Production and purification of a protease from an alkalophilic Bacillus sp. 2-5 strain isolated from soil

Document Type : Brief Report


1 Department of Food Technology Research, National Nutrition and Food Technology Research Institute, Shahid Beheshti University, M.C., P.O. Box 19395-4147, Tehran, I.R. Iran

2 Department of Nutrition and Biochemistry, School of Public Health, Tehran University of Medical Sciences, I.R. Iran


This research has focused on isolation and characterization of a strain of Bacillus sp. from alkaline soil, which was able to produce extracellular alkaline protease at pHs ranging from 8 to 11 and temperatures of 20 to 50ºC. Also the impact of different carbon and nitrogen sources were investigated. The yield and fold of enzyme purification was 24% and 50 times, respectively. Molecular weight of purified enzyme was measured by SDS-PAGE as 24.7 kDa. The alkaline protease produced by Bacillus sp. 2 - 5 showed the most caseinolytic activity (without any gelatinolytic activity) at pH>10


Proteases constitute one of the most important groups
of industrial enzymes and have applications in different
industries such as detergent, food, feed, pharmaceutical, leather, silk and for recovery of silver from used Xray films (Anisworth, 1994; Fujiwara, 1993). This
enzyme accounts for 30% of the total world enzyme
production (Horikoshi, 1996). Among bacteria,
Bacillus spp. are specific producers of extracellular
alkaline proteases (Godfrey and Reichelt, 1985). These
enzymes are quite often added to laundry detergents (to
facilitate the release of proteinaceous stains) (Masse
and Tilburg, 1983) and in detergent preparations used
in the dairy and food industries (to remove protein
foulants from ultrafiltration and reverse osmosis membrane systems). Given the wide application of this
enzyme, it was reported that in the year 2005, the global proteolytic enzyme demand increased dramatically
to 1.0-1.2 billion dollars (Godfrey and Reichelt, 1985).
Therefore, taking this demand into account and knowing the geographic richness and biodiversity of the
Iranian local environment, it is assumed that there is
potential for alkalophilic Bacillus species living in
these environments. In this paper isolation and characterization of a new strain of Bacillus sp. from alkaline
soil, and its ability to produce alkaline protease at pHs
ranging from 8 to 11 and temperature of 20 to 50ºC
have been reported. Also, purification and certain properties of the alkaline protease as well as the effect of
process variables such as carbon and nitrogen sources,
temperature, pH and time on alkaline protease activity
have been investigated.
Alkaline salty soil sample with a pH 10 was collected (surrounding regions of Yazd and Tehran, Iran), suspended in sterile saline water (100 g/l) and incubated
at 80°C for 20 minutes (Hitomi, et al., 1994). After
cooling, it was spread on specialized culture media
containing (g/l): glucose 11.1, peptone 5.5, yeast
extract 5.5, K2HPO4 11.1, MgSO4.7H2O 0.22, and
agar 16.6. The plates were incubated at 37°C for 24 h.
Pure colonies were transferred to a new medium containing (g/l): peptone 5, beef extract 3 (or yeast extract
1), agar 15 to prepare stock culture. By comparing the
ability of microorganisms to hydrolyze gelatin, casein
and starch at two different pHs (7 and 10), an alkaline
protease producer was selected for further experimental studies. A loopful of the prepared stock culture was

trifuged at 4°C. After resuspension of precipitated
phase in phosphate buffer, it was dialyzed under vacuum (Cut off<10 kDa). The concentrated enzyme was
applied to carboxy methyl cellulose (CMC) column
(2.5 × 30 cm). The bound enzyme was eluted using a
linear gradient of KCl (0.5 M). Fractions containing
alkaline protease were pooled (80 ml, 40 tubes) and
redialyzed. 1 ml of purified and concentrated enzyme
was analyzed for alkaline protease activity, specific
activity and protein content (Bollag and Edelstein,
Casamino acids (878 APU/ml, peptone (1022
APU/ml), yeast extract (1264 APU/ml), L-glutamate
(1118 APU/ml) and urea (684 APU/ml) individually
reduced protease activity (Table 1). These results are
similar to those reported by Joo et al. (2002) who
observed decreased protease activity of Bacillus sp. I-
312 after growth on peptone. Casein addition (1 g/l)
had a significant effect on biomass production and
enzyme activity, although it was still less than the
effect of mixed nitrogen sources. This observation is in
agreement with the results of reduced alkaline protease
activities of Bacillus horikoshii, Bacillus licheniformis
MIR29, Bacillus mojavensis and B. horikoshii 104 in
the presence of casein (Beg and Gupta, 2003; Joo et
al., 2002). But it was somewhat different from the
Bacillus spp. I-312 (Glazer and Nikaido, 1995). It
should be mentioned that application of synthetic and
unpurified nitrogen sources influence yield not only as
nitrogen sources but also as sources for excess carbon
during protease production. Data in Table 2 shown that
the highest protease production was achieved by addition of starch at a concentration of 5 g/l. Higher concentrations did not affect protease production, significantly. Similar results have been reported with respect
to the influence of corn, potato starch and wheat flour
as carbon sources on protease production by Bacillus
sp. I-312 (Glazer and Nikaido, 1995). It seems that the
“catabolite repression” mechanism, is the best possible
explanation for the reduce of protease production in
the presence of glucose (Glazer and Nikaido, 1995),
therefore, it is preferable to use complex carbon
sources. Glucose (1 g/l), sodium citrate and sodium
acetate (1 g/l) decreased protease production yield by
34%, 43% and 20%, respectively. Addition of glucose
(1 g/l) to basal media reduced alkaline protease production by B. horikoshii to 45% (Joo et al., 2002).
Gessesse and colleagues (2003) also reported that protease production in Bacillus pseudofirmus AL-89
increased in the presence of glucose, whereas in
Nesternkonia sp. AL-20 was suppressed. Table 3
shows the effect of purification steps on specific activ

ity, purification fold and percent of purified alkaline
protease recovery. Final purification yield and fold
was obtained as 24% and 50 times, respectively (Table
In this study, the alkalophilic Bacillus sp. 2-5 strain
showed higher protease production at pH 10.
Therefore, this isolate can be a potential source of
alkaline protease for use as an additive in industrial
applications. The objective of the future study is to
find the optimal conditions for Ca-alginate gel immobilization of the new isolated bacterium and to determine the operational stability of the resulting biocatalyst for the production of alkaline protease under semicontinuous cultivation conditions

Anisworth SJ (1994). Soap and detergents. Chem Eng News.
Beg QK, Gupta R (2003). Purification and characterization of an
oxidation stable thiol-dependent serine alkaline protease from
Bacillus mojavenesis. Enzyme Microbial Technol., 32, 294-
Bollag DM, Edelstein SJ (1991). Protein Methods. John Wiley and
Sons, New York, PP. 71-160.
Fujiwara N (1993). Production of thermopilic alkaline protease
from Bacillus sp. B18. J Biotechnol. 30, 245-256.
Gessesse A, Kabul RH, Gashe BA, Mattiasson B (2003). Novel
alkaline protease from alkalophilic bacteria grown on chicken
feather. Enz Microb Tech. 32, 519-524.
Glazer AG, Nikaido H (1995). Microbial Biotechnology In:
Fundamentals of Applied Microbiology, Freeman and
Company, Washington, PP. 256-259.
Godfrey TA, Reichelt J (1985). Industrial enzymology: the application of enzymes in industry. The Nature Press. London.
Hitomi J, Adachi S, Hakamada Y, Takaiawa M, Yoshimatsu T,
Watanabe Y, Kobayashi T, Kawai S, Ito S (1994). Alkaline
protease isolation from Bacillus sp. US patent No.
Horikoshi K (1996). Alkalophils from an industrial point of view.
FEMS Microbial Rev. 18: 259-270.
Joo HS, Kuma CG, Park CG, Paik SR, Chang CS (2002).
Optimization of the production of an extracellular alkaline
protease from Bacillus horikoshii. Process Biochem. 38: 155-
Kalisz HK (1988). Microbial proteinases. Adv Biochem Eng
Biotechnol. 36: 1-65.
Kobayashi T, Hakamada Y, Hitomi J (1996). Purification of alkaline proteases from Bacillus strain and their possible interrelationship. Appl Microbiol Biotechnol. 45: 63-71.
Lowry OH, Rosenberg NI. Farr AL. Randall RJ (1951). Protein
measurement by folin phenol reagent. J Biol Chem. 193: 265-
Masse FWJL, Tilburg RV (1983). The benefit of detergent
enzymes under changing washing conditions. J Am Oil Chem
Soc. 60:1672-1675.
Uyar F, Baysal Z (2004). Production and optimization of process
parameters for alkaline protease production by a newly isolated Bacillus sp. under solid state fermentation. Process
Biochem. 39:1893-1898