DK167882B1 - Alginate lyase used in cooling water systems - produced by cultivating strain of atypical Bacillus stearothermophilus - Google Patents

Abstract

The following are claimed: (A) a biologically pure culture of a strain of a typical Bacillus stearothermophilus (B.S) NRRL B-18394, mutants and variants characterised by the ability to produce an alignate lyase and by growth at pH 5.7, characterised also by having optimal temp. for growth at 55-60 deg.C with no growth below 30 deg.C. (B) a process for prepg. alignate lyase, characterised by cultivating an alignate lyase producing strain of atypical B.S. a mutant or variant or a transformant microorganism cell contg. a gene encoding for and expressing the alignate lyase aerobically under submerged conditions in a culture and then recovering the enzyme from the culture broth. (C) an alignate lyase native to an atypical B.S. strain characterised and by the ability to degrade algal cell wall and the alignate polymer produced by Pseudomonas aeruginosa, the alignate lyase retaining 100% activity after 4 hrs. heat-treatment at 60 deg.C.

Description

DK 167882 B1

This invention relates to a novel atypical strain of Bacillus stearothermophilus capable of producing alginate-lyase, a process for producing alginate-lyase, a novel alginate-lyase, an enzyme additive containing the alginate-lyase, and a method for controlling pollution. 5 of a cooling water system using the alginate lyase.

It is known that uncontrolled growth of microorganisms in a cooling water system (which recirculates the water) can lead to the formation of deposits which produce contamination, corrosion and coatings. Mucus can clog pipes, impede heat supply or interfere with the operation of the cooling water system at all. It has been found that 10 presence of the algae Chroococcus, Oscillatoria and Chlorococcus in cooling water systems has been the cause of pollution. Furthermore, it seems that algal mucus becomes a growing site for corrosive bacteria and perhaps pathogenic bacteria.

Alginic acid (alginate), which is a polysaccharide, is the major constituent of algae cell walls. In some algae species such as Fucus distichus, alginate accounts for up to 60% 15 of the total cell wall. In addition to algae, some bacteria commonly found in cold water form, e.g. Pseudomonas spp., An extracellular polysaccharide polymer (mucus), which results in contamination, gas formation, and protection of corrosive bacteria in cooling water systems. The extracellular polysaccharide polymer (mucus) made by some Pseudomonas spp. 20 has been identified as alginate (L. R. Evans et al., J. Bacteriol. 1973, 116: 915-924).

Alginate is a copolymer of three structural blocks, poly-D-mannuronic acid (poly-M), poly-aL-guluronic acid (poly-G), as well as blocks in which both uronic acids are present in what is considered an alternating sequence ( poly-MG) (A. Haug et al., Acta Chem.Scand. 1967, 21: 691-704).

Thus, the action of enzymes such as alginate lyases capable of depolymerizing alginate in the algae cell wall and mucus from Pseudomonas spp. May cause degradation of these organisms and / or increase their susceptibility to the chemical biocides commonly found in cooling water systems.

Any of the results render these nuisance microorganisms 30 non-viable, which is highly desirable for optimum operation of cooling water systems.

DK 167882 B1 2

The object of the invention is to provide an alginate lyase which has improved thermostability and is effective in bunk water systems.

Further objects and advantages of the invention will become apparent from the description thereof hereinafter.

All alginate-lyase enzymes characterized prior to the date of this specification, including those from non-bacterial sources, appear to catalyze an elimination reaction in which, during cleavage of the uronic acid polymer, a non-reducing Δ 4.5 is formed. unsaturated binding (IW Sutherland In I. Sutherland (ed.), "Surface Carbohydrates of the Prokaryotic Cell", 1977, pp. 209-245, Academic Press, Inc., London). Various alginate lyases show a preference for guluronate or mannuronate blocks in the alginate polymer; Thus, they can be characterized as guluronidase or mannuronidase (I. W. Davidson et al., J. Gen. Microbiol. 1977, 98: 223-229).

Several bacteria are known to produce enzymes that can degrade 15 alginate, but most of these alginate-lyase-producing bacteria are marine isolates (RS Doubet et al., Appl. Environ. Microbiol. 1982, 44: 754-756 and VL von Riesen , Appl.Environ.Microbiol., 1980, 39: 92-96). Some characterized alginate lyases have been isolated from bacteria of terrestrial origin: examples of these include those from Klebsiella aerogenes (J. Boyd et al., Carbohydr. Res., 1977,57: 163-171) 20 and Bacillus circulans (JB Hansen et al. al., Appl.Environ.Microbiol., 1984, 47: 704-709). So far, no thermophilic, gram-positive alginate-lyase producers have been reported.

The alginate lyase of the invention is prepared extracellularly from a thermophilic Bacillus sp. It has better thermostability than known alginate lyases and retains 100% of its activity after 4 hours of treatment at 60 ° C without the presence of substrate.

The preferred producer strain (W36-7-4) (NRRL B-18394) for the alginate lyase of the invention is taxonomically determined as a strain of Bacillus stearo-thermophilus that is atypical by growing at pH 5.7. The significant number of other B. stearothermophilus strains that have been tested does not produce alginate lyase. Ten strains were tested, namely NRRL B5407, ATCC 12980 type strain; ATCC 7953,

30 10149, 31783, 31195, 31196, 31197, 81198 and 31199. Further, others form Bacillus sp. strains tested, not alginate-lyase, i.e. B. coagulant ATCC

DK 167882 B1 3 7050; B. licheniformis ATCC 14580; B. subtilis ATCC 6633; and B. cereus ATCC 11778. Thus, the atypical S. stearothermophilus strain, wherein the alginate lyase is a free extracellular enzyme that can be delivered, is also atypical in that it can form the enzyme.

The detailed account of the alginate lyase of the invention relates to the use of the lyase as an algae pesticide and mucosal inhibitor in cooling water systems (in which the water recycles). Other applications of the alginate lyase may be found, e.g. extraction of cellular constituents from algae.

For further understanding of the invention, reference is made to the drawings in which: Figure 1 graphically shows the activity of the alginate lyase from atypical Bacillus stearothermophilus strain W36-7-4, (NRRL B-18394) as a function of pH and temperature.

Figure 2 graphically shows a comparison of the thermostability at 60 ° C between alginate lyases from Bacillus stearothermophilus strain W36-7-4 (·), Bacillus circulans (ATCC 15518) (□) and Xanthomonas maltophilia (ATCC 13637) (X).

As can be seen from Figure 2, the alginate lyase of the invention was superior in terms of thermostability to alginate lyases from Bacillus circulans (ATCC 15518) and from Xanthomonas maltophilia (ATCC 13637). After four hours of heat treatment at 60 ° C in the absence of substrate, the enzyme of the invention retained 100% of its original activity, whereas the enzyme from Bacillus circulans lost a total of 20 its original activity and the enzyme from Xanthomonas maltophilia lost 88% of its original activity.

The characteristics of the thermostability of the alginate lyase according to the invention, i.e. being stable at 60 ° C, are the main advantage as 60 ° C is at the high end of the usual temperature range at which cooling water systems are operated.

Thus, the ability to depolymerize the alginate component of algal cell walls and mucus is produced by Pseudomonas spp. in cooling water systems possessed by the alginate lyase of the invention is accompanied by a desirable high level of (thermo) stability in such systems.

When an algal cell suspension (obtained from a cooling water tower) was treated with the alginate lyase of the invention, a quantitative release of unsaturated uronic acid units was obtained (shown in Table I). The results show that the alginate component of algae cell wall is degraded by the alginate lyase. After 60 minutes of enzyme treatment 5, you can see under the microscope the algae cell wall with very small holes.

Thus, this invention further includes a method for controlling contamination of a cooling water system by algae and Pseudomonas spp., Characterized in that an effective amount of the alginate lyase of the invention is added.

10 It is not only algae that are affected by the alginate lyase. It is clearly seen (see Table III) that the alginate lyase can also depolymerize the alginate polymer prepared by Pseudomonas aeruginosa (ATCC 9027), which alginate polymer is also a troublesome microbe present in cooling water systems. P. aeruginosa's resistance to treatment of cooling water system with biocides is believed to be due to a membrane containing alginate. Treatment of Pseudomonas aeruginosa with the alginate lyase of the invention removes the polymer layer and it is believed to improve susceptibility to the organisms against the biocides.

In addition to alginate, algal cell walls also contain laminarin, a polysaccharide that can be depolymerized by island glucanase. You can obtain a 20 combination and possibly a synergistic effect for the degradation of algal cell walls by the addition of both a commercially available islet glucanase (e.g. Ceremix® 2X L) and the alginate lyase of the invention as shown in Table II. Indeed, Ceremix® 2X L is a mixture of enzyme activities, especially β-glucanase, α-amylase and protease activities, but commercially available enzyme products often contain a substantial portion of 25 bi-activity enzymes.

Accordingly, this invention further encompasses a method for controlling contamination of a cooling water system by algae and Pseudomonas spp., In which an effective amount of alginate lyase according to the invention is added and an additional effective amount of islet glucanase is added to the water. A preferred β-glucanase is 30 Ceremix®, which will benefit from the presence of other desirable enzyme activities. Possibly. for example, biocidal chemicals (eg glutaraldehyde, hydrazine) which do not inhibit the enzyme (s) may be present in conjunction with the two enzymes or for that matter.

Advantageously, the alginate lyase of the invention is compatible with the various chemicals commonly used nowadays when used for the treatment of cooling water systems, e.g., EDTA, glutaraldehyde. As indicated in Table IV, the alginate lyase retains 100% of its enzyme activity after 16 hours of incubation with chelating agents, various oxidants and typical corrosion inhibitors. The chemicals tested were at 10mM final concentration, which is approx. 10 times higher than the usual dose used in the treatment of cooling water systems (100 PPM).

Thus, it is possible to use the enzyme of the invention in combination with chemicals for treating water, and especially with the chemicals commonly heretofore used to solve the problem of microbes in cooling water systems.

According to a further aspect of the invention, there is provided a process for producing alginate lyase characterized by culturing the strain Bacillus stearothermophilus NRRL B-18394 or mutants thereof having substantially the same characteristics under submerged conditions in a culture medium containing suitable carbon. - and nitrogen sources and then extract the enzyme from the culture fluid.

As already mentioned, the alginate lyase of the invention is suitable for use in reducing pollution by microorganisms in systems with water, especially systems with recirculating water, such as cold water containers or recycled water in the paper and pulp industry (e.g., "white water" or " back water "). The enzyme can be used at temperatures from room temperature to 70 ° C, especially 40-60 ° C and at pH 4-9, especially 5-8. A solid or liquid concentrate of the alginate lyase 25 is added to the recirculating water in effective amounts. Good advice can be given regarding the minimum effective amount, as each system of water can be special, e.g. clean or highly contaminated water, low or high mineral content in the water (zinc and sodium chloride appear to partially inhibit the enzyme), frequency of dosing of the enzyme, e.g. continuous, weekly, monthly, possibly. content of biocide and about β-30 glucanase, e.g. Ceremix® 2X L is also present in the system. It is recommended to take a sample to determine the minimum effective enzyme dose. Usually, DK Tb / bbZ bl 6 will be sufficiently slightly smaller than the concentrations given in the examples later.

An effective amount of alginate lyase according to the invention, e.g. 2500 units per liters of water and maybe less to treat can be added to the water. Larger 5 amounts than 2500/1 alginate lyase can of course also be used if it is not too expensive. 2.5 ml to 25 ml Ceremix® 2X L in combination with the 2500 u / l alginate lyase should be quite effective. Commonly used biocides can also be used with the alginate lyase (since the enzyme is not inhibited by most biocides) at the concentration of 100 PPM, which is the usual recommended amount (1987 10 Guide to water treatment chemicals).

The microorganisms of the invention are an aerobic Bacillus isolate of atypical Bacillus stearothermophilus. The best producer strain W36-7-4 (NRRL B-18394) was deposited with the Agricultural Research Culture Collection (NRRL), Peoria, IL, U.S.A., under the terms of the Budapest Treaty.

Mutants of this strain, W36-7-4 (NRRL B-18394) with substantially the same properties are also within the scope of the invention. Other lyase-forming strains of the atypical Bacillus stearothermophilus have been studied (relatively superficially). The alginate lyase exemplified in this specification is the preferred embodiment. The alginate lyase of the invention can be prepared by a transformed host organism (cell). The native alginate lyase is an induced enzyme but can be constitutively produced in mutated or transformed cells.

The growth temperature of strain W36-7-4 is 50 ° C to 60 ° C, low growth at or above 60 ° C. Optimal pH for growth of strain W36-7-4 is 7. Zero growth occurs at or above pH 8.0.

On nutrient agar swabs, mature colonies of strain W36-7-4 are transparent and with smooth surfaces.

A rudimentary indication of alginate-lyase activity is an increase in viscosity of a 0.5% sodium alginate solution. Quantitative calculations of the alginate-30 lyase reaction have been performed by the following methods: (a) Increase in UV absorbance at 230 nm (J. Boyd et al., Carbohydr. Res. 1977, 57: 163- 171). To a 2 ml sodium algae acid solution (0.1%, 0.25% or 0.5%) prepared in 10mM sodium phosphate buffer, pH 7.0 containing 10mM MgCl 2, 0.1 ml of a suitably diluted enzyme liquid was added and The reaction mixture was incubated at 55 ° C for 2 hours with vigorous stirring. At the end of the incubation time, the increase in absorbance at 230 nm was measured by a spectrophotometer. One unit was defined as the amount of alginate lyase giving an increase of 0.001 OD unit (230 nm) per unit area. per minute at a specified temperature, usually 55 ° C.

1 o (b) Reducing force. The substrate solution was identical to the above.

Substrate solution (2 ml) and enzyme solution (1 ml) were incubated at 55 ° C for 2 hours, and dinitrosalicylate reagent (2 ml) was then added. The reduction power as mannuronic acid was determined as described by G. Noelting et al., (G. Noelting et al., Helv.Chim.Acta., 1948,31: 286-290). An activity unit was defined as the amount of enzyme that could release the reducing power corresponding to 0.123 mg of mannuronic acid under the degradation conditions mentioned.

(c) Thiobarbituric acid experiments. The substrate solution was identical to the above. The substrate solution (2 ml) and enzyme solution (1 ml) were incubated at 55 ° C for 2 hours. The yield of unsaturated uronic acid was determined by the periodate / -20 thiobarbituric acid method (TBA method) by Weissbach and Hurwitz (A. Weissbach et al., J. Biol. Chem. 1959,234: 705-709). The enzyme activity unit was defined as the amount of enzyme capable of releasing the equivalent of 1 pmol per ml. minute of formylpyruvic acid; 0.01 yesterday generated an OD549 of 0.29 in the sample.

As can be seen in Figure 1 (a), the optimum activity of alginate lyase 25 from Bacillus stearothermophilus strain W36-7-4 was observed at pH ca. 7. Surprisingly, the alginate lyase of the invention exhibits a temperature optimum around 50 ° C as shown in Figure 1 (b), as the microorganisms producing the enzyme are thermophilic.

The alginate lyase from strain W36-7-4 not only exhibited a high temperature optimum, but also exhibited good thermostability at 60 ° C as shown in Figure 2. Alginate lyase from Bacillus circulans ATCC15518) and Xanthomonas maltophilia (ATCC13637) DK Lost 55 and 29% of initial activity, respectively, after one hour of heat treatment at 60 ° C. The enzyme of the invention retained 100% of its enzyme activity through four hours of heat treatment.

Bacillus stearotherophilus strain W36-7-4 (NRRL B-18394) can be grown under aerobic conditions in a nutrient medium containing assimilable carbon and nitrogen along with other essential nutrients. The medium per se is not considered to be part of the invention and can be formulated according to the guidelines known in the art for growing Bacillus stearothermophilus strains.

If desired, sodium igate can be used as the carbon source and to induce the enzyme. The alginate concentration incorporated into the medium can vary widely, e.g. 0.1 to 1% with 0.5% concentration in the solution of the medium is appropriate.

The nitrogen source in the nutrient medium may be organic or inorganic. Among possible organic nitrogen sources, a number are regularly used in 15 fermentation processes (including cultivation of bacilli) such as soy flour, cotton seed cake, peanut flour, corn mold water and yeast extract. In addition, the nutrient medium should also contain the usual trace elements.

As the atypical B. stearothermophilus of the invention is thermophilic, the culture is carried out at relatively high temperatures (e.g., 55 ° C). For the cultivation of 20 strain W36-7-4 or a mutant thereof having substantially the same properties in fermentation tanks, artificial aeration is recommended. The aeration rate may be like that used in conventional fermentation tanks (submers).

After fermentation, the product alginate-lyase (which is formed extracellularly) can be prepared in liquid form by removing coarse material from the fermentation liquid and then by concentrating the liquid by conventional methods, e.g. evaporation at low temperature or by ultrafiltration. Finally, preservatives can be added to the concentrate.

The alginate lyase of the invention may also be prepared by culturing a transformed microorganism cell containing a gene encoding and expressing the alginate lyase formed by culturing the atypical Bacillus stearothermophilus of the invention, followed by the extraction of the enzyme from DK 167882 B1. 9 the culture fluid. Said transformed host organism comprises a host cell in which the gene for the alginate lyase of the invention and expression DNA is inserted by recombinant DNA technique. This technique is known in the literature and usually comprises the following steps: a) providing a suitable recombinant DNA cloning vector comprising DNA sequence encoding functions that facilitate gene expression and a DNA sequence encoding Bacillus alginate lyase ; b) transforming a suitable host organism with the cloning vector of step a); and then c) culturing the transformed host in a suitable culture medium and recovering the alginate lyase from the culture medium.

Preferred host organisms are strains of Bacillus, especially Bacillus subtilis.

Solid form enzyme preparations may, if desired, be prepared from the purified and / or concentrated fermentation liquid by precipitation with salt such as Na 2 SO 4 or with water miscible solvents such as ethanol or acetone. Removal of all water in the fermentation liquid by suitable drying methods such as spray drying may also be used. The activity of crude alginate-lyase preparations thus obtained in the cultivation of NRRL B-18394 is usually about 5000 to 20 units / g powder.

The enzyme additive according to the invention is characterized in that it comprises the alginate lyase according to the invention and said additive is in the form of a non-dusting granulate or a stabilized liquid.

Non-dusty enzyme granules are well known, and may e.g. are manufactured in accordance with Dutch Patent Application No. 167,993 (Novo Nordisk A / S), US Patent No. 4,106,991 (Novo Nordisk A / S) or US Patent No. 4,661,452 (Novo Nordisk A / S) and the granulate may be coated if desired according to known methods.

DK Ί67882 ΒΊ 10

A liquid alginate-lyase preparation may be stabilized, e.g. by the addition of propylene glycol, other polyols, sugars, sugar alcohols and boric acid. Other known enzyme stabilizers may be used.

The following examples describe the preparation of the enzyme, characterization thereof, and possible uses thereof.

EXAMPLE 1

Bacillus stearothermophilus strain W36-7-4 (NRRL B-18394) was grown at 55 ° C on a shaking table (250 rpm) in 250 ml Erlenmeyer flasks with triple baffle containing 50 ml of a medium of the following composition:

Composition of the medium in grams per gram l:

Tryptone 1.0 (NH 4) 2 SO 4 2.0 KH 2 PO 4 1.3 Na 2

MgCII2'2H2 ° ° · 2

CaCl2-2H2O 0.2

Sodium alginate 5.0

There was no need to adjust the pH of the medium. After 30 hours of 20 incubation, the alginate-lyase activity of the fermentation liquid was determined using the U.V. absorbance sample as previously described. The alginate-lyase activity of the W36-7-4 liquid was 21 U / ml with 0.1% sodium alginate as the substrate.

EXAMPLE 2 The pH-activity and temperature-activity curves of crude alginate lyase from Bacillus 25 stearothermophilus strain W36-7-4 CNRRL B-183941_

Alginate lyase activity (increasing OD value at 230 nm) at 55 ° C was studied with sodium alginate as the substrate. Constant amounts of substrate, DK167882 B111 enzyme and MgCl2 (10mM) were present in all reactions. Citrate-phosphate buffer was used between pH 5.0 and 6.5 and phosphate buffer was used between pH 6.0 and 8.0. The maximum activity was observed around pH 7.0 (see Figure 1a).

The temperature activity curve was prepared by examining the alginate-5 lyase activity at temperatures between 30 ° C to 70 ° C in 10 mM MgCl 2. The maximum activity was observed around 50 ° C, see Figure 1 b.

EXAMPLE 3

A comparison of the thermostability of alginate lyases from Bacillus stearo-thermophilus strain W36-7-4 (·), Bacillus circulans (ATCC 15518) (□) and 10 Xanthomonas maltoohilia (ATCC 13637) (XI_

All 2.0 ml of a crude enzyme solution was heat treated at 60 ° C using the same buffer used in the previous example. The heat treatment lasted for 4 hours. Immediately thereafter, the solution was cooled to room temperature in cold water bath and the residual enzyme activities were measured using sodium alginate as the substrate. After 4 hours of incubation, alginate lyase from strain W36-7-4 retained 100% of its original activity, whereas the enzyme from Bacillus circulans and Xanthomonas maltophilia lost 100% and 88% of their original activity respectively (see Figure 2).

Example 4 20 Release of Unsaturated Uric Acid Units from the Alginate Component of Algae Cell Walls Using Alainate Lvase from Bacillus stearothermophilus strain W36-7-4_ 2.0 ml of algae suspensions obtained from a factory cooling water tower brought to a final concentration 10mM phosphate buffer, pH 7.0, was treated with various amounts of alginate lyase from strain W36-7-4 at 37 ° C.

The release of 4-deoxy-1-erythro-hex-5-ulosuronic acid was investigated by measuring the U.V. absorbance at 230 nm; the results obtained are shown in Table I.

Table I

DK 167882 ΒΊ II Δ OD230nm 12 5 Incubation time 0 mg 3 mg 5 mg

Enzyme Enzyme * Enzyme -1- 3.5 hours || 0 0 0.7 15.0 hours || 0.05 0.4 1.30 10 22.5 hours || 0 1.25 3.05 * Enzyme was added as crude enzyme lyophils of approx. 5000 U / g EXAMPLE 5

The combination effect of β-glucanase, ie. Ceremix® 2X L, and the alginate lyase from Bacillus stearothermophilus strain W36-7-4 by degradation of alginate component 15 in ala cell cells.

The algae suspensions were similar to those used in Example 4. The alginate lyase was dosed in amounts of 5 mg of the crude enzyme lyophil per ml. 2 ml algal cell suspension and the commercial Novo Nordisk product Ceremix® 2X L was added at 50 µl per ml. sample. Degradation was investigated as described in Example 4.

20 A synergistic effect of Ceremix® 2X L and alginate-lyase upon degradation of algal cell walls is clearly seen, as shown in Table II.

13 Δ OD230nm DK 167882 Bl

Table II

5 mg of alginate lyase

Incuba- 5 mg 50 μΐ +

no alginate lyase Ceremix® 2X L 50 μ 50 time Enzyme alone Ceremix® 2X L

10 ----- 4 t II 0 I 0.75 I o I 0.81 23 t || 0 I 3.24 I 0.35 | 5.70 74 t || 0 I 5.49 I 0.85 | 7.13 Example 6 15 The Degradability of Alginate Lyase from Bacillus Stearothermophilus Strain W36-7-4 for Alginate Polymer Prepared by Pseudomonas aeruginosa (ATCC 9027) 1 ml Suspension of Freshly Cultivated Pseudomonas aeruginosa (ATCC 9027) with OD at 660 nm of 0.43 were mixed with 5 mg and 10 mg of alginate-lyase-20 lyophils and incubated at 37 ° C for 30 minutes, 1 hour and 2 hours. The release of the unsaturated uronic acid unit was studied by absorbance at 230 nm as described above. The blank test was performed under the same conditions without the addition of enzyme. The results are shown in Table III.

Δ OD230nm

Table XII

DK 167882 B1 14 5 Incubation time 0 mg 5 mg 10 mg

Enzyme Enzyme Enzyme -1 - = - 1/2 hour || 0 0.15 0.245 1 hours || 0 0.27 0.51 10 2 hours || 0 0.65 1/41 EXAMPLE 7

The effect of chemicals commonly used in cooling water systems on the activity of alainate lvase from Bacillus stearothermophilus strain W36-7-4_ EDTA (ethylenediaminetetracetate), NaCl, FeSO4, NaAlOa Na2MoO4, Zinc, the concentration to 10mM. The alginate-lyase activity was assayed as described in Example 2 during 16 hours of incubation. The results are summarized in Table IV. Alginate lyase from Bacillus stearothermophilus strain W36-7-4 is found to be reasonably stable in the presence of various oxidants, metal chelators and salts commonly used in 20 cooling water systems such as pollutants, biocides, coagulants / flocculants, corrosion inhibitors, etc.

DK 167882 B1 15

Table IV

Inhibition (%) of chemicals Chemicals on the enzyme activity

EDTA O.

NaCl 48.7

FeS04 O

NaAlO2 O

Na-MoCl2

Zinc 43.7

Glutaraldehyde 0

hydrazine O.

NaNO2 O

Claims (10)

1. Atypical strain of Bacillus stearothermophilus, characterized in that it has the ability to form aiginate lyase, has growth at pH 5.7 and has optimum temperature for growth at 55-60 ° C with near zero growth below 30 ° C , and is a biologically pure culture of the strain Bacillus stearothermophilus NRRL B-18394 or a mutant thereof having substantially the same properties. 2. A process for the preparation of aiginate lyase, characterized by cultivating the strain Bacillus stearothermophilus NRRL B-18394 or mutants thereof with substantially the same properties under submerged conditions in a culture medium 10 containing suitable carbon and nitrogen sources and then extracting the enzyme from the culture liquid. 3. Aiginate lyase, characterized in that it has the ability to degrade algae cell walls and degrade the alginate polymer prepared by Pseudomonas aeruginosa, and wherein said aiginate lyase retains 100% activity after 4 hours of heat treatment at 60 ° C, and is identical to the enzyme produced by the cultivation of Bacillus stearothermophilus NRRL B-18394. Aiginate lyase according to claim 3, characterized in that it is formed by the cultivation of Bacillus stearothermophilus NRRL B-18394. An enzyme additive for cooling water systems, characterized in that it comprises the alginate lyase according to claim 3 or 4, and said additive is in the form of a non-dusting granulate or stabilized liquid. DK 167882 B1 17 Enzyme additive according to claim 5, characterized in that the alginate lyase is formed by the cultivation of Bacillus stearothermophilus NRRL B-18394. Process for controlling contamination of a cooling water system by algae and Pseudomonas spp., Characterized in that an effective amount 5 of the alginate lyase according to claim 3 or 4 is added. Process according to claim 7, characterized in that the alginate lyase is formed by growing Bacillus stearothermophilus NRRL B-18394. Process according to claim 7 or 8, characterized in that an effective amount of islet glucanase is further added to the water. 1 o Process according to any one of claims 7 to 9, characterized in that an effective amount of biocide is further added to the water.