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The production of oxytetracycline in a date medium by different mutants of Streptomyces rimosus induced by dry and wet heat
Bioresource Technology 44 (1993) 259-261
Short Communication
METHODS
The Production of Oxytetracycline in a Date Medium by Different Mutants of Streptomyces rimosus Induced by Dry and Wet Heat
Mutant strains Auxotrophic mutants of S. rimosus were detected on minimal medium and isolated and maintained on slants of complete and sporulation media at 30°C. Detection and isolation of auxotrophic mutants This was done by a total-isolation procedure. A random sample of spores that survived each mutagen treatment was inoculated onto CM templates (37/ plate) and incubated for three days at 30"C. Each template plate was in turn replicated on MM plates in duplicate and incubated at 30°C for from 4 to 5 days in the dark. All colonies that failed to grow on MM at the end of the incubation period, or grew very weakly, were considered auxotrophic mutants and isolated from the original CM templates on CM/or SM slants.
Abstract Dry heat was more effective as a physical mutagen than wet heat in induction of Streptomyces rimosus. The optimal temperature for inducing auxotrophic mutants in spores of Streptomyces rimosus was 55 °C under drytreatment conditions. Only two mutants out of 138 dryand wet-heat mutants produced higher antibiotic titres than the parent organism, and these were obtained after exposure of spores to dry heat at 45 °Cfor 160 min. Key words: Oxytetracycline, mutants of Streptomyces rimosus, dry heat, wet heat.
Heat mutagenesis Different temperatures higher than the optimal growth temperature for S. rimosus (30°C) were used. The spores were suspended in tris buffer pH 8"5 at 40°C, 45°C, 50°C, 55°C, and 60°C and immediately immersed in water baths at the same temperatures. A 1-ml sample was immediately diluted in 9 ml sterile water to serve as untreated control, and these and subsequent samples were serially diluted in sterile distilled water to reach a final dilution of 100 spores per millilitre. Subsequent samples were taken at regular intervals for each temperature. Samples of the final dilutions were spread on CM plates to determine the survival percentage. In another set of experiments, the spores of S. rimosus were heated without being suspended in tris buffer to avoid any effect of a wet environment. Barnes (1928) was not able to induce mutation in Eurotium herbariorum when the spores were suspended in water, but, on exposing the spores to heat in dry chambers, he obtained mutants. Cultures of S. rimosus on plates of SM were exposed directly to temperatures of 40°C, 45°C, 50°C, 55°C, and 60°C in incubators. Spore samples were taken at the beginning of each treatment and then at regular intervals, and the percentage survival was determined after further incubation of plates at 30°C.
INTRODUCTION High-temperature or transient-temperature shifts have long been known to affect mutation rates in various organisms (Barnes, 1928; Burnett, 1975; Drake & Baltz, 1976). However, heat has received little attention as a mutagenic agent. Lindgren (1972) reviewed and summarized the mutagenic effects of heat treatment of Drosophila, bacteria, viruses, plants, and fungi and concluded that induced mutation rates seemed to be high in most systems at 38-40°C. It seemed worth trying heat in the induction of mutations affecting antibiotic activity in S. rimosus since no such study had been made previously. The mutants produced were used in a date medium to evaluate their efficiencies for the antibiotic outputs. Saudi dates are a local natural renewable resource. Hence Saudi-date components, especially the fleshy part (epi- and mesocarp) of the tamer stage, were utilized in the fermentation medium for the formation of oxytetracycline. Bioresource Technology 0960-8524/93/S06.00 © 1993 Elsevier Science Publishers Ltd, England. Printed in Great Britain 259
260
N. A. Baeshin, A. A. Abou-Zeid, A. O. Baghlaf
Sporulation medium (SM) The sporulation medium contained (g/litre): sucrose 3"0, dextrin 15.0, urea 0"1, peptone 5"0, beef extract 1"0, KH2PO 4 0"5, MgSOn.7H20 0"5, NaC1 0"5, FeSO4.7H20 0-01, agar 30; distilled water to 1000 ml.
Complete medium (CM) The complete medium contained (g/litre): glucose 10"0, yeast extract 2"0, peptone 2"0, hydrolysed casein 2.0, MgSO4.7H20 0-5, agar 20-0; distilled water to 1000 ml.
Minimal medium (MM) The minimal medium contained (g/hire): glucose 10-0, asparagine 0-5, KH2PO 4 0"5, K O H 0"3, FeSO 4. 7H20 0"05, agar 20"0; distilled water to 1000 ml.
Maintenance medium The different mutants produced were maintained on a medium containing (g/litre): glucose, 10"0; peptone 2-5; yeast extract 2-5; KH2PO4, 1"0; MgSO4.7H20, 0-25; MnSO 4 . 4H20, 0"025; FeSO 4 . 7HeO, 0"005; agar, 30"0; distilled water to 1000 ml.
Vegetative medium The vegetative medium used for growing the different mutants contained (g/litre): glucose, 10-0; peptone, 5"0; yeast extract, 5"0; KH2PO4, 1"0; MgSO4.7H20 , 0"25; MnSO4.4H20, 0-025; FeSOn.7H20, 0-005; distilled water to 1000 ml.
Fermentation medium The different mutants produced by dry- and wet-heat treatment were grown in the fermentation medium to determine their potencies for the formation of oxytetracycline. The fermentation medium contained (g/ litre): date-coat sugar extract, 20"0; date-seed hydrolysate, 10"0; urea, 1.5; KH2PO4, 1"0: date-seed lipid, 1.0; date-seed ash, 0"5; distilled water to 1000 ml (Abou-Zeid et al., 1991). The medium was portioned into Erlenmeyer flasks (capacities 250 ml), each containing 50 ml. The flasks were sterilized and inoculated when they attained room temperature with 0"5 ml of the mutant grown in the vegetative medium. The flasks were inserted in a shaker (200 r/min) at 30°C for 144 h. At the end of the fermentation process, oxytetracycline was determined.
Microbiological determination of oxytetracycline Oxytetracycline produced in the fermented media by the different mutants was determined by the method of Abou-Zeid and Shehata (1969) and Kavanagh (1976). RESULTS AND DISCUSSION
Heat mutagenesis The results showed that the percentage of survival decreased linearly, whereas the percentage of mutation
increased (confirmed by linear-regression computation). The total percentage of auxotrophic mutants resulting from a dry treatment was almost always as much as twice the total percentage of auxotrophic mutants resulting from a wet treatment. The opposite held true for the total percentage of survival, but the difference between wet and dry treatments was within the range from 5 to 20%. The highest total percentage of auxotrophic mutants was induced by a temperature of 55°C in the dry treatment: 3.06%. The optimal temperature for inducing auxotrophic mutants in S. rimosus would therefore be 55°C under dry-treatment conditions for 60 min. The potencies of 138 mutants induced by dry and wet heat at 40°C, 45°C, 50°C, 55°C, and 60°C for the formation of oxytetracycline were determined. The data showed that the mutants could be divided into three categories. The first category included active mutants that gave lower titres of the antibiotic than the initial micro-organism. The second category consisted of non-oxytetracycline producers. The third category contained only two mutants (M-12 and M-22) that gave higher titres for the antibiotic than the initial organism. The two mutants were produced when the organism was treated by dry heat (45°C) for 160 min. A temperature of 55°C was best for auxotrophic mutants, but 45°C seems best for antibiotic production.
ACKNOWLEDGEMENT The authors express their gratitude to King Abdulaziz City for Science and Technology (KACST) for financial support (AR-9-15).
REFERENCES Abou-Zeid, A. A. & Shehata, M. A. (1969). A simple technique for assaying antibiotic using methylene blue as an indicator. Indian J. Pharmacy, 11, 72-5. Abou-Zeid, A. A., Baeshin, N. A. & Baghlaf, A. O. (1991). The formation of oxytetracycline in a date-coat medium. Bioresource Technol., 37, 179-84. Barnes, B. (1928). Variation in Eurotium herbariorum (Wigg). Link induced by the action of high temperature. Ann. Bot., 168, 787-872. Burnett, J. H. (1975). Mycogenetics. John Wiley & Sons, London. Drake, J. W. & Bahz, R. H. (1976). The biochemistry of mutagenesis. Ann. Rev. Biochem., 45, 11-37. Kavanagh, F. (1976). Analytical Microbiology, cited by Miller, B. M. & Litsky, W. Industrial Microbiology. McGraw-Hill, New York. Lindgren, D. (1972). Temperature influence on the spontaneous mutation rate. 1. Literature Review. Hereditas, 70, 165-78.
The production of oxytetracycline in a date medium
Nabih A. Baeshin, Abou-Zeid A. Abou-Zeid* & Ahmed O. Baghlaf Biology, Biochemistry, Chemistry Departments, Faculty of Science, PO Box No 9028, King Abdulaziz University, Jeddah-21413, Saudi Arabia (Received 27 May 1992; revised version received 28 October 1992; accepted 5 November 1992) *To whom correspondence should be addressed.
261
Short Communication
METHODS
The Production of Oxytetracycline in a Date Medium by Different Mutants of Streptomyces rimosus Induced by Dry and Wet Heat
Mutant strains Auxotrophic mutants of S. rimosus were detected on minimal medium and isolated and maintained on slants of complete and sporulation media at 30°C. Detection and isolation of auxotrophic mutants This was done by a total-isolation procedure. A random sample of spores that survived each mutagen treatment was inoculated onto CM templates (37/ plate) and incubated for three days at 30"C. Each template plate was in turn replicated on MM plates in duplicate and incubated at 30°C for from 4 to 5 days in the dark. All colonies that failed to grow on MM at the end of the incubation period, or grew very weakly, were considered auxotrophic mutants and isolated from the original CM templates on CM/or SM slants.
Abstract Dry heat was more effective as a physical mutagen than wet heat in induction of Streptomyces rimosus. The optimal temperature for inducing auxotrophic mutants in spores of Streptomyces rimosus was 55 °C under drytreatment conditions. Only two mutants out of 138 dryand wet-heat mutants produced higher antibiotic titres than the parent organism, and these were obtained after exposure of spores to dry heat at 45 °Cfor 160 min. Key words: Oxytetracycline, mutants of Streptomyces rimosus, dry heat, wet heat.
Heat mutagenesis Different temperatures higher than the optimal growth temperature for S. rimosus (30°C) were used. The spores were suspended in tris buffer pH 8"5 at 40°C, 45°C, 50°C, 55°C, and 60°C and immediately immersed in water baths at the same temperatures. A 1-ml sample was immediately diluted in 9 ml sterile water to serve as untreated control, and these and subsequent samples were serially diluted in sterile distilled water to reach a final dilution of 100 spores per millilitre. Subsequent samples were taken at regular intervals for each temperature. Samples of the final dilutions were spread on CM plates to determine the survival percentage. In another set of experiments, the spores of S. rimosus were heated without being suspended in tris buffer to avoid any effect of a wet environment. Barnes (1928) was not able to induce mutation in Eurotium herbariorum when the spores were suspended in water, but, on exposing the spores to heat in dry chambers, he obtained mutants. Cultures of S. rimosus on plates of SM were exposed directly to temperatures of 40°C, 45°C, 50°C, 55°C, and 60°C in incubators. Spore samples were taken at the beginning of each treatment and then at regular intervals, and the percentage survival was determined after further incubation of plates at 30°C.
INTRODUCTION High-temperature or transient-temperature shifts have long been known to affect mutation rates in various organisms (Barnes, 1928; Burnett, 1975; Drake & Baltz, 1976). However, heat has received little attention as a mutagenic agent. Lindgren (1972) reviewed and summarized the mutagenic effects of heat treatment of Drosophila, bacteria, viruses, plants, and fungi and concluded that induced mutation rates seemed to be high in most systems at 38-40°C. It seemed worth trying heat in the induction of mutations affecting antibiotic activity in S. rimosus since no such study had been made previously. The mutants produced were used in a date medium to evaluate their efficiencies for the antibiotic outputs. Saudi dates are a local natural renewable resource. Hence Saudi-date components, especially the fleshy part (epi- and mesocarp) of the tamer stage, were utilized in the fermentation medium for the formation of oxytetracycline. Bioresource Technology 0960-8524/93/S06.00 © 1993 Elsevier Science Publishers Ltd, England. Printed in Great Britain 259
260
N. A. Baeshin, A. A. Abou-Zeid, A. O. Baghlaf
Sporulation medium (SM) The sporulation medium contained (g/litre): sucrose 3"0, dextrin 15.0, urea 0"1, peptone 5"0, beef extract 1"0, KH2PO 4 0"5, MgSOn.7H20 0"5, NaC1 0"5, FeSO4.7H20 0-01, agar 30; distilled water to 1000 ml.
Complete medium (CM) The complete medium contained (g/litre): glucose 10"0, yeast extract 2"0, peptone 2"0, hydrolysed casein 2.0, MgSO4.7H20 0-5, agar 20-0; distilled water to 1000 ml.
Minimal medium (MM) The minimal medium contained (g/hire): glucose 10-0, asparagine 0-5, KH2PO 4 0"5, K O H 0"3, FeSO 4. 7H20 0"05, agar 20"0; distilled water to 1000 ml.
Maintenance medium The different mutants produced were maintained on a medium containing (g/litre): glucose, 10"0; peptone 2-5; yeast extract 2-5; KH2PO4, 1"0; MgSO4.7H20, 0-25; MnSO 4 . 4H20, 0"025; FeSO 4 . 7HeO, 0"005; agar, 30"0; distilled water to 1000 ml.
Vegetative medium The vegetative medium used for growing the different mutants contained (g/litre): glucose, 10-0; peptone, 5"0; yeast extract, 5"0; KH2PO4, 1"0; MgSO4.7H20 , 0"25; MnSO4.4H20, 0-025; FeSOn.7H20, 0-005; distilled water to 1000 ml.
Fermentation medium The different mutants produced by dry- and wet-heat treatment were grown in the fermentation medium to determine their potencies for the formation of oxytetracycline. The fermentation medium contained (g/ litre): date-coat sugar extract, 20"0; date-seed hydrolysate, 10"0; urea, 1.5; KH2PO4, 1"0: date-seed lipid, 1.0; date-seed ash, 0"5; distilled water to 1000 ml (Abou-Zeid et al., 1991). The medium was portioned into Erlenmeyer flasks (capacities 250 ml), each containing 50 ml. The flasks were sterilized and inoculated when they attained room temperature with 0"5 ml of the mutant grown in the vegetative medium. The flasks were inserted in a shaker (200 r/min) at 30°C for 144 h. At the end of the fermentation process, oxytetracycline was determined.
Microbiological determination of oxytetracycline Oxytetracycline produced in the fermented media by the different mutants was determined by the method of Abou-Zeid and Shehata (1969) and Kavanagh (1976). RESULTS AND DISCUSSION
Heat mutagenesis The results showed that the percentage of survival decreased linearly, whereas the percentage of mutation
increased (confirmed by linear-regression computation). The total percentage of auxotrophic mutants resulting from a dry treatment was almost always as much as twice the total percentage of auxotrophic mutants resulting from a wet treatment. The opposite held true for the total percentage of survival, but the difference between wet and dry treatments was within the range from 5 to 20%. The highest total percentage of auxotrophic mutants was induced by a temperature of 55°C in the dry treatment: 3.06%. The optimal temperature for inducing auxotrophic mutants in S. rimosus would therefore be 55°C under dry-treatment conditions for 60 min. The potencies of 138 mutants induced by dry and wet heat at 40°C, 45°C, 50°C, 55°C, and 60°C for the formation of oxytetracycline were determined. The data showed that the mutants could be divided into three categories. The first category included active mutants that gave lower titres of the antibiotic than the initial micro-organism. The second category consisted of non-oxytetracycline producers. The third category contained only two mutants (M-12 and M-22) that gave higher titres for the antibiotic than the initial organism. The two mutants were produced when the organism was treated by dry heat (45°C) for 160 min. A temperature of 55°C was best for auxotrophic mutants, but 45°C seems best for antibiotic production.
ACKNOWLEDGEMENT The authors express their gratitude to King Abdulaziz City for Science and Technology (KACST) for financial support (AR-9-15).
REFERENCES Abou-Zeid, A. A. & Shehata, M. A. (1969). A simple technique for assaying antibiotic using methylene blue as an indicator. Indian J. Pharmacy, 11, 72-5. Abou-Zeid, A. A., Baeshin, N. A. & Baghlaf, A. O. (1991). The formation of oxytetracycline in a date-coat medium. Bioresource Technol., 37, 179-84. Barnes, B. (1928). Variation in Eurotium herbariorum (Wigg). Link induced by the action of high temperature. Ann. Bot., 168, 787-872. Burnett, J. H. (1975). Mycogenetics. John Wiley & Sons, London. Drake, J. W. & Bahz, R. H. (1976). The biochemistry of mutagenesis. Ann. Rev. Biochem., 45, 11-37. Kavanagh, F. (1976). Analytical Microbiology, cited by Miller, B. M. & Litsky, W. Industrial Microbiology. McGraw-Hill, New York. Lindgren, D. (1972). Temperature influence on the spontaneous mutation rate. 1. Literature Review. Hereditas, 70, 165-78.
The production of oxytetracycline in a date medium
Nabih A. Baeshin, Abou-Zeid A. Abou-Zeid* & Ahmed O. Baghlaf Biology, Biochemistry, Chemistry Departments, Faculty of Science, PO Box No 9028, King Abdulaziz University, Jeddah-21413, Saudi Arabia (Received 27 May 1992; revised version received 28 October 1992; accepted 5 November 1992) *To whom correspondence should be addressed.
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