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Process Biochemistry 35 (1999) 213–219

Thermostable alkaline protease from Bacillus bre6is and itscharacterization as a laundry detergent additive

Uttam Chand Banerjee *, Rajesh Kumar Sani, Wamik Azmi, Raman SoniBiochemical Engineering Research and Process De6elopment Center, Institute of Microbial Technology, Sector 39-A, Chandigarh 160036, India

Received 1 February 1999; accepted 9 April 1999

Abstract

An alkaline protease from a facultatively thermophilic and alkalophilic strain of Bacillus bre6is has been studied. The enzymefrom a shake flask culture displayed maximum activity at pH 10.5 and 37°C. The extracellular production of the enzyme, itsthermostable nature and compatibility with most commercial detergents are features which suggest its application in detergentindustry. The organism utilized several carbon sources for the production of proteases, lactose was the best substrate followed byglucose and sucrose. Among the various organic nitrogen sources, soyabean meal was found to be the best. The protease wasstable at 25°C for 288 h whereas, at 50 and 60°C, the half lives were 60 and 7 h, respectively. The thermostability of the proteasewas enhanced by modifying its microenvironment. Acetate salts of Ca2+ and Na+ increased thermostability and protected againstautolysis. Addition of Ca2+ (10 mM) and glycine (1 M) individually and in combination was found to be effective in increasingthe half life of protease by many folds. The enzyme retained more than 50% activity after 4 days at 60°C in the presence of bothCa2+ (10 mM) and glycine (1 M). The enzyme showed compatibility at 60°C with commercial detergents such as AerialMicroshine®, Surf excel®, Surf Ultra® and Rin® in the presence of Ca2+ and glycine. This enzyme improved the cleaning powerof various detergents. It could remove blood stains completely when used with detergents in the presence of Ca2+ and glycine.© 1999 Elsevier Science Ltd. All rights reserved.

Keywords: Alkaline protease; Thermostability; Bacillus bre6is ; Detergent compatibility

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1. Introduction

Proteases are one of the most important industrialenzymes, accounting for nearly 60% of total world wideenzyme sales [1–3]. Of these, alkaline proteases areemployed primarily as cleansing additives. Among thevarious proteases, bacterial proteases are the most sig-nificant, compared with animal and fungal proteases[1]. Ideally, proteases used in a detergent formulationshould have a high level of activity over a broad rangeof pH and temperatures. Alkaline proteases from highyielding strains have been studied extensively. The ma-jor draw back of enzymes recovered from thermophilesare their instability at alkaline pH, whereas enzymesfrom alkalophiles confer stability in a wide pH range

but are usually thermolabile [4,5]. Thus it is desirable tosearch for new proteases with novel properties from asmany different sources as possible. In this report weexamine the efficacy of the enzyme, recovered fromBacillus a newly isolated strain of bre6is, in the presenceof standard commercial detergents. Further, characteri-zation of this enzyme and the effect of various co-fac-tors or additives on the stability at higher temperatureand in alkaline pH range was carried out.

2. Materials and methods

2.1. Chemicals

All medium components were obtained from Hi-Me-dia, Bombay, India. Azocasein and glycine were pur-chased from Sigma Chemical Co., St. Louis, MO, USA.Other chemicals were of reagent grade and were com-

* Corresponding author. Tel.: +91-172-690908; fax: +91-172-690585.

E-mail address: [email protected] (U.C. Banerjee)

0032-9592/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.

PII: S 0032 -9592 (99 )00053 -9

U.C. Banerjee et al. / Process Biochemistry 35 (1999) 213–219214

mercially available. Various detergents used in thisstudy were purchased from a local market.

2.2. Microorganism and culture conditions

Bacillus bre6is used in the present study was isolatedfrom water samples collected from hot springs atManikaran (Himachal Pradesh, India) after extensivescreening at various temperatures (37, 50 and 60°C) andpHs (8.0, 9.0 and 10.0) on nutrient agar plates supple-mented with 0.2% skim milk powder. The strain wasidentified by the Microbial Type Culture Collection andGene Bank (MTCC), Chandigarh, India and given theaccession no. MTCC B0016. The organism was main-tained on nutrient agar plates and stored at 4°C, whilethe stock was maintained in 10% glycerol at 70°C.Various carbon sources (1%, w/v) including glucose,lactose, sucrose, maltose and starch and various organicnitrogen sources (1%, w/v) including yeast extract, beefextract, meat extract, peptone, biopeptone, yeast au-tolysate, soyabean meal, soyatose, soyapeptone andcasein were used in this study. The liquid medium usedfor the production of alkaline protease had the followingcomposition (%, w/v): sucrose 1.0, soyabean meal 1.0,calcium acetate 0.01, KH2PO4 0.1, MgSO4·7H2O 0.01and CaCl2·2H2O 0.01. The pH of the medium wasadjusted to 10.5 with 10% (w/v) Na2CO3 solution. Theeffect of temperature and pH on enzyme production wasstudied at 37 and 50°C and in a pH range of 9.0–11.0.Experiments were carried out in 500 ml Erlenmeyer flaskcontaining 100 ml medium. After inoculation with B.bre6is (12 h growth), the flasks were incubated for 96 hin a temperature controlled (37°C) shaking incubator(200 rpm). Duplicate flasks were withdrawn at regularintervals and analysed for growth (OD600) and pH. Thecontents were then centrifuged (10 000×g, 4°C, 20 min)and the cell free supernatant was used for determiningextracellular protease activity.

2.3. Assay methods

Alkaline protease activity was determined by themodified method of Longo et al. [6]. A total of 20 ml of5% (w/v) azocasein in NaOH/glycine buffer (50 mM, pH10.5) was incubated at 37°C with 460 ml of the samebuffer. After 5 min, 20 ml of appropriately dilutedenzyme was added to the above mixture and incubatedat 60°C for 30 min. The reaction was stopped by adding500 ml trichloroacetic acid (10%, w/v) and the mixtureallowed to cool for 15 min on ice. This mixture wascentrifuged and 800 ml supernatant was mixed with the200 ml NaOH (1.8 N) in a tube and the absorbance wasread at 420 nm. Enzyme activity was expressed as azounits, where one unit (U) is defined as the amount ofenzyme required to increase 0.1 absorbance at 420 nmper hour under assay conditions (60°C, pH 10.5).

2.4. Temperature and pH studies

The temperature-activity profile was studied by assay-ing protease activity in a temperature range of 25–75°Cat pH 10.5 while the pH-activity profile was studied ina pH range of 6.0–12.0 at 60°C using 50 mM buffers ofdifferent pHs (6.0–6.5, sodium acetate; 7.0–8.0, Tris–HCl and 8.5–12.0, NaOH/glycine). The thermostabilityprofile was studied by incubating the enzyme prepara-tion at various temperatures (25, 30, 37, 50 and 60°C) inNaOH/glycine buffer (50 mM, pH 10.5) for differenttime period and measured the residual activity at 60°C.Effect of various additives (inorganic salts and metalions) on the thermostability of protease activity wasdetermined by incubating the enzyme in the presence ofadditives at 50 and 60°C for 30 min. Samples withdrawnat various intervals were immediately ice cooled and theresidual activity (%) was determined under standardassay conditions (60°C, pH 10.5). pH stability profilewas determined by incubating equal volumes of enzymewith buffers in the pH range of 6.0–12.0 for 1 h at 60°Cand the residual activity (%) determined at pH 10.5 bystandard assay procedure. The effect of various metalions and inhibitors on enzyme activity was studied byincubating the enzyme preparation at 60°C with differ-ent concentrations of metal ions and inhibitors and theresidual activity was determined after 1 h.

2.5. Detergent stability

The stability of the enzyme with and without additivesin the presence of detergents was determined byincubating the enzyme (3000 U/ml) at 60°C with variouscommon detergent preparations (7 mg/ml) and theresidual activity determined at regular intervals up to 3h.

2.6. Washing test with protease preparation

Application of protease as a detergent additive wasstudied on white cotton cloth pieces (4×4 cm) stainedwith human blood. The stained cloth pieces were takenin separate flasks. The following sets were prepared:1. Flask with distilled water (100 ml)+stained cloth

(cloth stained with blood)2. Flask with distilled water (100 ml)+1 ml detergent

(7 mg/ml)3. Flask with distilled water (100 ml)+1 ml detergent

(7 mg/ml)+2 ml enzyme solutionThe above flasks were incubated at 50°C for 15 min.

After incubation, cloth pieces were taken out, rinsedwith water and dried. Visual examination of variouspieces exhibited the effect of enzyme in removal ofstains. Untreated cloth pieces stained with blood weretaken as control.

U.C. Banerjee et al. / Process Biochemistry 35 (1999) 213–219 215

3. Results and discussion

3.1. Isolation and characterization of alkaline proteaseproducing strain

Bacterial strains were isolated from a hot spring andscreened for hyper protease producers at pH 9.0, 10.0and 11.0. From 50 samples screened on nutrient agarplates supplemented with skim milk powder (0.2%),some bacterial strains that grew in the presence of skimmilk powder at various alkaline pHs, were selected onthe basis of zone of clearance around the colonies. Fivestrains were isolated from the samples screened. Theproteolytic activity of these strains were checked atvarious pHs and temperatures and finally one strainwas selected as the most potent producer of proteolyticenzyme (data not shown). The isolated alkalophilicthermostable proteolytic strain was a spore formingGram-positive rod and identified as Bacillus bre6is. This

strain seems to be a novel one being capable of growingover a wide pH and temperature range.

3.2. Optimization of en6ironmental conditions onenzyme production in shake flask

Among various carbon sources tested, lactose pro-duced the maximum (3455 U/ml) protease activity(Table 1). With glucose and sucrose as carbon source,the maximum activities obtained were 3125 and 3150U/ml, respectively. Moderate to good amount ofprotease activity was produced in the presence of starch(2688 U/ml) and maltose (2016 U/ml) Highest produc-tion of enzyme activity on various carbon sources wasachieved at different times of fermentation. Sucrose,being the cheapest carbon source was used for enzymeproduction in subsequent experiments. In a similarstudy Fujiwara and Yamamoto [7] showed that glucoseand starch were the best carbon sources for proteaseproduction by Bacillus sp. no. 221 while lactose andsucrose were not very effective. Fujiwara et al. [8] alsoreported glucose (1%, w/v) as the best carbon sourcefor protease production by Bacillus sp. B18. Phadatareet al. [9] reported that sucrose was used as a carbonsource for growth and protease production byConidobolus coronatus. Easalp et al. [10] reported thatalthough glucose could not support for maximumprotease production in B. subtilis, yet it emerged as abetter substrate than maltose, starch and galactose.

Enzyme production was also affected by the type ofnitrogen sources used. Among the various organic ni-trogen sources used, the maximum enzyme activity(2240 U/ml) was obtained when soyabean meal wasused in the medium (Fig. 1). Moderate levels of enzymeactivities were obtained when biopeptone, meat extract,tryptone, malt extract, yeast extract, tryptose and yeastautolysate were used as nitrogen source. Peptone wasthe poorest organic nitrogen source for alkalineprotease production. Our results corroborate the previ-ous reports showing the preference of Bacillus sp. forsoyabean meal [2,7,11]. On the other hand, Phadatareet al. [9] reported the enhancement of protease produc-tion in C. coronatus by organic nitrogen sources liketryptone, peptone, yeast extract, skim milk and soybeanmeal.

Enzyme production was substantially improved whencalcium acetate, CaCl2 and MgSO4 were used to supple-ment the medium along with KH2PO4. The increase ofenzyme production was small when these salts wereused individually. Enzyme production in the absence ofthese salts was sufficiently low, thereby indicating therequirements of acetate and some metal ions (Ca2+ andMg2+) for protease production by B. bre6is (data notshown). A combination of sodium acetate and calciumchloride not only produced better enzyme activity butacted as a protectant against autolysis and thereby

Table 1Production of alkaline protease by Bacillus bre6is using differentcarbon sources

Time (h)Carbon sources Maximum alkaline protease ac-tivity (U/ml)

72Glucose 312560Sucrose 315048Lactose 3455

Galactose 650722688Starch 60

60Maltose 2016

Fig. 1. Maximum alkaline protease production by B. bre6is usingvarious organic nitrogen sources (1%, w/v): (1, control; 2, peptone; 3,soyatose; 4, soyabean meal; 5, Soyapeptone; 6, tryptose; 7, yeastextract; 8, biopeptone; 9, meat extract; 10, yeast autolysate; 11, maltextract; and 12, tryptone).


Table 2Effect of initial pH and temperature on the production of alkalineprotease by Bacillus bre6is

Maximum alkaline protease activity (U/Initial pHml)

50°C37°C

9.0 1670 80510.0 2100 970

1135237010.529011.0 110

formis MIR 29. The maximum production of proteaseactivity (2370 U/ml) by B. bre6is was observed at 37°Cand pH 10.5 while at 50°C and pH 10.5, it produced(1135 U/ml) half of the enzyme activity (Table 2). Atthe lower and higher pH, the enzyme activity reducedconsiderably.

3.3. Characterization of the enzyme

Protease activity increased progressively with temper-ature and maximum activity was obtained at 60°C (Fig.2a). Enzyme activity decreased at temperatures higherthan 60°C. It is reported [14,15] that a number ofalkaline proteases isolated from Bacillus sp. have highoptimal temperatures for their activity. This is an im-portant characteristic for the use of alkaline proteasesas detergent additives. Similar to our results, two im-portant alkaline proteases of the detergent industry,subtilisin Carlberg, produced by B. licheniformis andsubtilisin Novo or Bacterial Protease Nagase (BPN),produced by B. amyloliquefaciens, also showed opti-mum temperature of 60°C [14]. The pH-activity profileof the enzyme is shown in Fig. 2(b). At pH 6.5, only22% of the maximum enzyme activity was obtained,increasing to 65 and 77% at pH 7.0 and 7.5, respec-tively. Maximum enzyme activity was observed at pH10.5 and an increase in pH beyond 10.5 brought abouta decline in protease activity resulting in 18% activity atpH 12.5. These findings are in accordance with severalearlier reports showing pH optima of 10–10.5 forprotease from Bacillus sp., Thermus aquaticus, Xan-thomonas maltophila and Vibrio metschniko6ii [15–18].The important detergent enzymes, substilisin Carlbergand substilisin Novo or BPN also showed maximumactivity at pH 10.5 [14].

Fig. 2. (a) Effect of temperature on alkaline protease activity by B.bre6is. (b) Effect of pH on alkaline protease activity by B. bre6is.

Fig. 3. Thermostability of alkaline protease activity from B. bre6is atvarious temperatures.

increased the shelf-life of the enzyme. These resultscorroborate the earlier findings of metal ions enhancingthe production and stabilizing the activity of proteaseenzyme [12]. Ferrero et al. [13] reported the use oftrisodium citrate along with MgSO4, CaCl2, MnSO4

and ZnSO4 for protease production by Bacillus licheni-


Fig. 4. Thermostability of alkaline protease activity from B. bre6is inpresence of CaCl2 and glycine at 60°C: (, control; , enzyme+CaCl2; �, enzyme+glycine; �, enzyme+glycine+CaCl2).

M), individually and in combination, improved thethermostability of the enzyme (Fig. 4). The enzymeretained more than 50% of its maximum activity after 4days at 60°C in the presence of CaCl2 and glycine.Thermostability studies of protease activity in the pub-lished literatures showed enzyme with a half-life of 25min at 60°C [15], 23 min at 60°C [18] and 10 min at60°C [14,19,20]. Yamagata et al. [20] also reportedthermostable protease having 100% stability upto 55°Cfor 10 min. Kobayashi et al. [21] showed that alkalineserine protease of Bacillus sp. KSM-K16 remained sta-ble for 10 min at 50 and 60°C. Ferrero et al. [13] alsoreported thermostable protease from B. licheriformisMIR 29 showing 100% stability upto 70°C for 10 min.Compared to these findings, the B. Bre6is proteaseshowed much higher thermostability at 60°C.

The enzyme showed high pH stability being 100%stable at pH 10.5 and 92% at pH 11 and 11.5 for 1 h(data not shown). Further increase in pH resulted in asignificant decline in protease activity. These resultscorroborate the earlier observations on protease fromBacillus alcalophilus sub sp. halodurans exhibiting 100%stability over a pH range of 5–10 [22]. They alsoreported that protease activity was reduced to 50% atpH 11 and was completely inactivated at pH 13 after 10min incubation at 55°C. Takami et al. [23] reportedalkaline serine protease from Bacillus sp. AH-101 show-ing stability over a wide range of pH 5–13 after 10 minincubation at 60°C. They reported 80% enzyme activityin the pH range 5–9 which increased to 90% at pH10–11.5. Compared to these findings, protease pro-duced by B. bre6is was much more stable at high pH. Invitro studies using different concentrations of metalions showed a pronounced effect on enzyme activity(Table 3). Ag+ resulted in the complete loss of activityat 1 mM concentration, followed by Zn2+ and Hg2+.Residual activity in the presence of urea was 95%whereas with SDS it was 40%. Activity was stimulatedby calcium acetate, CaCl2 and MnSO4. Addition ofmagnesium salt, however, resulted in some loss ofactivity but its presence during growth and enzymeproduction was essential. Ca2+ and Mn2+ have beenshown previously to enhance protease activity [24]while the role of Ca2+ in the stabilization of proteasehas been reported from Thermus sp. subtilisin andBacillus sp. [21,22,25,26].

Protease from B. bre6is showed stability and compat-ibility with a wide range of commercial detergents at 50and 60°C in the presence of CaCl2 and glycine asstabilizers (Table 4). The enzyme retained about 60%activity after 1.5 h in the presence of Surf Excel® at60°C and was completely inactivated after 3 h in theabsence of any stabilizer (Fig. 5). However, the addi-tion of CaCl2 (10 mM) and glycine (1 M), individuallyand in combination, was very effective in improving thestability where it retained 58% activity even after 3 h.

Table 3Effect of various metal ions and chemicals on alkaline proteaseactivity by Bacillus bre6isa

Residual alkaline protease activity (%)

Metal ions and chemicals Concentration (mM)

1 2 5 10

100 100 100 100ControlZnCl2 0103037

110105 12099CaCl264NaCl 86 80 75

91 90 82MgSO4 90100 92 90KCl 95

124120114MnSO4 1070AgNO3 10 0 0

43 15HgCl2 0 0FeSO4 3243 -5060

7072 6274CoCl2CdCl2 18 0 0 0

0 0 0NiCl2 1519 0CuSO4 0 0

100 100Ca(CH3COOH)2 140 13540 32SDS 30 18

162026EDTA 895 94 92 90Urea

a The activity is expressed as a percentage of the activity level in theabsence of metal ions and chemicals. The enzyme was preincubatedwith metal ion (60°C, 5 min). Separate blanks with individual metalions and chemicals were prepared.

Thermostability profile (Fig. 3) indicated that theenzyme was stable at 25°C for 288 h whereas it retainedabout 70% of its maximum activity at 30 and 37°C. Itexhibited half lives of 60 and 7 h at 50 and 60°C,respectively. Addition of CaCl2 (10 mM) and glycine (1


Table 4Compatibility of alkaline protease activity from Bacillus bre6is with commercial detergents in the presence of glycine and CaCl2

a

Relative residual alkaline protease activity (%)

ControlTime (h) Surf Excel® Surf Ultra® Aerial Microshine® Rin

100 1000 100100 100890.5 8892 85 8585 8490 801.0 80

851.5 81 78 76 78822.0 73 72 70 60

70 6580 652.5 53583.0 5575 55 44

a The activity is expressed as a percentage of the activity level in the absence of detergents. The enzyme was preincubated with individualdetergent (60°C, 5 min). Separate blanks with individual detergent in the presence of glycine and CaCl2 were prepared.

The enzyme retained more than 50% activity with mostof the detergents tested even after 3 h incubation at60°C after the supplementation of CaCl2 and glycine

(Table 4). Bhosale et al. [27] reported high activityalkaline protease from C. coronatus showing compati-bility at 50°C, in the presence of 25 mM CaCl2, with avariety of commercial detergents. They reported thattheir enzyme preparation retained 16% activity inRevel®, 11.4% activity in Ariel® and 6.6% activity inWheel®. Comparing these results, the B. bre6is enzymewas significantly more stable in commercial detergents.

As the protease produced by our isolate, B. bre6is,was stable over a wide range of pH and temperatureand also showed compatibility with various commercialdetergents tested, it was used as an additive in deter-gent, to check the contribution of the enzyme in im-proving the washing performance of the detergent. Thesupplementation of the enzyme preparation in two de-tergents i.e. Surf Excel® and Rin® could significantlyimprove the cleansing performance towards the bloodstains (Fig. 6).

Acknowledgements

Authors gratefully acknowledge the assistance pro-vided by the Department of Science and Technologyand the Council of Scientific and Industrial Research,India. Communication no. IMT-010/98.

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Fig. 6. Washing performance of alkaline protease from B. bre6is inpresence of detergent. (A, Cloth stained with blood; B, blood stainedcloth washed with detergent only; and C, blood stained cloth washedwith detergent and enzyme.)


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