Chemicals
of convenience, the hydrolytic power of the enzymes on casein was called caseinase activity.
(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 55C. 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
76
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.
Procedure
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
77
Table 3.1 Construction of standard graph for Tyrosine
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
78
79
Estimation of total protein:
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
Folin Ciocalteau reagent: The commercial reagent (LOBA) was used after diluting with equal
Standard protein solution: Five mg of Bovine serum albumin (BSA) was dissolved in 50 ml of
Procedure:
solution.(working solution) was added, mixed well and incubated at 55C for 10 min. To the above
mixture 0.5 ml Folin Ciocalteau reagent was added, mixed well and incubated at 55C for 30 min.
80
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
Blank 0.000
10 0.145
20 0.306
30 0.448
40 0.595
50 0.757
60 0.900
81
82
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
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
Sample III: It was a liquid sample and was collected from the vermicompost, waste water besides
Sample IV: Sample was collected from rice fields, N.R.I Junior College at Gujjanagundla, Guntur.
Sample V: Soil sample was collected from the drainage of a slaughter house at Ramnagar,
Sample VI: The liquid sample was collected from Lam, Guntur. It is rich in organic matter.
83
Sample VII: The sample was collected from the J.K.C. College waste dumping yard at Guntur,
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,
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.
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 45C. Mixed thoroughly and plated in 10 cm dia. sterile Petri dishes and
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
84
starch, 10; casein, 3; KNO3, 2; NaCl, 2; K2HPO4, 2; MgSO4.7H2O, 0.05; CaCO3, 0.01;
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.
Primary screening:
The selected isolates were initially screened for their proteolytic activities i.e.
Casein hydrolysis:
The caseinolytic activity of the isolates was evaluated using casein-agar plate
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 28C
for 96 h. The diameters of hydrolyzed zones around the colonies and the growth zones were
85
measured. The ratio of hydrolysis zone/growth zone was calculated (Table 3.4) which gives a
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 28C 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.
86
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
87
Table 3.4 Growth pattern and Proteolytic activities of selected isolates
1 25 + ++ 5.3 4.1
2 11 + ++ 4.9 3.0
3 29 + ++ 4.5 2.2
5 07 + ++ 5.4 3.8
6 02 + + 4.8 2.9
7 10 + + 4.9 3.0
12 02 + + 3.2 2.0
88
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
89
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.
No.
I Glucose, 6.0; soyabean meal, 2.0; CaCl2, 0.04; MgCl2, 0.2. Lee et al (1990)
III Glucose, 0.1; yeast extract, 0.5; tryptone, 0.5 Frankena et al (1986)
90
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
28C 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.
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.
91
Design of suitable basal medium:
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
No. GAS-04
Tiyptone 0.5%
Tiyptone 0.5%
92
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.
Protease U/ml
I 70.9
II 79.7
III 78.5
IV 92.0
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
93
pH 4.0: Commercially available buffer Titrisol was used. It is a citrate / hydrochloric acid
pH 7.0: Commercially available buffer - Convol (pH- 7.0 0.05 at 27C) was used. It contains
These commercially available buffers were diluted to 500 ml and then used for preparing
casein solution.
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 55C.
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.
94
Table: 3.8 Protease activity at different pH values:
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
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
95
Table 3.9 Dehairing activities of Protease produced by Isolate GAS-4
GAS-04 ++
Control GAS-4
96
TAXONOMIC STUDIES
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
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
1. Three or four complex natural media such as potato plug, gelatin or milk.
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
97
1. Yeast extract malt extract agar (ISP-2).
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
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 28C 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.
98
Preparation of media:
Detailed compositions of all the media employed in this work are given in the
Appendix I.
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
Micro-morphology:
To study the aerial mycelium and its sporulation characteristics, the following two methods
were used.
1. Direct method
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.
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
99
where the upper surface of the cover slip meets the medium. After full sporulation, the cover slips
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
India.
Physiological Characteristics:
1. Gram - staining .
To study the Gram's reaction of the culture, a 48 h culture was used. The
sulphide producing cultures are grown on media containing salts of iron resulting in the formation
100
of a black or bluish black precipitate. The use of H2S production as a taxonomic implement was
at 28C 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.
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
For this test, the isolates were grown on gelatin agar plates for two days at
55C. 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
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
101
4.. Casein hydrolysis (Salle, 1948):
measuring the hydrolyzed zones after incubating the inoculated plates at 28C 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.
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.
Potassium iodide : 3g
Iodine : 1g
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
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.
102
5ml of nitrate broth medium was inoculated with a loopful of spores and
incubated at 28C 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:
-Naphthylamine : 5.0g
Distilled water : 1L
To the diluted sulphuric acid, -naphthylamine was added and stirred until solution was effected.
Conc. H2SO4 : 48 ml
Distilled water : 1L
Sulfuric acid was added to 500 ml of water. Then sulphanilic acid was added followed by
Procedure:
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
103
examination was confirmed by adding a pinch of zinc dust, after the addition of the reagents, when
25C, 37C and 40C. 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
10.0 were inoculated and incubated for3 to 5 days. Then the tubes were examined for the extent of
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-
was supplemented with graded amounts of sodium chloride (1,4,7,10 and 13%). The above medium
104
was inoculated with spore suspensions of the organisms. After incubation for 3-5 days, the
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)
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:
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 55C
and observed for growth on 3rd and 5th day. The results were recorded as per the extent of growth in
105
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
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 Bergeys Manual of Systematic
Microbiological Abstracts and all other relevant journals. The isolate GAS-04 was further identified
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
107
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 110C 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
108
RESULTS AND DISCUSSIONS:
GAS-4
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
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
(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
109
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
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
110
(GAS-4) was selected for detailed taxonomic studies. The following taxonomic properties were
Growth rate was moderately rapid with a colony diameter ranging from 0.5 to 1 cm.
The color was white becoming yellowish white or pale pinkish while pale on the reverse
Microscopic appearance:
Conidiogenous cells on the hyphae were inflated at the base and were typically flask-shaped
Conidia were produced from each bending point of the filament, this type of conidium
Conidia were hyaline, one-celled and globose to ellipsoid in shape and diameter ranges from
2 to 3 m;
The condiogenous cells formed dense clusters which appeared as small powdery balls in the
111
Microscopic morphology:
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
Fig. 3.6 Microscopic morphology of the isolate GAS-4 under 40x magnification
112
Macroscopic observation:
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
113
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.
114
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% +
Starch hydrolysis +
Casein hydrolysis +
Gelatin Liquefaction +
H2S production -
MR +
115
VP -
Nitrate Reduction -
Indole -
Catalase -
Urease -
Arabinose -
Galactose -
Glucose +
Mannitol -
Raffinose +
Salicin -
Xylose -
Sucrose -
Rhamnose -
Meso-inositol -
Fructose +
116
Carbon sources
Utilization
Doubtful Sucrose
Table 3.13 Growth of isolate GAS-4 in the presence of various nitrogen sources
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
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
118
GCCGCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCGAAGGCTAAAACTCAAAGGAATTG
ACGGGGGCCCGCACAAGCAGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGC
TTGACATACACCGGAAAGCATTAGAGATAGTGCCCCCCTTGTGGTCGGTGTACAGGTGGTGCATGGCT
GTGCTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAAGCAGCGCAACCCTTGTCCCGTGTTGC
CAGCAACTCTTCGGAGGTTGGGGACTCACGGGAGACCGCCGGGGTCAACTCGGAGGAAGGTGGGGACG
ACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTACAATGAGCTG
CGATACCGCGAGGTGGAGCGAATCTCAAAAAACCGCTCTCAGTTCGGATTGGGGTCTGCAACTCGACC
CCATGAAGTCGGAGTTGCTAGTAATCGCAGATCACCCCCCCCTTTCTTGAATACGTTCCCGGCCTTGT
ACAC
119
Table 3.14 Top 9 Sequence Producing Significant Alignments
(Krassilnkov
Streptomyces LMG
2 1941) AJ781330 96.741 44/1350 2303 2284
globosus 19896(T)
Waksman 1953
(Preobrazhenskaya
et.al. 1958)
(Konev and
Streptomyces NBRC
4 Tsyganov 1962) AB184349 96.557 46/1336 2268 2240
xantholitucus 13354(T)
Pridham 1970)
(Kudrina 1957)
Streptomyces IFO
5 Pridham et.al. AY999864 96.557 46/1336 2262 0
Rubiginosohelvolus 12912(T)
1958
Streptomyces
achromogenes
Streptomyces LMG
8 Hata et.al.1952 AJ781362 96.437 48/1347 2266 2218
tranashiensis 20274(T)
Streptomyces NBRC
9 Tresner et.al.1961 AB184652 96.413 48/1338 2264 2230
crystallinus 15401(T)
120
121
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
We have carried out a detailed comparison of the biochemical properties of our isolate
Isolate GAS-4 xylose (-), nitrate reduction (-) and mannitol (-). Isolate 1H32-1(T) xylose
Based on the differences of these biochemical characters we assign our isolate GAS-4 to be a new
Moreover our strain has been isolated from terrestrial source whereas Streptomyces indicus 1H32-
122
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