SCREENING AND SELECTION OF STRAINS FOR

ALKALINE PROTEASE PRODUCTION

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.