermaking, Florence, 12-14 oct.

1998,93-101.

Sambrook, J., Fritsch, E. F. and Maniais, T. (1989) Molecular Cloning, A Laboratory Manual 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Sjöström, E. (1981) Extractives. In: Wood Chemistry. Fundamentals and applications. 2 eds. Academic Press. San Diego. pp. 90-108.

Ståhlbrand, H., Siika-aho, M., Tenkanen, M. and Viikari, L. (1993) Purification and characterization of two endo- (3-mannanases from Trichoderma reesei. J. Biotechnol. 29:

229-242.

Zurbriggen, B. Z., Bailey, M. J., Penttilä, M. E., Poutanen, K. and Linko, M. (1990) Pilot scale production of a heterologous Trichoderma reesei cellulase in Saccharomyces cerevisiae. J. Biotechnol. 3: 267-278.

Örså, F. and Holmbom, B. (1994) A convenient method for determination of wood extractives in papermaking process waters and effluents. J. Pulp Paper Sci. 20 (1994) J361- 366. INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule 13bis) A. The indications maat bClow rcIate to the microorganism reterred to in the description on page 6 line 31 Furiher deposits are identifie on an additional sheet O Name of depositary inscitution DSMZ-Deutsche Samnlung von Mikroorganisnten und Zellkulturen (3nbH Address of deposiLuY institution finciuding posrai coae and counirvi Mascheroder Weg 1 b, D-38124 Braunschweig, (3ermany Date of deposh | Accession Number December 16,1998 DSM 12587 C. 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