76

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.

77

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).

78

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.

79

80

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.

81

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

82

83

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.

84

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

85

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

86

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.

87

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

88

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)

90

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%.

91

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.

92

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

93

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:

94

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.

95

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.

96

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

97

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.

98

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.

99

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

100

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

101

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.

102

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.

103

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

105

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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