studies on alkaline-thermostable protease from an...
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INTERNATIONAL JOURNAL OF ENVIRONEMNTAL SCIENCES
Volume 5, No 2, 2014
© Copyright by the authors - Licensee IPA- Under Creative Commons license 3.0
Research article ISSN 0976 – 4399
Received on August 2014 Published on September 2014 353
Studies on Alkaline-thermostable protease from an alkalophilic bacterium:
Production, characterization and applications Aditya Bharadwaj1, Neena Puri2, Prakram Singh Chauhan1, Balneek Singh Cheema1, Naveen
Gupta1
1- Department of Microbiology, South Campus Panjab University, Sec-25, Chandigarh, India.
2- Department of Industrial Microbiology, Guru Nanak Khalsa College, Yamunanagar,
Haryana, India.
doi: 10.6088/ijes.2014050100031
ABSTRACT
Alkalophilic organisms producing alkaline thermostable protease(s) were isolated from the
industry waste, corpses and soil. Out of them Isolate no. NB-34 was found to produce
maximum alkaline thermostable protease. Optimization studies on protease production from
isolate NB-34 revealed that protease production was maximum : With 0.5% gelatin as
nitrogen source, pH 11.2 (2% sodium carbonate, 0.5% glucose, 24 hours incubation period,
37⁰C incubation temperature, 150 rpm agitation. The alkaline thermostable protease from
isolate NB-34 had pH and temperature optima of 9.5 and 50⁰C respectively. The Km value for
the enzyme was calculated to be 0.909 mg casein/ml and Vmax 5.55. The alkaline
thermostable protease from isolate NB-34 was found to be compatible with most of the
commercial detergents. When the alkaline thermos table protease from isolate NB-34 was
supplemented with the commercial detergents, it improved the destaining capacity of the
detergent.
Keys word: Optimization, thermostable protease, alkaline stable mannanase, detergent
industry.
1 Introduction
Proteases are probably the most important class of industrial enzymes worldwide, accounting
for nearly 60% of total enzyme sales. The two-third of proteases produced commercially are
by microorganism (Kalisz, 1988; Khan, 2013; Rani et al., 2012). Protease find their
application in a wide range of industrial process viz; detergent, brewing, baking,
pharmaceuticals, leather tanning, meat tenderization, peptide synthesis and medical diagnosis
(Dayanandan et al., 2003; Cowan et al., 1985; Kalisz, 1988; 1985; Thomas et al., 2007;
Chauhan et al., 2012; Chauhan et al., 2014a).
Though plants and animals also produce extracellular protease, microorganism are preferred
source of protease because of their rapid growth, limited space required for their cultivation,
longer shelf life, the ease with they can be genetically manipulated to generate improved
enzymes (George et al., 2014a; George et al., 2014b; Chauhan et al., 2014b; Chauhan et al.,
2014c; Chauhan et al., 2014d; Chauhan et al., 2014e; Kumar et al., 2014). Microorganisms
elaborate a large array of proteases that are intracellular and/or extracellular. Intracellular
proteases are important for different metabolic function like sporulation and differentiation,
protein turnover, maturation of enzymes and hormones and maintenance of cellular protein
pool whereas extracellular proteases help in hydrolysis of protein in the cell free environment
and their cellular uptake (Kaliz 1988). The hydrolytic property of extracellular proteases has
Studies on Alkaline-thermostable protease from an alkalophilic bacterium: Production, characterization and
applications
Aditya Bharadwaj et al.,
International Journal of Environmental Sciences Volume 5 No.2, 2014 354
been commercially exploited in various industrial processes (Ramasami et al., 1999;
Saravanabhavan et al., 2005; Singh et al., 2011; Yadav et al., 2013; George et al., 2014c;
Sondhi et al., 2014). lthough alkaline proteases are produced by bacteria, fungi,
actinomycetes and yeast yet bacteria are the most dominant group of alakaline protease
producers with the genus Bacillus being the most predominant source followed by
Pseudomonas (Rao et al., 1988; Gupta et al., 2002; Thomas et al., 2007). Beside these
Flavobacterium and Arthrobacter are also known to produce alkaline serine protease. In
fungi Aspergillus is the most exploited group (Chakarbarti et al., 2000) while Condiobacillus
sp. and Rhizopus sp. also known to produce alkaline protease producer (Poza et al., 2001)
whereas strains of Streptomyces are the preferred source among actinomycetes.
The first alkaline protease from Bacillus licheniformis named subtilisin was developed in
Denmark in 1960 and named BIOTEX. After this a number of commercial alkaline proteases
belonging to Bacillus , Pseudomonas etc. have been reported such as subtilisin Carlsberg ,
subtilisin BPL and savinase , with their application as detergent enzymes. Though a number
of alkaline proteases for their potential application has been characterized and patented, the
industry is still in search of efficient alkaline protease with respect to their temperature and
pH optima (Gupta et al 2002). Thus, there is a need to isolate and characterize bacterial
strains with increasing potential of producing alkaline proteases.
2. Material and methods
2.1 Isolation of the organisms
Soil samples were collected from milk industry waste, corpses and soil. 1 gram of the sample
was suspended in 100 ml of sterile distilled water and suitable dilutions were plated on the
Horikoshi Medium for the isolation of alkalophiles.
2.2 Screening for the production of protease
The isolates were grown in 1% casein incorporated Horikoshi Medium and incubated at 37°C
for 24 hrs. The protease production was observed by visualization of clear halo zones around
the colonies after the plates flooded with 1N HCl.
2.3 Protease production in liquid medium
100ml broth of Horikoshi Medium Ph 10 was taken in a 250 ml flask. It was inoculated with
0.1% inoculums of log phase grown cells of alkalophilic isolate NB34 and incubated in
shaker (150rpm) at 37°C for 24hrs. The culture was centrifuged at 10,000rpm for 10 mins
Protease activity was assayed in the cell free supernatant (standard curve for protease
appendix 1)
2.4 Optimization of parameters for alkaline protease production
The effect of various physico- chemical cultural parameters i.e. carbon source , nitrogen
source , temperature , pH and agitation rate was studied for optimum protease production in
selected proteolytic bacteria.
2.5 Effect of different nitrogen source
Studies on Alkaline-thermostable protease from an alkalophilic bacterium: Production, characterization and
applications
Aditya Bharadwaj et al.,
International Journal of Environmental Sciences Volume 5 No.2, 2014 355
The alkaline protease production was studied by using different nitrogen source (peptone ,
soya, bean, casein and gelatin ) at the concentration of 0.5%.
2.6 Different concentration of Na₂₂₂₂CO₃₃₃₃ which corresponds to pH change
Alkaline protease production was studied by preparing HK medium having different
concentrations of Na₂CO₃ 0.25% (pH7.0) 0.5% (pH9.3), 1.0% (pH10.2), 1.5% (pH 10.8),
2.0% (pH11.2), 3.0% (pH11.8) and inoculating with 1% inoculoums of log phase grown
culture NB34. The inoculated flasks were incubated under shake flask conditions and
samples were drawn after 24hrs for protease assay.
2.7 Effect of different carbon source
The alkaline protease production was studied by using different carbon source (glucose,
fructose, maltose and starch) at the concentration of 0.5%.
2.8 Effect of incubation time period
The effect of time period was studied by incubating the inoculated HK medium flasks for 12h,
24h, 48h, 72h.
2.9 Effect of incubation temperature
The effect of temperature was studied by incubating the inoculated HK medium flasks 30°C,
37°C and 40°C.
2.10 Effect of agricultural by-products
Three agricultural by products viz, wheat bran, rice bran and deoiled cotton meal at the
concentration of 0.5% were supplemented in the HK medium . the effect of agricultural by
products was tested without gelatin and with 0.5%
2.11 Characterization of alkaline protease
The alkaline protease of the isolateNB34 was produced under optimum conditions. This
enzyme used to study the effect of various parameters on enzyme activity.
2.12 Hydrogen ion concentration
The optimal pH for enzyme activity was determined by preparing the substrates (0.5%
casein) in the buffers of different pH range 7 -11 and performing the assay under standard
conditions. The pH stability of protease was determined by diluting the enzyme in buffers of
pH 7.5-9.5 and then measuring the residual activity after different time intervals (15-240 min).
buffers used were phosphate buffer pH 7-8.5, Tris –HCl buffer pH 8.4 and carbonate –
bicarbonate pH 8.5-9.5.
2.13 Temperature
The optimal temperature for enzyme activity was determined by incubating the assay mixture
in the temperature range 40-70°C for 2 min. The thermal stability of the protease was
Studies on Alkaline-thermostable protease from an alkalophilic bacterium: Production, characterization and
applications
Aditya Bharadwaj et al.,
International Journal of Environmental Sciences Volume 5 No.2, 2014 356
determined in the temperature range 50-70°C by assaying the residual protease activity. The
enzyme was incubated at the respected temperature, aliquots were withdrawn sequentially
and residual protease activity was measured under standard conditions (pH9.5,
temperature50°C)
2.14 Effect of substate concentration
The concentrations of casein 0.1%, 0.2%,0.5%,0.75%,1.0%,1.25%,1.50% (1 to 15 mg/ml)
were varied to study the effect of substrate concentration on a alkaline protease activity
2.15 Effect of enzyme concentration
The effect of using different quantities of enzyme. the total volume of the enzyme mixture
was set 1.0 by adding distilled water.
2.16 Effect of chelating agents
Inhibitors were added in the assay mixture at 1, 10 and 100mM concentration and the
protease activity was assayed under standard assay conditions.
2.17 Effect of oxidizing and reducing agent
Oxidizing and reducing agents were added in the assay mixture at 1, 10 and 100 Mm
concentration and the protease activity was assayed under standard assay conditions.
2.18 Effect of metal ions
The effect of metal ions (Ba²ᶧ cu²ᶧ, Hg²ᶧ, Mn²ᶧ, ca²ᶧ, Zn²ᶧ and Co²ᶧ) on alkaline protease activity
was studied by incubating them in reaction mixtures at the concentration of 1Mm, 10Mm,
100Mm. Some of the ions are insoluble in distill water they were dissolved in their
corresponding buffer simultaneous control was also run which lack the ion.
2.19 Studies on compatibility of alkaline protease with detergents
5 detergents (surf excel, ariel. Tide, rin, fena were used for studying compatibility of alkaline
protease under buffered and unbuffered condition. Detergent solutions were prepared as per
directions given on their respective sache. Casein solution (0.5%) (used as substrate) was
prepared either in buffer (carbonate-bicarbonate,0.1M,pH-9.5) or in distilled water. Both
buffered and unbuffered solutions were used in reaction mixture comprising of 2ml of casein
solution, 0.9ml of detergent solution and 0.1 ml of crude extract alkaline protease activities
were measured as described above.
2.20 Stain removal activity of alkaline protease
The blood stain removal activity of alkaline protease was determined by dipping the blood
stained muslin cloths for10min in 100 ml of detergent solution in tap water containing crude
alkaline protease at the concentration of 2%. The control was done with the addition of the
enzyme in the detergent.
Studies on Alkaline-thermostable protease from an alkalophilic bacterium: Production, characterization and
applications
Aditya Bharadwaj et al.,
International Journal of Environmental Sciences Volume 5 No.2, 2014 357
3 Results and discussion
3.1 Isolation and Screening of Alkaline Protease Producing Bacteria
In the present study, samples were collected from milk industry, waste, corpses and soil.
Alkalophilic organisms were isolated from these samples. Out of the isolated alkalophiles, six
bacterial isolates (NB13, NB 21, NB 34, NB 42, NB 53, NB 64) were found to be potential
alkaline protease producers by plate clear zone method on casein agar plate. These six strains
were screened for alkaline protease production by submerged fermentation (as explained in
section 3.2.3) and isolate NB4 was found to produce maximum protease. When assayed at pH
9.5 and temperature 50°C (Table 1). This isolate was selected for further studies.
Table 1: Alkaline protease production by different isolate
Isolate number Enzyme activity (U/ml)
NB 13 1.80
NB 21 1.20
NB 34 2.50
NB 42 1.00
NB 53 0.70
NB 64 1.00
3.2 Morphological characteristics of NB 34
Broth culture was used for detecting the Gram behaviour of NB 34, it revealed that NB 34 is
a Gram positive small sized rod, present singly, or in pairs. It was non motile and produced
white, rough, opaque, convex colonies. The physiological characteristics of NB 34 showed it
tto be an obligate alkalophilie growing at pH 10, the organism was mesophile as it was not
able to grow at temperature above 37°C. These results indicate that isolate NB 34 is an
obligate alkalophile and most probably a Bacillus sp.
3.3 Optimization of Alkaline Protease Production
The protease production with isolate NB-34 was optimized with respect to previous
parameters:
3.4 Effect of different nitrogen sources
NB34 was screened for the effect of different nitrogen sources viz (peptone, casein, soyabean,
gelatine) at a concentration of 0.5% in HK medium, results showed that out of four nitrogen
sources ,gelatin is the best nitrogen source as it gave maximum enzyme production (Figure.
1). In literature, organic nitrogen source like soyabean , casein , gelatine, peptone, yeast
extract, tryptone, etc. have been reported best for optimal production of bacterial alkaline
proteases (Hameed et al 1999; Banerjee et al 1999; Kim et al 2004). Thomas et al (2007)
reported the maximum protease production with casein from Virgibacillus pantothenticus and
Studies on Alkaline-thermostable protease from an alkalophilic bacterium: Production, characterization and
applications
Aditya Bharadwaj et al.,
International Journal of Environmental Sciences Volume 5 No.2, 2014 358
reported soyabean meal to be the best for the optimal production of alkaline protease using
Bacillus sp RJ14. For further experiments, gelatine was used as nitrogen source in this study.
Figure 1: Protease production by alkalophilic isolate NB-34 with different nitrogen sources.
3.5 Effect of pH
The protease production was done in HK medium and effect of pH was tested by adding
different amounts of sodium carbonate 0.25% (pH 7.0), 0.5% (pH 9.3), 1.0% (pH 10.2),
1.25 % (pH 10.5), 1.5% (pH 10.8), 2.0% (pH 11.2), 2.5 % (pH 11.6).Being an obligate
alkalophile , negligible growth was observed at neutral pH. Maximum enzyme production
was seen with 2.0% sodium carbonate (pH~11.2). Enzyme production decreased at pH values
below and above this (Figure. 2). Different Bacillus spp. have been reported to produce
alkaline protease in alkaline range, eg. Virgibacillus pantothenticus (Thomas et al 2007) and
Bacillus sp isolate K30) at pH 9.0. Bacillus brevis at pH 10.5 (Banerjee et al 1999), Bacillus
clausii at pH 9.6 For, futher experiments, 2% sodium carbonate was used in this study.
Figure 2: Protease production by alkalophilic isolate NB34 with different concentration of
sodium carbonate pH range (7.6 – 11.5)
Studies on Alkaline-thermostable protease from an alkalophilic bacterium: Production, characterization and
applications
Aditya Bharadwaj et al.,
International Journal of Environmental Sciences Volume 5 No.2, 2014 359
3.6 Effect of different carbon sources
NB34 was screened for the effect of different carbon source viz (glucose, fructose, maltose,
starch) at a concentration of 0.5% in HK medium. The result showed that the maximum
production of enzyme was with glucose, however with maltose the enzyme production was
also comparable (Figure. 3). Glucose and maltose have been reported to be the best carbon
sources for the production of alkaline proteases by Virgibacillus pantothenticus (Thomas et al
2007), Bacillus cereus strain 146 (Sallah et al 2005), Bacillus sp RJ-14. For further
experiments, glucose was used as carbon source in this study.
Figure 3: Protease production by alkalophilic isolate NB-34 with different carbon source.
3.7 Effect of different incubation time period
The protease production by alkalophilic isolate NB 34 was done by doing the submerged
fermentation for different time period (12h, 24h, 48 h, 72h). The maximum enzyme
production was seen after 24 h (Figure.4), the enzyme activity decreased on further
incubation. In the reports on alkaline protease production by Bacillus spp., an incubation
period of 18h to 96 h has been observed to be optimum for enzyme production(Singh et al
2001; Banerjee et al 1999). Besides,other bacteria like Alcaligenes fecalis, Serratia
marcescens etc have also been reported to produce alkaline protease in 16 to 48 h (Thangam
and Rajkumar 2000; Romero et al 2001). For further experiments, 24 h was used as
incubation time period in this study.
3.8 Effect of different incubation temperature
HK medium having 0.5% gelatine, 2% sodium carbonate was inoculated with NB34 culture
and incubated at different incubation temperature viz 30°C, 37°C,40°C for 24 h and the
enzyme production was found to be maximum at 37°C (Figure. 5.) The alkaline protease
producing bacteria showed wide range of variation with respect to incubation temperature,
Thermomicrobium sp KN-22 produced thermostable alkaline protease at 70°C (Takami et al,
1989), while Bacillus sp. isolated from glaciers showed maximum protease production at
70°C (Gordon, 1982). For further experiments, 37°C was used as incubation temperature in
this study.
Studies on Alkaline-thermostable protease from an alkalophilic bacterium: Production, characterization and
applications
Aditya Bharadwaj et al.,
International Journal of Environmental Sciences Volume 5 No.2, 2014 360
Figure 4: Protease production by alkalophilic isolate NB 34 with different incubation time
period (12h – 72h)
Figure 5: Protease production by alkalophilic isolate NB-34 at different incubation
temperature (30-40°C)
3.9 Effect of agricultural by-products
Three different agro-industrial waste viz wheat bran, rice bran and deoiled cotton meal were
used for alkaline protease production with a view to improve enzyme yield. All the three
products when used without the standard nitrogen source (gelatin) produced significantly
higher alkaline protease enzyme as compared to when they were used in combination with
gelatin as the nitrogen source. Among the three, wheat bran alone produced the highest
alkaline protease activity. (Figure 6)
3.10 Characterization of Alkaline Protease
The alkaline thermostable protease from alkalophilic isolate NB34 was produced under the
conditions optimized in section 4.3. This crude enzyme was characterized for different
parameters:
3.11 pH optima
Studies on Alkaline-thermostable protease from an alkalophilic bacterium: Production, characterization and
applications
Aditya Bharadwaj et al.,
International Journal of Environmental Sciences Volume 5 No.2, 2014 361
The enzyme assay was done by preparing the substrate (casein) in the buffer of different pH
values (7, 7.5, 8.5, 9.5, 10.0, 10.5, 11), the enzyme was found to be optimally active at pH 9.5.
However, enzyme could retain 67% of activity at pH 10.0 and 40% activity even at pH 11.
However, activity declined sharply near neutral pH (Figure 7). Similar pH optimum has been
reported for other proteases in literarure eg: optimum pH of 9 has been reported for alkaline
protease from Bacillus sp. K-30 (Devi et al 2005), Virgibacillus pantothenticus (Thomas et al
2007).
Figure 6: Effect of agricultural raw material on alkaline protease production in isolate NB-34.
Figure 7: Activity of protease from alkalophilic isolate NB-34 at different pH values
3.12 pH stability
The pH stability of protease was examined by diluting the enzyme in buffers of pH 7.5-9.5
and then measuring the residual activity at different time intervals of 15-240 minutes. The
enzyme was maximum stable at pH 9.5, it could retain 90% of its activity after 30 minutes
and 75% of its activity even after 2 h. At pH 8.5, the enzyme could retain 675 of its activity
after 2 h but afterwards there was a sudden fall. At pH 7.5 the enzyme could retain 67% of its
activity but after 1 h there was a sudden fall of the activity (Figure 8). The stability of enzyme
Studies on Alkaline-thermostable protease from an alkalophilic bacterium: Production, characterization and
applications
Aditya Bharadwaj et al.,
International Journal of Environmental Sciences Volume 5 No.2, 2014 362
in alkaline range indicate its potential use in detergent formulation. Similar stability has been
reported from alkaline protease Virgibacillus pantothenticus (Thomas et al 2007).
Figure 8: pH stability of protease from alkalophilic isolates NB-34 at different pH values
3.13 Temperature optima
The enzyme activity of proteolytic isolate NB-34 was studied in the temperature range 40°C-
70°C by incubating the enzyme mixture at different pH temperatures under standard assay
conditions. The results revealed that it is a broad range (50°C-70°C) enzyme with optimum
activity at 50°C. However, the enzyme could retain 90% of its activity at 60°C and 70% of its
activity at 70°C (Figure 9). Elsewhere, reports from literature also suggests the alkaline
proteases displaying maximum activity between 37°C-70°C (El-Sawah and El- Din 2000;
Eftekhar et al 2003; Thomas et al 2007).
3.14 Temperature stability
The thermostability of proteases was examined in temperature range of 50°C-70°C. The
enzyme was incubated at the respective temperatures, aliquots was withdrawn suddenly and
residual protease activity was measured under standard assay conditions. The enzyme showed
maximum stability at 50°C where it could retain 80% of its activity even after 1 h and at
60°C and 70°C also the enzyme was reasonably stable where it could retain 80% of its
activity after 20 minutes (Figureure 10). The similar temperature stabilites has been reported
by alkaline protease from Virgibacillus pantothenticus (Thomas et al 2007) and thermo
Bacillus sp. Strain SMAI-2 (Lata et al 2002).
3.15 Substrate concentration
The effect of substrate concentration studied by using varying concentration of casein (1.25
mg to 10 mg/ml) revealed a typical hyperbolic curve showing 7.5 mg/ml casein as the best
concentration (Figure. 11). A doubtful reciprocal plot was prepared that revealed an apparent
Km of the enzyme as 0.99 mg casein per ml and a Vmax of 5.55 (Figureure 4.12). Elsewhere,
Manchini et al (1998) reported Km value of 2.5mM in Bacillus thermoruber, Sinha and
Satyanarayana (1991) found Km of 9.1 and Vmax of 0.33mM in Bacillus licheniformis, gave
Km value of 3.7 mg/ml in Bacillus polymyxa and Thangam and Rajkumar (2002) reported a
Km value of 1.66 mg/ml and Vmax of 562 U for alkaline protease of Alcaligenes faecalis,
Studies on Alkaline-thermostable protease from an alkalophilic bacterium: Production, characterization and
applications
Aditya Bharadwaj et al.,
International Journal of Environmental Sciences Volume 5 No.2, 2014 363
using casein as substrate. This suggest that even with same substrate, alkaline protease of
different Bacillus sp. Show variation in their enzymatic rates.
Figure 9: Activity of proteases from alkalophilic isolate NB-34 at different temperatures
Figure 10: Temperature stability of proteases from alkalophilic NB-34 at different
temperatures
3.16 Enzyme concentration
The effect of enzyme concentration (0.05-0.5 ml) in the reaction mixture of 3 ml was studied
to determine the optimum enzyme concentration required for maximum casein hydrolysis.
The results presented in Figure 12 revealed that 0.1 ml of the enzyme is sufficient to digest
the substrate reported 33% enzyme and Huang et al (2003) revealed 50% protease (of the
reaction mixture) as optimum for enzyme activity.
3.17 Effect of chelating agents
EDTA strongly inhibited the enzyme activity causing a decrease in 62%, 91% and 92% at
concentration of 1 mm, 10 mm and 100 mm, respectively while ammonium hydroxide causes
Studies on Alkaline-thermostable protease from an alkalophilic bacterium: Production, characterization and
applications
Aditya Bharadwaj et al.,
International Journal of Environmental Sciences Volume 5 No.2, 2014 364
a fall of 24%, 62% and 37% at concentration of 1 mm, 10 mm and 100 mm, respectively
(Table 2) Similar effects have been reported by chelating agents on alkaline protease from
Vergibacillus pantothenticus (Thomas et al 2007).
Figure 11: Activity of proteases from alkalophilic isolate NB-34 at different substrate
concentrations
Figure 12: Activity of proteases from alkalophilic isolate NB-34 at different enzyme
concentration
Table 2: Effect of chelating agents on the activity of protease from alkalophilic isolate NB-
34
Chelating agents Concentration
(mM) Specific activity
Percent change in
activity
Control - 3.5 -
EDTA
1 1.33 (-)62.21
10 0.33 (-)91.02
100 0.33 (-)91.02
Sodium Hydroxide
1 2.66 (-)24.01
10 1.33 (-)62.21
100 0.97 (-)73.42
Studies on Alkaline-thermostable protease from an alkalophilic bacterium: Production, characterization and
applications
Aditya Bharadwaj et al.,
International Journal of Environmental Sciences Volume 5 No.2, 2014 365
3.18 Effect of oxidizing and reducing agent
The effect of various oxidizing and reducing agents on the enzyme activity was studied under
standard assay conditions. The results are shown in Table 3. Hydrogen peroxide, ammonium
persulphate, acetic anhydride, iodo acetamide and potassium iodate showed variable
inhibitory effect at 1mM, 10mM and 100mM as represented in Table 3.
Table 3: Effect of oxidizing and reducing agents on the activity of protease from alkalophilic
isolate NB-34
Concentration
mM Specific activity
Percent change in
activity
Control - 3.5 -
Oxidizing agent
Ammonium
persulphate
1 3.63 (-)10.25
10 2.5 (-)25.5
100 2.66 (-)28.5
Acetic anhydride
1 1.0 (-)25
10 1.33 (-)62
100 0.66 (-)83.5
Iodoacetamide
1 1.0 (-)75
10 1.02 (-)72
100 0.66 (-)83.5
Hydrogen
peroxide
1 1.66 (-)58.5
10 1.66 (-)58.5
100 1.33 (-)62
Potassium iodate
1 2.66 (-)24.2
10 2.16 (-)46
100 2.66 (-)24.2
Reducing agents
Mercaptoethanol
1 2.66 (-)24.2
10 2.50 (-)28.5
100 2.50 (-)28.5
Dithioethral
1 2.50 (-)28.5
10 1.33 (-)62
100 1.33 (-)62
3.19 Effect of heavy metals
Heavy metals like Zn2+, Hg2+, Cd2+ and Mn2+ strongly inhibited the protease activity while
Cu2+, Co2+, K+, Ca2+ and Ba2+ partially inhibited the protease activity at 10mM concentration,
whereas metals like Na+ and Fe2+ increased the enzyme activity as shown in Table 4.Similar
results have been reported by alkalophilic protease from Vergibacillus pantothenticus
(Thomas et al 2007).
Studies on Alkaline-thermostable protease from an alkalophilic bacterium: Production, characterization and
applications
Aditya Bharadwaj et al.,
International Journal of Environmental Sciences Volume 5 No.2, 2014 366
Table 4: Effect of various metal ions on the activity of protease from alkalophilic isolate NB-
34
Metal ion Concentration mM Specific activity Percent change in
activity
Control - 3.5 -
Zn2+
1 1.33 (-)62
10 1.66 (-)52.58
100 1.33 (-)62
Hg2+
1 0.33 (-)90.6
10 0.33 (-)90.6
100 0.33 (-)90.6
Cd2+
1 1.66 (-)52.8
10 1.66 (-)52.8
100 1.33 (-)62
Mn2+
1 2.16 (-)38.79
10 1.66 (-)52.58
100 1.83 (-)47.72
Cu2+
1 2.66 (-)24
10 0.97 (-)72.79
100 1.33 (-)62
4 Application Prospects of alkaline protease of Bacillus sp. NB 34
The alkaline protease of Bacillus sp. NB 34 was tested for its applicability in detergent
industry.
4.1 Compatibility with detergents
For the commercial exploitation of enzyme in detergent industry, the crude alkaline protease
was tested for its compatibility with five different detergents. The enzyme incubated with
detergent solution (either in water or in buffer) revealed that when used in water-aerial, surf
excel and tide showed maximum compatibility of alkaline protease with rin and fena (Figure
13) (Table 5). This result showed that this enzyme is compatible with various detergents
which make it a potential candidate for use in detergent industry.
Table 5: Compatibility of alkaline protease with commercial detergents
Commercial detergents Tap water Buffer
Control 3.50 3.50
Ariel 2.50 2.66
Surf excel 2.66 2.83
Tide 2.50 2.16
Rin 2.66 3.01
Fena 1.83 1.33
Studies on Alkaline-thermostable protease from an alkalophilic bacterium: Production, characterization and
applications
Aditya Bharadwaj et al.,
International Journal of Environmental Sciences Volume 5 No.2, 2014 367
0
0.5
1
1.5
2
2.5
3
3.5
4
Ariel Surf excel Rin Tide Fena Control
Series 1
Series 2
Figure 13: Compatibility of alkaline protease with commercial detergents.
4.2 De-staining properties
Two pieces of cloth artificially stained with blood were dipped in either detergent solution or
detergent solution supplemented with enzyme and incubated for 10 minutes at 60⁰C. The
results (Figure 14) showed the complete removal of stain in detergent solution supplemented
with enzyme whereas bloodstain was not completely removed from cloth dipped in detergent
solution only.
Figure 14: Blood destaining activity of alkaline thermostable protease of isolate no. NB 34 a)
detergent solution without enzyme; b) detergent solution with enzyme
5. Conclusion
Protease find their application in a wide range of industrial process viz; detergent, brewing,
baking, pharmaceuticals, leather tanning, meat tenderization, peptide synthesis and medical
diagnosis (Karcia-Carreno, 1991; Outrun et al., 1991). Out of these processes proteases
Studies on Alkaline-thermostable protease from an alkalophilic bacterium: Production, characterization and
applications
Aditya Bharadwaj et al.,
International Journal of Environmental Sciences Volume 5 No.2, 2014 368
specially alkaline thermostable protease carry a vast potential in detergent industry.
Alkalophilic organisms producing alkaline thermostable protease(s) were isolated from the
industry waste, corpses and soil. Isolate number NB-34 was found to produce maximum
alkaline thermostable protease. Optimization studies on protease production from isolate NB-
34 revealed that protease production was maximum : With 0.5% gelatin as nitrogen source,
pH 11.2 (2% sodium carbonate, 0.5% glucose, 24 hours incubation period, 37⁰C incubation
temperature, 150 rpm agitation. The alkaline thermostable protease from alkalophilic
thermostable isolate NB-34 had pH and temperature optima of 9.5 and 50⁰C respectively.
The Km value for the enzyme was calculated to be 0.909 mg casein/ml and Vmax 5.55. The
alkaline thermostable protease from isolate NB-34 was found to be compatible with most of
the commercial detergents. When the alkaline thermostable protease from isolate NB-34 was
supplemented with the commercial detergents, it improved the destaining capacity of the
detergent.
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