analytical methods all the chemicals used in this study...
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ANALYTICAL METHODS
Chemicals
All the chemicals used in this study were of analytical grade.
Determination of proteolytic activity
The proteolytic activity was determined with casein as substrate. As a matter
of convenience, the hydrolytic power of the enzymes on casein was called caseinase activity.
Caseinase determination (Bergkvist, 1963)
Caseinase activity was assessed by the modified procedure of Tsuchida et al.
(1986) using 2% casein (Hammerstan casein, Merck, Germany) in 0.2 M carbonate buffer (Varley
et al, 1995) (pH 10) as substrate. Casein solution (0.5 ml) with an equal volume of suitably diluted
enzyme solution was incubated at 55°C. After 10 min, the reaction was terminated by the addition
of 1 ml of 10% trichloroacetic acid. The mixture was centrifuged and to the supernatant was added
5 ml of 0.44 M Na2CO3 and 1ml of two-fold diluted Folin Ciocalteau reagent. After 30 min, the
colour developed was read at 660 nm against a reagent blank prepared in the same manner.
Tyrosine served as the reference standard. The optical density of these solutions was measured in a
Shimadzu (Japan) spectrophotometer. One unit of enzyme activity was defined as the amount of
enzyme that released one ug of tyrosine per ml per min.
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Preparation of 0.44M Na2CO3
0.2 M solutions each of sodium carbonate anhydrous (21.2 g/L) and sodium
hydrogen carbonate (16.8 g/L) were prepared. 50 ml of 0.2 M sodium carbonate solution was
pipette into a 100 ml volumetric flask and made upto the mark with 0.2 M sodium hydrogen
carbonates.
Construction of standard graph for Tyrosine
Stock solution: 50 mg of Tyrosine was dissolved in distilled water to make
100 ml in a volumetric flask, which resulted in 500 µg/ml.
Procedure
Into a series of 10 ml volumetric flasks 1,2, 3,4, and 5 ml of standard stock
solution of tyrosine was taken and distilled water was added to make upto 10 ml mark in each
volumetric flask. Mixed well and the optical density was measured at 660 nm after developing the
colour as described above, against a reagent blank prepared in same manner. The results are shown
in the Table 3.1.
A standard curve was constructed taking concentration of Tyrosine µg/ml
on X-axis and corresponding optical density on Y-axis (Fig. 3.1).
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Table 3.1 Construction of standard graph for Tyrosine
Tyrosine concentration (µg/ml) Optical density
0(Blank) 0.00
20 0.18
40 0.36
60 0.55
80 0.72
100 0.90
Unit: One protease unit (PU) is defined as the amount of enzyme that released 1ug of tyrosine per
ml per minute under the above assay conditions.
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Estimation of total protein:
Estimation of extracellular protein:
Extracellular protein was estimated by Lowry method (Lowry et al. 1951).
The protein reacts with the Folin Ciocalteau reagent to give a coloured complex. The color formed
is due to the reaction of alkaline copper with protein as in the Biuret reaction and the reduction of
the phosphomolybdate by tyrosine and tryptophan present in the protein. The intensity of "the
colour depends on the amount of these aromatic amino acids and thus vary with different proteins.
Preparation of reagents
Solution A (alkaline sodium carbonate): 2% Na2CO3 in 0.1 N NaOH.
Solution B (copper sulphate solution): 1% CuSC4 5H2O in distilled water.
Solution C (Rochelle 's salt): 2% sodium potassium tartarate.
Alkaline solution (working solution): 1 ml CuSC4 5H2O solution + 1 ml Rochelle's salt + 98 ml
alkaline Na2CO3 solution
Folin Ciocalteau reagent: The commercial reagent (LOBA) was used after diluting with equal
volume of water on the day of use.
Standard protein solution: Five mg of Bovine serum albumin (BSA) was dissolved in 50 ml of
distilled water to get a final concentration of 0.1 mg/ml (100 µg/ml).
Procedure:
To 1 ml properly diluted protein sample (10-60 fig/ml), 5 ml alkaline
solution.(working solution) was added, mixed well and incubated at 55°C for 10 min. To the above
mixture 0.5 ml Folin Ciocalteau reagent was added, mixed well and incubated at 55°C for 30 min.
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The absorbence of the colour was measured at 680 nm in a
spectrophotometer. The results are presented in Table 3.2 and Fig 3.2. The amount of protein
present in the sample was calculated from the standard curve.
Table 3.2 Construction of standard graph for protein estimation
Con. of BSA(µg/ml) OD (at 680 nm)
Blank 0.000
10 0.145
20 0.306
30 0.448
40 0.595
50 0.757
60 0.900
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SCREENING AND ISOLATION OF ALKALINE PROTEASE PRODUCING
ACTINOMYCETES
Experimental
Chemicals
All chemicals and medium constituents used in this study were of analytical grade.
Screening program
The following samples were collected at various places from Prakasam,
Khammam and Guntur districts in Andhra Pradesh with a view to isolate potent protease producing
actinomycetes.
Sample Collection:
Sample I: Sample was collected from the underneath the compost from Ratnagiri Nagar, Guntur.
Sample II: Sample was collected from the dumping yard of Municipal High School at Kurnool
Road, Ongole which was rich in Organic matter.
Sample III: It was a liquid sample and was collected from the vermicompost, waste water besides
Vignan College at Palakaluru, Guntur. It was turbid and blackish in color.
Sample IV: Sample was collected from rice fields, N.R.I Junior College at Gujjanagundla, Guntur.
Soil having organic matter and black in color.
Sample V: Soil sample was collected from the drainage of a slaughter house at Ramnagar,
Khammam district which was black in color.
Sample VI: The liquid sample was collected from Lam, Guntur. It is rich in organic matter.
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Sample VII: The sample was collected from the J.K.C. College waste dumping yard at Guntur,
which was black in color.
Sample VIII: The sample was liquid collected from A.P.S.R.T.C. Garage at Vijayawada.
Sample IX: Sample was collected from the dumping yard of Sugarcane industry near Nallapadu,
Guntur which was grey in color.
Sample X: Sample was liquid and collected from the dumping yard of Mother Diary Industry at
Pernamitta, Ongole.
Sample XI: Sample was collected from Sangam Diary Industry at Sangam Jaagarlamudi, Guntur.
Sample XII: Sample was collected from sandy soil at Mangaladas nagar, Guntur.
All the samples were collected in sterile screw capped tubes and care was
taken to see that the points of collection had as widely varying characteristics as possible with
regard to the organic matter, particle size, colour of soil and geographical distribution.
Isolation of Proteolytic actinomycetes from soil samples
About 1 gm of each of the above samples was taken into separate conical
flasks each containing 100 ml of sterile water. The suspension was kept on rotary shaker for 30 min
and kept aside to settle the suspending matter. One ml of the supernatant was serially diluted with
sterile water. One ml each, of these dilutions was added to 20 ml of sterile molten starch casein-
agar medium maintained at 45°C. Mixed thoroughly and plated in 10 cm dia. sterile Petri dishes and
incubated at 28°C. Antifungal (Flucanazole-75 µg/ml) and antibacterial (Refampicin-25 µg/ml)
agents were incorporated to control the fungal and bacterial contamination. After 96 h of
incubation, the actinomycete colonies with clear hydrolyzed zones around them were picked up and
transferred onto starch casein agar slants. The composition of starch casein agar is (g/L): Soluble
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starch, 10; casein, 3; KNO3, 2; NaCl, 2; K2HPO4, 2; MgSO4.7H2O, 0.05; CaCO3, 0.01;
FeSO4.7H2O, 0.01 and agar, 50ml in distilled water.
A total of 18 colonies were isolated from all the samples. The number of
isolates from each sample is given in Table 3.3. The slants were incubated for 48 to 96 h. Out of 18
isolates, 3 were selected based on their macroscopic characters, eliminating those that appeared
close to each other. These 3 isolates were sub-cultured onto two different types of media: Skimmed
milk agar, Starch Casein Agar and tested for their proteolytic activity. The results are presented in
Table 3.4.
Screening of isolates for proteolytic activity
Primary screening:
The selected isolates were initially screened for their proteolytic activities i.e.
caseinolytic and gelatinolytic activities.
Casein hydrolysis:
The caseinolytic activity of the isolates was evaluated using casein-agar plate
technique (Williams and Cross, 1971) as described below:
Composition of milk-agar base
Peptone : 0.1%
Agar : 2.0%
To the sterilized milk agar base, 10% of pasteurized skimmed milk was
added aseptically and this medium was transferred into 10 cm dia. sterile petridish and kept aside
for solidification. Then a loopful of each culture was streaked onto the medium, incubated at 28°C
for 96 h. The diameters of hydrolyzed zones around the colonies and the growth zones were
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measured. The ratio of hydrolysis zone/growth zone was calculated (Table 3.4) which gives a
measure of the caseinolytic activities of the isolates.
Gelatinolytic activity:
For this purpose, 20 ml of sterile nutrient gelatin agar medium (Williams and
Cross, 1971) was poured in sterile petridishes and spot inoculated with a loop full of spores from 48
h old cultures and incubated at 28°C for 96 h. The plates were flooded with mercuric chloride
reagent with the following composition: mercuric chloride 15% and concentrated HCl 20% in
distilled water (Williams and Cross, 1971). After treating with mercuric chloride-HCl solution, the
hydrolysis zone and growth zones were noted and the results are presented in Table 3.4.
The actinomycetes with promising proteolytic activity were selected for further studies.
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Table 3.3 Number of Actinomycetes from various samples
Sample No. No. of cells present per gm. of the sample No. of selected isolates
I 3x105 3
II 2x105 2
III 2x105 2
IV 1x105 1
V 1x105 1
VI 1x105 1
VII 2x105 2
VIII 1x105 1
IX 1x105 1
X 1x105 1
XI 2x105 2
XII 1x105 1
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Table 3.4 Growth pattern and Proteolytic activities of selected isolates
Sample
No.
Isolate No.
Extent of growth (96h) Proteolytic activity *
YEME Starch Casein
Agar Medium
Gelatinolytic
Activity
Caseinolytic
Activity
1
25
+
++
5.3
4.1
2
11
+
+ +
4.9
3.0
3
29
+
++
4.5
2.2
4
05
++
+++
7.1
5.6
5
07
+
++
5.4
3.8
6
02
+
+
4.8
2.9
7
10
+
+
4.9
3.0
10
23
++
+++
6.9
5.1
11
17
++
+++
6.6
4.4
12
02
+
+
3.2
2.0
89
13
15
+
++
2.1
1.2
14
33
++
++
5.1
4.5
15
06
+
++
4.4
3.4
16
24
+
++
4.6
3.8
17
19
+
++
4.8
4.0
18
28
+
++
4.7
3.1
*Ratio= Hydrolyzed zone (mm)/growth zone(mm)
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Secondary Screening
Among the 18 isolates, 3 isolates (Nos. 04, 10 and 11) were selected for
secondary screening and designated as GAS-04, GAS-10 and GAS-11. They were tested for
extracellular protease production, in shake flasks on rotary shaker at 180 rpm. The following six
different types of media were used for the study. These media were selected based on the literature
survey.
Composition of different media for protease production:
Medium
No.
Composition (g/100ml) Reference
I Glucose, 6.0; soyabean meal, 2.0; CaCl2, 0.04; MgCl2, 0.2. Lee et al (1990)
II Glucose, 1.5; yeast extract, 0.5; CaCl2, 0.2. Manachini et al (1988)
III Glucose, 0.1; yeast extract, 0.5; tryptone, 0.5 Frankena et al (1986)
IV Glucose 0.2; Caseine 0.15; salt solution, 5 ml*. Ellaiah et al(2003)
V Glucose 0.2; peptone 0.15; salt solution 5 ml*. Ellaiah et al (2003)
VI
Soluble starch, 1; Casein, 0.3; KNO3, 0.2;
K2HPO4, 0.2; MgSO4.7H2O, 0.005;
CaCC-3, 0.002; FeSO4.7H2O, 0.001.
*Salt solution: MgSO4,0.5%; KH2PO4, 0.5%; FeSO4,0.5%.
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Shake flask fermentation:
5 ml of sterile water was added to 96 h old slant of above isolates. The cells
were scrapped from the slant into sterile water and from the resultant 5 ml cell suspension, 1 ml
suspension was transferred aseptically into 250 ml Erlenmeyer flasks containing 50 ml each of
sterile medium as mentioned above. The flasks were incubated on a rotary shaker (180 rpm) at
28°C for 96 h. The contents of the flasks were centrifuged at 3000 rpm for 10 min and the
supernatant solution were tested for proteolytic activity by modified method of Tsuchida et al.
(1986) as described earlier. The results are presented in Table 3.5.
It is clear from the results that isolate GAS-4 is the best protease producer
and in all the six media selected .Hence this isolate GAS-4 was selected for further studies.
Table 3.5. Production of protease (U/ml) by selected isolates in shake flask*
Medium No.
Isolate GAS-04 Isolate GAS-10
Isolate GAS-11
I 64.7 65.3 50.6
II 58.2 59.9 63.9
III 80.1 78.2 75.5
IV 53.5 63.2 52.3
V 62.9 62.4 62.1
VI 69.5 68.3 66.7
* Protease activity expressed in U/ml.
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Design of suitable basal medium:
The composition of the medium I and III were slightly changed in
their glucose concentration and used for fermentative production of protease by isolate GAS-4 to
compare and design a suitable basal medium for efficient production. The results are presented in
Table 3.6. Maximum yield of 80.1 U/ml was obtained in medium No III The composition of which
was Glucose, 0.1; yeast extract, 0.5; tryptone, 0.5. This was designated as the basal medium and
used for further studies.Medium I and III were slightly modified and the composition of the
modified media were given in Table 3.6
Table 3.6 Production of protease in modified media
Medium
No.
Composition of media Enzyme yield (U/ml)
GAS-04
I Glucose 6%, Soybean meal 2%,
CaCl2 0.04% and MgCl2 0.2%
70.9 ± 1.5
II Glucose 1%, Soybean meal 2%,
CaCl2 0.04% and MgCl2 0.2%
79.7 ± 1.0
III Glucose 0.1%, Yeast extract 0.5%,
Tiyptone 0.5%
78.5 ± 2.0
IV Glucose 1%, Yeast extract 0.5%,
Tiyptone 0.5%
92.0 ±1.0
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The yields of protease produced in the modified media by isolate GAS-4 are
presented in Table 3.7.It was observed that maximum protease of 92.0 U/m L was produced in the
modified media No. IV .Hence this media was selected for further optimization studies.
Table 3.7 Production of protease in modified media
Modified media Isolate-GAS-4
Protease U/ml
I
70.9
II
79.7
III
78.5
IV
92.0
Determination of type of Protease produced by the isolates
To find out whether the enzyme secreted by the isolates belong to alkaline or
acidic or neutral protease the following experiments were conducted. Protease activity in the
harvested broth was assayed by adding 0.5 ml culture broth to 0.5 ml of 2% casein solution. In
order to distinguish between acid, neutral and alkaline protease, the reactions were carried out at pH
4 (Citrate buffer), pH 7 (Phosphate buffer) and pH 10 (Carbonate buffer), by dissolving the casein
in respective buffers. Buffer solutions:
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pH 4.0: Commercially available buffer – Titrisol® was used. It is a citrate / hydrochloric acid
buffer manufactured by Merck.
pH 7.0: Commercially available buffer - Convol® (pH- 7.0 ± 0.05 at 27°C) was used. It contains
potassium dihydrogen orthophosphate.
These commercially available buffers were diluted to 500 ml and then used for preparing
casein solution.
Preparation of carbonate buffer pH 10.0:
0.2 M solutions of each, anhydrous sodium carbonate (21.2 g/L) and sodium
hydrogen carbonate (16.8 g/L) were prepared. 50 ml of 0.2 M sodium carbonate solution was
pipetted into a 100 ml volumetric flask and made up to the mark with 0.2 M sodium hydrogen
carbonate.
Each enzymatic reaction was carried out in duplicate for 10 min. at 55°C.
The amount of Tyrosine released was measured by the modified method of Tsuchida et al (1986) as
described earlier.
The results are shown in Table 3.7. The results indicated that the enzyme
activities by isolate (GAS-04) was high at pH 10 indicating that the enzyme is an alkaline protease.
Further the isolate GAS-04 showed comparatively good enzyme productivity.
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Table: 3.8 Protease activity at different pH values:
Isolate Protease activity (U/ml)
- pH 9.0 pH 7.0 pH 5.0
GAS-4 96.0 70.2 26.1
The result obtained indicated that the protease produced by isolate GAS-4 is
more active at an alkaline pH of 9.0 where maximum protease of 96 U/m l was produced, at pH 7.0
and 8.0 the yield of protease decreased to 70 U/m l and 26 U/m l respectively .These observations
suggested that isolate GAS -4 Produced an alkaline protease.
Capacity of proteases to dehair goat skin:
The isolate GAS-4 was grown in basal medium, the culture broth was
centrifuged at 3000 rpm for 10 min, and the supernatant was used as source of enzyme. Skin pieces
(area about 4 × 4cm) from freshly slaughtered goat were procured from the local meat shop. The
enzyme (20 ml) in the form of a paste, after mixing with kaolin (10 gm) and streptomycin sulphate
(100 mg) was painted on the flesh side of paired goatskin pieces and incubated for 1 h. In control
experiment, water was used in place of enzyme solution. After incubation, ease of unhairing was
noted by removing-the hairs with a blunt scalpel. The results are presented in Table 3.9 and Fig
3.3.
The promising two isolate (GAS-04) was selected for further studies and subjected to
taxonomic studies for identification.
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Table 3.9 Dehairing activities of Protease produced by Isolate GAS-4
Strain Loosening of hairs in 6 hr.
GAS-04 ++
+ loosening of hair by applying maximum force.
++ loosening of hair by applying moderate force.
Fig. 3.3 Dehairing capacities of isolate GAS-04
Control GAS-4
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TAXONOMIC STUDIES
Selection of media for taxonomic studies
Culture media used for characterization and identification of species consists
of both synthetic and organic forms. Synthetic media have found extensive application in the study
of morphology, physiology and cultural properties of the organism while organic media are used for
obtaining supplementary cultural evidence.
Media used for characterization:
Waksman (1958) and others recommended the inclusion of following media
for characterization of actinomycetes:
1. At least three synthetic media, preferably sucrose nitrate salt agar or sucrose ammonium salt
agar, glucose or glycerol asparagine agar and calcium malate or citrate agar.
2. Two to three organic media such as nutrient agar, yeast extract malt extract agar, potato
glycerol glutamate agar or oatmeal agar.
1. Three or four complex natural media such as potato plug, gelatin or milk.
2. Peptone iron yeast extract agar for H2S production.
3. Tyrosine medium for tyrosinase reaction.
4. A synthetic medium for carbohydrate utilization.
Experimental
In the present work the morphological studies and colour determinations of the selected
isolate GAS -4 was studied by following International Streptomycetes Project (ISP) procedures
(Shirling and Gottlieb, 1966). The following media as recommended by ISP were used for
the morphological studies and colour determinations.
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1. Yeast extract malt extract agar (ISP-2).
2. Oatmeal agar (ISP-3).
3. Inorganic salts starch agar medium (ISP-4) and
4. Glycerol asparagine agar medium (ISP-5)
Further, the following biochemical reactions were determined employing the
prescribed media: melanin formation, H2S production, tyrosine reaction, gelatin hydrolysis,
coagulation and peptization of milk, casein hydrolysis, starch hydrolysis, nitrate reduction, carbon
source utilization, sodium chloride tolerance, effect of various nitrogen sources on growth, growth
temperature range, chemical tolerance and cell wall composition.
Preparation of inoculum:
In general, the agar media favouring abundant sporulation are those with a high C/N ratio
such as jowar starch agar, oatmeal agar (ISP) and starch-casein agar.
In the present study starch-casein agar was used for the isolates. These slants were
inoculated from the stock cultures and incubated at 28°C for 1-5 days to get maximum sporulation.
Spore suspension was prepared by transferring a loopful of spores from these slants into sterile
distilled water and shaking thoroughly. For gelatin liquefaction, starch hydrolysis and casein
hydrolysis, a loopful of spores taken from the stock culture was used for inoculation. For all other
tests, spore suspensions prepared as above were used employing equal volumes of the suspension in
each case.
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Preparation of media:
Detailed compositions of all the media employed in this work are given in the
Appendix I.
Morphological and Cultural Studies
The color of aerial mycelium, substrate mycelium and soluble pigment when
grown on different media were observed and recorded. The macro and micro-morphological
features of the colonies and the color determinations of the aerial mycelium, substrate mycelium
and soluble pigment were examined after 96 h of incubation. Macro-morphology was noted by the
naked eye and by observation with magnifying lens.
Micro-morphology:
To study the aerial mycelium and its sporulation characteristics, the following two methods
were used.
1. Direct method
Very thin layer of the respective solidified media in petridishes, were
inoculated with 0.05 ml of the spore suspension. This was placed near the edge of the plate to serve
as a pool of inoculum. Using a sterile loop, four to five equally spaced streaks were made. A
number of plates were inoculated in this manner to facilitate observations on different days.
Observations were recorded from next day onwards up to 4 days.
2. Inclined cover slip method (Kawato and Shinobu, 1959;Williams and Davis 1967):
Sterile cover slips were placed at an angle of 45° into solidified agar medium
in petridish such that half of the cover slip was in medium. Inoculum was spread along the line
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where the upper surface of the cover slip meets the medium. After full sporulation, the cover slips
were removed and examined directly under the microscope.
3. Electron microscopy (Williams and Davis, 1967):
For scanning electron microscopy, slides were cut into 1cm pieces and
sterilized. The pieces were dipped at an angle of 45° into solidified starch casein agar medium. The
inoculum was spread along the glass-agar medium interface. During incubation, the organisms grew
over the surface of the glass pieces. The growth from the glass pieces were removed carefully using
a brush and affixed onto the copper stud, washed with serial grades of 30,50,70 and 90% alcohol.
The studs were then kept in a dessicator for final drying. The surface containing organisms was
coated under vacuum, with a film of gold about 150-200A thickness and observed under Scanning
Electron Microscope (PSEM 500) for spore surface ornamentation. The scanning electron
microscope was provided by Advanced Analytical Laboratory, Andhra University, Visakhapatnam,
India.
Physiological Characteristics:
1. Gram - staining .
To study the Gram's reaction of the culture, a 48 h culture was used. The
experimental protocol as described by Salle (1948) was followed
2. H2S production (Shirling and Gottlieb, 1966):
Many organisms, in their metabolism of sulphur containing organic
compounds, liberate hydrogen sulphide in considerable amounts. This can be demonstrated if
sulphide producing cultures are grown on media containing salts of iron resulting in the formation
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of a black or bluish black precipitate. The use of H2S production as a taxonomic implement was
suggested by many authors.
The inoculated peptone-yeast extract-iron agar (ISP-6) slants were incubated
at 28°C for 5 days. Observations for the presence of characteristic greenish brown, brown, blackish
brown, bluish black or black colour of the substrate, indicative of H2S production, were made every
24 h upto 5 days.
3.Gelatin hydrolysis (Gordon and Mihm, 1957):
This represents the ability of microorganisms to hydrolyze or liquefy gelatin.
Most of the species of Streptomyces bring about liquefaction of gelatin but the rapidity of
liquefaction varies with the species. The non-pigmented forms are most active while the pigmented
forms are least active.
For this test, the isolates were grown on gelatin agar plates for two days at
55°C. At the end of incubation period, the plates were flooded with 1 ml of the following solution.
Mercuric chloride : 15 g
Conc. HCl : 20 ml
Distilled water : 100 ml
The extent of hydrolysis was noted by comparing the width of the clear zone
around the growth. The widths of the hydrolyzed zone and growth zone were measured and the
ratio of hydrolyzed zone and growth zone was calculated.
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4.. Casein hydrolysis (Salle, 1948):
The proteolytic activity was studied with milk-casein agar medium by
measuring the hydrolyzed zones after incubating the inoculated plates at 28°C for 48 h. The extra-
cellular protease (caseinase) activity of the isolates was determined qualitatively as the ratio of the
diameter of the hydrolyzed zone and that of the growth on the milk-casein agar medium.
5. Starch hydrolysis (Salle, 1948):
Numerous actinomycetes are able to hydrolyze starch rapidly by the action of
amylolytic enzymes. For this test, the selected isolates were grown for 5 days on starch agar plates.
At the end of incubation period, the plates were flooded with weak iodine solution. The width of
hydrolyzed zone around the growth versus the width of the growth was measured.
Composition of iodine solution:
Potassium iodide : 3g
Iodine : 1g
Distilled water : 100mL.
6. Nitrate reduction test (Salle, 1948):
The reduction of nitrate to nitrite has been universally used among the
criteria for species differentiation. The reduction is the result of the use of NaNO3 or KNO3 as an
electron acceptor with some organisms. Nitrate (NO3) and NO2 serve as sources of nitrogen for the
synthesis of organic nitrogen compounds or they may function as H+ acceptors in reactions
concerned with the organisms energy metabolism. The first step in the reduction of NO3 involves its
conversion to NO2 by an enzyme system, which is adaptive in nature and is known as nitratase.
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5ml of nitrate broth medium was inoculated with a loopful of spores and
incubated at 28°C for 5 days. Controls were also run without inoculation. After 5 days, the clear
broth was tested for the presence of nitrite in the following way.
Reagents:
a) α -Naphthylamine test solution:
α-Naphthylamine : 5.0g
Conc. H2SO4 : 8.0 ml
Distilled water : 1 L
To the diluted sulphuric acid, α-naphthylamine was added and stirred until solution was effected.
b) Sulphanilic acid test solution:
Sulphanilic acid : 8.0 g
Conc. H2SO4 : 48 ml
Distilled water : 1L
Sulfuric acid was added to 500 ml of water. Then sulphanilic acid was added followed by
water to make up the volume.
Procedure:
To 1 ml of the broth under examination and 1 ml of control, two drops of
sulphanilic acid solution followed by two drops of a-naphthylamine solution were added. The
presence of nitrite was indicated by a pink, red or orange colour and absence of colour change was
considered as nitrite negative. In the later case presence or absence of nitrate in the broth under
104
examination was confirmed by adding a pinch of zinc dust, after the addition of the reagents, when
the unreduced nitrate, if present, gave a pink, red or orange colour.
7. Growth temperature (Williams et al. 1989):
Ability of the isolates to grow at different temperatures was studied at 15° C,
25°C, 37°C and 40°C. The selected isolates were inoculated on starch casein agar medium and
jowar starch medium slants and incubated at the different temperatures as mentioned above. Results
were recorded on 3rd day and 5th day.
8. Chemical tolerance pH(Williams et al, 1989):
10 ml quantities of glycerol-nutrient broth with pH levels of 5.2, 8.0, 9.0, and
10.0 were inoculated and incubated for3 to 5 days. Then the tubes were examined for the extent of
growth of the organism.
9. Sodium chloride tolerance (Pridham et al. 1956):
The ability of certain types of actinomycetes to tolerate and to adapt to high
concentrations of sodium chloride is well known. This adaptability is particularly marked in
organisms found in marine water and salt lake mud. Klevenskaya (1960) found that Streptomyces
isolated from dry and saline soils tolerated upto 7% NaCl. Waksman's literature also indicated that
different Streptomyces species vary widely in their sodium chloride tolerance. Tressener et al.
(1968) surveyed approximately 1300 strains of Streptomyces for tolerance to sodium chloride in the
growth medium. Their results indicated that higher tolerance was found with the yellow and white-
spored Streptomycetes, whereas red series have less tolerance.
For the determination of sodium chloride tolerance, Bennet's agar medium
was supplemented with graded amounts of sodium chloride (1,4,7,10 and 13%). The above medium
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was inoculated with spore suspensions of the organisms. After incubation for 3-5 days, the
maximum salt concentration supporting growth was recorded.
10. Utilization of carbon sources (Shirling and Gottlieb, 1966):
The ability of Streptomyces species to utilize various carbohydrates,
alcohols, salts of organic acids, fats and amino compounds can be of considerable diagnostic value
(Hata et al. 1953). Waksman (1961) in his early work, employed synthetic solution with various
carbon sources, while others (Shirling and Gottlieb, 1966; Hata et al. 1953; Waksman, 1961;
Benedict et al. 1955) indicated that solid media were more suitable. Pridham and Gottleib's (1948)
basal medium is widely used for this purpose.
The ability of thermoactinomycete isolates to utilize various carbon
compounds as source of energy was studied using Pridham and Gottleib's basal salts medium (ISP-
9). The following chemically pure carbon sources were employed in the present study:
D-glucose, L-arabinose, galactose, sucrose, ribose, meso-inositol, D-mannitol, D-fructose,
rhamnose, raffinose, cellulose, lactose, maltose, salicin, trehalose and D-xylose.
A 10% solution of the above were prepared and sterilized except cellulose
and inositol by filtration using bacteriological filters. Cellulose and inositol were sterilized by ether
sterilization technique. Sterilized carbon sources were added to the Pridham and Gottleib's basal
mineral salts agar to give a final concentration of l%. The inoculated tubes were incubated at 55°C
and observed for growth on 3rd and 5th day. The results were recorded as per the extent of growth in
the respective slants.
106
Good growth : +++
Moderate growth : ++
Poor growth : +
Doubtful growth : ±
No growth : –
11. Growth in the presence of different nitrogen sources (Williams et al. 1989):
The ability of isolates to use different nitrogen sources was studied by the
following method. Each nitrogen source was incorporated into the basal medium at 0.1% level. The
prepared slants were inoculated and incubated at 28oC. Results were recorded after 2-4 days. The
growth on each source was compared with that on the un-supplemented basal medium and on a
positive control containing L-asparagine. The following nitrogen sources were used in the present
study: L-asparagine (positive control), L-arginine, L-cysteine HCl, L-histidine, L-valine, phenyl
alanine, threonine, hydroxyl praline, methionine, serine,arginine and potassium nitrate.
Identification and characterization of the selected isolate:
Proper identification and characterization of microorganisms is very important
because it expands the scope for exploitation of industrially important products. To establish the
novelty or otherwise of the present isolate with those of reported in the literature, the various
morphological, physiological and biochemical characteristics of the isolate GAS-04 was done with
the description cited in the literature. The literature survey includes Bergey’s Manual of Systematic
Bacteriology (1992), Bergey’s Manual of Determinative Bacteriology (1974), Biological Abstracts,
Microbiological Abstracts and all other relevant journals. The isolate GAS-04 was further identified
by MTCC, Chandigarh and results were shown in Table 3.3
107
Bio-chemical characteristics:
The biochemical characteristics of the isolates were studied by analyzing the
cell wall composition and physiological characteristics.
1. Analysis of cell Walls:
Cell Wall compositions were analyzed according to the method of Boone and
Pine (1968). Cultures were grown for 3 days in 50 ml yeast extract malt extract (YEME) broth in
250 ml conical flasks, the mycelia were collected by centrifugation at 10,000 rpm for 15 min and
washed thrice with sterile distilled water. Five hundred milligram of the mycelia was extracted with
5 ml of 0. IN NaOH, in tightly sealed screw capped tubes for 1 h, in a boiling water bath. The
mixture was cooled and centrifuged. The alkali extract was discarded. The cell walls were then re-
suspended in 1 ml of water and was used for detection of sugars and amino acids. Identification of
sugars:
The cell wall sample (0.7 ml) was taken and HCl was added to it (to give a
final concentration of 2N HCl) in screw capped tubes, sealed tightly and placed in a boiling water
bath for 2 h. The hydrolyzed materials were transferred to small beakers and dried over a boiling
water bath. To this water was again added, the process was repeated four times and the materials
were finally suspended in 0.5 ml of water and used for chromatography. One-tenth ml sample was
spotted on Whatman No. 1 paper and ascending chromatography was run using the solvent system
n-butanol, acetic acid and water (3:1:1). The chromatogram was sprayed with a solution containing
0.5 g silver nitrate, 1 ml water and 25 ml of 95% ethanol. The papers were then dried for 2 to 3 min
until the appearance of dark spots.
108
2. Identification of amino acids:
The cell wall samples (0.3 ml) were taken in a sealed tube and hydrolyzed
with HC1 (to give a final concentration 6N HC1) at 110°C for 18 h. The hydrolyzed material was
dried in the same way as mentioned for sugar identification procedure. One-tenth ml of the same
sample was spotted on Whatman No. 1 paper and ascending chromatography was run using the
solvent n-butanol, acetic acid and water (4:1:1). Amino acids were detected by spraying the
chromatograms with 0.25% ninhydrin and drying at 100°C for 5 min.
109
RESULTS AND DISCUSSIONS:
IDENTIFICATION AND CHARACTERISATION OF THE SELECTED ISOLATE OF
GAS-4
Proper identification and characterization of microorganisms is very
important because it expands the scope for exploitation of industrially important products. In the
present investigation, criteria laid down by the International Streptomyces Project (ISP) were
followed for the identification and characterization of the selected isolates.
To establish the novelty or otherwise of the present isolates with those
reported in the literature, the various morphological, cultural and biochemical characteristics of the
isolated organisms were compared with the descriptions of the numerous Thermoactinomyces
species cited in the literature. The literature survey includes: Bergey's Manual of Determinative
Bacteriology (Buchanan and Gibbons, 1974), Bergey's Manual of Systematic Bacteriology
(Williams et al. 1989), The Actinomycetes (Vol. H) by Waksman (1961), The International
Streptomyces Project Reports (ISP) (Shirling and Gottlieb, 1966, 1968, 1969 and 1972), Biological
and Microbiological abstracts and all other relevant journals.
Fig. 3.4 growth of isolate GAS -4 on starch casein agar
110
Fig. 3.5 Screening of isolates with caseinolytic activity by skimmed milk agar plate
Fig .3.5.1 Screening of isolates with gelatenolytic activity by gelatin agar medium
The 18 isolates selected after primary screening for caseinolytic activity in
skimmed milk upon were employed to assess their gelatinolytic activity in gelatin agar medium.It
was observed that in general all these isolates had higher gelatinolytic activity compared to
caseinolytic activity which was evident from the extent of the zones of the hydrolysis
Among 3 isolates isolates which exhibited significant caseinolytic and
gelatenolytic activities,isolate GAS-4.Showed most promising caseinolytic activity.Hence isolate
111
(GAS-4) was selected for detailed taxonomic studies. The following taxonomic properties were
investigated for the characterization of the isolate GAS-4.
Section A: Macroscopic appearance:
� Growth rate was moderately rapid with a colony diameter ranging from 0.5 to 1 cm.
� The colony texture was cottony to powdery to mealy and
� The color was white becoming yellowish white or pale pinkish while pale on the reverse
Microscopic appearance:
� The hyphae were hyaline, narrow and septate
� Conidiogenous cells on the hyphae were inflated at the base and were typically flask-shaped
and terminated in a thin zigzagging filaments;
� Conidia were produced from each bending point of the filament, this type of conidium
production is called sympodial geniculate growth;
� Conidia were hyaline, one-celled and globose to ellipsoid in shape and diameter ranges from
2 to 3 µm;
� The condiogenous cells formed dense clusters which appeared as small powdery balls in the
aerial hyphae when viewed under a dissecting microscope.
112
Microscopic morphology:
Mount showed hyaline and septate hyphae. Conidiophores were
globose to elliptical. The non motile, elliptical spores with smooth surface are straight to
flexuous chains with compact coils at the ends .On the test media ,aerial mycelium color was
white and no diffusible pigment produced as shown in Fig. 3.6
Fig. 3.6 Microscopic morphology of the isolate GAS-4 under 40x magnification
Fig.3.7 Scanning electron Microscope photograph of isolate GAS-4
A.X 6500 Magnification B.X 9500 Magnification
113
Macroscopic observation:
For the macroscopic observation, the isolate GAS-4 was inoculated
on the plates containing the following differential media. Yeast extract Malt extract, Glucose
aspargine Agar, Nutrient Agar, Half strength Nutrient Agar, Gelatin Agar, Starch Agar, L.C.Agar,
Potato Dextrose Agar, Oat meal Agar, Inorganic salts- Starch Agar. The culture characteristics were
shown in Table 3.10.
Table 3.10 Cultural Characteristics of isolate GAS-04
Medium Cultural Characteristics
Yeast extract-Malt extract agar (ISP-2) G : Abundant
AM : White
R : Nil
SP : None
Glucose Aspargine Agar G : Moderate
AM : White
R : Nil
SP : None
Nutrient Agar G : Moderate
AM : Dull white
R : Nil
SP : None
Half strength Nutrient Agar G : Moderate
AM : White
R : Nil
SP : None
Gelatin Agar G : Poor
AM : Dull white, Little
aerial mycelia
R : Nil
114
SP : None
Starch Agar G : Good
AM : White
R : Nil
SP : None
Lactobacillus casei agar (L.C.Agar) G : Good
AM : White
R : Nil
SP : None
Potato Dextrose Agar G : Moderate
AM : Dull white
R : Nil
SP : None
Oat meal Agar (ISP-3) G : Good
AM : White
R : Nil
SP : None
Inorganic Salts-Starch Agar (ISP-4) G : Good
AM : Dull white, moderate
aerial mycelia
R : Nil
SP : None
Glycerol-aspargine Agar (ISP-5) G : Moderate, wrinkled
colonies
AM : White
R : Nil
SP : None
G: growth, AM: Aerial mycelium, R: Reverse Color and SP: Soluble Pigment.
115
Table 3.11 Physiological and biochemical properties of isolate GAS-4
Reaction Result
Gram staining +
Growth at 150C +
Growth at 250C +
Growth at 370C +
Growth at 400C +
Growth at pH 5.2 +
Growth at pH 8.0 +
Growth at pH 9.0 +
Growth at pH 10.0 +
Growth on NaCl 2% +
Growth on NaCl 5% +
Growth on NaCl 7% +
Growth on NaCl 10% -
Starch hydrolysis +
Casein hydrolysis +
Citrate utilization test +
Gelatin Liquefaction +
H2S production -
MR +
116
Table 3.12 Carbon source utilization pattern of isolate GAS-4
VP -
Nitrate Reduction -
Indole -
Catalase -
Urease -
Acid Production from
Arabinose -
Galactose -
Glucose +
Mannitol -
Raffinose +
Salicin -
Xylose -
Sucrose -
Rhamnose -
Meso-inositol -
Fructose +
Cell wall composition Cell Wall Type-I
117
Utilization
Carbon sources
Positive
D-glucose(+++), D-fructose (++)
Raffinose (+)
Doubtful
Sucrose
Negative
L(+) arabinose, Cellulose, Galactose,
D-Mannitol, D-xylose, Meso-inositol,
Sucrose, Salicin, Rhamnose
+++: Good growth; '++: Moderate growth; +: Poor growth;
Table 3.13 Growth of isolate GAS-4 in the presence of various nitrogen sources
Nitrogen source (0.1% w/v)
Growth response
L-asparagine (positive control)
++
Methionine, Hydroxy proline,
Valine, Threonine & cysteine HC1
++
Phenylalanine, Serine, Arginine,
Histidine, Potassium nitrate
-
-: No growth; +: Poor growth ++: Moderate growth;
16S r RNA sequencing of GAS-04
118
The 16S rDNA gene sequence of the isolate GAS-04 was used as a query to search
for homologous sequence in the nucleotide sequence databases by running BLASTIN programme.
The high scoring similar to 16S rDNA gene sequences were identified from the BLASTIN result
and retrieved from Gene Bank database. Phylogenetic trees were inferred by using the neighbor
joining Bootstrap analysis. Isolate GAS -4 was sent to IMTECH Chandigarh for detrming the
biochemical properties and sequencing the 16s r RNA and its comparison with closely related
species by BLASTINA score and construction of the phylogenetic tree.
The sequence results were trimed and assembled. The assembly of the sequences is as follows
>GAS-4
ATCCTGGCTCAGGACGAACACTAGCGGCGTGCTTAACACATGCAAGTCGAACGATGAACCTCCTTCGG
GAGGGGATTAGTGGCGAACGGGTGAGTAACACGTGGGCAATCTGCCCTTCACTCTGGGACAAGCCCTG
GAAACGGGGTCTAATACCGGATATGACACGGGATCGCATGGTCTCCGTGTGGAAAGCTCCGGCGGTGA
AGGATGAGCCCGCGCCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCG
GCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTG
GGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTG
TAAACCTCTTTCAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGT
GCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAG
GCGGCCAGTCGCGTCGGGTGTGAAAGACCGGGGCTTAACCCCGGTTCCTGCATTCGATACGGGCTGGCT
AGAGTGTGGTAGGGGAGATCGGAATTCCTGGTGTACGGTGAAATGCGCAGATATCAGGAGGAACAACC
GGTGGCGAAGGCGGATCTCTGGGCCATTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGAT
TAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGAACTAGGTGTTGGTCACATTCCACGTGATCGGT
119
GCCGCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCGAAGGCTAAAACTCAAAGGAATTG
ACGGGGGCCCGCACAAGCAGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGC
TTGACATACACCGGAAAGCATTAGAGATAGTGCCCCCCTTGTGGTCGGTGTACAGGTGGTGCATGGCT
GTGCTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAAGCAGCGCAACCCTTGTCCCGTGTTGC
CAGCAACTCTTCGGAGGTTGGGGACTCACGGGAGACCGCCGGGGTCAACTCGGAGGAAGGTGGGGACG
ACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTACAATGAGCTG
CGATACCGCGAGGTGGAGCGAATCTCAAAAAACCGCTCTCAGTTCGGATTGGGGTCTGCAACTCGACC
CCATGAAGTCGGAGTTGCTAGTAATCGCAGATCACCCCCCCCTTTCTTGAATACGTTCCCGGCCTTGT
ACAC
120
Table 3.14 Top 9 Sequence Producing Significant Alignments
Rank Name/Title Authors Strain Accession Pairwise
Similarity
Diff/Total
nt
MegaBLASTIN
score
BLASTINN
score
1 Streptomyces
indicus
Luo et.al. (in
press)
IH32-
1(T) EF157833 99.120 12/1363 2605 2605
2 Streptomyces
globosus
(Krassilnkov
1941)
Waksman 1953
LMG
19896(T) AJ781330 96.741 44/1350 2303 2284
3 Streptomyces
toxytricini
(Preobrazhenskaya
and Sveshnikoya
1957) Pridham
et.al. 1958)
NBRC
12823(T) AB184173 96.733 44/1357 2297 2278
4 Streptomyces
xantholitucus
(Konev and
Tsyganov 1962)
Pridham 1970)
NBRC
13354(T) AB184349 96.557 46/1336 2268 2240
5 Streptomyces
Rubiginosohelvolus
(Kudrina 1957)
Pridham et.al.
1958
IFO
12912(T) AY999864 96.557 46/1336 2262 0
6 Streptomyces
Iucensis
Arcamone et.al.
1957
NBRC
13056(T) AB184280 96.456 47/1326 2250 0
7
Streptomyces
achromogenes
subsp.
achromogenes
Okami and
Umezawa 1953
NBRC
12735(T) AB184109 96.444 48/1350 2272 2252
8 Streptomyces
tranashiensis Hata et.al.1952
LMG
20274(T) AJ781362 96.437 48/1347 2266 2218
9 Streptomyces
crystallinus Tresner et.al.1961
NBRC
15401(T) AB184652 96.413 48/1338 2264 2230
121
122
Based on the results obtained in these studies our isolate GAS-4 was
identified as Streptomyces indicus. It possessed 99.120 pair wise similarity with Streptomyces
indicus 1H32-1(T) reported by Luo et al (2011).
We have carried out a detailed comparison of the biochemical properties of our isolate
GAS-4 with Streptomyces indicus 1H32-1(T)(2011)reportred by L uo et al (2011).This revealed the
following differiences in the biochemical characters between the two isolates.
Isolate GAS-4 xylose (-), nitrate reduction (-) and mannitol (-). Isolate 1H32-1(T) xylose
(+), nitrate reduction (+) and mannitol (+).
Based on the differences of these biochemical characters we assign our isolate GAS-4 to be a new
variant and designate it as Streptomyces indicus var.GAS-4.
Moreover our strain has been isolated from terrestrial source whereas Streptomyces indicus 1H32-
1(T) was isolated from a deep sea sediment.
123
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