BIOSYNTHESIS OF ANTIBIOTIC THROUGH METABOLISM

OF ACTINOMYCETES STRAIN MH-9 THROUGH

SHAKE FLASK FERMENTATION

SAIRA ABBAS*, MUHAMMAD SUBHAN*, FARAN DURRANI*, SULTAN MEHMOOD*,

HIDAYATULLAH KHAN* and ABDUL HAMEED**

* Department of Botany and Biotechnology, University of Science &Technology, Bannu – Pakistan.

** Department of Microbiology, Quaid-i-Azam University, Islamabad – Pakistan.

ABSTRACT
A strain of MH-9 (Actinomycetes) was isolated from desert soil and manipulated to detect its
ability to produce antibiotics. Optimization of different parameters reflected that the antibiotic production
was maximum at pH 7 and 37°C. The optimum antibiotic production was obtained after 120- hours of
incubation at 150 rpm. Antibiotic activity was recorded as maximum i.e. 38mm against Bacillus subtilis,
while increase of E.coli, Pseudomonas and Staphylococcus aureus zone of 30,30 and 31mm were shaped
respectively. Extraction of sample with different solvents was carried out and antibiotic which was
synthesized as a result of metabolic activities of MH-9 was found to be water soluble. The effect of proteins
on antibiotic was observed by column chromatography and noted the activity in different fractions.
Concentration for streptomycin (4473µg/ml) and (228µg/ml) were determined in the fractions and further
analysis was confirmed by IR spectrophotometery and TLC method. Moreover the activity of crude
antibiotic mixture with the standard streptomycin and kanamycin was also compared. Antibiotic Activity
was also checked and was found best against Gram-positive and found good against Gram-negative
bacteria.

Key Words: Strain-MH-9, (Actinomycetes), parameters, optimization, streptomycin, and kanamycin.

Citation: Abbas, S., M. Subhan, F. Durrani, S. Mehmood, H. Khan and A. Hameed. 2010. Biosynthesis of
antibiotic through metabolism of actinomycetes strain MH-9 through shake flask fermentation. Sarhad J.
Agric. 26(1): 7-18.

INTRODUCTION
Antibiotics are the antimicrobial agents, which are produced by some micro-organisms to inhibit
or to kill many other micro-organisms including different bacteria, viruses and eukaryotic cells. It can be
purified from microbial fermentation and modified chemically or enzymatically. Antibiotics are secondary
metabolites and these secondary metabolites have also been termed “idiolites” (Walker, 1974). The best-
known genus of actinomycetes; Streptomycetes (Order Actinomycetales, Family Streptomycetaceae) are
gram-positive, filamentous bacteria that are ubiquitous in soil and produce the majority (>70%) of known
antibiotics (Tanaka and Omura, 1990). There is a large class of β-lactam antibiotics produced by bacteria,
fungi and streptomyces. They all possess unusual chemical linkage β-lactam core (Bayer et al. 1990).
Sometime it shows the “selective toxicity” which means that compound inhibits or kills the microorganism
without having a similar effect on the host organism (e.g., humans) (Robbers et al. 1996). More than
10,000 antibiotics have been isolated from fermentation, plant materials, animal tissues and various marine
sources. Commercially important antibiotics are used in agriculture, medicine and food preservation. More
than 5000 different antibiotics have been isolated from cultures of bacteria, fungi and plant cells, 60% of
them are contributed by the genus streptomyces. Antibiotics, as secondary metabolites, are generally
produced by multistep biosynthetic pathways starting from intermediates of primary metabolism to specific
moieties. Actinomycetes are aerobic, gram-positive bacteria that form branching filaments or hyphae
(usually 0.5-1.0μm) and asexual spores. Streptomycetes are best known for their synthesis of a vast array of
antibiotics, some of which are useful in medicine and biological research. Examples include amphotericin
B, chloramphenicol, erythromycin, neomycin, nystatin, streptomycin, tetracycline and so forth. Although
most streptomyces are non-pathogenic saprophytes, a few are associated with plant and animal diseases
(Prescott et al. 1999). They are placed in the order Actinomycetales (Prescott et al. 1999). Actinomycetes
Saira Abbas et al. Biosynthesis of antibiotic through metabolism of actinomycetes strain… 8

are also found in fresh water, seawater, and cold and warm blooded animals and composts. Actinomycetes
produce over 6,000 chemically different antibiotics of microbial origin, and they continue to be an excellent
source of novel compounds. Many of these natural products are commercially important medicinal
compounds with a variety of therapeutic uses (Butler et al. 2002). The majority of antibiotics and
substances with diverse biological activity used in medicine are produced by actinomycetes, however, other
microorganisms, such as Myxobacteria, Pseudomonads, Nocardias, Enterobacteria, Halobacteria,
hyperthermophiles etc. are investigated for new biologically active metabolites by Behal (2003). As soil
bacteria can metabolize a variety of carbon source and their activity and regulation substantially determine
the nutritional status of the cell and therefore, influence antibiotic production (Bertram et al. 2004)

MATERIALS AND METHODS

Actinomycetes strain (MH-9) was used as the source of antibiotic production. The strain was
obtained in pre-isolated form in April 02, 2004 from Microbiology Research Laboratory, Department of
Biological Sciences; Qaid-i-Azam University, Islamabad. Identification of the test organisms was made on
the basis of standard morphological and biochemical tests. Cultures of (MH-9) stored at 4°C and refreshed
periodically for the study purposes. Optimization of Parameters for normal growth like pH level,
temperature range, incubation period, optimization of size of inoculum, optimization of media were
conducted. Arginine glycerol salts medium (AGS) (el-Nakeeb and Lechevalier, 1963) was used as growth
medium for actinomycetes. The ability of the strain MH-9 to produce antibiotics was tested using AGS
medium by agar diffusion assay. After that by using Shake Flask Fermentation method, 150ml of AGS
medium was adjusted to pH 7 and 10% inoculum was added in to the medium. Flasks were incubated at
37°C in a shaking incubator. 10ml of sample was taken after every 24hours interval. Samples were
centrifuged at 4°C, and 10,000 rpm (revolution/minute) for 30min to get cell free supernatant. The
supernatant was utilized in well diffusion method to check antibiotic activity against bacteria, noted as
positive test for antibiotic production. Bacteria were separated from the fermentation medium by using
centrifugation technique. The medium was centrifuged at 10,000 rpm for 30 minutes at 4°C in a Kokusan
model H-251 centrifuge. The supernatant was filtered through Whatmann filter paper No 1, and stored in
the cold room. Furthermore the precipitation of proteins was carried out using Ammonium sulfate. 70g of
ammonium sulfate were added to 100 ml of crude antibiotic solution at 4°C and mixture was stirred. After
sufficient shaking, the precipitates were collected by centrifugation at 10,000 rpm and 4°C for 30 minutes.

Now the Gel Filtration using Sephadex-G-75 (70 x 2.5cm) was used for the separation of proteins.
The buffer utilized was 0.1 M KH2PO4.NaOH (pH 7) while Dextran Blue solution was kept at (0.5% w/v).
The flow rate was maintained at 5ml/15minutes. Thin layer chromatography (TLC) with ethanol as a
mobile phase was also adopted for the detection of Proteins. After detection, the fractions from the column
were compared with the standard solutions of streptomycin and kanamycin to detect the presence of these
antibiotics through UV spectrophotometer. After chromatographic purification, pooled samples were
concentrated by lypholization. Lypholized material was treated with different solvents including
acetonitrile, ethanol, acetone and chloroform and antibiotic activity was tested through agar diffusion assay.

Finally pooled samples were concentrated by lypholization. Lypholized material was run on IR
spectrophotometer for the analysis of the product.

RESULTS AND DISCUSSION

The present study was carried out to handle the production of antibiotics from strain of MH-9,
mesophilic actinomycetes. The isolates were morphologically characterized and identified as Streptomyces
sp. The microorganisms were cultured in petri plates using arginine glycerol salts medium (AGS) as
suggested by el-Nakeeb and Lechevalier (1963). Optimization of following parameters for the production
of antibiotics from strain MH- in Actinomycetes were tested along with the Purification of antibiotic and
Separation of protein by column Chromatography in the present study.
Sarhad J. Agric. Vol. 26, No. 1, 2010 9

PH Level
It was observed that at neutral pH (pH 7) antibiotic activity was maximum while at pH 6 and 8 no
activity was found due to acidic or alkaline conditions. Chattopadhyay and Sen (1997) showed maximum
antibiotic production by Streptomyces at pH 7 which is in confirmation to the findings concluded as
highlighted in the following Table I. To induce higher amount of antifungal antibiotic production from
Strepytomyces rochei- G164 by variation of cultural parmeters have been studied by Sen and
Chattopadhyay (1997) and achieved maximum affectivity while added sucrose as carbon source and
peptone as nitrogen source at pH 7.0 which is the clear cut finding of our concluded data.

Table I PH for Antibiotic production by Actinomycetes MH-9 (+ Presence of activity) ( - Absence of activity)
pH Time (hrs) E. coli Bacillus subtilis Pseudomonas sp. Staphylococcus aureus
6 48 - - - -
72 - - - -
96 - - - -
120 - - - -
144 - - - -
7 48 - - - -
72 + + - -
96 ++ ++ + +
120 +++ +++ ++ ++
144 ++ ++ + +
8 48 - - - -
72 - - - -
96 - - - -
120 - - - -
144 - - - -

Temperature Range
During recording of temperature at 28°C, 30°C, 37°C, 40°C and 47°C, , it was observed that
maximum activity was noted at 37°C that is mesophilic range while at high temperature less antibiotic
production was observed. It might be due to degradation of essential constituents at high temperature which
are required for the production of antibiotics in shake flask fermentation. It means actinomycetes strain
MH-9 showed maximum antibiotic production at pH 7 and temperature 37°C. The same results were
concluded by Raytapadar and Paul (2001) for antibiotic production at 37 ˚C which are clearly matching our
observations as shown in Table II. The optimum temperature for the production of antibiotic, hongoquercin
A & B from the fungus LL-23G277, was approximately 22oC. Growth was declined when the temperature
was lowered down to 15 oC appeared to reach higher level of production when the temperature was kept at
22 oC agian. It was also observed that addition of dextrose to growth media increased the production of
hongoquercin B at 22 oC as reported by Abbanat et al. (1998) which is in confirmation to our findings.

Incubation Period
It was observed that maximum antibiotic production or activity was noted after 120 hours for the
optimization of incubation period when the data was recorded from 48 to 168 hours, because in early hours
actiniomycete growth remains in lag phase in which micro organisms take time to adjust themselves with
the experimental conditions (Table III). At this stage, cells are immature. After 48-72 hours they followed
the exponential phase of their growth and more new cells were produced and immature cells now become
matured. It was also observed that young cells showed maximum activity. Maximum growth production
was also observed after 192 hours. This is because after 120 hours antibiotic production was declined and
less activity was shown by the crude filtrate. It means that production hongoquercin B is inversely
proportional to the growth of bacteria. However at 192 hours again log phase appeared by the germination
of spores in the medium and new and young cells showed increase in antibiotic production which was
indicated by a remarkably enhanced antibiotic activity of crude filtrate. Production of antibiotics in
synthetic medium reached the maximum on the 5th day of incubation at 30oC. Glucose and starch were
found to be the best carbon sources while NH4NO3 was preferred as nitrogen source. These results achieved
Saira Abbas et al. Biosynthesis of antibiotic through metabolism of actinomycetes strain… 10

are correlated with an antifungal antibiotic by Streptomyces having incubation time of 120 hours and pH
7.4. as concluded and reported by Raytapadar and Paul (2001).

Optimization of size of Inoculum

During the optimization of inoculum size which was 10%, 20%, and 30%. Maximum activity was
observed with 10% inoculum (Table IV). Although increased activity was also observed with 30%
inoculum size under specific conditions when incubation period was extended up to 192 hrs. As the
concentration of inoculum increases, it is followed by an increase in cell mass and after a certain period,
metabolic waste interfere with the production of metabolites due to which degradation of the product
occurs.

Table II Optimization of temperature for Antibiotic production by Actinomyctes MH-9

Temperature oC Time (hrs) Ecoli Bacillus subitils Pseudomonas sp. Staphylococcus aureus
28 48 - - - -
72 - - - -
96 - - - -
120 - - - -
144 - - - -
30 48 - - - -
72 - - - -
96 - - -
120 + + - -
144 + + - -
37 48 - - - -
72 + + - -
96 ++ ++ + +
120 +++ +++ ++ ++
144 ++ ++ + +
40 48 - - - -
72 - - - -
96 - - -
120 + + - -
144 + + - -
42 48 - - - -
72 - - - -
96 - - - -
120 - - - -
144 - - - -

Table III Optimization of incubation time for antibiotic production by strain MH-9

Time (hrs) E. coli Bacillus subtilis Pseudomonas sp. Staphylococcus aureus

48 - - - -
72 + + - -
96 ++ ++ + +
120 +++ +++ + +
144 ++ ++ + +
168 ++ ++ + +
Sarhad J. Agric. Vol. 26, No. 1, 2010 11

Table IV Optimization of size of Inoculum for Antibiotic Production by Strain MH- 9

Inoculum Time (hrs) E. coli Bacillus subtilis Pseudomonas sp. Staphylococcus aureus
size
10 48 - - - -
72 + + - -
96 ++ ++ + +
120 +++ +++ ++ ++
144 ++ ++ - -
20 48 - - - -
72 - - - -
96 - - - -
120 - - - -
144 - - - -
30 48 - - - -
72 - - - -
96 - - - -
120 - - - -
144 - - - -

Optimization of Media
During the optimization of different media for antibiotic production with Ariginine glycerol salts
medium (AGS), Tryptone yeast extract agar media (TYM) and Czapeks plus yeast extract media (C+Y),
the best activity was observed in AGS medium while in TYM very scarce growth was noted as highlighted
in Table IV. It was due to glycerol as the most excellent source of carbon and arginine as the best source of
nitrogen. This is in accordance with another study by Haque et al. (1995), where antibiotic production from
Streptomyces with glycerol and arginine as best sources of carbon and nitrogen respectively was observed.
To induce higher amount of antifungal antibiotic production from Strepytomyces rochei G164 by variation
of cultural parmeters have been studied and maximum affectivity was found in sucrose as carbon source,
peptone as nitrogen source and at pH 7.0 Sen and Chattopadhyay (1997). Growth and antibiotic production
is dependent on the medium composition particularly on the carbon and nitrogen sources and other
fermentation conditions. The best results with respect to antibiotic productivity were achieved using a
chemically defined medium with glycerol and lysine as carbon and nitrogen sources respectively giving
clarification of our medium utilized as concluded by Theobald et al. (2000). Glucose and Potassium
dihydrogen phosphate are required for the antibiotic production by MH-9 strain. The same findings with
glucose and potassium dihydrogen phosphate as the best sources for antibiotic production from
Streptomyces natalensis were also achieved by Farid (2000). Moreover, equivalent findings were reported
by Gupte and Kulkarni et al. (2002) in case of glucose as carbon source and soybean meal as nitrogen
source involved in maximum production of antifungal antibiotic by a strain of Streptomyces
chattanoogensis MTCC 3423.

Table V Optimization of Media for Antibiotic Production by Actinomycetes MH-9

Media Time (hrs) E. coli Bacillus subtilis Pseudomonas sp. Staphylococcus aureus
AGS 48 - - - -
72 + + - -
96 ++ ++ + +
120 +++ +++ ++ ++
144 ++ ++ + +
TYM 48 - - - -
72 - - - -
96 - - - -
120 + + + +
144 - - - -
C+Y 48 - - - -
72 - - - -
96 - - - -
120 - - - -
144 - - - -
Saira Abbas et al. Biosynthesis of antibiotic through metabolism of actinomycetes strain… 12

Purification of Antibiotic and Separation of Protein by Column Chromatography

The first step in the purification of the antibiotic was separation of crude antibiotic from the
microbial growth followed by precipitation of proteins by 70% ammonium sulfate (Shimogki et al. 1991).
Then sample was passed from the column to get different fractions of protein because these proteins cause
interference with antibiotic activity of crude filtrate. In order to minimize the interference caused by protein
present in the crude filtrate different fraction of proteins were separated by gel filtration through Sephadex
G-75-120. The precipitates with the maximum activity was suspended in KH2PO4 NaOH buffer of pH 7.0,
were subjected to Gel-filtration. Sephadex G-75-120 column was prepared and 20 fractions each of 3ml
were collected. The absorbance of separate protein fractions present in crude mixture of antibiotics was
observed at 280nm in spectrophotometer.

The fractions obtained from column chromatography were also analysed by TLC method and
compared with standard solutions of streptomycin. Comparison of crude extract of antibiotics with standard
solution of kanamycin showed an RF value of 0.45 and for streptomycin was 4.9. The same study of
Lincomycin production was monitored by TLC method in which different proteins were assayed in the cell
free extract of Streptomyces lineolnensis (Hola et al. 2003). Besides antibiotic activity was also assayed by
agar diffusion method. Fraction no. 5, 8, 12, having more proteins showed maximum antibiotic activity
(Table IV). The same shows remarkable antibiotic activity even in the presence of proteins, which caused
least interference with microbial activity of the crude filtrate. Each fraction was assayed for the presence of
proteins at 280 nm and antibiotic activity was tested by agar diffusion assay.

Table V Activity of fractions against E coil, Bacillus, Staphylococcus aureus and Pseudomonas sp.
Fractions E. coli Bacillus Pseudomonas sp. Staphylococcus
0 - - - -
1 7 9 5 5
2 10 11 7 6
3 10 12 8 8
4 12 14 8 7
5 30 34 10 11
6 28 30 10 10
7 35 38 12 11
8 25 30 12 12
9 14 15 7 7
10 13 14 8 8
11 20 30 9 9
12 20 32 10 11
13 15 20 9 9
14 15 20 8 8
15 12 15 10 7
16 10 14 9 8
17 10 15 7 7
18 12 14 7 8
19 10 14 9 9
20 10 12 7 6
Sarhad J. Agric. Vol. 26, No. 1, 2010 13

2.5

Absorbance (280nm)
1.5

0.5

0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Fractions

Fig. 1 Separations of protein by column chromatography

Separation of Protein by Column Chromatography

In the process of extraction, lypholized samples were treated with different solvents including
acetonitrile, ethanol, acetone and chloroform. However, the antibiotic was found to be water soluble.
Fractions of separate proteins in the mixture of antibiotics were obtained by column chromatography and
compared with different standard solutions of antibiotics like streptomycin, kanamycin as proposed by
Pang et al. 2004. It was observed that maximum streptomycin was present in fraction no.5, however, it was
minimum in fraction no.6. Concentration of streptomycin was increased again in fraction no. 7, 8 and 12.
The activity of fractions was also observed for the presence of kanamycin. It was also observed that
fractions with maximum concentration of streptomycin showed maximum antibiotic activity as compared
to the fractions containing kanamycin (Fig. 02 & 03). The presence of kanamycin was confirmed with the
help of IR spectrophotometer (Satoh et al., 1976).

250
Concentration of Kanamycin

200

150
(ug/ml)

100

50

0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Fractions

Fig. 2. Concentrations of kanamycin in various fractions obtained by column Chromatography

Saira Abbas et al. Biosynthesis of antibiotic through metabolism of actinomycetes strain… 14

5000

Concentration of Streptomycin
4500
4000
3500
3000

(ug/ml)
2500
2000
1500
1000
500
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Fractions

Fig. 3 Concentrations of streptomycin in various fractions obtained by column Chromatography

Activity of antibiotic against pathogens

Activity of antibiotic produced from strain MH-9 was observed with different pathogens i.e.
E.coli, Bacillus subtilis, Pseudomanas sp. and Staphylococcus aureus by well diffusion assay. Maximum
antibiotic activity was found by fraction 7 against Bacillus indicated by zone of inhibition of 38mm.
Moreover, it showed good activity against E.coli, Pseudomonas sp and Staphylococcus aureus i.e. zones of
29, 32 and 31 mm, respectively. Activity showed by fraction 5, 7 and 12 is highlighted in fraction graph
Fig. 1. The best activity was observed against Bacillus which showed that cell free extract of actinomycetes
strain MH-9 was more effective against Gram positive bacteria. Activity was also good against E.coli while
less activity was observed against Pseudomonas sp. and Staphylococcus aureus as indicated in Fig. 4, 5, 6,
7, 8 and 9. In case of E. coli and Bacillus more activity was observed while Pseudomonas and
Staphylococcus showed less susceptibility against all the fractions. Two new antibiotics, hungoquercins A
and B were isolated from blue fungus LL-23G227, which exhibited moderate activity against Gram-
positive bacteria as reported by (Abbanat et al. 1998).

E.coli
Pseudomonas sp.

40
Zone of Inhibition (mm)

35
30
25
20
15
10
5
0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20

Fractions

Fig. 4 Zone of inhibition of various fractions against E.coli and Pseudomonas sp.
Sarhad J. Agric. Vol. 26, No. 1, 2010 15

Fig. 5 Activity of crude antibiotic mixture after separation of proteins against E.coli

Fig. 6 Activity of crude antibiotic mixture after separation of proteins against Pseudomonas sp.
Saira Abbas et al. Biosynthesis of antibiotic through metabolism of actinomycetes strain… 16

B.subtilis

Staphylococcus aureus
40
35

Zone of Inhibition (mm)

30
25
20
15
10
5
0
0
1
2
3
4
5
6
7
8
190
11
12
13
14
15
16
17
18
19
20
Fractions

Fig. 7 Zone of inhibition of various fractions against B. subtilis and Staphylococcus aureus

Fig. 8 Activity of crude antibiotic mixture after separation of proteins against Bacillus subtilis
Sarhad J. Agric. Vol. 26, No. 1, 2010 17

Fig. 9 Activity of crude antibiotic mixture after separation of proteins against Staphylococcus aurous

REFERENCES
Abbant, D.A., M.P. Singh and M. Greenstein. 1998. Hongoquercins, new antibacterial agents from the fungus LL-
23G227: fermentation and biol activ. J. Antibiot. (Tokyo). 51(8): 708-714.
Bayer, D., W. Zhou, K. Holzhauer and K. Schuegar. 1990. Investigation cephalosporin C production in an airlift loop
towel loop reactor. Appl. Microbiol. Biotech. 30(1): 26-33.
Behal, V. 2003. Alternative sources of biologically active substance. Folia. Microbiol (Paraha). 48(5): 563-571.
Bertram, R., M. Schlicht, K. Mahr, H. Nothaft, M.H. Saier and F. Titgemeyer. 2002. Insilico and transcriptional
analysis of carbohydrate uptake systems of streptomyces coelicolor A3 (2). J. Bacteriol. 186(5): 1362-1373.
Butler, M.J., P. Broheim, S. Jovetic, F. Marinelli, P.W. Postma and M.J. Bibb. 2002. Engineering of primary carbon
metabolism for improved antibiotic production in streptomyces lividans. Appld. Envir. Microbiol. 68(10):
4731-4739.
Chattopadhyay, D. and S.K. Sen. 1997. Optimization of cultural condition for antifungal antibiotic accumulation by
streptomyces rochei G164. Hindustan Antibiotic. Bullet. 39(1-4): 64-71.
Nakeeb, M.A. and H.A. Lechevalier. 1963. Selective isolation of Aerobic Actinomycetes. Appld. Microbiol. 11: 75-77.
Farid, M.A., el-Enshasy, H.A. el-Diwany and A.I. el-Sayed. 2000. Optimization of the cultivation medium for
natamucin production by Streptomyces natalensis. J. Basic Microbiol. 40 (3): 157-161.
Gupte, M.D. and P.R. Kulkarni. 2002. A study of antifungal antibiotic production by Streptomyces chattanougensis
MTCC 3423 using full factorial design Lett. Appld. Microbiol. 35 (1): 22-26.
Hague, S.F., S.K. Sen and S.C. Pal. 1995. Nutrient optimization for production of broad spectrum antibiotic by
streptomyces antibioticus SR 15. 4. Acta- Microbial. Immunol. Hune. 42 (2): 155-162.
Pang, X., B. Aigle, J.M. Girardet, S. Mangenot, J.L. Pernodet, B. Dccaris and P. Leblond. 2004. Antimocrob Agents-
Chemother. 48(2): 575-588.
Prescot, M.L., J.P. Harley and D.K. Klein.1999. Procryotic cell structure and function. Antimicirobial Chemother.
677-889.
Rubbers, J.E., M.K. Speedie and V.E. Tyler. 1996. Pharmacognosy and Pharmacobiotechnology. Williams and Wilkina
Co. 219p.
Raytapadar, S. and A.K. Paul. 2001. Production of an antifungal antibiotic by streptomyces aburaviensis IDA-28.
Microbiol. Res. 155 (4): 315-323.
Satoh, A., H. Ogawa and Y. Satomura. 1976. Regulation of N-acetylkanamycin amidohydrolase in the idiophase
kanamycin fermentation. Agric. Biol. Chem. 40:191-196.
Saira Abbas et al. Biosynthesis of antibiotic through metabolism of actinomycetes strain… 18

Sen, S.K. and D. Chattopodhyay. 1997. Optimization of cultural conditions for antifungal ntibiotic accumlation by
Streptomyces rochei G176. Hindustan Antibiotic Bullet. 39(1-4): 64-71.
Shimogki, H., K. Takeuchi, T. Nishino, M. Ohdera, T. Kudo, K. Ohba, M. Iwnma and M. Irie. 1991. Purification and
properties of a Novel surface active agent and Alkaline resistant protease from Bacillus sp. Agric. Biol.
Chem. 55(9): 2251-2258.
Tanaka, Y. and S. Omura. 1990. Metabolism and products of actinomycetes: an introduction. Actinomycetologica 4:
13-14.
Theobald, U., J. Schiwana and H.P. Fiedler. 2000. Microbial growth and production kinetics of streptomyces
antibioticus Tu 6040. Antonic. Van. Leeuwenhoek. 78 (3): 307- 313.
Walker, J.B. 1974. Biosynthesis of the monoguanidinated inositol moiety of bluensomycin, a possible evolutionary
precursor of Streptomycin. J. Biol. Chem. 249: 2397-2404.
Hola, K., J. Janata, J. Kopecky and J. Spizek. 2003. protein levels correlate with lincomycin production in
Streptomyces lincolnensis. Actinomycetes, Instt. of Microbiol. Acad. of Sci. of the Czech Republic, Videnska
1083, Prague 4, CZ-14220, the Czech Republic Copyright 2003. The Soc. for Appld. Microbiol.