studies on alkaline-thermostable protease from an...

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.

[email protected]

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


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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


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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.


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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


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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)


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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.


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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


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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


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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,


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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


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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


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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).


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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


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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


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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.

6. References

1. Banerjee, U.C., Sani, R.K., Azmi, W, and Soni, R. (1999), Thermostable alkaline

protease from Bacillus brevis and its characterization as laundry detergent additive.

Process Biochemistry, 35, pp 213-19.

2. Chauhan, PS., George, N., Sondhi, S., Puri, N., and Gupta, N., (2014a), An overview

of purification strategies for microbial mannanases. International Journal of Pharma

and Bio Sciences, 5 (1), pp 176-192.

3. Chauhan, PS., Puri, N., Sharma, P, and Gupta., N. 2012. Mannanases: microbial

sources, production, properties and potential biotechnological applications. Applied

Microbiology Biotechnology, 93 (5), pp 1817-1830.

4. Chauhan, PS., Soni, SK., Sharma, P., Saini., A, and Gupta, N. 2014b. A mannanase

from Bacillus nealsonii PN-11: Statistical optimization of production and application

in biobleaching of pulp in combination with xylanase. International Journal of Pharma

and Bio Sciences, 5 (1), pp 237-251.

5. Chauhan, P.S., Sharma, P., Puri, N., and Gupta, N. 2014c Purification and

Characterization of an Alkali-Thermostable β-Mannanase from Bacillus nealsonii PN-

11 and Its Application In Manno-oligosaccharides Preparation Having Prebiotic

Potential. European Food Research and Technology, 238 (6), pp 927-936.

6. Chauhan, P.S., Sharma, P., Puri, N., and Gupta, N. 2014d A Process for Reduction in

Viscosity of Coffee Extract by Enzymatic Hydrolysis of Mannan. Bioprocess and

Biosystems engineering, 37 (7), pp 1459-1467.

7. Chauhan, P.S., Bharadwaj, A., Puri, N. and Gupta N. 2014e. Optimization of medium

composition for alkali-thermostable mannanase production by Bacillus nealsonii PN-

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