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Patent survey
ProcessBiochemistry Vol. 31, No. 7, pp. 719-721, 1996 Copyright © 1996Elsevier Science Ltd Printed in Great Britain. All rights reserved 0032-9592/96 $15.00+ 0.00
~ ! ~ ~tl ~"" l j i"" i ELSEVIER
Patent Survey This is a regular series of articles covering a selection of recent patents and patent applications from Europe, the USA and Japan. Though many patent applications and patents on biotechnological processes have been published in Western Europe, USA and Japan during the last few months, the following are deemed to earn particular attention. Improvement biological aerated filters
(British patent application 9405871, now intemational patent application WO 95/25695 to Thames Water Utilities Ltd, Reasling, UK) Alan J. Smith, assignor to the above company, has developed a biological aerated filter as shown in Fig. 1. The apparatus (1) comprises a treatment vessel with a high voidage bed (2) situated above a low voidage granular media bed (3). Fluid to be treated (4), combined with an effluent recycle flow stream (5) and intermediate recycle flow stream (6), flows down through the flooded aerated high voidage media (2), through an intermediate section (7) containing a collector in the form of a trough (8) and down through the aerated packed bed of granular media (3). The trough (8) is known as a washout trough.
The high voidage media is supported on a grille or grid (9). During a cycle any surplus treated water passes out of the intermediate section (7) at (10), while backwash liquors pass out of the vessel at (11). The usual operating level of the liquid in the vessel is shown at (12), treated liquor or effluent passing out of the vessel at (13). Process air passes into the vessel (1) at (14). During a wash cycle of the media, the high voidage media (2) is drained down to a level below the washout trough (8) prior to the commencement of the wash. Only the granular media bed (3) is subjected to a combined air and water wash. During another operation the high voidage media (2) is subject to an air scour prior to draining or both media are washed at the same time. Figure 2 shows a treatment vessel (20) comprising a combination of a flooded anoxic packed media bed (21) and aerated high voidage media (2) situated above an aerated low voidage granular media bed (3). The fluid (4) to be treated, combined with the effluent recycle flow stream (5), passes down through the anoxic packed bed (21) which is separated from the lower (as viewed) aerated portion of the treatment vessel by a trough (22) in intermediate section (7). The flow exits the base of the anoxic filter (21) and is recycled (23) to the top of the aerated high voidage media (2) passing down through it, into and through the aerated
S
...12
-.-Zl ~w2
i
-3 14
/
1f
I
2O
Fig. 1.
Fig. 2. 719
1]
packed bed. During the wash cycle both anoxic and aerated filters can be drained via the washout trough (8) prior to washing the lower granular media bed. The high voidage media bed (2) is toroidal and surrounds the packed media bed (21).
References German patent publications 3 423 285, 3 428 798, 4 032 234. European patent publications 240 929, 497 214, 121 851,100 029. US patent publication 4 032 234.
A new method for producing heparinase through Bacillus sp. BHIO0
(Japanese patent application 63-297807 corresponding to US patent 5 455 162 to Research Development Corporation, 7bkyo, Japan) Bacillus sp. strain BH100 (FERM BP2613 and P-10408) has been used by Robert W. Bellamy and Kouki Horikoshi, both biochemists working for the above company, to develop a new process for the production of extracellular heparinase which has an optimum temperature of activity between 45 and 50°C. Heparinase is an enzyme which cleaves certain glycosidic linkages in heparin, a sulfated mucopolysaccharide with the major repeating unit -(4)2-deoxy-2-sulfamino-~-o-glucopyranoseo- sulfate - (1 - 4) - ~ - idopyranosylyronic acid-2-sulfate-(1-). The products of this enzyme activity are chain-shortened fragments of heparin. Heparinase also cleaves heparitin (otherwise known as heparin monosulfate or heparan sulfate), a sulfated mucopolysaccharide with a chemical structure similar to that of heparin, producing chain-shortened fragments of heparitin. The substrate, heparin, is widely used as an anticoagulant drug and consequently heparinase has numerous applications in the study of heparin structure, the investigation of the blood coagulation mechanism and in bioassays for detection of heparin in body tissues and fluids. Heparinase also has use in the preparation of low molecular weight heparin fragments which have
Patent Survey
720
potential therapeutic values as antithrombotic or anti-tumour agents. Bellamy and Horikoshi isolated a large number of microorganisms from soils by selection for growth at 45°C in a liquid medium containing mineral salts, trace amounts of vitamins and heparin as the major nutrient. From those samples which exhibited evidence of microbial growth after 1 week of cultivation, the microorganisms were isolated in a biologically pure form and tested for production of heparinase with activity at 45°C. One bacterial isolate was discovered which produced the desired heparinase activity and further, which, released the heparinase into the culture medium. This new heparinaseproducing isolate was designated strain BH100. The taxonomic properties of strain BH100 were examined in accordance with methods described in Bergey's
Manual of Systematic Bacteriology Vol. 2, and the strain was found to be Grampositive, spore-forming, rod-shaped, facultatively anaerobic, motile, catalasepositive and had a growth temperature range of 20-55°C. Based on these characteristics, it was determined that the strain was a microorganism belonging to the genus Bacillus. Isolation of the microorganisms from the soil was conducted as follows. Approximately 1"0 g of soil sample was added to 1'5 ml of medium which consisted of per litre: 2.0 g heparin, 0"35 g K2HPO4, 0.27 g NHaCI, 0.2 g MgCI2-6H20, 0"001 g each of lysine, histidine and methionine, 0.0025rag ferrous citrate, 0.1ml trace element solution (containing per litre: 0.12g ZnSO4, 0.5g MnSO4, 0.13g H3BO3, 0.004 g CuSO4, 0.006 g Na2MoO4, 0.012g COC12"6H20), and 0.1ml of vitamin solution containing per litre: 10mg each of folic acid and biotin, 25mg each of riboflavin, thiamine, nicotine acid, calcium pantothenate and para-aminobenzoic acid, and 50 mg pyridoxine hydrochloride. The cultures were held in sterile 24-well microtiter trays, sealed with plastic wrap to minimize evaporation, at 45°C without shaking. From those samples which exhibited evidence of microbial growth after 1 week of cultivation, the microorganisms were isolated in biologically pure form by repeated subculture of cells on a solid medium containing per litre: 2"0g heparin, 0.11 g K2HPO4, 1.0 g MgClz. 6H20, 8.0 g gellan gum, at pH 8.0 with incubation at 45°C. Figure 3 shows the influence of pH on the activity of the enzyme and Fig. 4 shows the influence of temperature on its activity.
100
•
?5-
~
2S"
Fig. 3. I00
-
7~j,
~o
\ o
30
35
40 45 TEMPERATURE
50
55
60
('C]
Fig. 4.
References B6hmer et al., J. Biol. Chem., 265 (1990) 13609-17. Can. J. Microbiol., 26 (1980) 337-84. Linhardt et al., Appl. Biochem. Biotechnol., 12 (1986) 135-76. Nader et al., J. Biol. Chem., 265 (1990) 16807-13. Nakamura et al., J. Clinical Microbiol., 26 (1988) 1070-1. US patent publications 5 145 778, 5 362 645. International examined patent publication WO 82/00659.
A process and apparatus for biological remediation of vaporous pollutants
(European patent 693 959 corresponding to international patent application 94/23825 to Alliedsignal Inc., NJ, USA) During the last 5 years there has been much development in the use of carbon-coated substrates in the support of pollutant-remediating microorganisms, and indeed reports have appeared in Process Biochemistry. Louis J. Defilippi, Francis S. Lupton and Mansour Mashayekhi, all bioengineers with the above company, have designed a very simple and practical process for this, the apparatus of which is shown in Fig.
5, a cross-sectional side view of their vertical reactor. The process involves passing a vaporous-stream with one or more of pollutants through a bioreactor (1) comprising a packed column (2) with a plurality of biologically active bodies. The biologically active bodies (3) comprise a macroporous substrate and one or more of microorganisms which are capable of metabolizing at least one of the pollutants contained in the vaporous stream. The bioreactor may also contain open or substantially open space bodies (4) that are intermixed with the biologically active bodies. The porous substrate and open space bodies are coated with an absorbent that is capable of absorbing one or more of the pollutants contained in the influent stream. The biologically active bodies and the open space bodies are placed on top of a vented support layer (5), which may be a perforated metal or plastic plate. The gas inlet (6) may be placed at the lower end of the bioreactor, as shown in Fig. 5, or at the top of the reactor, and the gas outlet (7) should be positioned at the opposite end to the inlet. During operation of the bioreactor, nutrients and water need to be provided. Nutrients, such as carbon and minerals, may be added in the form of fish meal peptine, soybean flour, peanut oil, etc. and usually salts capable of providing phosphate, sodium, potassium, ammonium, calcium, sulphate, etc. This feed mixture, containing nutrients and buffers, is provided in aqueous solution through an inlet (8). The feed mixture is fed into the bioreactor at the top through sprayers (10) and allowed to flow down the column and accumulate at the bottom of the reactor. Alternatively, the feed mixtures may be fed at different locations in the reactor through any inlet positioned in such a way that effectively and evenly distributes the feed mixture to the biologically active bodies. The accumulated feed mixture is withdrawn through a conduit or an outlet (9) to an external reservoir (11) in which the pH and the nutrient concentration of the collected feed mixture are analysed. Based on the analyses of the mixture, a pH controlling agent, such as a solution of an acid or a base, and nutrients are added to the external reservoir. The re-conditioned feed mixture is then recycled to the top of the bioreactor.
References 3 German patent publications. 4 European patent publications. 1 US patent publication.
721
Patent Survey
DO,Glucasc
O.D.,HA
120
10 /
60 40 20 0 0
RLOWER
10
5
Culture Tima +Glucose(g/L)*
-0.D.
(US patent 5 455 174 based on 3 equivalent Japanese patent applications 3-113591; 3-262943 and 3-289958 to Mitsubishi Rayon Company, Ltd, Tokyo, Japan)
Keiichi Sakashita and his co-biochemwith the above ists, cooperating Japanese company, have found a process for producing an organic acid ester of ascorbic acid or erythorbic acid, by reacting these acids with an organic acid enol ester in an organic solvent with a solubility of those acids of more than 0.3% at 25°C in the presence of an active lipase. Their work is exemplified by the following laboratory test which formed part of the research. 0.4 g of Amano Lipase PS (produced by Amano Seiyaku K.K.) was dissolved in 10 ml of a l/20 M aqueous phosphate buffer with a pH value of 7, and the solution was then evaporated to dryness under a reduced pressure to obtain a pretreated Lipase PS. 2 g of ascorbic acid and 6.7 g of stearic acid vinylester were dissolved in 40 ml of dried pyridine (water content 1200 ppm) and the pretreated Lipase PS was added to the mixture. The reaction was carried out at 40°C with vigorous stirring. After 4 h the concentration of ascorbic acid6-stearate in the reaction mixture was found to be 115% according to HPLC analysis. The reaction mixture was filtered and the filtrate poured into
20
25
DO+HA(g/L)
Fig. 6.
Fig. 5. Stereoselective esterification of ascorbic or erythorbic acids with long-chain enol esters and with the aid of Amano Lipase PS
15
200 ml of ice-cooled 6 N aqueous hydrochloric acid. The precipitate was filtered, washed with water and then dried under reduced pressure; 6 g of a crude product was obtained. The crude product was then washed with hot hexane to obtain 52 g of a yellowish-white product. The content of ascorbic acid6-stearate in the product was 96%. References 4 US patent publications.
3 European patent publications. 2 Japanese patent publications. A novel strain of Streptococcus zaaepidetnicus for producing hyaluronic acid (US patent 5 496 726 to Lucky Ltd. Seoul, Republic of Korea)
Myoung G. Park and his co-biochemists have discovered that high molecular weight hyaluronic acid can be produced by using a strain of Streptococcus and, furthermore, they have found a medium suitable for the culture of the microorganism. The microorganism lacks hyaluronidase activity and hemolytic activity, and is obtained by mutating a known strain of Streptococcus zooepidemicus (ATCC 35246). The process of mutagenesis includes treatment of the parent microorganism at least three times with N-methyl - N’ - nitro - N - nitrosoguanidine (NTG). Mutant strains lacking in hemolytic activity and are produced and selected after the first NTG treatment of the known microorganism. The
second NTG treatment thereof selects strains lacking hyaluronidase, which catalyzes the decomposition of hyaluronic acid. Thereafter, the selected strains are subjected to a third NTG treatment and then incubated on a solid medium to collect large rapidly-growing, highly mucous colonies as the mutant strains have a superior productivity of hyaluronic acid. One of the strains found has been registered with accession number KCTC 0075BP under the terms of the Budapest Treaty. In general, microorganisms belonging to the genus Streptococcus are incubated on media containing a carbon source, such as starch, glucose or sucrose, a nitrogen source, such as ammonium sulfate, ammonium nitrate, sodium nitrate, casamino acid, yeast extract, peptone or tryptone, and an inorganic salt, for example sodium chloride, sodium dihydrogenphosphate or disodium hydrogenphosphate. Figure 6 depicts the fermentation profiles of Streptococcus zooepidemicus (ATCC 35246) and Streptococcus zooepidemicus (LBF 707). References
Beaty,
N.
B., Anal.
Biochem.,
147
(1985) 387-95.
Chabreck, P., Chromatographia, 30 (1990) 201-4. Dische, Z., J. Biol. Chem., 189 (1947) 167. Hinodedo, A., J. Sot. Cosmet. Chem. Japan, 22 (1988) 35-42. Motohashi, N., .I. Chromatographia, 299 (1984) 508-12. 3 US patent publications.
3 European patent publications.
~ ! ~ ~tl ~"" l j i"" i ELSEVIER
Patent Survey This is a regular series of articles covering a selection of recent patents and patent applications from Europe, the USA and Japan. Though many patent applications and patents on biotechnological processes have been published in Western Europe, USA and Japan during the last few months, the following are deemed to earn particular attention. Improvement biological aerated filters
(British patent application 9405871, now intemational patent application WO 95/25695 to Thames Water Utilities Ltd, Reasling, UK) Alan J. Smith, assignor to the above company, has developed a biological aerated filter as shown in Fig. 1. The apparatus (1) comprises a treatment vessel with a high voidage bed (2) situated above a low voidage granular media bed (3). Fluid to be treated (4), combined with an effluent recycle flow stream (5) and intermediate recycle flow stream (6), flows down through the flooded aerated high voidage media (2), through an intermediate section (7) containing a collector in the form of a trough (8) and down through the aerated packed bed of granular media (3). The trough (8) is known as a washout trough.
The high voidage media is supported on a grille or grid (9). During a cycle any surplus treated water passes out of the intermediate section (7) at (10), while backwash liquors pass out of the vessel at (11). The usual operating level of the liquid in the vessel is shown at (12), treated liquor or effluent passing out of the vessel at (13). Process air passes into the vessel (1) at (14). During a wash cycle of the media, the high voidage media (2) is drained down to a level below the washout trough (8) prior to the commencement of the wash. Only the granular media bed (3) is subjected to a combined air and water wash. During another operation the high voidage media (2) is subject to an air scour prior to draining or both media are washed at the same time. Figure 2 shows a treatment vessel (20) comprising a combination of a flooded anoxic packed media bed (21) and aerated high voidage media (2) situated above an aerated low voidage granular media bed (3). The fluid (4) to be treated, combined with the effluent recycle flow stream (5), passes down through the anoxic packed bed (21) which is separated from the lower (as viewed) aerated portion of the treatment vessel by a trough (22) in intermediate section (7). The flow exits the base of the anoxic filter (21) and is recycled (23) to the top of the aerated high voidage media (2) passing down through it, into and through the aerated
S
...12
-.-Zl ~w2
i
-3 14
/
1f
I
2O
Fig. 1.
Fig. 2. 719
1]
packed bed. During the wash cycle both anoxic and aerated filters can be drained via the washout trough (8) prior to washing the lower granular media bed. The high voidage media bed (2) is toroidal and surrounds the packed media bed (21).
References German patent publications 3 423 285, 3 428 798, 4 032 234. European patent publications 240 929, 497 214, 121 851,100 029. US patent publication 4 032 234.
A new method for producing heparinase through Bacillus sp. BHIO0
(Japanese patent application 63-297807 corresponding to US patent 5 455 162 to Research Development Corporation, 7bkyo, Japan) Bacillus sp. strain BH100 (FERM BP2613 and P-10408) has been used by Robert W. Bellamy and Kouki Horikoshi, both biochemists working for the above company, to develop a new process for the production of extracellular heparinase which has an optimum temperature of activity between 45 and 50°C. Heparinase is an enzyme which cleaves certain glycosidic linkages in heparin, a sulfated mucopolysaccharide with the major repeating unit -(4)2-deoxy-2-sulfamino-~-o-glucopyranoseo- sulfate - (1 - 4) - ~ - idopyranosylyronic acid-2-sulfate-(1-). The products of this enzyme activity are chain-shortened fragments of heparin. Heparinase also cleaves heparitin (otherwise known as heparin monosulfate or heparan sulfate), a sulfated mucopolysaccharide with a chemical structure similar to that of heparin, producing chain-shortened fragments of heparitin. The substrate, heparin, is widely used as an anticoagulant drug and consequently heparinase has numerous applications in the study of heparin structure, the investigation of the blood coagulation mechanism and in bioassays for detection of heparin in body tissues and fluids. Heparinase also has use in the preparation of low molecular weight heparin fragments which have
Patent Survey
720
potential therapeutic values as antithrombotic or anti-tumour agents. Bellamy and Horikoshi isolated a large number of microorganisms from soils by selection for growth at 45°C in a liquid medium containing mineral salts, trace amounts of vitamins and heparin as the major nutrient. From those samples which exhibited evidence of microbial growth after 1 week of cultivation, the microorganisms were isolated in a biologically pure form and tested for production of heparinase with activity at 45°C. One bacterial isolate was discovered which produced the desired heparinase activity and further, which, released the heparinase into the culture medium. This new heparinaseproducing isolate was designated strain BH100. The taxonomic properties of strain BH100 were examined in accordance with methods described in Bergey's
Manual of Systematic Bacteriology Vol. 2, and the strain was found to be Grampositive, spore-forming, rod-shaped, facultatively anaerobic, motile, catalasepositive and had a growth temperature range of 20-55°C. Based on these characteristics, it was determined that the strain was a microorganism belonging to the genus Bacillus. Isolation of the microorganisms from the soil was conducted as follows. Approximately 1"0 g of soil sample was added to 1'5 ml of medium which consisted of per litre: 2.0 g heparin, 0"35 g K2HPO4, 0.27 g NHaCI, 0.2 g MgCI2-6H20, 0"001 g each of lysine, histidine and methionine, 0.0025rag ferrous citrate, 0.1ml trace element solution (containing per litre: 0.12g ZnSO4, 0.5g MnSO4, 0.13g H3BO3, 0.004 g CuSO4, 0.006 g Na2MoO4, 0.012g COC12"6H20), and 0.1ml of vitamin solution containing per litre: 10mg each of folic acid and biotin, 25mg each of riboflavin, thiamine, nicotine acid, calcium pantothenate and para-aminobenzoic acid, and 50 mg pyridoxine hydrochloride. The cultures were held in sterile 24-well microtiter trays, sealed with plastic wrap to minimize evaporation, at 45°C without shaking. From those samples which exhibited evidence of microbial growth after 1 week of cultivation, the microorganisms were isolated in biologically pure form by repeated subculture of cells on a solid medium containing per litre: 2"0g heparin, 0.11 g K2HPO4, 1.0 g MgClz. 6H20, 8.0 g gellan gum, at pH 8.0 with incubation at 45°C. Figure 3 shows the influence of pH on the activity of the enzyme and Fig. 4 shows the influence of temperature on its activity.
100
•
?5-
~
2S"
Fig. 3. I00
-
7~j,
~o
\ o
30
35
40 45 TEMPERATURE
50
55
60
('C]
Fig. 4.
References B6hmer et al., J. Biol. Chem., 265 (1990) 13609-17. Can. J. Microbiol., 26 (1980) 337-84. Linhardt et al., Appl. Biochem. Biotechnol., 12 (1986) 135-76. Nader et al., J. Biol. Chem., 265 (1990) 16807-13. Nakamura et al., J. Clinical Microbiol., 26 (1988) 1070-1. US patent publications 5 145 778, 5 362 645. International examined patent publication WO 82/00659.
A process and apparatus for biological remediation of vaporous pollutants
(European patent 693 959 corresponding to international patent application 94/23825 to Alliedsignal Inc., NJ, USA) During the last 5 years there has been much development in the use of carbon-coated substrates in the support of pollutant-remediating microorganisms, and indeed reports have appeared in Process Biochemistry. Louis J. Defilippi, Francis S. Lupton and Mansour Mashayekhi, all bioengineers with the above company, have designed a very simple and practical process for this, the apparatus of which is shown in Fig.
5, a cross-sectional side view of their vertical reactor. The process involves passing a vaporous-stream with one or more of pollutants through a bioreactor (1) comprising a packed column (2) with a plurality of biologically active bodies. The biologically active bodies (3) comprise a macroporous substrate and one or more of microorganisms which are capable of metabolizing at least one of the pollutants contained in the vaporous stream. The bioreactor may also contain open or substantially open space bodies (4) that are intermixed with the biologically active bodies. The porous substrate and open space bodies are coated with an absorbent that is capable of absorbing one or more of the pollutants contained in the influent stream. The biologically active bodies and the open space bodies are placed on top of a vented support layer (5), which may be a perforated metal or plastic plate. The gas inlet (6) may be placed at the lower end of the bioreactor, as shown in Fig. 5, or at the top of the reactor, and the gas outlet (7) should be positioned at the opposite end to the inlet. During operation of the bioreactor, nutrients and water need to be provided. Nutrients, such as carbon and minerals, may be added in the form of fish meal peptine, soybean flour, peanut oil, etc. and usually salts capable of providing phosphate, sodium, potassium, ammonium, calcium, sulphate, etc. This feed mixture, containing nutrients and buffers, is provided in aqueous solution through an inlet (8). The feed mixture is fed into the bioreactor at the top through sprayers (10) and allowed to flow down the column and accumulate at the bottom of the reactor. Alternatively, the feed mixtures may be fed at different locations in the reactor through any inlet positioned in such a way that effectively and evenly distributes the feed mixture to the biologically active bodies. The accumulated feed mixture is withdrawn through a conduit or an outlet (9) to an external reservoir (11) in which the pH and the nutrient concentration of the collected feed mixture are analysed. Based on the analyses of the mixture, a pH controlling agent, such as a solution of an acid or a base, and nutrients are added to the external reservoir. The re-conditioned feed mixture is then recycled to the top of the bioreactor.
References 3 German patent publications. 4 European patent publications. 1 US patent publication.
721
Patent Survey
DO,Glucasc
O.D.,HA
120
10 /
60 40 20 0 0
RLOWER
10
5
Culture Tima +Glucose(g/L)*
-0.D.
(US patent 5 455 174 based on 3 equivalent Japanese patent applications 3-113591; 3-262943 and 3-289958 to Mitsubishi Rayon Company, Ltd, Tokyo, Japan)
Keiichi Sakashita and his co-biochemwith the above ists, cooperating Japanese company, have found a process for producing an organic acid ester of ascorbic acid or erythorbic acid, by reacting these acids with an organic acid enol ester in an organic solvent with a solubility of those acids of more than 0.3% at 25°C in the presence of an active lipase. Their work is exemplified by the following laboratory test which formed part of the research. 0.4 g of Amano Lipase PS (produced by Amano Seiyaku K.K.) was dissolved in 10 ml of a l/20 M aqueous phosphate buffer with a pH value of 7, and the solution was then evaporated to dryness under a reduced pressure to obtain a pretreated Lipase PS. 2 g of ascorbic acid and 6.7 g of stearic acid vinylester were dissolved in 40 ml of dried pyridine (water content 1200 ppm) and the pretreated Lipase PS was added to the mixture. The reaction was carried out at 40°C with vigorous stirring. After 4 h the concentration of ascorbic acid6-stearate in the reaction mixture was found to be 115% according to HPLC analysis. The reaction mixture was filtered and the filtrate poured into
20
25
DO+HA(g/L)
Fig. 6.
Fig. 5. Stereoselective esterification of ascorbic or erythorbic acids with long-chain enol esters and with the aid of Amano Lipase PS
15
200 ml of ice-cooled 6 N aqueous hydrochloric acid. The precipitate was filtered, washed with water and then dried under reduced pressure; 6 g of a crude product was obtained. The crude product was then washed with hot hexane to obtain 52 g of a yellowish-white product. The content of ascorbic acid6-stearate in the product was 96%. References 4 US patent publications.
3 European patent publications. 2 Japanese patent publications. A novel strain of Streptococcus zaaepidetnicus for producing hyaluronic acid (US patent 5 496 726 to Lucky Ltd. Seoul, Republic of Korea)
Myoung G. Park and his co-biochemists have discovered that high molecular weight hyaluronic acid can be produced by using a strain of Streptococcus and, furthermore, they have found a medium suitable for the culture of the microorganism. The microorganism lacks hyaluronidase activity and hemolytic activity, and is obtained by mutating a known strain of Streptococcus zooepidemicus (ATCC 35246). The process of mutagenesis includes treatment of the parent microorganism at least three times with N-methyl - N’ - nitro - N - nitrosoguanidine (NTG). Mutant strains lacking in hemolytic activity and are produced and selected after the first NTG treatment of the known microorganism. The
second NTG treatment thereof selects strains lacking hyaluronidase, which catalyzes the decomposition of hyaluronic acid. Thereafter, the selected strains are subjected to a third NTG treatment and then incubated on a solid medium to collect large rapidly-growing, highly mucous colonies as the mutant strains have a superior productivity of hyaluronic acid. One of the strains found has been registered with accession number KCTC 0075BP under the terms of the Budapest Treaty. In general, microorganisms belonging to the genus Streptococcus are incubated on media containing a carbon source, such as starch, glucose or sucrose, a nitrogen source, such as ammonium sulfate, ammonium nitrate, sodium nitrate, casamino acid, yeast extract, peptone or tryptone, and an inorganic salt, for example sodium chloride, sodium dihydrogenphosphate or disodium hydrogenphosphate. Figure 6 depicts the fermentation profiles of Streptococcus zooepidemicus (ATCC 35246) and Streptococcus zooepidemicus (LBF 707). References
Beaty,
N.
B., Anal.
Biochem.,
147
(1985) 387-95.
Chabreck, P., Chromatographia, 30 (1990) 201-4. Dische, Z., J. Biol. Chem., 189 (1947) 167. Hinodedo, A., J. Sot. Cosmet. Chem. Japan, 22 (1988) 35-42. Motohashi, N., .I. Chromatographia, 299 (1984) 508-12. 3 US patent publications.
3 European patent publications.