Extracellular alkaline protease from a newly isolated haloalkaliphilic

Bacillus sp.: Production and optimization

Rajesh Patel a, Mital Dodia b, Satya P. Singh b,*

aDepartment of Life Sciences, University of North Gujarat, Patan, IndiabDepartment of Biosciences, Saurashtra University, Rajkot 360005, Gujarat, India

Received 18 October 2004; received in revised form 11 March 2005; accepted 25 March 2005

Abstract

A newly isolated haloalkaliphilic Bacillus sp., Ve1, produced substantial levels of extracellular alkaline protease. Enzyme production

corresponded with growth and reached a maximum level (410 U/ml) during the early stationary phase along with a brick red pigmentation.

Protease production was maximum (397 U/ml) in gelatin broth. While growth and protease production was optimum at 10% (w/v) NaCl, only

marginal growth without enzyme production was evident in the absences of salt. Ve1 could grow and produce protease at pH 7–9, the optimum

being at 8 and 9, respectively. Among the organic nitrogen sources used, growth was best supported by a combination of peptone and yeast

extract, while the optimum protease production was with casamino acid followed by gelatin. Enzyme production was highly reduced in soya

peptone, trader’s protein and tryptone. Inorganic nitrogen sources proved to be less favourable. Strong catabolic repression on protease

production was observed with glucose and ammonia. The study assumes significance in the light of increasing emphasis on biocatalysis under

more than one extreme condition.

# 2005 Elsevier Ltd. All rights reserved.

Keywords: Alkaline protease; Haloalkaliphiles; Catabolite repression; Extremophiles; Bacillus sp.; Protease optimization

www.elsevier.com/locate/procbio

Process Biochemistry 40 (2005) 3569–3575

1. Introduction

Enzymes have attracted attention from researchers all

over the world because of the wide range of physiological,

analytical and industrial applications, especially, from

microorganisms, because of their broad biochemical

diversity, feasibility of mass culture and ease of genetic

manipulation. Despite the fact that more than 3000 different

enzymes have been identified and many of them have found

their way into biotechnological and industrial applications,

the present enzymes toolbox is not sufficient to meet most of

the industrial demands. In view of these restrictions,

researchers have diverted their attention for isolation and

characterization of enzymes from extremophiles. Extremo-

philes are valuable sources of novel enzymes [1–3].

* Corresponding author. Tel.: +91 281 2586419; fax: +91 281 2586419.

E-mail addresses: satyapsingh@yahoo.com,

satyapra58@indiatimes.com (S.P. Singh).

1359-5113/$ – see front matter # 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.procbio.2005.03.049

Microbial alkaline proteases dominate the worldwide

enzyme market, accounting for a two-third share of the

detergent industry [4,5]. Although protease production is an

inherent property of all organisms, only those microbes that

produce a substantial amount of extracellular protease have

been exploited commercially. In view of its possible

applications, alkaline protease from extreme organisms

should be produced commercially in high yield at a low-cost

method. Recently, production of extracellular alkaline

proteases has been optimized from alkaliphiles [6,7].

Amongst the extremophiles, most of the studies on

alkaline protease have been concentrated either from

halophiles or from alkaliphiles; haloalkaliphilic bacteria

have been relatively less explored. Haloalkaliphilic

bacteria require not only high salt but also alkaline pH

for growth and enzyme secretion. Their extracellular

enzymes must be active under such extreme conditions.

Recently, a novel alkaliphilic and moderately halophilic

Gram-positive coccus Salinicoccus alkaliphiles sp. Nov.

was reported to grow in the presence of 0–25% (w/v) NaCl

R. Patel et al. / Process Biochemistry 40 (2005) 3569–35753570

at pH 6.5–11.5, with optimum growth at 10% (w/v) and

pH 9.0 [8]. Two different groups of haloalkaliphilic, obliga-

tory autotrophic, sulphur-oxidizing bacteria have recently

been isolated which are found to be a true alkaliphilic in

nature [9]. A serine protease from the haloalkaliphilic

archaeon Natronococcus occultus has also been character-

ized, where the optimum salt for enzyme activity was

1–2 M at 60 8C [10]. Recently, serine proteases from

Atrialba magadii [11] and the haloalkalophilic archaeon

N. occultus [12] have been studied with reference to their

production and characterization.

While most of the studies on haloalkaliphilic bacteria

have focused on molecular phylogeny [13–17]; only a few

reports are on their enzymic potential [10,12,18]. As the

alkaline proteases from haloalkaliphilic bacteria and

archaebacteria are relatively less explored, it is of potential

value to optimize the conditions required for growth and

protease production. In a view of this realization, the present

work has focused on the production of extracellular alkaline

protease from haloalkaliphilic bacteria, Ve-1, which relates

to Bacillus pseudofirmus.

2. Materials and methods

2.1. Organism

A haloalkaliphilic bacteria, Ve-1, identifiable with B.

pseudofirmus was isolated by enrichment culture techni-

ques from salt enriched soil sample collected from the

natural saline and hyper saline habitats from the Veraval

costal region of the Gujarat, India. The soil sample (1 g)

was added to a CMB medium containing: glucose, 10 g/l;

peptone, 5 g/l; yeast extract, 5 g/l; KH2PO4, 5 g/l; with

NaCl (20%, w/v). The pH of the medium was adjusted to

10 by adding separately autoclaved Na2CO3 (20% , w/v)

and incubated at 37 8C for 48 h under shake flask

conditions. After 48 h, a loopful culture was streaked on

the same media and based on colony characters different

organisms were selected. The potential strain, Ve1 was

further identified by 16S rRNA amplification and nucleo-

tide sequencing.

2.2. Inoculum preparation and protease production

The inoculum was prepared by adding a loop full of pure

culture into 25 ml of sterile CMB medium and incubated at

37 8C on a rotary shaker for 24 h. A 10% inoculum from the

culture (At A660; 1.0) was added to gelatin broth (GB)

containing: gelatin, 10 g/l; casein enzymatic hydrolysate,

10 g/l; NaCl (w/v), 100 g/l and pH 9. After incubation of

48 h at 37 8C under shaking condition (100 rpm), the culture

was harvested and growth was measured at A660. The

cultures were centrifuged at 10,000 rpm for 10 min at 4 8C.

The cell free extract was used as crude preparation to

measure protease activity.

2.3. Enzyme assay

Alkaline protease activity was measured by Anson–

Hagihara’s method [19]. The enzyme (0.5 ml) was added to

3.0 ml casein (0.6%, w/v in 20 mM Borax–NaOH buffer, pH

10) and the reaction mixture was incubated at 37 8C for

10 min before the addition of 3.2 ml of TCA mixture (0.11 M

trichloro acetic acid, 0.22 M sodium acetate, 0.33 M acetic

acid). The terminated reaction mixture was incubated for

30 min at room temperature. The precipitates were removed

by filtration through Whatman-1 filter paper and absorbance

of the filtrate was measured at 280 nm. One unit of alkaline

protease activity was defined as the amount of enzyme

liberating 1 mg of tyrosine/min under assay conditions.

Enzyme units were measured using tyrosine (0–100 mg) as

standard.

2.4. Growth kinetics and protease production

The kinetics of growth and enzyme secretion was followed

at different time intervals. The Ve1 culture was inoculated in

GB medium (NaCl, 10%, w/v; pH 9) and incubated at 37 8Cunder shaking conditions (100 rpm). Culture samples were

withdrawn aseptically every 4 h and cell density along with

enzyme activity was monitored as describe above.

2.5. Effect of different media composition on protease

production

Growth and enzyme production was compared in three

different media; complete (CMB), GB and haloalkaliphilic

medium. While the composition of the first 2 media are

given above, the haloalkaliphilic medium contained;

solution A (500 ml) + solution B (500 ml). Solution A

included: NaCl, 200 g/l and Na2CO3�10H2O, 50 g/l. Solu-

tion B had: yeast extract, 10 g/l; casamino acids, 7.5 g/l;

trisodium citrate, 3 g/l; KCl, 2 g/l; MgSO4�H2O, 1 g/l;

FeSO4, 50 mg/l; MnCl2�4H2O, 0.36 g/l. Inoculum was

added and incubated for 72 h at 37 8C under shake flask

conditions (100 rpm).

2.6. Optimization of production medium for growth

and protease secretion

2.6.1. Effect of NaCl

The effect of salt on growth and protease secretion was

studied by varying the NaCl concentrations (0–20%, w/v)

and a constant pH 9 in GB (describe above). Growth and

enzyme activity were quantified after incubation for 66 h at

37 8C under shaking at 100 rpm.

2.6.2. Effect of pH

In order to investigate the influence of pH on growth and

protease production, the isolate, Ve1 was grown in a GB

medium at varying pH (7–10) and constant NaCl

concentration; 10% (w/v). After incubation of 66 h at

R. Patel et al. / Process Biochemistry 40 (2005) 3569–3575 3571

Fig. 1. Growth kinetics of Ve1 with references to protease production.

Samples were withdrawn at 4 h interval for the determination of cell growth

(OD660) (*) and protease activity (~).

37 8C under shaking conditions at 100 rpm, growth and

protease activity were quantified.

2.6.3. Effect of gelatin and casamino acid

To study the effect of gelatin, the medium was

supplemented with gelatin (0–2%, w/v). Similarly, the

effect of casamino acid was also investigated at different

concentrations (0–2%, w/v). The growth and enzyme

activity were monitored after 66 h growth at 37 8C.

2.7. Effect of nitrogen sources

Organic nitrogen sources included soya peptone, tryptone,

caseitone, gelatin, casamino acid, trader’s protein, peptone

and yeast extract, while inorganic nitrogen sources included

ammonium nitrate, ammonium chloride and potassium

nitrate. The respective nitrogen sources were added as a

sole source of nitrogen (0.5%, w/v). The growth and enzyme

activity were monitored after 66 h growth at 37 8C.

2.8. Repression studies with glucose and inorganic

phosphate

To study the repression of enzyme secretion by glucose

and inorganic phosphate (K2HP04), different concentrations

(0–2%, w/v) were incorporated into the production medium.

Growth and enzyme activity were monitored after 66 h

growth at 37 8C.

2.9. Enzyme production with molasses and wheat flour

The molasses and wheat flour in the range of 0.25–1%

(w/v) with NaCl (10%, w/v) at pH 9 were used as sole source

of carbon and nitrogen. After incubation of 48 h at 37 8Cunder shaking conditions (100 rpm), the growth and

protease activity were quantified.

3. Results and discussion

Most of the halophiles and alkaliphiles reported are from

Soda Lakes, while the present study exhibited the presence

of haloalkaliphilic bacteria from natural and man made

saline habitats. On the basis of 16S rRNA studies, the isolate,

Ve1 was found to be a Bacillus sp. having 95% similarity

with Bacillus peudofirmus.

By studying the growth kinetics with reference to protease

production from Ve1, it was clear that the organism

maintained a slow growth up to 10 h, after which the growth

was exponential up to 40 h followed by stationary phase.

Protease secretion nearly corresponded with the growth and

maximal in early stationary phase (Fig. 1). Brick red

pigmentation was observed in stationary phase. The relation-

ship between growth and enzyme secretion has also been

studied in some recently published work with halophilic

organisms. In case of a moderate halophile, Pseudoalter-

omonas,maximal protease production was detected at the end

of the exponential growth phase [20]. However, with the Ve1,

optimum enzyme production occurred during the early

stationary phase. In B. sphaericus, an obligate alkalophile, a

major portion of the alkaline protease was secreted in post

exponential phase [21]. These observations were comparable

to other reports on Bacillus sp. and other haloalkaliphilic

organisms [10,22,23]. These results clearly suggested the

prominent role of extracellular proteases in both primary

metabolism and ecological sustenance of these organisms.

The comparative production of enzyme in CMB, GB and

haloalkaliphilic medium revealed that while growth was

similar in all cases, enzyme production varied extensively.

The enzyme level was maximal in GB medium (397 U/ml)

that may be due to induction of protease production by

casamino acid and gelatin (data not shown). Similar results

were described for Bacillus mojavensis that produced 440 U/

ml of an alkaline protease during batch-fermentation at 50 8Cin a minimal medium containing casamino acids. Protease

production was also shown to be induced in the presence of

organic nitrogen, casein and casamino acids [24].

3.1. Effect of salt (NaCl) and pH

Protease production was maximal (186 U/ml) at 10% (w/

v) salt followed by 172 U/ml at 15% and 158 U/ml at 20% (w/

v) salt. There was only marginally reduced activity at 15 and

20% as compared to optimal enzyme secretion with 10% salt.

Enzyme secretion, however, was significantly suppressed at

5 % (w/v) salt, while there was no enzyme production in the

absence of salt (Fig. 2). These results clearly indicated the

halophilic nature of the enzyme where salt appears to be a

prerequisite for enzyme production. However, while Ve1

could grow in the absence of salt, optimum growth was at

10% NaCl (w/v). Another trend was comparable reduction in

growth at either side of the optimum salt concentration

(Fig. 2). These results could be compared with haloalk-

alophilic archaeon N. occultus in which protease secretion

was maximum at 1–2 M NaCl [12]. A much higher salt

requirement (25%, w/v) for a serine protease secretion was

R. Patel et al. / Process Biochemistry 40 (2005) 3569–35753572

Fig. 2. Effect of NaCl (%, w/v) on growth and protease production by Ve1.

Samples were taken after incubation of 66 h at 37 8C under shaking

conditions (100 rpm), for the determination of cell growth (OD660) (&)

and protease activity ( ).

Fig. 4. Effect of gelatin concentration (0–2%, w/v) on growth (*) and

protease activity (~). Samples were withdrawn after incubation of 66 h at

37 8C.

reported in an archaebacterium Halobacterium mediterranei

[25]. An obligatory alkaliphilic Bacillus sp. P-2 produced a

thermostable alkaline protease tolerating 20% (w/v) NaCl.

This bacterium was also able to produce protease at this salt

level, though at a lesser extent [26].

Ve1 could grow in the range of pH 7–9 with an optimum

at 8. The growth at pH 8 and 9 was quite comparable;

however, the growth was substantially reduced at pH 7. This

indicated the alkaliphilic nature of the isolate. A similar

response on growth has been observed in some haloalk-

aliphilic archaea such as a Natronoincul [27] and

Natronorubrum bangense [28], where the optimum pH

for growth was 9–9.5. The isolate under study showed

gradual increase in the protease production with increasing

pH, optimum being at 9 (185 U/ml) (data not shown). The

optimum pH range between 9 and 10 for growth and

protease production is common among alkaliphilic and

haloalkaliphilic organisms [2,12,29–31].

3.2. Effect of nitrogen sources

In microorganisms, nitrogen (both organic and inorganic

forms) is metabolized to produce primarily amino acids,

Fig. 3. Effect of different organic nitrogen sources (O.5 %, w/v). SP, soya

peptone; TT, tryptone; CT, caseitone; GA, gelatin; CA, casamino acid; TP,

trader’s protein; PP, peptone; YE, yeast extract. After incubation of 66 h at

37 8C under shaking conditions at 100 rpm, the growth (&) and protease

activity ( ) were quantified.

nucleic acids, proteins and cell wall components. Alkaline

protease production heavily depends on the availability of

both carbon and nitrogen sources in the medium. Both have

regulatory effects on the enzyme synthesis [32,33].

The organic nitrogen sources used in our study supported

growth, while the maximum effect on growth was observed

with a combination of peptone and yeast extract (Fig. 3).

Among the organic nitrogen sources, protease production

ranged between 15 and 110 U/ml. However, optimum activity

(110 U/ml) was achieved with casamino acids followed by

gelatin, peptone, peptone plus yeast extract, yeast extract and

caseitone. Enzyme production was highly reduced in soya

peptone, trader’s protein and tryptone (Fig. 3).

Gelatin and casamino acid, the best nitrogen sources,

were further investigated at varied concentrations (0–2%, w/

v). Enzyme production steadily increased from 60 to 106 U/

ml in the range of 0–2% (w/v) gelatin, the optimum being at

broader range of 0.5–2%. However, there was a distinct

optimum concentration of gelatin at 1.5% (w/v) for growth

(Fig. 4). Enzyme production gradually increased with

increase in casamino acid concentrations from 0 to 2% (w/

v), with an optimum at 1.5–2%. Growth was also increased

up to 1.5% followed by suppression at higher concentrations

of casamino acids (Fig. 5). From these results, it could be

concluded that casamino acids proved to be the best organic

Fig. 5. Effect of caseamino acid concentration (0–2%, w/v) on growth (*)

and protease activity (~). Samples were withdrawn after incubation of 66 h

at 37 8C.

R. Patel et al. / Process Biochemistry 40 (2005) 3569–3575 3573

Fig. 6. Effect of different inorganic nitrogen sources (O.5 %, w/v) on

growth (&) and protease activity ( ). Samples were withdrawn after

incubation of 48 h at 37 8C.

Fig. 8. Effect of K2HPO4 concentration (0–2%, w/v) on growth (*) and

protease activity (~). Samples were withdrawn after incubation of 66 h at

37 8C.

nitrogen source which acted as inducer for enzyme

production. This has also been reported earlier in a marine

microorganism where the proteolytic activity was induced

by casamino acid [34]. In another case, Bacillus sp. RGR-14,

protease production was repressed in the presence of high

concentrations of casamino acid [35].

Inorganic nitrogen sources proved less favourable

towards both growth and enzyme secretion. The range of

enzyme production was between 1 and 34 U/ml, much

reduced as compared to the secretion levels (15–110 U/ml)

with organic nitrogen sources. Maximum growth and

enzyme production was observed in potassium nitrate

followed by ammonium chloride, whereas ammonium

sulphate and ammonium nitrate were not able to support

enzyme secretion beyond 5 U/ml (Fig. 6). These findings

supported the phenomenon of repression of the growth and

enzyme production by ammonium [22,30,36,37].

3.3. Repression study with glucose and inorganic

phosphate

Strong catabolic repression was observed with glucose.

Enzyme production was repressed from 120 to 4 U/ml with

increase in glucose concentration from 0.5–2 % (w/v).

Fig. 7. Effect of glucose concentration (0–2 %, w/v) on growth (*) and pro-

tease activity (~). Samples were withdrawn after incubation of 66 h at 37 8C.

Growth was, however, slightly increased up to 1.5% (w/v)

glucose (Fig. 7). A similar relationship is reported in the case

of alkaline protease from an alkaliphilic Bacillus sp. where

glucose repressed both growth and protease production [26].

Earlier, protease synthesis was demonstrated to be

modulated by an inducer catabolite repression system,

where glucose and ammonia repressed enzyme production

[38].

Enzyme production was gradually suppressed from 140

to 64 U/ml with increasing phosphate concentrations.

Growth was decreased by 50% at 0.5% (w/v) K2HP04

and remained almost at the same level up to 2% phosphate

concentration (Fig. 8). This effect is in contrast to the result

for alkaline protease production by Bacillus firmis in which

an increased supply of nitrogen and phosphorus stimulated

protease synthesis up to certain threshold levels [33]. This

contrasting effect may be due to the difference in the

production and secretion of the alkaline protease in

haloalkaliphilic bacteria as compared to alkaliphilic organ-

isms. So far, we have not come across with any particular

reference relating to protease repression in haloalkaliphilic

bacteria. With the isolates under study, maximum enzyme

production was achieved in the late exponential and early

stationary phases that correspond to secondary metabolism.

Therefore, enzyme production may not be supported by

phosphate, which is usually known to assist primary

metabolism.

3.4. Enzyme production on cheap sources

Cheaper sources of both carbon and nitrogen sources are

the key attraction for commercialization of the production

processes and thus, ability of the microbial agent to grow

and produce enzymes using these sources has been arguably

a point of interest [39,40]. Molasses, a byproduct of the

sugar cane industry, supported both, enzyme production and

growth maximum at 1% (v/v). Decreased levels of molasses

at less than 1% (v/v), however, were not as effective for

growth and enzyme production. At 0.25% molasses, there

R. Patel et al. / Process Biochemistry 40 (2005) 3569–35753574

Fig. 9. Effect of molasses concentration (0.25–1%, w/v) on growth (&) and

protease activity ( ). Samples were withdrawn after incubation of 48 h at

37 8C.

was a significantly reduced level of growth with no enzyme

secretion (Fig. 9). In experiments with wheat flour as the sole

source of carbon and nitrogen, the maximum growth and

enzyme production was at 1% (w/v). With decreasing

concentrations of wheat flour, both growth and enzyme

production was substantially reduced (Fig. 10). Enzyme

production, however, was sensitive to the concentration of

molasses and wheat flour indicating that a threshold level of

carbon and nitrogen is required for the optimum enzyme

production. Wheat flour was a more favoured candidate as

compared to molasses. In literature, there are few instances

of the usefulness of the cheap carbon and nitrogen for the

alkaline protease production. For example, an extracellular

alkaline protease from Bacillus horikoshii, was optimally

produced when grown in soybean meal (1.5%, w/v) and

casein (1%, w/v) [41]. This feature was also observed in

some earlier reports [30,31]. More recently, alkaline

protease production from Bacillus sp. was investigated in

solid-state fermentation using wheat bran and lentil husk as

carbon and nitrogen sources, where wheat bran was found to

be a better source [42].

The study on optimization of alkaline protease from

haloalkaliphilic bacteria has not been explored. Therefore, it

is significant to optimize the conditions for the production of

this enzyme using these organisms. The present study on

Fig. 10. Effect of wheat four concentration (0.25–1 %, w/v) on growth (&)

and protease activity ( ). Samples were withdrawn after incubation of 48 h

at 37 8C.

growth and protease production from haloalkaliphilic

bacteria clearly indicates the importances of these organisms

in the field of industrial enzyme production.

Acknowledgements

Financial Assistance from University Grants Commis-

sion (New Delhi, India) is acknowledged. The assistance

provided by Dr. B. K. C. Patel from Griffith University,

Brisbane (Australia) for the 16S rRNA gene sequencing is

also gratefully acknowledged.

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