Int.J.Curr.Microbiol.App.

Sci (2014) 3(6): 36-44

ISSN: 2319-7706 Volume 3 Number 6 (2014) pp. 36-44

http://www.ijcmas.com

Original Research Article

Optimization and Production of Alkaline Protease enzyme from
Bacillus subtilis 168 isolated from food industry waste
N.Vanitha1*, S. Rajan2 and A. G. Murugesan3
1
Research Scholar, Manonmaniam Sundaranar University, Tirunelveli, India
2
Research Department of Microbiology, M. R. Government Arts college, Mannargudi, India
3
SPKCES, Alwarkurichi, Manonmaniam Sundaranar University, Tirunelveli, India
*Corresponding author

ABSTRACT

The demand for alkaline proteases in industries is increasing worldwide. The

present study intended to isolate a suitable higher alkaline protease producing
bacteria and its identification, optimize conditions for alkaline protease production
Keywords
such as carbon sources, nitrogen sources, pH, temperature, inoculums size and
mass production of alkaline protease by submerged fermentation. In order to isolate
Bacillus the protease producing organisms, solid wastes from industries were collected and
subtilis, 16S primary screening was achieved by skim milk casein hydrolysis method. The
rRNA analysis, organism showing maximum (17 mm) hydrolysis was selected and identified by
alkaline microscopic, biochemical and 16S rRNA phylogenetic analysis as Bacillus subtilis
protease, 168. Purification of crude enzyme was carried out by ammonium sulphate
optimization, precipitation and dialysis. The apparent molecular weight of purified enzyme was
submerged determined as 55 kDa. The maximum alkaline protease production was achieved
fermentation. with 2% inoculums size at pH 13 and 35ºC temperature, maltose as best carbon
source, yeast extract as good nitrogen source, and using wheat bran as important
substrate. According to our knowledge, this study demonstrated the first report on
alkaline protease producing B. subtilis 168 isolated from food industry waste.
Purified enzyme can be used in textile, leather and food industries.

Introduction

Proteases, one among the three largest compared with animal, fungi and plant
groups of industrial enzymes, accounts for protease. Bacterial alkaline proteases are
about 60% of the total worldwide sale of characterized by their high activity at
enzymes from biological sources since alkaline pH. Optimal temperature required
they possess almost all characteristics for alkaline protease production is around
desired for their biotechnological 60°C. Due to these properties of bacterial
applications (Adinarayana et al., 2003). alkaline proteases make them suitable for
Among the various proteases, bacterial industrial applications. Bacillus species
protease was the most significant were specific producers of extracellular

36
 Int.J.Curr.Microbiol.App.Sci (2014) 3(6): 36-44

protease under extremes of conditions culture conditions promoting cell growth.

using simple carbon sources (Gupta et al., In the industrial production of alkaline
2002). proteases, technical media were usually
employed that contained very high
Alkaline proteases of microbial origin concentrations (100 150 g dry
posses considerable industrial potential weight/liter) of complex carbohydrates,
due to their biochemical diversity and proteins, and other media components
wide applications in detergents, leather (Aunstrup, 1980). With a view to develop
processing, silver recovery, medical an economically feasible technology,
purposes, food processing, feeds, and research efforts are mainly focused on to
chemical industries, as well as tannery improve the yields of alkaline proteases
waste treatment (Vijay et al., 2010). and to optimize the fermentation medium
and production conditions. The present
Production of alkaline protease was study was aimed to evaluate the usage
carried out generally using submerged potency of Bacillus sp., and to optimize
fermentation. It is advantageous than other various parameters for alkaline protease
methods due to its consistent enzyme production under submerged fermentation.
production with defined medium, better
process conditions and improved Materials and Methods
downstream processing (Prakasham et al.,
2006). Microbial proteases producing Collection of samples
industries are always in search of new and
cheaper methods to enhance the protease Three different solid samples (leather,
production as well as to decrease the food industrial waste and slaughter house
market price of this enzyme (Mukherjee et waste) were collected in sterile container
al., 2008). The use of cost effective growth according to microbiological procedures
medium for the production of alkaline and shifted to the laboratory for further
proteases from an alkalophilic analysis.
microorganisms is especially important,
because these enzyme account for Screening of protease producers
approximately 25% of the world wide
enzyme consumption (Gessesse, 1997). The collected samples were serially
diluted and streaked on skin milk agar
The growth and enzyme production of the plates. The plates were incubated for 48h
organism are strongly influenced by at 37 C and protease producers were
medium components like carbon and selected by observation of zone of
nitrogen sources. Besides the nutritional hydrolysis around the colonies (Genkal et
factors the cultural parameters is the al., 2006).
primary task in a biological process. So,
the media components and cultural Identification of isolates
conditions are need be optimized. It is
essential that these organisms be provided All the screened organisms were identified
with optimal growth conditions to increase based on morphology, cultural and
enzyme production. The culture conditions biochemical characteristics (Koneman et
that promote protease production were al., 1994). The higher yielding strain was
found to be significantly different from the identified by making use of 16S rRNA

37
 Int.J.Curr.Microbiol.App.Sci (2014) 3(6): 36-44

sequencing using forward (5 determined. One unit of protease activity

AGAGTTTGATCCTGGCTCAG-3 ) and was defined as the amount of enzyme
reverse (5 TACCTTGTTACGACTT 3 ) required to liberate 1 g/ml tyrosine under
primers. The sequence similarity search the experimental conditions.
was done for the 16S rRNA sequence
using online search tool called BLAST Enzyme activity = OD value X amount of
(http://www.ncbi.nlm.nih.gov/blast). The protein released ( g)/ concentration of
unknown organism was identified using substrate X time of incubation X weight of
the maximum aligned sequence through the sample
BLAST search (Krishnan et al., 2012).
Optimization of conditions for enhanced
Characterization of protease enzyme enzyme production (Das and Prasad,
2010)
The total protein contents of the samples
were determined according to the method Standard methods were adopted to
described by Lowry s method using optimize the parameters like cultural
Bovine Serum Albumin (BSA) as conditions, carbon, nitrogen, temperature,
standard. Enzyme activity was determined pH, inoculum size and substrates.
using culture supernatant collected by
centrifuging culture broth at 10, 000 rpm Mass production of alkaline protease
for 15min. Protease activity was measured
by standard assay procedure proposed by The fermentation was carried out in a
Akcan and Uyar, 2011. About 0.5ml of sterile Stirred Bed Reactor (SBR). The
0.5% casein and 1.25ml of tris buffer (pH- vessel was maintained at optimized
8.0 to 14.0) was added into 0.2ml of each temperature, pH and other & incubated for
of the culture supernatant separately. 48h in a shaking incubator. At the end of
Mixture was incubated for 30 min at 370C. fermentation period, the whole culture
About 3ml of trichloroacetic acid was both was centrifuged at 10,000 rpm for 15
added and incubated at 400C for 10 min to minutes, to remove the cellular debris and
form precipitate. The mixture was the clear supernatant was used for enzyme
centrifuged at 10,000rpm for 15min and analysis.
0.5ml of supernatant was collected.
Characterization of partially purified
Reagent containing sodium carbonate, alkaline protease
copper sulphate, sodium potassium
tartarate was mixed with 1ml of Folin- The culture filtrate (crude protease) was
phenol reagent. The mixture was collected aseptically after upstream
incubated at dark for 30 minutes to form production in a SBR under controlled
blue colour. The absorbance was read at conditions. The required volume of the
660 nm to determine the optical density of spent media was centrifuged at 10,000 rpm
each sample. The obtained OD was for 15 min at 4ºC in order to obtain a cell
extrapolated in the standard graph. The free filtrate. About 200 ml of the cell free
standard curve was obtained for series of filtrate containing protease were collected
known concentrations of bovine serum and their proteolytic activity was
albumin. From the graph, the amount of determined. Protease enzyme was purified
protein liberated due to the action of by ammonium sulfate fractionation the
enzyme protease in the supernatant was concentration of ammonium sulphate
38
 Int.J.Curr.Microbiol.App.Sci (2014) 3(6): 36-44

required for precipitation varies from showed maximum of 17 mm diameter of

protein to protein and should be zone of hydrolysis.
determined empirically. The two milliliter
of the crude protease enzyme was first The zone of hydrolysis was due to
brought to 20% (w/v) saturation with solid protease enzyme produced by the isolates
ammonium sulfate (enzyme grade) and on Skim milk agar media. Narendra et al.,
100% saturated dialysis against distilled (2012) reported that about 25 organisms
water in a dialysis bag (cut off 30) for 3 h, were recovered from different fields near
followed by dialysis against phosphate to Ravulapalem village, East Godavari
buffer at pH 7.0. The obtained protease district, Andhra Pradesh, India. Five
enzyme preparation was concentrated isolates were considered as protease
against crystals of sucrose and kept in the positive strain. These researchers
refrigerator at 4ºC. The enzyme activity explained that, Indian soil is best for
and protein content was determined for alkaline protease producing bacteria. The
salted out dialyzed enzyme fractions. The isolated proteolytic strain (PD4) was
enzyme activity of the purified fractions of spore-forming, gram-positive rod, which
the alkaline protease after harvesting, was identified as Bacillus sp.
ammonium sulfate precipitation and Siddalingeshwara et al., (2010) similarly
dialysis was determined by the method of isolated fifty-three bacterial isolates from
Gomori (1955). Separation and size various fields of cosmopolitan city of
determination of enzyme was performed Karachi, out of which 25 were alkaline
by SDS-PAGE (Joo et al., 2002). protease producers.

Results and Discussion Alkaline protease activity of the

protease producing isolates
Alkaline proteases has considerable
industrial potential in detergents, leather After isolating five different bacterial
processing, silver recovery, medical strains, each isolates was screened for their
purposes, food processing, feeds and enzymatic activity at different alkaline pH
chemical industries, as well as tannery conditions (pH 8.0 to 14.0). During this
waste treatment . At present, the largest screening step, PD4 exhibited more
part of the hydrolytic enzyme market is enzymatic activity in all pH conditions
occupied by the alkali proteases. Extreme when compared to all other isolates. The
environments are important sources for maximum alkaline proteolytic activity was
isolation of microorganisms for novel exhibited at pH 13.0 (170.32 ± 1.5IU/ml).
industries and enzymes production. The difference in enzymatic activity at
different pH among the five different
Hence, in this present study the protease isolates was presented in Figure 1.
producing bacteria were isolated from
tannery, food industry; and slaughter Optimization of conditions for enhanced
house industrial effluent discharge site. enzyme production
Five different protease producers were
selected based on zone of hydrolysis and The enzyme production was optimized
named as PD 1, PD 2, PD 3, PD 4 under different parameters like growth in
and PD 5. Among the five isolates, the different carbon sources, nitrogen sources,
isolate PD - 4 isolated from food industry pH, temperature, and substrates. All the

39
 Int.J.Curr.Microbiol.App.Sci (2014) 3(6): 36-44

parameters were analyzed using PD- 4 activity of 585.5 ± 3.3 IU /ml. In the
strain to determine the enzymatic activity present study, wheat bran was found to be
of protease (Table. 1). the best inducer of protease enzyme
production.
Of various carbon sources used, maltose
exhibited maximum enzyme activity of Since the organism has industrial
about 493.5 ± 5.0IU/ml. Previous works potentiality it was identified by 16S rRNA
substantiates that depending on the species method, which is found to be the novel
and source of the organism carbon source and accurate method for identifying
requirement varies. Isolated organism unknown species. The DNA from the
prefers to utilize the maltose as the carbon isolate PD4 was isolated and the 16s
source. Next to the carbon, nitrogen was rRNA was amplified and sequenced. The
served as important nutrient source for the BLAST analysis of PD4 using its 16S
protease production. Of the various rRNA sequence data showed that strain
nitrogen sources used; yeast extract had highest homology (100%) with
exhibited the highest enzyme activity of Bacillus subtilis 168. The sequence has
about 696.3 ± 4.4IU / ml for PD-4. Atalo been submitted to the Genbank
and Gashe (1993) showed that yeast (KJ668820).
extract and peptone can induce the
alkaline protease production in glucose Bulk production of B. subtilis (PD4) was
medium. PD4 strain exhibited best carried out under controlled cultural
production of protease was at 35 C with conditions in a stirred bed reactor. At the
the yield of 696.3±4.41 IU/ml. two end of each fermentation period, the
percentage inoculum yielded good enzyme culture broth was harvested to remove the
productivity. cellular debris and the clear supernatant
was used for characterization of enzyme.
Irfan et al., (2009) found that the optimum Harvested media containing the crude
inoculum size of 2% showed the protease was partially purified by
maximum activity. However a further ammonium sulfate precipitation and
increase in inoculum size decreased dialysis. The supernatant collected after
enzyme production by B. megaterium. centrifugation of harvest media was
During the recent years, efforts have been precipitated with 100% saturated
directed to explore the means to reduce the ammonium sulfate. Very sharp needle-
protease production cost through shaped precipitates were obtained during
improving the yield, and the use of either the incubation of crude enzyme and
cost free or low cost feed stocks or ammonium salt mixtures (Plate- 2). In the
agricultural by products as substrate like present study, the enzyme alkaline
green gram husk which are highly protease from Bacillus subtilis was
involved in the protease enzyme purified using ammonium sulphate
production (Prakasham et al., 2006). The precipitation method. The needle shaped
effect of agro based by products as protein precipitate exhibited 55kDa in
alternative substrate on bacterial protease size. The molecular weight of the protease
production under submerged fermentation reported by Sousa et al., 2007 that the
was studied using five different substrates purified enzyme migrated as a single band
(Rice bran, wheat bran, sugarcane with an apparent molecular weight of 46
molasses, sago waste and Black gram bral) kDa in SDS-PAGE.
wheat Bram exhibited maximum enzyme
40
 Int.J.Curr.Microbiol.App.Sci (2014) 3(6): 36-44

Figure.1 Alkaline protease activity of the isolates at various pH

180

160

140

120
PD1
Enzym e activity IU /m l

100 PD2

80 PD3

60 PD4

PD5
40

20

0
8 9 10 11 12 13 14
pH

Table.1 Optimization of conditions for alkaline protease production using B. subtilis 168
Parameters Enzyme activity (IU/ml))
Carbon sources Glucose 344.7± 1.5
Sucrose 182.2± 6.0
Maltose 493.5± 5.0
Lactose 211.7± 2.1
Fructose 126.0±4.0
Nitrogen sources Peptone 495.6 ± 4.7
Yeast extract 696.3 ± 4.4
Ammonium sulphate 195.2 ± 2.5
Ammonium chloride 354.2 ± 3.5
Beef extract 208.3 ± 5.0
Temperature 300 275.5 ± 3.9
350 442.6 ± 7.4
400 174.3 ± 6.0
450 75.4 ± 4.7
500 137.0 ± 8.4
Inoculum size (%) 2 464.9 ± 1.5
4 238.6 ± 1.7
6 163.8 ± 4.5
8 50.9 ± 1.5
10 20.8 ± 1.0
Substrates Rice bran 11.9 ± 1.8
Wheat bran 585.5 ± 3.3
Sugarcane molasses 208.2 ± 1.3
Sago waste 190.8 ± 2.9
Black gram bran 460.7 ± 1.6

41
 Int.J.Curr.Microbiol.App.Sci (2014) 3(6): 36-44

Figure.3 Needle shaped enzyme precipitate

A B C D

79KDa

66KDa

43KDa

29KDa

16KDa

Figure.4 SDS PAGE pattern of Protease enzyme

These results were in accordance with Bacillus sp. KSM-K16 has been reported
literature reports, where most of the to be retaining 70% of its original activity
molecular mass of protease from Bacillus after incubation for three weeks in a
genusis was less than 50 kDa. The commercial heavy-duty liquid detergent
alkaline proteases of some bacteria such as with pH 9.6 at 40°C (Kwon et al., 1994).
Bacillus subtilis RM 615, Bacillus sp. Y, The study therefore concludes that because
Bacillus sp. KSM-K16, Vibrio of the higher enzyme stability under
metschnikovii RH530 and various conditions and production at lower
Fiennoac6nomyces sp. HS 682 have been cost can be commercialized for industrial
reported to be stable towards detergents to applications
some extent. Of these the M-protease of

42
 Int.J.Curr.Microbiol.App.Sci (2014) 3(6): 36-44

References Gomori, G. 1955. Preparation of buffers

for use in enzyme active studies, in
Adinarayana, K, Ellaiah, P, Prasad, D, methods in Enzymology, Vol.I. Eds.
2003. Purification and partial (Colwick, S.P. and Kaplan, N.O)
characterization of thermo stable Gupta, R., Beg, Q.K. and Lovenz, P. 2002.
serine alkaline protease from a newly Bacterial alkaline Proteases.
isolated Bacillus subtilis PE-II, AAPS Molecular approaches and industrial
Pharma Sci Tech, 4: 440 448. applications, Applied Microbiology
Akcan, N and Uyar, F., 2011. Production and Biotechnol. 59 : 15 32
of extracellular alkaline protease from Irfan, M., Gulsher. M., Nadeem, M., Syed.
Bacillus subtilis RSKK96 with solid Q and Baig. S., 2009. Evaluation of
state fermentation, Eur. Asian Journal Cultural conditions for thermostable
of BioSciences, 5, 64-72. -Amylase production by Bacillus sp.
Atalo, K and Gashe, B. A., 1993. Protease Pak. J. Biochem. Mol. Biol, 42:43-48.
production by a thermophilic Bacillus Joo. H.S., Kumar, C.G. Park, G.C., Kim,
species (P-001A) which degrades K.M., Paik, S.R. and Chang, C.S.
various kinds of fibrous proteins. 2002. optimization of the production
Biotechnol, Lett, 11, 1151-1156. of an extracellular alkaline protease
Aunstrup, K., 1980. Proteinases, In: Rose from Bacillus Horikoshi. Process
AH (Ed) Microbial enzymes and Biochemistry 38: 155 159.
bioconversions, Academic Press, Koneman, E.W., William, M. J., Stephen,
New York, 50 114. D. A., Schreeken, B., Washington,
Darani, K., Falahatpishe. H.R., and Jalai. C.W. 1994. Laboratory and clinical
M. 2008. Alkaline protease diagnosis of infections diseases.
production on date waste by an In.Introduction to diagnostic
alkalophillic Bacillus sp. 2-5 isolated Microbiology. J.B. Lippincott
from soil. African J. Biotechnol., Company, 1 Philadelphia: 1 -19.
7:1536-1542. Krishnan, R. Muthusamy.A and
Das, G and Prasad, M.P. 2010. Isolation, Saravanan, K. 2012. Isolation of
Purification and Mass production of alkaline protease from Bacillus
protease enzyme from Bacillus subtilis AKRS3, African J. Biotech,
subtilis. International Research 11(69) : 13415 13427.
Journals of Microbiology 1(2): 026 Kwon, Y, T., Kim, J, O., Moon, S, Y.,
031 Lee, H, H., and Rho, H, M, 1994.
Genkal, H., and Tari. C., 2006. Alkaline Extracellular alkaline proteases from
Protease production from alkalophilic alkalophilic LG brio rnetschnikovii
Bacillus sp., isolated from natural strain RH 530, Biotechnol, Lett., 16,
habitats. Enzyme and Microbial 413 - 418.
Technology 39: 703 710. Lowry, O.H., N.J. Rosebrough, A.L. Farr
Gessesse, A, 1997. The use of nugmeal as and R.L. Randall, 1951. Protein
low cost substrate for the measurement with the folin Phenol
production of alkaline protease by the reagent. J. Biological Chemistry, 193:
alkalophilic Bacillus sp. AR 009 265 273.
and same properties of the enzyme, Mukherjee, P.K. Das, P and Rao, P.S.S.
Bioresource Technology, 62, 59 61. 2008. Time trends in MB-PB ratio
among untreated leprosy patients

43
 Int.J.Curr.Microbiol.App.Sci (2014) 3(6): 36-44

attending a referral hospital in UP,

India during 2001 to 2010. Indian J
Lepr, 85, 59 64.
Narendra, G., Ramalakshmi, M., Roja R.,
Archana, B and Maanasa. F, 2012.
Isolation and characterization of
protease producing bacterial from soil
And estimation of protease by
spectrophotometer, The experiment,1
(1) , 1-7.
Prakasham, R, S., Rao, C, S and Sarma, P,
N, 2006. Green gram husk - An
inexpensive substrate for alkaline
protease production by Bacillus sp. in
solid-state fermentation, Biosensors.
Technol, 97(1), 1449-1454.
Siddalingeshwara, K., Uday, J., Huchesh,
C., Puttaraju, H, P., Karthic, J.,
Sudipta, M., Pramod, T and
Vishwanatha, T., 2010. Screening
and characterization of protease from
Bacillus sp, International Journal of
applied biology and pharmaceutical
technology, 1(2): 575-576.
Sousa. F., Ju. S, Erbel, A., Kokol. V.,
Paulo. C.A and Gubitz, G, M. 2007.
A novel metallo protease from
Bacillus cereus for protein fibre
processing. Enzyme Microb.
Technol. 40(1):1772 1781.
Vijay, K, M., Jesse, D., Aitken, F.,
Carvalho, A., Tyler, C, C., Simon,
M., Shanthi, S., Shanthi, V, S., Rob
K., Ruth, E, L and Andrew, T. G.,
2010. Metabolic Syndrome and
Altered Gut Microbiota in Mice
Lacking Toll-Like Receptor, Science,
5:228-328.

44