Catalase in growth media

Abstract

The present invention relates to the use of a secreted fungal catalase, for hydrogen-peroxide neutralization in growth media for the detection of microorganisms as well as to a method for detecting microorganisms in hydrogen peroxide-bearing aerosol or on a hydrogen peroxide bearing surface, said method comprising contacting said aerosol or surface with a growth medium comprising a secreted fungal catalase, and detecting growth of microorganisms in said medium.

Claims

1. A hydrogen-peroxide neutralizing growth medium comprising at least 0.3 U/ml of a secreted fungal catalase in admixture with a growth medium, between 0.2 g/l and 4 g/l sodium pyruvate, and between 0.005 g/l and 0.05 g/l sodium thiosulfate, wherein the growth medium is a solid agar growth medium.

2. The medium according to claim 1, wherein said medium is sterilized.

3. The medium according to claim 1, wherein said catalase is derived from a species of the genus Magnaporthe or Scytalidium.

4. The medium according to claim 3, wherein the catalase is derived from Magnaporthe grisea or from Scytalidium thermophilum.

5. The medium according to claim 1, wherein the concentration of catalase in the growth medium is between 0.3 U/ml and 90 U/ml.

6. The medium according to claim 1 wherein the concentration of sodium pyruvate in the growth medium is between 0.2 g/l and 2 g/l.

7. A method for detecting microorganisms in a hydrogen peroxide-bearing aerosol or on a hydrogen peroxide-bearing surface, said method comprising contacting said aerosol or surface with a medium according to claim 1 and detecting growth of microorganisms in said medium.

8. The method according to claim 7, comprising the steps of: a. bringing a hydrogen peroxide-bearing aerosol or a hydrogen peroxide-bearing surface into contact with a growth medium comprising the catalase according to claim 1; b. placing the growth medium in an environment allowing the development of colonies of microorganisms; and c. determining if colonies of microorganisms which may have developed during step (b) are present.

9. The method according to claim 7, wherein the hydrogen peroxide-bearing aerosol comprises air.

10. The method of claim 7, further comprising the step of sterilizing the medium with gamma-radiation.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) In the context of this invention the term aerosol is defined as a gas that may contain solid particles and/or liquid droplets and/or biological cells and/or spores and/or conidia and/or virus particles.

(2) In the context of this invention the term secreted fungal catalase is defined as an enzyme that derived from a catalase that originated from a fungus that secreted said catalase. The secreted fungal catalase can be isolated from its original fungus, or it can be produced as secreted or as intracellular protein in an expression host, for example Escherichia coli, Bacillus subtilis, Pichia pastoris, Aspergillus oryzae. A gene encoding a catalase according to the invention can be derived from a natural fungal gene or from a synthetic gene with sequence information from a fungal gene. Examples of fungi where said secreted fungal catalase or its gene can originate from are Magnaporthe grisea (Pyricularia grisea, Magnaporthe oryzae), Scytalidium thermophilum, Humicola insolens, Trichoderma atroviride, Colletotrichum graminicola, Nectria haematococca, Gibberella zeae (Fusarium graminearum), Fusarium oxysporum, Gibberella moniliformis, Cryphonectria parasitica, Claviceps purpurea, Oidiodendron maius, Phytophthora sojae, Pythium ultimum, Thielavia terrestris (Acremonium alabamense), Verticillium albo-atrum or Verticillium dahliae.

(3) The term medium refers to a sterilisabel growth or nutrient medium designed to support the growth of microorganisms. The most common growth media for microorganisms are nutrient broths and agar plates. Microorganisms can grow in liquid growth media and on the surface of solid growth media.

(4) Surprisingly, the hydrogen peroxide-neutralising action of the media according to the invention can be boosted by the addition of at least 40 U/plate of a secreted fungal catalase; optionally sodium pyruvate and/or sodium thiosulfate are added in small amounts.

(5) Media according to the invention with the addition of a secreted fungal catalase and optionally sodium pyruvate and/or sodium thiosulfate are stable for 6 month, which substantially facilitates storage and shipping and guarantees that the customer has a long potential period of use.

(6) The subject matter of the following definitions is considered embodiments of the present invention:

(7) 1. A hydrogen-peroxide neutralizing medium comprising at least 0.3 U/ml of a secreted fungal catalase.

(8) 2. The medium according to claim 1 further comprising up to 4 g/l, preferably up to 2 g/l, more preferably up to 0.2 g/l sodium pyruvate.

(9) 3. The medium according to claim 2, wherein said pyruvate prevents the formation of cracks or bubbles in the agar medium after the addition of hydrogen peroxide.

(10) 4. The medium according to any one of claim 1 or 3 further comprising up to 0.05 g/l, preferably up to 0.005 g/l sodium thiosulfate.

(11) 5. The medium according to any one of claims 1 to 4, wherein said medium is sterilized, preferably gamma-irradiated.

(12) 6. The medium according to any one of claims 1 to 5, wherein said catalase is derived from a species of Magnaporthe or Scytalidium, preferably from Magnaporthe grisea or from Scytalidium thermophilum.

(13) 7. Use of a secreted fungal catalase, for hydrogen-peroxide neutralization in a medium for the detection of microorganisms.

(14) 8. The use according to claim 7, wherein the catalase is derived from a species of Magnaporthe or Scytalidium, preferably from Magnaporthe grisea or from Scytalidium thermophilum.

(15) 9. The use according to claim 7 or 8, wherein the catalase exhibits an activity of at least 25%, preferably of at least 35%, more preferred of at least 50% after incubation at 50 C. for 4 h.

(16) 10. The use according to any one of claims 7 to 9, wherein the catalase exhibits an activity of at least 25%, preferably of at least 35%, more preferred of at least 50% after sterilization of the medium by gamma-irradiation with at least 10 kGy, preferably at least 15 kGy, more preferably at least 20 kGy.

(17) 11. The use according to any one of claims 7 to 10, wherein the catalase exhibits an activity of at least 25% preferably of at least 35%, more preferred of at least 50% after storage in the medium for at least 6 months.

(18) 12. The use according to any one of claims 7 to 11, wherein the catalase is used in a concentration of about 0.3 to 90 U/ml medium.

(19) 13. The use according to any one of claims 7 to 12, comprising up to 4 g/l, preferably up to 2 g/l, more preferably up to 0.2 g/l sodium pyruvate and optionally up to 0.05 g/l, preferably up to 0.005 g/l sodium thiosulfate.

(20) 14. A method for detecting microorganisms in hydrogen peroxide-bearing aerosol or on a hydrogen peroxide-bearing surface, said method comprising contacting said aerosol or surface with a medium according to any one of claims 1 to 6, and detecting growth of microorganisms in said medium.

(21) 15. The method according to claim 14 comprising the steps of a. bringing a hydrogen peroxide-bearing aerosol or a hydrogen peroxide-bearing surface into contact with a growth medium comprising the catalase according to any one of claims 1 to 6; b. placing the growth medium in an environment allowing the development of colonies of microorganisms; c. determining if colonies of microorganisms which may have developed during step (b) are present.

(22) 16. The method according to claim 14 or 15, wherein the hydrogen peroxide-bearing aerosol comprises air.

Example 1 Preparation of Agar Plates

(23) TABLE-US-00001 TABLE 1 Components of TSA-LT agar growth medium Ingredient g/l Casein peptone 15.0 Soy peptone 5.0 Sodium chloride 5.0 Agar 15.0 Lecithin 0.7 Tween80 5.0
TSA-LT Stands for Tryptic Soy Agar with Lecithin and Tween.

(24) TSA-LT agar growth medium was prepared by adding into each half-liter screw-cap bottle 16 g CASO agar powder, which contains the first four components of Table 1 premixed, 0.28 g lecithin, 2 g Tween80 and 400 ml pure water. After autoclaving at 121 C. for 20 minutes, the medium was cooled down to 50 C. in a water bath. Catalase solutions, or other solutions with additives, were added with sterile filtration. After mixing, 30 ml growth medium were poured into each petri dish with a diameter of 90 millimeter. The next day the agar plates were packed in plastic foil and some plates were gamma-irradiated with 20.5 kGy.

(25) Catalase solutions were prepared as described below, sterile filtered, stored at 4 C. and their concentration measured as described in Example 4.

(26) Catalase from Magnaporthe was prepared as described (and called MagKatG2) by Zmock et al. (Biochimie, 2012, pp 673-683) and the purification process stopped before the hydroxyapatite column.

(27) Catalase from Scytalidium was prepared from the commercial product (Terminox Ultra 50 L from Novozymes) by ultrafiltration with a 30,000 MWCO (Molecular Weight Cut Off) and buffer exchange to 0.1 M potassium phosphate with 0.1 M sodium chloride at a pH of 7.0.

(28) Catalase from bovine liver was prepared from the commercial product (C9322 from Sigma) by dissolving in 0.1 M potassium phosphate at a pH of 7.0.

Example 2 Agar Diffusion Test

(29) TABLE-US-00002 TABLE 2 Components of LB-T growth medium Ingredient g/l Tryptone 10.0 Yeast extract 5.0 Sodium chloride 5.0 Tween20 5.0

(30) Bacillus subtilis ATCC 6633 was grown in LB-T growth medium (see Table 2) overnight at 37 C. The Bacillus suspension was diluted with LB-T growth medium to an OD600 (optical density at 600 nm) of approximately 0.1. This diluted Bacillus suspension (200 l/plate) was spread onto dry TSA-LT agar plates from Example 1. Four paper discs were put onto each agar plate. The paper discs had a diameter of 6 mm and were prepared from gel blot paper GB003 from Whatman with a hole punch. 10 l of an aqueous solution that contained hydrogen peroxide at a concentration of 2%, 5%, 8% and 10%, respectively, were pipetted onto the four paper discs on each agar plate. Afterwards the plates were incubated overnight at 37 C. The diameter of the inhibition zone was measured and the diameter of the paper disc subtracted and the result divided by two. Thus a reported value in the following table is the distances from the edge of the paper disc to the end of the inhibition zone. A value of zero means no inhibition observed. The larger the value, the stronger the undesired inhibition.

(31) TABLE-US-00003 TABLE 3 Inhibition on agar plates mm mm mm mm Additives Concentration (2%) (5%) (8%) (10%) Catalase from Magnaporthe 300 U/plate 0 0 0 0.5 Catalase from Magnaporthe 150 U/plate 0 0 0.5 1 Catalase from Magnaporthe 80 U/plate 0 0.5 1.5 2.5 Catalase from Magnaporthe 40 U/plate 0 2 3.5 4 Catalase from Magnaporthe 20 U/plate 0 3.5 5 5.5 Catalase from Magnaporthe 10 U/plate 1.5 5 6 7 Catalase from Scytalidium 300 U/plate 0 0 0 0.5 Catalase from Scytalidium 150 U/plate 0 0 0.5 1 Catalase from Scytalidium 80 U/plate 0 0.5 1.5 2 Catalase from Scytalidium 40 U/plate 0 1.5 2.5 3.5 Catalase from Scytalidium 20 U/plate 0 3 4 5 Catalase from Scytalidium 10 U/plate 1 4.5 5.5 6. Catalase from bovine liver 300 U/plate 0 2 4 4.5 Catalase from bovine liver 150 U/plate 0.5 4 5 6 Catalase from bovine liver 80 U/plate 1 5 6 7 Sodium pyruvate 2.00 g/l 0 1 2 2.5 Sodium thiosulfate 0.05 g/l Without additives 4.5 7 8 8.5

(32) TABLE-US-00004 TABLE 4 Inhibition on gamma-irradiated agar plates mm mm mm mm Additives Concentration (2%) (5%) (8%) (10%) Catalase from Magnaporthe 300 U/plate 0 0 0.5 0.5 Catalase from Magnaporthe 150 U/plate 0 0.5 1 2 Catalase from Magnaporthe 80 U/plate 0 1 2.5 3 Catalase from Magnaporthe 40 U/plate 0 3 4.5 4.5 Catalase from Magnaporthe 20 U/plate 0.5 4.5 5.5 6 Catalase from Magnaporthe 10 U/plate 1.5 5.5 7 7.5 Catalase from Scytalidium 300 U/plate 0 0 0.5 0.5 Catalase from Scytalidium 150 U/plate 0 0 0.5 1.5 Catalase from Scytalidium 80 U/plate 0 1 1.5 2.5 Catalase from Scytalidium 40 U/plate 0 2.5 3.5 4 Catalase from Scytalidium 20 U/plate 0.5 3.5 5 5.5 Catalase from Scytalidium 10 U/plate 1.5 5.5 7 7.5 Catalase from bovine liver 300 U/plate 1.5 5.5 6.5 7.5 Catalase from bovine liver 150 U/plate 2 6 7.5 8 Catalase from bovine liver 80 U/plate 3.5 7 8 8.5 Sodium pyruvate 2.00 g/l 0 1 2 3 Sodium thiosulfate 0.05 g/l Without additives 4.5 7.5 8.5 9

(33) Because 30 ml growth medium was used per plate, a concentration of 300 U/plate equals 10 U/ml, 40 U/plate equals 1.3 U/ml. and 10 U/plate equals 0.3 U/ml.

Example 3 Growth Promotion Test

(34) Bacillus subtilis ATCC 6633 was grown in LB-T growth medium (see Table 2) overnight at 37 C. The Bacillus suspension was put on ice and diluted to an OD600 of approximately 0.1 with a PBS solution that has a pH of 7.4 and contains 8 g/l sodium chloride, 0.2 g/l potassium chloride, 0.2 g/l potassium dihydrogen phosphate and 0.918 g/l disodium hydrogen phosphate. This diluted Bacillus suspension was further diluted 10,000 times with the PBS solution and kept on ice. Meanwhile, 300 l 1% hydrogen peroxide solution was spread onto a dry plate, which contains 30 ml TSA-LT agar growth medium (see Example 1). Thus a concentration in the growth medium of 100 ppm (parts per million) hydrogen peroxide was applied. The plate was dried for 20 minutes at room temperature (20-25 C.). Then 200 l 10,000 times diluted Bacillus suspension was spread onto the agar plate. Afterwards the plates were incubated overnight at 37 C.

(35) TABLE-US-00005 TABLE 5 Colonies on agar plates Additives Concentration CFU* Catalase from Magnaporthe 300 U/plate 189 Catalase from Magnaporthe 150 U/plate 210 Catalase from Magnaporthe 80 U/plate 169 Catalase from Magnaporthe 40 U/plate 140 Catalase from Magnaporthe 20 U/plate 8 Catalase from Magnaporthe 10 U/plate 0 Catalase from Scytalidium 300 U/plate 163 Catalase from Scytalidium 150 U/plate 178 Catalase from Scytalidium 80 U/plate 162 Catalase from Scytalidium 40 U/plate 154 Catalase from Scytalidium 20 U/plate 6 Catalase from Scytalidium 10 U/plate 0 Catalase from bovine liver 300 U/plate 0 Catalase from bovine liver 150 U/plate 0 Catalase from bovine liver 80 U/plate 0 Sodium pyruvate 2.00 g/l 161 Sodium thiosulfate 0.05 g/l Without additives 0 Without additives and H.sub.2O.sub.2 164 *CFU stands for colony-forming units.

(36) TABLE-US-00006 TABLE 6 Colonies on gamma-irradiated agar Additives Concentration CFU Catalase from Magnaporthe 300 U/plate 205 Catalase from Magnaporthe 150 U/plate 190 Catalase from Magnaporthe 80 U/plate 171 Catalase from Magnaporthe 40 U/plate 58 Catalase from Magnaporthe 20 U/plate 1 Catalase from Magnaporthe 10 U/plate 0 Catalase from Scytalidium 300 U/plate 161 Catalase from Scytalidium 150 U/plate 205 Catalase from Scytalidium 80 U/plate 109 Catalase from Scytalidium 40 U/plate 6 Catalase from Scytalidium 20 U/plate 0 Catalase from Scytalidium 10 U/plate 0 Catalase from bovine liver 300 U/plate 0 Catalase from bovine liver 150 U/plate 0 Catalase from bovine liver 80 U/plate 0 Sodium pyruvate 2.00 g/l 229 Sodium thiosulfate 0.05 g/l Without additives 0

(37) As shown in Table 5, catalase from Magnaporthe and catalase from Scytalidium at concentrations of 40 U/plate and at higher concentrations in not irradiated medium, lead to a recovery of much more than 50% of the applied colony forming units. However, catalase from bovine liver, even at the highest tested concentration of 300 U/plate, leads to 0% recovery of the applied colony forming units.

(38) As shown in table 6, catalase from Magnaporthe and catalase from Scytalidium at concentrations of 80 U/plate and at higher concentrations in gamma-irradiated medium, lead to a recovery of more than 50% of the applied colony forming units. After gamma-irradiation, agar plates that contain catalase from Magnaporthe or catalase from Scytalidium remain effective in degrading hydrogen peroxide.

Example 4 Measurement of Catalase Activity

(39) The definition of 1 catalase unit (1 U) is the amount of enzyme decomposing 1 micromole of hydrogen peroxide per minute at 25 C. at a pH of 7.0 and with an initial hydrogen peroxide concentration of 0.010 M (M stands for molar, which is mol/l).

(40) The pH of 7.0 was ensured with a 0.1 M phosphate buffer, which was prepared by mixing 61.5 ml 1 M dipotassium hydrogen phosphate and 38.5 ml 1 M potassium dihydrogen phosphate with 900 ml pure water. A 0.01034 M hydrogen peroxide solution was prepared by mixing 42.3 l 30 weight % hydrogen peroxide solution, which has a density of 1.11 g/ml, with 40 ml 0.1 M phosphate buffer. The temperature was brought to 25 C. in a water bath, and 2.9 ml of this solution was pipetted into a quartz cuvette. Then 100 l diluted catalase solution was added, mixed and the kinetic measurement quickly started. The absorption decrease per minute was measured with a photometer at 240 nm within the first minute in the linear range.

(41) Before this measurement, catalase solutions were diluted with 0.1 M phosphate buffer. Different dilutions were prepared, for example with a dilution factor of 20, 50 and 100. The dilution with the factor 20 was prepared by mixing 50 l catalase solution with 950 l 0.1 M phosphate buffer. The catalase dilution that gave around 0.1 absorption decrease per minute was used for the following calculation. The absorption decrease per minute was multiplied with the catalase dilution factor and multiplied with 688, which gives the concentration in the undiluted catalase solution in U/ml. The factor 688 includes the extinction coefficient (43.6 M.sup.1cm.sup.1) of hydrogen peroxide at 240 nm and the dilution in the cuvette.

(42) FIG. 1: shows the measurement of the activity of Magnaporthe catalase

Example 5 Crack formation Test

(43) Equal amounts of hydrogen peroxide, either 100 l 3% or 300 l 1% hydrogen peroxide were spread onto dry agar plates, which were prepared as described in Example 1 without gamma-irradiation. Then the plates were dried for 20 minutes at room temperature (20-25 C.). Afterwards 200 l sterile PBS solution, which was prepared as described in Example 3, were spread onto the agar plate. The plates were incubated overnight at 37 C.

(44) TABLE-US-00007 TABLE 7 Crack formation in agar plates and its prevention Cracks Cracks and bubbles and bubbles 100 l 100 l Additives Concentration 3% H.sub.2O.sub.2 1% H.sub.2O.sub.2 Catalase from Magnaporthe 338 U/plate many 0 Catalase from Magnaporthe 75 U/plate many 0 Catalase from Scytalidium 338 U/plate many few Catalase from Scytalidium 75 U/plate many few Catalase from Magnaporthe 75 U/plate 0 0 sodium pyruvate 2.00 g/l sodium thiosulfate 0.05 g/l Catalase from Magnaporthe 75 U/plate 0 0 sodium pyruvate 0.20 g/l sodium thiosulfate 0.005 g/l Catalase from Scytalidium 75 U/plate 0 0 sodium pyruvate 2.00 g/l sodium thiosulfate 0.05 g/l Catalase from Scytalidium 75 U/plate 0 0 sodium pyruvate 0.20 g/l sodium thiosulfate 0.005 g/l

(45) The formation of cracks and bubbles was completely prevented by the addition of pyruvate, even at low concentrations of pyruvate.

(46) Even if the same amount of hydrogen peroxide per plate had been applied, fewer cracks were formed with a more diluted solution of hydrogen peroxide. Furthermore we observed that cracks predominantly appeared in that part of the agar plated, where the hydrogen peroxide had been first applied before it was spread on the whole agar surface.

Example 6 Growth Promotion Test, Further Examples

(47) As described in Example 3, growth promotion tests were conducted with the agar plates as used in Example 5.

(48) TABLE-US-00008 TABLE 8 Colonies and cracks on agar Additives Concentration CFU Cracks and bubbles Catalase from Magnaporthe 338 U/plate 137 1-2 Catalase from Magnaporthe 75 U/plate 97 1-2 Catalase from Scytalidium 338 U/plate 83 3-4 Catalase from Scytalidium 75 U/plate 68 3-4 Catalase from Magnaporthe 75 U/plate 120 0 sodium pyruvate 2.00 g/l sodium thiosulfate 0.05 g/l Catalase from Magnaporthe 75 U/plate 108 0 sodium pyruvate 0.20 g/l sodium thiosulfate 0.005 g/l Catalase from Scytalidium 75 U/plate 114 0 sodium pyruvate 2.00 g/l sodium thiosulfate 0.05 g/l Catalase from Scytalidium 75 U/plate 99 0 sodium pyruvate 0.20 g/l sodium thiosulfate 0.005 g/l sodium pyruvate 2.00 g/l 125 0 sodium thiosulfate 0.05 g/l sodium pyruvate 0.20 g/l 0 0 sodium thiosulfate 0.005 g/l without additives 0 0 without additives and H.sub.2O.sub.2 83 0

(49) The combination of catalases with low concentrations of chemical additives is advantageous when compared to each component alone.

(50) Catalases without chemical additives may lead to cracks or bubbles in agar plates with hydrogen peroxide.

(51) Low concentrations of chemical additives without catalase, e.g. 0.2 g/l sodium pyruvate and 0.005 g/l sodium thiosulfate, did not support growth in agar plates with hydrogen peroxide.

(52) However, the combination of a catalase with low concentrations of chemical additives, e.g. 75 U/plate catalase from Magnaporthe and 0.2 g/l sodium pyruvate and 0.005 g/l sodium thiosulfate, did support growth and prevented the formation of cracks and bubbles.