Biotechnology Letters21: 135–138, 1999.© 1999Kluwer Academic Publishers. Printed in the Netherlands.

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Bleach-stable, alkaline protease fromBacillus sp.

Rani Gupta∗, Komal Gupta, R. K. Saxena & Seema KhanDepartment of Microbiology, University of Delhi, South Campus, Benito Juarez Road, Dhaula Kuan, New Delhi,110 021, India∗Author for correspondence (Fax: 91-11-6886427/6885270; E-mail: micro@dusc.ernet.in)

Received 6 October 1998; Accepted 22 December 1998

Key words:bleach-stable, thermotolerant, alkaline protease,Bacillussp.

Abstract

A bleach-stable, thermotolerant, alkaline protease for detergent formulation from a newly isolatedBacillusSB5 isreported. Most (85%) activity of the enzyme was retained in the presence of 10% (v/v) H2O2 and 1% SDS (w/v) at40◦C, after 1 h. The enzyme was optimal at pH 10 and 60◦C to 70◦C. Enzyme activity was enhanced 30 to 80%in presence of ionic and non-ionic detergents, surfactants and commercial detergents or bleach.

Introduction

Proteases constitute one of the most important groupsof industrial enzymes and find applications in differentindustries viz. detergent, food, pharmaceutical, leatherand silk (Outtrupet al.1988, 1993, 1995, Wolffet al.1996). This enzyme accounts for 20% of the worldmarket (approximately US$ 160 million), with pre-dominant use in detergents where particularly alkalineproteases are used (Outtrupet al. 1988, 1993, 1995,Boguslawski & Shultz 1992, Wolffet al.1996).

Important commercial detergent proteases avail-able are Subtilisin Carlsberg, Subtilisin BPN’, Al-calase, Esparase and Savinase, where the last threeare marketed by Novo industri A/S, Denmark (Out-trup et al. 1988, 1993, 1995). All these enzymes arestable in presence of various components of detergentsand are active at washing temperatures and pH values.However, the majority of them are unstable in pres-ence of peroxide agents. Therefore, various attemptshave been made to enhance stability of alkaline pro-teases by site directed mutagenesis (Boguslawski &Shultz 1992, Tuschiyaet al. 1992) and protein engi-neering (Wolffet al. 1996).‘Subtilisin Carlsberg’ hasbeen protein engineered to obtain a bleach-stable, al-kaline protease by molecular modification (Outtrupet al. 1988, Wolff et al. 1996) but still, there is al-ways a need for newer thermostable alkaline proteases

which can withstand oxidizing agents present in deter-gents. Here, we report bleach-stable, thermotolerant,alkaline protease from a new variant ofBacillus sp.,having potential application in detergent formulations.

Materials and methods

All analytical reagents and media components werepurchased from SRL or Hi-Media, India. Casein forprotease assay was from Sigma.

Bacterial strain and inoculum preparation

Bacillus SB5 was isolated at 50◦C from soil by en-richment. It was grown in nutrient broth (pH 7) at50◦C and 200 rpm in a shaker. One ml of 5 h oldculture (3×108 c.f.u./ml)) was used to inoculate theproduction flasks.

Enzyme production

The enzyme was produced by growing the organismin medium containing casein (0.5%, w/v) for 72 h at50◦C and 200 rpm. The culture was centrifuged at10,000 g for 20 min at 4◦C. The supernatant frac-tionated to ammonium sulfate precipitation (80%) wasdialyzed against 0.05 M glycine/NaOH buffer (pH 10)for 24 h, with three changes. The dialyzed sample

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served as the enzyme source for characterization andits possible application in detergent formulation.

Protease assay

Protease assay was carried out according to Meyersand Ahearn method (1977) using casein (1% w/v)as the substrate at pH 10 and 60◦C, unless men-tioned otherwise. One international unit of proteaseactivity is defined as amount of enzyme required toliberate 1µmol tyrosine per ml per min, under assayconditions.

Characterization of the enzyme

pH and temperature tolerance / stability

Enzyme assay was carried out at different pH (3–12)and temperature (30◦C–100◦C) and relative activitywas determined by caseinolytic assay. pH stability ofthe enzyme was also determined by incubating the en-zyme with buffers of different pH (3–12) for 1 h at40◦C. Various activity buffers (0.05 M) viz., citratephosphate buffer pH (3–6), phosphate buffer (pH 7–8), glycine/NaOH buffer (pH 9–11) and NaCl/NaOHbuffer for pH 12 were used. Residual activity was cal-culated in each sample by caseinolytic assay againstenzyme control sample at pH 7. Temperature stabilityof the enzyme was studied by incubating the enzymeat different temperatures for 1 h, at pH 10.

Effect of oxidizing agent

The enzyme was incubated with different concentra-tions of hydrogen peroxide (5–15%) at 40◦C for 15to 90 min (at regular intervals of 15 min) and enzymestability was calculated as residual activity followingcaseinolytic assay.

Detergents, surfactants and bleach compatibility

The enzyme sample was incubated with different ionicand non-ionic detergents and surfactants viz. SDS,saponin, Tween 20, Tween 80, sodium choleate andsodium taurocholate and different commercial deter-gents like Surf, Fena and Wheel and Robin liquidbleach (1% w/v or v/v) for 30 and 60 min at 40◦Cand enzyme was assayed.

Fig. 1. Thermal inactivation curve of alkaline protease fromBacil-lus SB5 at pH 10. Caseinolytic activity measured using casein (1%w/v) at pH 10 and 60◦C after 20 min of incubation and residualactivity (%) is calculated against enzyme control incubated in buffer(100% activity≡ 4.6 U/ml). Data represents an average of threereplicates repeated twice.

Results and discussion

An extracellular protease producingBacillussp. wasisolated from soil by enrichment and was identifiedat Microchek, Inc. USA. Based on the identificationresults the strain was found to be standard deviation(SI/SD) ratio of 0.416, 0.393 and 0.268, respectively.

The reported protease is casein inducible and has apH and temperature optima at pH 10 and 60◦C–70◦C.The enzyme is stable in a wide pH (5–12) and tem-perature (upto 60◦C) range. It is almost 85% stableat pH 7 to 12 after 1 h at 40◦C. In general, all de-tergent compatible enzymes are alkaline thermostablein nature with a high pH optima because the pH oflaundry detergents is generally in the range of 9–12and varying thermostability at laundry temperatures,(50◦C/60◦C) (Takamiet al. 1987, Manachiniet al.1988, Outtrupet al.1988, Tsaiet al. 1988, Phadatareet al.1993, Bhosaleet al.1995, Kobayashiet al.1995,Ferreroet al. 1996). The present enzyme has goodthermostability as it retains its 80% activity at 60◦C, inalkaline conditions (pH 10) after 1 h and also exhibitshalf life of more than 1 h at 60◦C and 7 min at 70◦C,respectively.

However, a well established commercial deter-gent protease, namely Substilisin Carlsberg, has ahalf life of 2.5 min at 60◦C (Durhamet al. 1987).Thus, the reported protease outstands as regards to pH

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Table 1. Effect of various surfactants, detergents and commercial de-tergents/bleach (1% w/v or v/v) on protease activity ofBacillus sp. at40◦C.

Detergents/surfactans/local detergents Residual activity (%)

and bleach (1%, w/v or v/v)

After 30 min After 60 min

Saponin (w/v) 182 180

SDS (w/v) 88 60

Tween 20 (v/v) 122 144

Tween 80 (v/v) 136 168

Sodium choleate (w/v) 131 153

Sodium taurocholate (w/v) 111 171

Surf (w/v) 66 35

Wheel (w/v) 132 119

Fena (w/v) 156 157

Robin liquid bleach (v/v) 140 156

Caseinolytic activity measured using casein (1% w/v) at pH 10 and 60◦Cafter 20 min of incubation and residual activity (%) is calculated againstenzyme control incubated in buffer (100% activity≡ 4.6 U/ml). Datarepresents an average of three replicates repeated twice.

and temperature characteristics relevant to detergentformulation.

Besides pH and temperature stability, a good de-tergent protease should also be stable in the presenceof surfactants, detergents and oxidizing agents. Inter-estingly, in the present case, enzyme activity of theprotease fromBacillusSB5 is stimulated in the pres-ence of all ionic (saponin, sodium choleate and sodiumtaurocholate) and non-ionic (Tween 20 and Tween 80)detergents and surfactants (Table 1). However, maxi-mum stimulation was with saponin where the activitydoubled, after 1 h incubation. The enzyme exhibitedunusual stability in presence of 1% SDS with 50%residual activity after 1 h at 40◦C. The enzyme alsoexhibited enhanced activity in Robin liquid bleach andmany commercial detergents.

The bleach stability of the enzyme was furtherconfirmed in the presence of hydrogen peroxide. Theperoxide inactivation curve, shows that the enzyme isstable even at high concentrations of H2O2 (10% v/v)for 1 h with 85% residual activity. This is an impor-tant property because bleach-stable enzymes are notgenerally available except for a few reports (Tuschiyaet al. 1992). Hence, bleach stability has been at-tained either by site directed mutagenesis (Outtrupet al. 1988, 1995, Tuschiyaet al. 1992) or pro-tein engineering (Boguslawski & Shultz 1992, Wolffet al. 1996) whereas the present protease ofBacillus

Fig. 2. Peroxide inactivation curve of alkaline protease fromBacil-lus SB5 in presence of varying concentration of H2O2 at 40◦C.Caseinolytic activity measured using casein (1% w/v) at pH 10 and60◦C after 20 min of incubation and residual activity (%) is calcu-lated against enzyme control incubated in buffer (100% activity≡4.6 U/ml). Data represents an average of three replicates repeatedtwice.

SB5 shows inherent stability in the presence of highconcentrations of hydrogen peroxide.

Considering the overall property of different alka-line proteases of microbial origin and their evaluation,the reported alkaline protease is excellent with regardsto pH and temperature stability, detergent compat-

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ibility and above all bleach stability, for its futureapplication in detergent formulation.

Acknowledgements

One of us (S. K.) acknowledges the financial supportfrom University Grants Commission, India, to carryout the present investigation.

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