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w ith the objective of selecting the strains for alkaline protease
production, the proteolytic bacteria isolated from alkaline soils were
screened. The separate screening procedures carried out for the selection of
strains for the enzyme production by the submerged and solid state
fermentations are described under section A and B of this chapter respectively.
Section A
SCREENING AND SELECTION OF STRAIN FOR ALKALINE PROTEASE PRODUCTION
BY SUBMERGED FERMENTATION
MATERIALS AND METHODS
Bacterial strains
Proteolytic bacteria were isolated from alkaline soil samples collected
from Karimannu region at Kozhinjampara village in Chittoor taluk of Palakkad
district, Kerala. The soil samples were suspended in water by vigorous
vortexing. Appropriate dilutions of soil solution were pour plated directly in
the casein agar medium, pH 10.0, which contained (per litre) 10 g of casein,
1 g of dibasic potassium phosphate, 20 g of agar, 0.5 g of calcium chloride
and 5 g of sodium chloride. After incubation at 45°C for 24 h, colonies with
zone of clearance were picked and purified by streaking on casein agar. The
purified proteolyhc isolates were stored and maintained in nutrient agar slants
(pH 8.0) by subculturing at monthly intervals. More than 250 isolates were
thus collected and stored. Two Bacjllus /ichenifonnis strains (NCIM 2042 and
NCIM 2044) procured from National Collection of Industrial Microorganisms,
National Chemical Laboratoy, Pune were also included in the study.
Enzyme assay
The alkaline protease activity was assayed by the method of Meyers
and Ahearn (1977) with some modifications. The protocol followed is given
below. The procedure followed for all the enzyme assays throughout this
study was same except for the modifications which are specifically mentioned.
1. Reaction mixtures were prepared as follows:
Test - Control
a. Enzyme - 0.5 ml Enzyme - 0.5 ml
b. Glycine-NaOH buffer - 0.5 ml Trichloroacetic acid (5%)-0.5 ml
(0.2 M, pH 10.0)
c. Casein solution - 1 ml Casein solution - 1 ml
(1% in 0.2 M glycine-NaOH (1% in 0.2 M glycine-NaOH
buffer, pH 10.0) buffer, pH 10.0)
2. Reaction mixtures were incubated for 20 min at 45°C.
3. Terminated the reaction by adding 4 ml of 5% TCA to both test and
control preparations. The tubes were incubated for one hour at room
temperature.
4. Filtered through Whatrnan no. 1 filter paper and the filtrate was
collected.
5. For the colour development for the assay of tyrosine in the filtrate, 5 ml
of 0.4 M sodium carbonate and 0.5 ml of Folin phenol reagent ( IN)
were added to 1 ml of filtrate. Vortexed immediately and incubated for
20 min at room temperature.
6. 0 D was taken at 660 nm. Concentrations of tyrosine in the filtrate was
read from a standard curve for tyrosine already prepared.
One unit of alkaline protease activity was defined as the amount of
enzyme that liberated one micromole of tyrosine per ml per minute under
experimental conditions.
Selection of high yielding strains
The proteolytic bacterial strains were tested for the yield of alkaline
protease by submerged fermentation.
The medium for submerged fermentation, peptone yeast extract (PYE)
medium contained (per litre) 10 g of peptone, 5 g of yeast extract, 5 g of
sodium chloride, 1 g of potassium dihydrogen phosphate, 2 g of dipotassium
hydrogen phosphate, 0.2 g of magnesium sulphate and 0.5 g of calcium
chloride. pH of the medium was adjusted to 8.0.
For the preparation of inoculum, the bacterium was first grown on the
nutrient agar slants (pH 8.0) for 24 h. A loopful of the growth was then
transferred to nutrient broth (pH 8.0) and was allowed to grow at room
temperature (28 k 2°C) for 24 h, with agitation at 150 r.p.m. The culture with
cell concentration adjusted to get an ODm corresponding to 2 mg d y cell
weight per ml was used as the inoculum.
Fifty ml production medium in 250 ml Erlenmeyer flask was inoculated
with 1 ml inoculum and incubated for 72 h at room temperature agitating at
150 r.p.m. After incubation the culture broth was centrifuged at 10,000 r.p.m.
for 20 min. The supernatant was used as the crude enzyme for the assay of
alkaline protease activity. The activity was expressed in u ml-'.
Identification of the highest yielding strains
Various cultural, morphological, physiological and biochemical
properties of the two highest yielding strains, K 147 and K 25, were studied.
Identification was done according to the guidelines in BergeyS Manual of
Systematic Baden'ofogy (Hensyl, 1989).
Monitoring the stability of the highest yielding strains
The ability of the highest yielding strains, K147 and K25 to maintain
the high yielding nature was assessed by subculturing and testing the yields at
monthly intervals. While the strain K147 was tested after 5th, 6th and 7th
subcultures, the other strain K25 was tested after 5th, 6th, 7th and 8th
subcultures. (Four subcultures of both the strains were over before starting this
experiment.) The yields were determined using WE medium as mentioned
earlier.
The strain K25 which was found to be showing no signs of instability in
monthly tests was selected for further SmF studies. Monitoring of the stability
of this strain was continued till the end of this study, testing the yields at six
months intervals.
RESULTS
Selection of high yielding strains
The extracellular alkaline protease activity of the isolates was found to
be ranging from the very low levels of -0.005 to more than 1 u ml-'. Table 3
shows the yield of alkaline protease by the top five high yielding strains.
Activities shown by the NClM strains are also given for comparison.
Table 3 Extracellular alkaline protease production by the top five high yielding strains and NCIM strains
Strain Alkaline protease activity in the culture supernatant (u ml-')
K 147 1.170
K 25 1.081
K 41 0.877
K 116 0.718
K 213 0.662
NCIM 2042 0.034
NCIM 2044 0.172
The highest activity was shown by K 147 and K 25. The yield by these
strains were 1.170 and 1.081 u ml-' respectively. The NCIM strains tested in
parallel were found to have only very low activity.
Identification of the highest yielding strains
The highest yielding strains K 147 and K 25 were identified as
belonging to genus Bacillus. While the strain K 147 could be identified as
Bacillus lichenifomis the other strain K 25 could not be identified up to
species level. The cultural, mo~hological, physiological and the biochemical
characters shown by the strain is given in Table 4 .
Table 4
Characters shown by Bacillus sp. K 25
Gram reaction and morphology Gram positive rod
Endospore production + Motility + Growth on nutrient agar + colony chamden Rough, d y and flat colonies with
irregular margin
Growth at 30°C + 40°C + 50°C + 55°C + 65°C -
Growth at pH 6.8 nutrient broth + 5.7 nutrient broth +
Growth in NaCl
2% + 5% + 7% + 10% -
Catalase + Methyl red - Voyes-Proskauer test + Acid from
D-glucose + L-arabinose - D-xylose - D-mannitol +
Gas from glucose - Hydrolysis of starch + Hydolysis of casein + Utiliition of citrate + Nitrate reduction + Indole production -
Stability of the highest yielding strains
Repeated subculturing was found to be affecting the yield by
B. lichenifomis K147. The original activity shown by this strain which was
determined after 3rd subculture was 1.170 u ml-'. The yields obtained in the
subsequent tests performed after 5th, 6th and 7th subcultures were 0.948,
0.874 and 0.523 u mi-' respectively. On the other hand, the repeated
subculturing was not affecting the yield by Bacillussp. K25. The original yield
shown by this strain which was determined after the second subculture was
1.081 u ml-'. More or less similar yields were obtained in the tests performed
after 5th, 6th, 7th and 8th subculture also. Since no signs of instability was
shown by the strain, it was selected for further SmF studies. The monitoring of
the stability of this strain was continued till the end of this study by testing the
yields at 6 months intervals. Results were suggestive of the stable high
yielding nature of the strain.
DISCUSSION
The naturally occurring alkaline environments comprise alkaline soda
lakes, alkaline springs, alkaline soils etc. Isolation and screening of bacteria
from these natural environments can be supposed to be useful for obtaining
bacterial strains with the potential of yielding alkaline enzymes. So in this
study, for obtaining the suitable strains for alkaline protease production, the
proteolytic bacteria from alkaline soils were isolated and screened. The soil
samples for this purpose were collected from alkaline soils in Chittoor taluk in
Palakkad district of Kerala state.
The diverse group of bacteria that thrive well in alkaline environments
can be categorized into two broad groups alkalotolerants and alkalophiles.
Alkalotolerants show optimal growth between pH 7.0 and 9.0, but cannot
grow above pH 9.5. The alkalophiles can be further divided into two groups,
facultative alkalophiles and obligate alkalophiles. Facultative alkalophiles can
grow at neutral pH while obligate alkalophiles cannot. Both these groups will
grow at pH 10.0 (Krulwich and Guffanti, 1989). So in a medium with
pH 10.0 or above only the alkalophiles are supposed to grow. Since the
casein agar used in this study was having a pH 10.0, the isolates obtained
could be supposed to be alkalophiles, either facultative or obligate. The use of
alkaline casein or milk agar for the isolation of alkaline protease producing
bacteria has been reported by some workers (Durham eta/., 1987; Nihalani
and Satyanarayana, 1992; Gessesse and Gashe, 1997).
Since the incubation of plates was performed at 45"C, the isolates
obtained could be supposed to be thermotolerant or thermophilic also. So the
method used in this study can be considered to be an easy and simple one for
the isolation of proteolytic strains which can possibly be the good sources of
thermostable alkaline proteases.
The proteolytic strains isolated from alkaline soils and the two procured
NClM strains were tested for the yield of alkaline protease by submerged
fermentation. The yield of alkaline protease by the isolates was found to be
ranging from the very low levels of -0.005 to more than 1 u ml-'. There were
only two isolates producing alkaline protease at a level more than 1 u ml-'.
The isolates K 147 and K 25 which produced 1.170 and 1.081 u ml-' alkaline
protease were the highest yielding strains. The NCIM strains, NCIM 2042 and
NClM 2044 tested in parallel showed only very low activity, i.e. 0.034 and
0.172 u ml-' respectively.
Of the two highest yielding strains, K 147 was identified as Bacillus
lichenifonnis. The other strain K 25 was also belonging to genus Bacillus.
Since the characters shown by this strain were not conforming to the
characters exhibited by any of the well characterized species under the genus
Bacilus, it could not be identified up to the species level.
Before selecting strain(s) for SmF studies, their ability to maintain the
high yielding nature was studied by subculturing and testing the yields at
monthly intervals. Repeated subculturing was found to be affecting the high
yielding nature of B. lichenifonnis K147, indicating the instability of the strain.
The instability of Bacillus lichenifonnis due to the repeated subculturing as
observed in this study has already been reported by Nihete et a/. (1985).
It has been opined by them that such instability of cultures was due to the
formation of low yielding variants (Nihete et a/., 1986). A similar loss of
elastase producing ability of Pseudomonas aeruginosa with continued
subcultures, w& reported by Morihara (1964). Since the instability of the
culture, as observed in this study can lead to the poor yield and the dismal
performance of the fermentation systems, the strain B. lichenifomis K147 was
exempted from the further SmF studies. On the other hand repeated
subculturing was not having an effect on the stability of Bacillus sp. K25.
So this strain was selected for further SmF studies. The periodical monitoring
of the stability of this strain was continued till the end of this study testing
yields at six months intervals. Results were suggestive of the stability of the
strain.
Section B
SCREENING AND SELECTION OF STRAIN FOR ALKALINE PROTEASE PRODUCTION
BY SOLID STATE FERMENTATION
MATERIALS AND METHODS
Bacterial strains
The same proteolytic bacterial strains isolated from alkaline soils and
the NCIM strains mentioned under section A of this chapter were subjected to
SSF studies.
Selection of high yieldlng strains
The bacterial strains were tested for the yield of alkaline protease by
solid state fermentation in hvo steps.
Step 1 -Preliminary SSF studies
Preliminary SSF studies with the strains, were performed using wheat
bran medium moistened with salt solution in the ratio 1:2. The salt solution
used contained (dl) dipotassium hydrogen phosphate, 2; potassium
dihydrogen phosphate, 1; magnesium sulphate, 0.1; calcium chloride, 0.1 and
zinc sulphate, 0.01. pH was adjusted to 9.0 with sodium carbonate solution.
10 g wheat bran was thoroughly mixed with 20 ml salt solution in 250 ml
Erlenmeyer flasks. Sterilization was done by autoclaving at 15 lb for 30 min.
The inoculum was prepared as described under section A of
this chapter and the moistened substrate was inoculated with the same at
5% (V/W of moistened substrate) level. After inoculation the contents of the
flasks were mixed thoroughly and incubated at 37°C for 72 h in an incubator
with 60.70% relative humidity. After incubation enzyme was extracted from
the bacterial bran with 0.01 M phosphate buffer, at a bran:buffer ratio of 1:10.
The contact time was 30 minutes. The supernatant of the extract obtained
after centrifugation at 10000 r.p.m. for 10 min was used as the crude enzyme.
The activity of crude enzyme was assayed and expressed in u/Q d y bacterial
bran (DBB). The d y weight of the baderial bran was determined
gravimetrically by dying the sample at 100°C to constant weight.
Step 2 -Detailed SSF studies
K 242, K 11, K 54, K 228 and K 2, the top five strains found to be high
yielding by preliminary SSF studies were tested in SSF systems with different
moisture levels incubated for different periods. Wheat bran medium for this
purpose was prepared as in the preliminary SSF studies described earlier,
except that the moisture levels were varied by mixing commercial wheat bran
and salt solutions at different ratios (1:1, 1:1.5, 1:2, 1:2.5). The media with
different moisture levels were inoculated with the high yielding strains and
incubated for different periods, 48, 72, 96 and 120 h. Enzyme activity was
determined as in the preliminary SSF studies.
identification of the highest yielding strain
Cultural, morphological, physiological and biochemical properties of
the highest yielding strain K 242 were studied. The strain was identified
according to the guidelines in Bergeyk Manual of Systematic Bacteriology
(Hensyl, 1989).
Monitoring the stability of the highest yielding strain
The ability of the highest yielding strain K242 to maintain the high
yielding nature was studied by subculturing and testing the yields at monthly
intervals for a period of six months after selecting the stmin (13 subcultures
were over by the time the strain was selected). The yields were tested in the
wheat bran medium having wheat bran:salt solution ratio of 1:1.5. The other
conditions were same as mentioned under the preliminaxy SSF studies.
Since no signs of instability was shown by Bacillus pumilus K242 it was
used for further SSF studies. Monitoring of the stability of this strain was
continued till the end of this study by testing the yields at six months intervals.
RESULTS
Selection of high yielding strains
Preliminary SSF studies
In the preliminary SSF studies, most of the bacterial strains gave visible
growth in solid substrate medium. The alkaline protease production by these
strains varied from -0.2 u/g DBB to more than 50 u/g DBB. The activity
shown by the top five high yielding strains are shown in Table 5. Activities
shown by the NCIM strains are also given for comparison.
Table 5
Alkaline protease production by top five high yielding strains and NCIM strains in preliminary SSF studies
Strain Alkaline protease production (u/g DBB)
K 242 52.80 K 11 34.86 K 54 32.47 K 228 31.91 K 2 30.80 NCIM 2042 21.86 NCIM 2044 7.25
The highest yield (52.80 u/g DBB) was shown by the strain K 242. The
yields by the other high yielding strains were comparatively lesser. Low
activities were shown by the NCIM strains.
Detailed SSF studies
Results of detailed SSF studies performed with the high yielding strains
are shown in Table 6.
Table 6
Alkaline protease production by the high yielding strains in solid state fermentation systems with different moisture levels,
incubated for different periods
Bacterial Wheat bran, salt Alkaline protease produdion (dg DBB) when strain solution ratio incubated for different periods
48 h 72 h 96 h 120 h -
1:l 33.86 37.93 38.94 35.19 K 242 1:1.5 45.98 55.66 46.18 38.51
1:2 48.40 48.66 47.77 38.83 1:2.5 35.72 35.83 32.68 36.35 1:l 24.20 32.19 37.51 30.25
K 11 1:1.5 26.41 42.35 41.16 38.90 1:2 32.19 34.51 37.64 39.13 1:2.5 38.83 40.14 44.70 38.42 1:l 21.28 30.88 30.04 28.25
K 54 1:1.5 22.89 33.60 25.41 21.00 1:2 28.95 31.46 26.60 29.07 1:2.5 18.15 21.78 19.10 22.31 1:l 16.94 22.40 19.36 22.09
K 228 1:1.5 32.67 40.95 43.56 36.66 1:2 18.45 33.80 39.90 35.09 12.5 37.93 40.88 33.80 38.08 1:l 29.01 26.62 27.14 30.10
K 2 1:1.5 35.88 40.50 36.30 30.67 1:2 21.07 28.60 26.00 34.40 1:2.5 21.22 32.04 24.29 31.15
The strain K 242 was found to be the best producer. It could produce
55.66 u/g DBB when grown in solid substrate medium with wheat bran:salt
solution ratio 1:1.5, for 72 h. The maximum yield shown by K 11, K 54,
K 228 and K 2 were 44.70,33.60,43.56 and 40.50 u/g DBB respectively.
Identification of the highed yielding strain
The highest yielding strain K 242 was identified as Bacilluspurnilus.
Stability of the highest yielding strain
Repetition of subculturing was not having an effect on the stability of
B. purnilus K 242. The original yield by this strain which was determined after
the 13th subculture was 55.66 u/g DBB. More or less similar yields were
obtained in all the monthly tests performed thereafter upto the 19th
subculture. Since the strain B. purnilus K 242 was not showing any signs of
instability in the monthly tests it was employed for further SSF studies.
Monitoring of the stability of this strain was continued till the end of this study,
by testing the yields at six months intervals. No signs of instability were seen
in these tests also.
DISCUSSION
Since the high yielding strains in submerged fermentation might not
give good yield in solid state fermentation also, separate extensive screening
was carried out for selecting a strain suitable for alkaline protease production
by solid state fermentation.
With the purpose of testing a large number of strains, a preliminary
screening programme was carried out providing arbitrarily selected conditions
presumed to be suitable for the majority of the isolates. The strains were
tested for the yield in moistened wheat bran medium with a moderate
moisture level. The wheat bran:saIt solution ratio was 1:2. In various
bacterial SSF systems reported so far, the ratio of solid substrate:moistening
solution was in the range 1:l to 1:2.5 (Qadeer et a/., 1980; Raimbault and
Alazard, 1980; Dipti, 1994; Babu and Satyanarayana, 1995; Sen, 1995).
So the ratio 1:2 could reasonably be moderate supporting the growth of
maximum number of strains. In conformity with this assumption, most of the
tested strains were giving the growth in the preliminary SSF attempts. In this
study wheat bran was used as the substrate for SSF. Earlier reports in this
field indicate the universal suitability of commercial wheat bran for use in SSF
systems. It has been reported to be containing 8.5% starch and 9.5% protein
in addition to several minerals (Park and Rivera, 1982).
The yield of alkaline protease by the strains which gave visible growth
on solid substrate medium ranged from 0.2 to 50 u/g DBB. K 242, K 11,
K 54, K 228 and K2 were the top five strains yielding high in the preliminary
SSF studies. It can be noticed that none of these strains were high yielding
under the submerged fermentation conditions. This observation confirms the
necessity of performing separate screening for the selection of strains for
submerged and solid state fermentation processes.
In order to select the best strain from the high yielding stmins, detailed
SSF studies were canied out varying the moisture levels and incubation
periods. In this study also, the highest yield was shown by the strain K 242.
It produced 55.66 u/g DBB in the medium with wheat bransalt solution ratio
1:1.5, after 72 h incubation. The maximum yield (u/g DBB) shown by the
strains, K11, W, K228 and K2 were 44.70, 33.60, 43.56 and 40.50
respectively.
The highest yielding strain K 242 could be identified as Bacillus
pumilus, based on its cultural, morphological, physiological and biochemical
characters.
The ability of the highest yielding strain B, pumilus K 242 to maintain
the high yielding nature was assessed by subculturing and testing the yields at
monthly intervals. Repeated subculturing was found to be not affecting the
stability of the strain. So this strain was used for further SSF studies. The
periodical testing of the yield by this strain was continued till the end of this
study, at an interval of six months. Results were suggestive of the stable high
yielding nature of the strain.
A survey of literature indicates that the alkaline protease production by
SSF using 5. pumilus has not yet been reported.
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