Isolation, Purification, Partial Characterization And Antibacterial Activities of Compound Produced by some Actinomycetes from Sedimented Waters
Department of Pharmaceutical Biotechnology, Nandha College of Pharmacy, Erode, Tamilnadu, India.
*Corresponding Author E-mail: sumoraji@rediffmail.com
ABSTRACT
KEYWORDS:
INTRODUCTION:
Screening of microorganisms for the production of novel antibiotics has been intensively pursued for many years by scientists. Antibiotics have been used in many fields including pharmaceutical and veterinary. These molecules present original and unexpected structures and are selective inhibitors of their molecular targets. The discovery of new bioactive molecules is mostly carried out through the exploitation of a proprietary strain collection of over 50,000 strains. Mostly unusual general of actinomycetes and uncommon filamentous fungi1.
The screening is mostly conducted in an automated fashion and is referred to as high throughput screening (HTS). HTS is finding increased applications thanks to the availability of large libraries and to the large number of targets that are being identified through genome sequencing.
The development and application of molecular methods for the detection of uncommon genera of actinomycetes. The actinomycetes are the most widely distributed groups of microorganisms in nature. The actinomycetes represent the main source of secondary metabolites. The species belonging to the genus streptomyces constitute 50% of the total population of water actinomycetes and 75% molecules with antibiotic activity are produced by this genus. The actinomycetes bacteria called rare constitute an important potential source and little explored of new secondary metabolites with antibacterial and antifungal activity.
Based on several studies among bacteria, the actinomycetes are noteworthy as antibiotic producers, making three quarters of all known products, the Streptomyces are especially prolific. Investigations can possibly reveal actinomycetes species that produce novel antibiotics. It is anticipated that the isolation, characterization and the study on actinomycetes can be useful in the discovery of antibiotic and novel species of actinomycetyes.
Microbially produced secondary metabolites are extremely important to our health and nutrition. The best known secondary metabolites are the antibiotics. As a group they have tremendous economic importance. The antibiotic market amounts to almost 30 billion dollars and includes about 160 antibiotics towards microorganisms is the problem of fight against certain bacterial diseases. So it is necessary for searching of new lead structures to play their role in such resistant cases. Most of the soil microorganisms from family actinomycetales are having the capacity to produce large number of secondary metabolites. Recently actinomycete plays an important role in biotechnological processes.
Today the very vast research work is going on actinomycetes soil screening and antibiotic production from the actinomycetes. My aim is to isolate new actinomycete species strain for production of antibiotic from isolated actinomycetes strain by fermentation technology. Today the ever antibiotics exist in the market are very costly as these are originate from the synthetic and complex bioprocess methods.
The increasing cost of antibiotics is one of the major problems in developing countries like India in treating bacterial infections, which is not affordable to poor people in our country. So my objective behind this study is to produce antibiotics with minimum cost so that it will be easily available to people.
METHODOLOGY:
Collection of Water Samples: Sedimented water samples were collected from the region of Tamilnadu, Uttar Pradesh and Madhya Pradesh in a readymade sterile bottle and stored at 4°C until further examination. Mean while PH and texture of water samples can be tested 4, 10.
Isolation of Actinomycetes from Water Samples:
Isolation and enumeration of Actinomycetes was performed by tenfold serial dilution technique. 1ml of sediment water samples was taken in 10 ml of distilled water. Shake well and from the above solution, take 1ml and transferred to 9ml sterile water (10-1). Again it was serially diluted to make up to 10-10 dilution. From the above diluted samples 10-3, 10-5 and 10-6 where randomly selected and it was for isolation of actinomycetes. 0.2ml of each dilution was plated on Glucose-Yeast extract malt agar medium6 (GLM), Luria- Bertani agar medium (LB) and ISP solid medium8 separately by pour plate method. The plates were prepared in triplicate manner. The plates were incubated at 27°C for 7 to 14 days.
Purification of Actinomycetes:
Selected colonies of actinomycetes were transferred into their respective liquid medium. The plates were incubated at 27°C for 7 days. The slant cultures of respective medium were prepared and stored at 4°C until examination.
Cultural Characteristics10:
The cultural characteristics of isolated actinomycetes were studied in which color of an aerial mycelia, color of the substrate mycelia and fragmentation of the isolates were observed.
Antibacterial Assay4,10:
The selected actinomycetes strains were tested for its antibacterial activity against gram-positive and gram-negative bacteria. For antibacterial screening, the Muller-hinton agar medium was used spectra-plak method. The isolated actinomycetes strains were streaked on solid Muller-Hinton agar medium and incubated for 7 days at 27°C. To the above actinomycetes strain, the gram positive and gram-negative bacteria were streaked at a 90° angle and incubated at 37°C for 24 hours. After incubation plates were examined for the zone of inhibition.
Morphological Characteristics:
Based on antibacterial assay the actinomycetes strain (ST6) is morphological characterized by Gram staining11, motility staining11, acid fast staining11 and spore staining11.
Bio-chemical Characteristics:7, 8, 9
a) Test for melanoid formation: The isolates were streaked on the slant culture of Waksman medium and incubated for 4 days. The slant was observed for melanoid formation for every 12 hrs. A positive reaction shows the production of pigments.
b) Test for nitrate reduction: To the sterilized organic nitrate broth a loopful of the isolates was transferred and incubated at 28°C. From the fifth day, the tubes were observed for nitrate reduction. α-napthalein solution (a), sulphonic acid solution (b) were the reagents used. To 1ml of the broth, 2 drops of reagent (a) and 2 drops of reagent (b) were added. A positive reaction shows pink color and the observation was recorded.
c) Test for hydrogen sulphide: The isolates was streaked on nutrient broth medium for hydrogen sulphide and incubated for 24 hrs at 28°C. The positive reaction shows greenish brown or bluish black at every 12 hrs for 4 days.
d) Test for acid production: The test organism was inoculated in to glucose nutrient broth and incubated at 25°C for 4 days. At every 12 hours interval the color changes from blue to yellow indicate the presence of acid production. Bromophenol blue was used as indicator.
e) Growth at 4°C: Actinomycetes strains was incubated at 4°C for 7 days and observed for the growth of strain.
f) Growth at 40°C: The actinomycete strain was incubated at 40°C for 7 days and observed for the growth of strain.
g) Growth on MacConkey’s agar medium: The actinomycete strain was streaked on MacConkey’s agar medium and incubated at 27°C for 24 hrs and observed for the growth of strain.
|
S. No. |
Isolation Medium |
Water Code |
Isolate Code |
Color of Aerial mycelia |
Color of substrate mycelia |
Fragmentation |
|
1 |
GLM |
W1 |
ST1 |
White |
Brown |
None |
|
2 |
GLM |
W7 |
ST2 |
White |
White |
None |
|
3 |
GLM |
W8 |
ST3 |
Yellow |
White |
None |
|
4 |
GLM |
W12 |
ST4 |
Pink |
Yellow |
None |
|
5 |
LB |
W14 |
ST5 |
Yellow |
Light Brown |
Very Little |
|
6 |
LB |
W16 |
ST6 |
White |
Yellow |
None |
|
7 |
LB |
W17 |
ST7 |
Brownish |
White |
None |
|
8 |
ISP |
W20 |
ST8 |
Light Grey |
White |
Very Little |
|
9 |
ISP |
W9 |
ST9 |
Yellow |
Light Grey |
None |
|
10 |
ISP |
W22 |
ST10 |
White |
Yellow |
None |
|
S. No. |
Isolate
|
BIOASSAYORGANISM |
|
||||
|
EC |
ST |
ML |
SA |
SF |
KP |
||
|
1 |
ST1 |
- |
- |
- |
+ |
- |
+ |
|
2 |
ST2 |
+ |
- |
++ |
++ |
+ |
- |
|
3 |
ST3 |
+ |
+ |
- |
+ |
++ |
- |
|
4 |
ST4 |
+ |
+ |
+ |
++ |
+ |
+ |
|
5 |
ST5 |
- |
+ |
+ |
_ |
+ |
+ |
|
6 |
ST6 |
+++ |
++ |
+++ |
+++ |
+ |
+ |
|
7 |
ST7 |
+ |
- |
+ |
+ |
+ |
- |
|
8 |
ST8 |
++ |
- |
+ |
+ |
+ |
- |
|
9 |
ST9 |
- |
- |
- |
- |
- |
+ |
|
10 |
ST10 |
++ |
+ |
- |
+ |
- |
+ |
EC – Escherichia coli ATCC 8739, ST – Salmonella typhi ATCC 23564, SA – Staphylococcus aureus ATCC 29737, KP – Klebsiella pneumonia ATCC 10031, ML – Micrococcus luteus ATCC 11880, SF – Shigella flexneri ATCC 4924
+++ = strong (6-10mm), ++ = moderate (2-6mm), + = weak (1-2mm), - = none
Table 3: Morphological characteristics of actinoecetes ST6 strain:
|
S. No. |
TEST |
RESPONSE |
|
1 |
Gram staining |
Gram positive |
|
2 |
Motility |
Non-motile |
|
3 |
Spore staining |
Spores present |
|
4 |
Acid fast staining |
Non acid – fast. |
h) Growth on Nutrient agar medium: The actinomycete strain was streaked on nutrient agar medium and incubated at 37°C for 24 hrs and observed for the growth of strain.
Production of Active Compound by Shake Flask Method 10,3 :
For the production of active compound, the synthetic medium-1 was used. The strain was selected for production of antibiotics which was previously seeded in glucose yeast extract malt agar medium. This broth was used for fermentation by shake flask method.10% of culture of seeded broth was transferred to synthetic medium-1. It was kept for fermentation on mechanical shaker for 5 days at 200 rpm at 30°C.
Recovery of the Fermentation Product 10:
After the completion of fermentation process, the fermentation broth was filtered through vacuum filtration to remove the excess of mycelia. The filtrate was further centrifuged at 8000 rpm for 20 minutes to remove traces of mycelia if present. After centrifugation, the supernatants S1, S2 were separated by addition of solvents (ethyl acetate, chloroform). The layers of S1, S2 were concentrated by evaporation and the active components extracted.
Extraction of Active Compound:
a) Extraction of active compound from S1 10,3: The supernatant S1 was mixed with the double volume of solvent ethyl acetate. The compound organic layer was evaporated and collected (SAM-1), which was used for further confirmation. (Antibacterial study, UV, TLC, FT-IR)
b) Extraction of active compound from S2 3: The supernatant S2 was mixed with the double volume of solvent chloroform. The combined organic layer was evaporated and extract (SAM-11) was collected.
Table 4: Biochemical characteristics of actinomycete ST6 strain
|
S. No. |
Test |
Response |
|
1 |
Melonoid formation |
- |
|
2 |
Nitrate reduction |
- |
|
3 |
Hydrogen sulphide |
- |
|
4 |
Gelation liquefaction |
- |
|
5 |
Acid production |
+ |
|
6 |
Growth at 4°C |
- |
|
7 |
Growth at 40°C |
+ |
|
8 |
Range of temp for growth |
25-40°C |
|
9 |
Optimum pH |
7.2 |
|
10 |
Growth of MacConkey’s agar |
No Growth |
|
11 |
Growth on nutrient agar |
Good growth |
Antibacterial Activity 5,9,12:
The cup plate method was used for antibacterial screening. The nutrient agar medium was prepared, sterilized and transferred into a sterile petridish and allow to solidify. The cup was filled with the test sample (SAM-1, SAM-11). The well was developed by sterile borer. The plates were kept for 2 hrs for diffusion in room temperature. The plates were incubated at 37°C for 24 hrs. After which the zone of inhibition was measured in mm.
Analysis of the Compounds:
a) Chromatography: The resulting dry extracts were recaptured in dimethyl sulfoxide (DMSO). Analyzed by thin layer chromatography using silica gel plates. The organic solvents were 5% chloroform, 95% methanol. TLC was run for the extracts and visualized with UV light and iodine vapors. The retention factor (Rf) was calculated for the extract. The active compounds separated were observed.
b) UV spectroscopy: The λmax of the compound SAM-1, SAM-11 were determined by UV spectroscopy. For the compound SAM-1, SAM-11. 0.1N HCl was used as solvent. The λmax was recorded for the SAM-1, SAM-11.
c) IR Spectroscopy: The infra red spectrum of the force extracts (SAM-1, SAM-11 were recorded). The KBr pellets were prepared by pressed pellet technique and these pellets were used for IR studies.
Extraction of Microbial DNA from Actinomycete Strain 2,6:
2ml of broth was taken and centrifuge at 12,000 rpm at room temperature pellets were collected. Suspend the pellets into 2ml of NaCl buffer and 250ml of SDS mixed well and incubated at 60°C for 10 minutes. To the above mixture added phenol:chloroform (1:2) mixture. Mixed well and centrifuged 12,000 rpm at 4°C for 10 minutes then collected the supernatant. To the supernatant added ice cold isopropanal twice the volume incubated for 30 minutes. Spin it at 12,000 rpm for 10 minutes at 4°C and suspended the pellet in 300µl of TE buffer.
Purification of DNA 6:
Sealed the edges of a clean dry glass plated with tape to form a mold set the mold on a horizontal section of the bench. Sufficient electrophoresis buffer was prepared (usually 0.5×TBE) to fill the electrophoresis tank and to cast the gel. A solution of agarose in electrophoresis buffer was prepared at a concentration appropriate for separating the particular size fragments expected in the DNA sample. Added the correct amount of powdered agarose (0.8%) to a measured quantity of electrophoresis buffer in an Erlenmeyer flask or a glass bottle. Loosely plug the neck of the Erlenmeyer flask. Heat the slurry on a boiling water bath until the agarose dissolved. Used insulated gloves to transfer the flask into water bath at 55°C. Mixed the gel solution thoroughly by gentle swirling. While the agarose solution was cooling, choose an appropriate comb for forming the appropriate slots in the gel. Position the comb 0.5-1 mm above the plate, so that complete well was formed when agarose was added to the mold. Poured the warm agarose solution into the mold. Allowed the gel to set completely. Then pour a small amount of electrophoresis buffer on top of the gel and carefully removed the comb. Poured off the electrophoresis buffer and then removed tape mounted the gel in the electrophoresis tank. Added just enough electrophoresis buffers to cover the gel to a depth of 1mm. Mixed the samples of DNA with 0.20 volume of the desired 5X gel loading buffer. Slowly load the sample mixture into the slot of the submerged gel using a disposable micropipette. Loaded the size standard into first and last slot of the gel. Close the lid of the gel tank and attached the electrical leads so that the DNA will migrate towards the positive anode (red lead). Apply a voltage of 1-5v/cm attached correctly, bubbles should be generated at the anode and cathode and within a few minutes, the bromophenol blue should migrated from the wells into the body of the gel. Run the gel until the bromophenol blue has migrated to an appropriate distance, through the gel. When the DNA samples or dyes have migrated a sufficient distance through the gel, the electric current was turned off and removed the leads and lid from the gel tank if ethidium bromide present in the gel and electrophoresis buffer. Examine the gel by UV light. In order to detect the DNA which were separated by gel electrophoresis, the gel was placed on the gel documentation system under transilluminator and bands were observed.
REULTS AND DISCUSSION:
Isolation of Actinomycetes:
10 actinomycetes strains were recovered from the water samples collected from the regions of Tamilnadu, Madhya Pradesh and Utter Pradesh by using the glucose-yeast extract malt agar medium, Luria Bertani agar medium, ISP solid medium. From the 10 isolates the 4 actinomycetes strains were recovered from glucose yeast extract malt agar medium, 3 actinomycetes strains were recovered from Luria Bertani medium, 3 actinomycetes strains were recovered from ISP solid medium.
Purification of Actinomycetes:
10 actinomycetes strains were purified by transfer of strains to their respective medium after incubation of 7 days, the pure actinomycetes strains were recovered (ST1 to ST10)
Cultural Characteristics:
The cultural characteristics of the actinomycetes strains ST1 to ST10 were studied if which the color of the aerial mycelia observed as pinkish, yellow, white, light grey and the color of substrate mycelia also varied for each actinomycetes strains. The fragmentation was not shown by most of the actinomycetes strains except few one with little fragmentation. The cultural characteristics of isolate ST1 to ST10 were given in Table -1.
Antibacterial Assay:
The antibacterial activity of the ten isolated actinomycetes (ST1 to ST10) was varied. The ST6 strain was shown to have very potent In vitro antibacterial activity against Micrococcus luteus ATCC 11880, Staphylococcus aureus ATCC 29737, Salmonella typhi ATCC 23564, Escherichia coli ATCC 8739 etc., out of which the isolate ST6 was selected for further study depending on its activity. The antibacterial activities of ten isolated actinomycetes were given in Table -2.
|
S. No |
Bio assay Organism |
Diameter of Zone Inhibition |
|||||
|
*SAM 1 |
*SAM 11 |
||||||
|
50 µg/ml |
100 µg/ml |
150 µg/ml |
50 µg/ml |
100 µg/ml |
150 µg/ml |
||
|
1 |
E.coli ATCC 8739 |
- |
7.0 |
8.0 |
- |
7.o |
12 |
|
2 |
S.typhi ATCC 23564 |
- |
7.0 |
8.0 |
- |
8.0 |
13 |
|
3 |
S.aureus ATCC 29737 |
- |
8.0 |
8.0 |
- |
8.0 |
11 |
|
4 |
M.luteus ATCC 11880 |
- |
8.0 |
9.0 |
10 |
12 |
14 |
* - Zone of inhibition in mm
Table 6: λmax of SAM - 1
|
S. No. |
Wave length in nm |
Absorbance |
|
1 |
220 |
0.279 |
|
2 |
222 |
0.280 |
|
3 |
224 |
0.283 |
|
4 |
226 |
0.286 |
|
5 |
228 |
0.290 |
|
6 |
230 |
0.295 |
|
7 |
232 |
0.291 |
|
8 |
234 |
0.289 |
|
9 |
236 |
0.283 |
|
10 |
238 |
0.279 |
|
11 |
240 |
0.274 |
|
12 |
242 |
0.269 |
|
13 |
244 |
0.266 |
|
14 |
246 |
0.261 |
|
15 |
248 |
0.253 |
Morphological characteristics:
The Morphological characteristics of the isolate ST6 was studied by gram staining, motility staining, Acid fast staining, and spore staining. The stain ST6 was the gram positive, filamentous bacteria non motile, non acid fast with few spores. The Morphological characteristics were indicated has compared with Bergey’s manual value 4 that the stain ST6 was related to streptomyces sp.
The Morphological characteristics were summarized in Table no-3.
Biochemical characteristics:
The various Biochemical characteristics were studied for the selected ST6 actinomycetes strain. The ST6 was not show melonoid production. It shows negative response for gelatin liquefaction, nitrate reduction and production of hydrogen sulphide. The acid was produced by the strain ST6 as it shows yellow color when bromophenol blue was used as indicator. The growth parameter for the strain was studied; the strain was not grown when kept at 4°c from the study it was shown that range of temperature for growth of actionmycetes strain ST6 was 25-40°c. The PH 7.2 was used while using different media for isolation of actinomycetes strain ST6 was 7.2. The strain ST6 showed no growth on MacConkey’s agar medium and good growth on nutrient agar medium. The results of biochemical characteristics are summarized in Table no-4.
Production and Extraction of Active Compounds:
The ST6 strain was used for the fermentation by using synthetic medium 1. After completion of fermentation process the supernatants solutions of S1, S2 were concentrated by evaporation, the two extracts were obtained by using the solvents (ethyl acetate, chloroform) out of which SAM-1 was brown in color, SAM-11 was yellow powder.
Antibacterial Activity:
The antibacterial activities were studied by using gram positive bacteria and gram negative bacteria. The SAM -11 was shown the good zone of inhibition as compared with SAM -1, which was indicate the SAM -11 as antibacterial agent against Micrococcus luteus ATCC 11880, Salmonella typhi ATCC 23564, Escherichia coli ATCC 8739, Staphylococcus aureus ATCC 297377.
Table 7: λmax of SAM - 11
|
S. No. |
Wavelength in mm |
Absorbance |
|
1 |
240 |
0.343 |
|
2 |
242 |
0.354 |
|
3 |
244 |
0.355 |
|
4 |
246 |
0.357 |
|
5 |
248 |
0.358 |
|
6 |
250 |
0.360 |
|
7 |
252 |
0.356 |
|
8 |
254 |
0.351 |
|
9 |
256 |
0.341 |
|
10 |
258 |
0.338 |
|
11 |
260 |
0.335 |
|
12 |
262 |
0.329 |
|
13 |
264 |
0.325 |
|
14 |
266 |
0.323 |
|
15 |
268 |
0.319 |
Analytical Methods:
The two extracts were analyzed by thin layer chromatography and the one active component was shown by SAM 1, SAM 11 and the Rf value were found as 0.81 (SAM 1), 0.79 (SAM 11). By using Elico 20 double beam UV spectrometer the λmax of the SAM 1, SAM 11 was found to be 230 nm, 250nm respectively. The λmax plots of SAM 1, SAM 11 were given in Table no-5. 6. The functional groups present in SAM 1 are C=O (1708 cm-1, 1624 cm-1), C-O (1271 cm-1), NH (3362 cm-1) and aromatic substituted compound (1494 cm-1, 1469 cm-1, 1450 cm-1).
Extraction of DNA:
The DNA of actinomyceytes strain ST6 was isolated by cell lysis method. The DNA bands were purified by Agarose gel electrophoresis. Using UV light transilluminator the bands were of DNA of ST6 strains were separated according to their free of contaminants like humic acid, but few contaminants of phenolic substances are present.
CONCLUSION:
From the present work it is concluded that the water samples of the region of Tamilnadu, Uttar Pradesh and Madhya Pradesh have the capability to produce Actinomycetes. The water sample which was collected from Tamilnadu had shown potential for producing Actinomycetes. Luria-Bertani medium is one of the best medium for isolation of Actinomycetes from sedimented water. From antibacterial assay, the Actinomycetes strain showed in vitro antibacterial activity. The synthetic medium 1 was one of the best medium for fermentation of Actinomycetes strain ST6. The antibacterial activity studies of SAM 11 was the antibiotic with broad spectrum activity showing more zone of inhibition compared to SAM 1. The DNA obtained from Actinomycetes was free of contaminants like humic acid due to isopropanol which was preferred instead of PEG and ethanol. The progress of current study will continue in future for determination of structure of antibiotic by mass and NMR studies.
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Received on 12.03.2009 Modified on 24.06.2009
Accepted on 12.07.2009 © RJPT All right reserved
Research J. Pharm. and Tech.2 (3): July-Sept. 2009,;Page 521-526