Alkaline serine protease from halotolerantBacillus licheniformis BA17

Annals of Microbiology, 59 (1) 83-90 (2009)

Alkaline serine protease from halotolerant Bacillus licheniformis BA17

Selçuk ÖZTÜRK1, Müşerref ÖZEREN-MORGAN1, Aydan Salman DILGIMEN2, Aziz Akın DENIZCI3, Burhan ARIKAN4, Dilek KAZAN2,3*

1Marmara University, Faculty of Art and Science, Department of Biology, Göztepe Campus, 81040 Ziverbey, Kadıköy, Istanbul; 2Marmara University, Faculty of Engineering, Bioengineering Department, Göztepe Campus, 34722 Kadıköy, Istanbul; 3The Scientific and Technological Research Council of Turkey (TÜBITAK), Genetic Engineering and Biotechnology Institute (GEBI), Marmara Research Center (MRC), P.O. Box 21, 41470 Gebze, Kocaeli; 4Çukurova University, Faculty of Art and Science, Department of Biology, Balcalı, Adana, Turkey

Received 7 July 2008 / Accepted 12 January 2009

Abstract - An alkaline protease from halotolerant Bacillus licheniformis BA17, isolated from Van Lake in Turkey, was purified 5.4 fold with 58% yield. The molecular weight was 19.7 kDa and the optimum temperature and pH were 60 °C and 10, respectively. The half-life of the pure enzyme was 38 h, 93 min, 14 min and 6 min at 40, 50, 60 and 70 °C, respectively. BA17 protease is very active at 30 °C between pH 8.0 and 10. Enzyme activity increased in the presence of Cu+2, Mg+2, Mn+2 and K+1 ions. Enzyme retained activity with 5% SDS (w/v) and 1% Triton X-100 (v/v). Inhibition with PMSF and EDTA suggested that the enzyme is a serine protease and is a metal-activated enzyme. Based on the N-terminal sequence of the first 13 amino acids, B. licheniformis BA17 alkaline protease did not show identity to any of those from other Bacillus species.

Key words: alkaline protease; Bacillus licheniformis; enzyme purification and characterization.

INTRODUCTION

One of the most important groups of hydrolytic enzymes is microbial alkaline proteases, since alkaline proteases have different industrial applications such as in peptide synthesis, detergents, laundry, food, leather and silk industries (Gessesse, 1997; Dodia et al., 2008). Accounting for about 35% of the total microbial enzyme sales, alkaline proteases are extensively used in the detergent industry. Halophiles have the ability to survive in hypersaline environments, being able to grow in the presence of 1 to 20% NaCl (w/v) and some can even grow in NaCl saturated waters (> 30% w/v) (Ventosa et al., 1998). The low solubility of halophilic enzymes has been used advantageously by applying them in aqueous/organic and non-aqueous media (Gomes and Steiner, 2004). Although there are many reports on proteases from alkaliphiles and thermophiles, proteases from halophiles and moderate halophiles have not been investigated extensively (Manachini

and Fortina, 1998; Ventosa et al., 1998; Gimènez et al., 2000; Joo and Chang, 2005; Amoozegar et al., 2007; Dodia et al., 2008). Since the microorganisms in soda lakes are mostly alkaliphilic, it is to be expected that the extracellular enzymes produced by these microorganisms would be active under alkaline conditions, moreover, active in the virtual absence of significant levels of Mg2+ and Ca2+. This is indeed the case, and such enzymes are of biotechnological interest, particularly as detergent additives, since detergents used in domestic and industrial washing processes are alkaline and contain sequestering agents to remove Ca2+ (which adversely affects the water hardness and foam characteristics). In this paper, we are reporting the purification and characterization of alkaline serine protease from a halotolerant Bacillus licheniformis BA17 isolated from Van (Soda) Lake, in Turkey.

MATERIALS AND METHODS

Chemicals. Chemicals used in the cultivation of the microor-ganism were supplied by Oxoid Ltd (Hampshire, England) and Merck AG (Darmstadt, Germany). All other chemicals used were obtained either from Merck AG, FLUKA (Switzerland), DIFCO (Michigan, USA) or Sigma Chem. Ltd. (St Louis, USA).

* Corresponding Author. Address: Marmara University, Faculty of Engineering, Department of Bioengineering, Göztepe Campus, 34722 Kadıköy, Istanbul, Turkey. Phone: +90 (216) 348 02 92; Fax: +90 (216) 348 02 93; E-mail: [email protected]

84 S ÖZTÜRK et al.

Microorganism and protease production. Halotolerant Bacillus licheniformis BA17 was isolated and identified on the basis of 16S rRNA gene sequencing as described previously (Ates et al., 2007). 16S rDNA sequence of the strain BA17 has been deposited in the NCBI databases under the accession number DQ176435. Alkaline protease production from B. licheniformis BA17 was carried out in the medium containing (% w/v) 1.0 maltose, 0.5 yeast extract, 0.1 K2HPO4, 1 Na2NO3 and 0.02 MgSO4·7H2O at 37 °C and 180 rpm (Ates et al., 2007). Medium pH was adjusted to 10.0 by aseptic addition of 10% Na2CO3 solu-tion after sterilization.

Purification of alkaline protease. After 24 h cultivation, Bacillus licheniformis BA17 cells were removed from the fermen-tation medium by centrifugation (10000 rpm, 15 min and 4 °C). The cell free culture supernatant was concentrated using stirred ultrafiltration cell (Millipore, Bedford, MA, USA). The molecular cut-off value for the membrane filter (Millipore) was 10000. Then, the upper phase containing 0.127 mg ml-1 protein was loaded onto DEAE-cellulose column (10 cm x 1.5 cm) equilibri-ated with 50 mM glycine-NaOH buffer (pH 10.5) and elution was carried out with the same buffer. At the subsequent step, the linear elution with NaCl at a concentration between 0.05 and 0.5 M in the same buffer was carried out on the column in order to elute the bounded proteins. All fractions were collected and assayed for enzyme activity. The fractions which exhibited pro-tease activity were pooled and used for characterization studies. Protein amount was measured by Coomassie Blue G-250 bind-ing method (Sedmak and Grosberg, 1978; Spector, 1978) using bovine serum albumin as the standard.

Enzyme assay. The method described by Takami et al. (1989) was used for the determination of alkaline protease activity. One unit of alkaline protease activity was defined as the amount of enzyme able to produce 1 µg tyrosine per minute under the assay conditions as 30 °C and pH 10.5.

Examination of purity and estimation of the molecular weight of protease. Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the method of Laemmli (1970) using 5% (w/v) stacking gel and 12% (w/v) resolving gel. Electrophoresis was performed at a constant 100 V for 15 min and 120 V for 75 min in Tris-glycine buffer, pH 8.3. Enzyme samples were denatured by boiling for 3 min in the presence of 5 mM phenylmethylsulfonylfluoride (PMSF) before loading onto the gel. After electrophoresis, proteins in the separating gel were visualized by silver staining (Damerval et al., 1978). Fermentas Protein Molecular Weight Marker SM0431 (MBI Fermentas, St. Leon Rot., Germany) containing seven proteins within 14.4-116 kDa range was used in order to determine the molecular weight of enzyme by Image Analyzer System (UVI, BTS-20.M). Native gel electrophoresis was also performed using 5% (w/v) stacking gel and 8% (w/v) acidic (pH 4.3) and basic (pH 8.8) separating gels. Electrophoresis was performed at 100 V for 30 min and 120 V for 150 min at 4 °C. The protein bands were visualized by silver staining (Damerval et al., 1978).

Zymogram. To prepare a zymogram, SDS-PAGE and native electrophoresis were carried out. SDS-PAGE was carried out according to a method described by Kazan et al. (2005). A clear zone on the destained gel indicates the presence of alkaline protease activity.

Native gel electrophoresis was performed using 5% (w/v) stacking and 8% (w/v) acidic (pH 4.3) separating gels at 100 V for 30 min and 120 V for 150 min at 4 °C. This gel was placed on a 1%(w/v) agarose gel containing 1% (w/v) skim milk and was then dipped into 50 mM glycine-NaOH buffer, pH 10.5, con-taining 0.25% (v/v) Tween-20. After 2 days of incubation at 37 °C, the native gel was separated from the agarose gel and TCA buffer was poured onto the agarose gel. A clear zone on the gel indicated the presence of alkaline protease activity.

Effect of temperature on activity and stability of enzyme. The effect of temperature on enzyme activity was determined by carrying out the enzyme assay in a temperature range between 20-80 °C in the presence and absence of 5 mM Ca+2 ions. Enzyme activity at each temperature was measured as described above. In order to determine the thermal stability profile of the enzyme, 0.5 ml of enzyme solution was incubated at 40, 50, 60 and 70 °C at different time intervals and the residual activity at each temperature was determined. The thermal stability was expressed as percent residual activity by taking the initial activity of the enzyme as 100% at each temperature studied. The half-life was calculated from the in:

(Ei/E0) = - kit linear plot by placing E0 = 2Ei

where E0 is the initial activity of enzyme before heat treatment, Ei is the initial activity of enzyme after heat treatment.

Effect of pH on activity and stability of enzyme. The effect of pH on activity of the enzyme was evaluated with 50 mM glycine-NaOH buffer (pH 8.0-12.0) and 200 mM glycine-NaOH buffer (pH 12.5 and 13.0). Enzyme activity at each pH was measured as described above. In the case of pH stability profile estimation of the enzyme, 0.1 ml of the enzyme solution in 50 mM glycine-NaOH buffer, pH 10.5 was mixed with 0.4 ml of buffers at pH ranging from 8.0-13.0. The mixtures were incubated for 2.5 h at room temperature and the residual activity at each pH value was measured. The pH stability was expressed as percent residual activity by taking the initial activity of the enzyme as 100% at each pH studied.

Effect of different ions on alkaline protease activity. The effects of Mn+2, Ca+2, Fe+2, Co+2, Mg+2, Cu+2 and K+1 ions on alkaline protease activity were investigated by adding these ions to the reaction mixture at a concentration of 5 mM. Relative enzyme activities were measured at 30 °C.

Effect of active site inhibitors on enzyme activity. The effects of active site inhibitors on protease activity were studied using diethyl-pyrocarbonate (DEPC), Np-tosyl-L-lysinechloro-methylketone (TLCK) and Np-tosyl-Lphenylalaninechloro-methyl ketone (TPCK), phenylmethylsulfonylfluoride (PMSF), ethyl-acetimidate, phenylglyoxal, N-bromosuccinimide, N-ethyl-5-phenyl-iso-oxasolium-3-sulfonate, iodoacetic acid, iodoacetimi-date, N-ethylmaleimidate and ethylene diamine tetra acetic acid (EDTA). Active site inhibitors were dissolved in 50 mM NaOH-glycine-NaCl buffer, pH 10.5. Enzyme solution was preincubated with 1, 5 or 10 mM of each inhibitor for 2 h at 30 °C. The percent residual activity of enzyme solution was measured by using casein as the substrate. The percentage of activity was expressed as percent residual activity by taking activity as 100% prior to incubation.

Ann. Microbiol., 59 (1), 83-90 (2009) 85

Effect of surface-active agents on alkaline protease activ-ity. The effects of 0.5% (w/v) SDS, 1% Tween-20 and 1% Tritone X 100 on alkaline protease activity were investigated by incubating the reaction mixtures for 2.5 h at 30 °C. The percent-age of activity was expressed as percent residual activity by tak-ing activity as 100% prior to incubation.

Substrate specificity of enzyme. p-nitroanilide (pNA)-conju-gated synthetic peptide substrates N-Suc-Ala-Ala-Pro-Leu-pNA, N-α-benzyl-L-Arg-pNA (L-BAPNA), N-Suc-Ala-Ala-Pro-Phe-pNA, N-Suc-Gly-Gly-Phe-pNA and L-Leu-pNA were used to determine the substrate specificity of the enzyme. Enzyme activity with these substrates was determined according to method of Singh et al. (2001). One unit of enzyme activity is defined as the amount of enzyme that liberates 1 µmol pNA per minute at 37 °C and pH 10.5.

Determination of the kinetic parameters of enzyme. The initial reaction rates for casein were measured in the 0.1-2.00 mg ml-1 concentration range of this substrate at 30 °C and pH 10.5. The Km and Vmax values of the purified enzyme for both substrates were determined according to Michaelis-Menten kinet-ics using a Lineweaver-Burk plot.

N-terminal amino acid sequence analysis. N-terminal amino acid sequence of the purified protease was performed by Edman Sequencer (ABI Model 492A precise sequencer, Model 785A detector and Model 140C microgradient systems sequencer) in WITA GMBH (Germany).

RESULTS AND DISCUSSION

Purification and identification of the alkaline protease of Bacillus licheniformis BA17The purification profile of alkaline protease from Bacillus licheniformis BA17 and the elution profile of the enzyme from DEAE-cellulose anion exchange chromatography column are given in Table 1 and Fig. 1, respectively. In this work, ammonium sulphate, acetone and ethanol at different concentrations were used to precipitate proteins from crude enzyme preparation (data not shown). Ultrafiltration was also used to concentrate the protein solution. Since, the best yield (88%) was obtained by ultrafiltration using 10000 cut-off ultrafilter, the upper phase obtained from ultrafiltration was applied to a DEAE-cellulose ion exchange column (10 x 1.5 cm diameter) and elution was carried out by 50 mM glycine-NaOH buffer, pH 10.5. Although a linear elution was also applied using NaCl at a concentration between 0.05 and 0.5 M, alkaline protease of B. licheniformis BA17 was eluted in the unbounded fraction of the anion exchanger DEAE-cellulose column (Fig. 1). A 5.4-fold purification of the enzyme was obtained with 58% yield and 225.46 U mg-1

specific activity. Comparing our results with the purification yield of three thermostable alkaline proteases (Prt I, Prt II and Prt III) as 71, 21 and 8% from a halotolerant strain of B. licheniformis (Manachini and Fortina, 1998), the yield obtained in this work was higher than Prt II and Prt III. Joo and Chang (2005) reported the purification of an alkaline protease from the halotolerant Bacillus clausii I-52 using Diaion HPA75, phenylsepharose and DEAE sepharose colomn chromatography. The purification yield as 78.9% and purification fold as 9.6 obtained for B. clausii I-52 alkaline protease was higher than that of our work. Singh et al. (2001) reported a similar procedure for the purification of serine alkaline protease from Bacillus sp. SSR1. Their purification yield of 36.9% was slightly lower than that of our work but their purification was significantly lower. Dodia et al. (2008) applied a single step purification method with phenyl sepharose 6FF for the purification of alkaline serine protease from haloalkaliphilic sp. AH-6. The 23-fold purification obtained by Dodia et al. (2008) was considerably higher than our result (5.4-fold), but the purification yield as 28% was lower than our process (58%). Silver staining after SDS-PAGE analysis of the alkaline pro-tease of B. licheniformis BA17 showed that the molecular weight of enzyme was 19.4 kDa (Fig. 2). The molecular weight of alka-line proteases generally ranges from 26-130 kDa (Gupta et al., 2005; Joo and Chang, 2005; Kazan et al., 2005; Dodia et al., 2008). However the molecular weight of B. licheniformis BA17 as 19.4 kDa was lower than that of other alkalophilic and halophilic proteases except for protease I from halotolerant B. licheniformis (Manachini and Fortina, 1998) which was 15 kDa. The zymogram revealed the higher level of protease activity on the gel, which corresponded to a single band, was obtained in SDS-PAGE and

TABLE 1 - Purification of alkaline protease from Bacillus licheniformis BA17 (250 ml fermentation medium)

Step Total activity (U)

Total protein (mg)

Specific activity (U/mg)

Yield (%)

Purification fold

Crude enzyme 793.8 19 41.77 100 1Ultrafiltration 705 5.09 138 88 3.31Pooled fractions of DEAE-cellulose 465 2.06 225 58 5.4

302520151050

1

0,8

0,6

0,4

0,2

0

12

10

8

6

4

2

0

Fraction No

OD

280

Enz

yme

acti

vity

, (U

ml-1

min

-1)OD280

Activity

FIG. 1 - Purification profile of the alkaline protease from Bacillus licheniformis BA17 by DEAE-cellulose column (10 cm x 1.5 cm) equilibrated with 50 mM glycine-NaOH buffer (pH 10.5), elution was carried out with the same buffer.

86 S ÖZTÜRK et al.

native PAGE gel electrophoresis (Fig. 2). Alkaline proteases do not bind to anion-exchange column materials due to their positive surface charges (Gupta et al., 2002). In accordance to this feature, BA17 alkaline protease was eluted in the unbounded fraction of the anion exchanger DEAE-cellulose column and the BA17 pro-tease band was detected on a pH 4.3 gel in native PAGE (Fig. 2). The N-terminal sequence of first 13 amino acid residues of the purified alkaline protease was found to be A-S-P-X-X-G-A-I-V-T-Q-T-D and the N-terminal sequences of alkaline proteases from other Bacillus strains such as B. mojavensis, B. licheniformis, B. subtilis, B. stearothermophilus, B. amyloliquefaciens, B. lentus,

Bacillus sp. G-825-6, Bacillus sp., B. alcalophilus PB92, Bacillus sphaericus, and halotolerant B. subtilis FP-133 (Beg and Gupta, 2003; Setyorini et al., 2006) are given in Table 2. There were only two common residues with between BA17 and the other alkaline proteases. This finding, in addition to the comparison made using the search program of the Gen-Bank database, showed that there was no close homology between BA17 alkaline protease and other proteases. The B. licheniformis BA17 alkaline protease is presumably a novel protease but the novelty remains to be verified by determining the complete amino acid sequence of this protease.

FIG. 2 - A: SDS-PAGE of Bacillus licheniformis BA17 alkaline protease. Lane 1: protein molecular weight markers, lane 2: fermenta-tion fluid, lane 3: retentate of ultrafiltration, lane 4: DEAE-cellulose column eluate, lane 5: SDS-PAGE zymogram of purified protease. B: Native PAGE of B. licheniformis BA17 alkaline protease. Lane 1: DEAE-cellulose column eluate, lane 2: native PAGE zymogram of purified alkaline protease.

TABLE 2 - Comparison of N-terminal amino acid sequence of alkaline protease from Bacillus licheniformis BA17 with other types of alkaline proteases from other Bacillus strains and two halotolerant proteases

Type and microbial source N-terminal amino acid sequence

B. licheniformis BA17 alkaline protease A S P X X G A I V P Q T DAlkaline protease (B. mojavensis)* A Q T B P H G I P L I K ASubtilisin Carlsberg (B. licheniformis)* A Q T B P Y G I P L I K ASubtilisin AML (B. subtilis. var. amylosacchariticus)* A Q S B P Y G I S Q I K KSubtilisin E (B. subtilis)* A Q S V P Y G I S Q I K KDubtilisin BPN (B. amyloliquefaciens)* A Q S V P Y G V S Q I K KSubtilisin J (B. stearothermophilus)* A Q S V P Y G I S Q I K KSubtilisin NAT (B. subtilis, natto)* A Q S V P W G I S Q I K KSubtilisin Sendai (Bacillus sp. G-825-6)* A Q V T P W G I T R V Q QSavinase (B. lentus)* A Q S V P W G I S R V Q QNo. 221 protease (Bacillus sp.)* A Q S V P W G I S R V Q QPB 92 protease (B. alcalophilus PB92)* A Q S V P W G I S R V Q QEsperase (B. lentus)* - Q W V P W G I S F I N NAH-101 protease (Bacillus sp.)* - Q T V P W G I S F I S SProtease B (B. sphaericus)* - G T V P W G I P Y I Y YProtease BYA (Bacillus sp.)* - N P V A R G I - - V K KEnastase Ya-B (Bacillus sp.)* - Q T V P W G I N R V Q QExpro-I (Halotolerant B. subtilis FP-133)** A E S V P Y G V S E I K AExpro-II (Halotolerant B. subtilis FP-133)** A D A T G X G G N Q X T G

* Data obtained from Beg and Gupta (2003); ** Data obtained from Setyorini et al. (2006).

1 2 3 4 5

A B

1 2

116.0 kDa

66.2 kDa

45.0 kDa

35.0 kDa

25.0 kDa

18.4 kDa

Ann. Microbiol., 59 (1), 83-90 (2009) 87

Effect of temperature and pH on BA17 alkaline protease activity and stabilityThe temperature profiles of the purified alkaline protease of B. licheniformis BA17 are shown in Fig. 3A. The half-life of the enzyme was 39 h at 40 °C, 1.5 h at 50 °C and 12 min at 60 °C (Fig. 3B). Similar results were obtained for proteases from halophilic Salinivibrio sp. AF-2004 (Amoozegar et al., 2007), halotolerant B. clausii I-52 (Joo et al., 2003), B. licheniformis MIR29 (Ferrero et al., 1996), various Bacillus species (Beg and Gupta, 2003) and B. subtilis FP-133 (Setyorini et al., 2006). A higher temperature optima as 70 °C has been reported for three proteases from halotolerant B. licheniformis by Manachini and Fortina (1998) but the optimum temperature of alkaline serine protease from haloalkaliphilic bacterium sp. AH-6 was as low as 37 °C. At 70 °C, the purified BA17 alkaline protease retained 23% of its original activity for 10 min incubation. However, the bulk BA17 protease has not lost its activity for 5 h at 70 °C (data not shown). The purified BA17 alkaline protease was

active in a relatively lower temperature range compared to the alkaline protease of haloterolant B. clausii I-52 (Joo et al., 2003) and of B. majovensis (Beg and Gupta, 2003). However, the crude BA17 protease retained its activity for 5 h at 50, 60 and 70 °C, respectively (data not shown). Considering these findings, our results showed that the alkaline protease of B. licheniformis BA17 has a higher thermostability in the 30-60 °C temperature range than many other alkaline proteases. The pH optimum of enzyme was found to be 10.0 (Fig. 4). The purified alkaline protease has not lost its activity at pH 10.0 for 2.5 h, however, 15% and 50% activity losses were measured at pH values 11.0 and 12.0, respectively (Fig. 4). Similar results to these findings were obtained for alkaline proteases from halotolerant B. clausii I-52 (Joo et al., 2003), Bacillus sp. (Patel et al., 2006), and B. licheniformis RP1 (Sellami-Kamoun et al., 2008). The optimum pH as 9.0 for three alkaline proteases from B. licheniformis reported by Manachini and Fortina (1998) was lower than that of BA17 alkaline protease. Sellami-Kamoun et al. (2008) discovered that the alkaline protease from B. licheniformis RP1 is stable at 40 °C for 1 h at pH 8.0-10.0; however, its stability dropped to 72 and 96% at pH 11.0 and 12.0, respectively. Comparing the pH stability of B. licheniformis RP1 protease with BA17 protease, BA17 protease has higher pH stability than RP1 protease. In general, all currently used detergent compatible enzymes have a high optimum pH and are thermostable in nature, since the pH of the laundry detergents is generally in the range of 9.0-12.0 and with varying thermo-stabilities at temperature ranges between 50 and 70 °C (Beg and Gupta, 2003). Consequently, considering its pH and temperature optima, the alkaline protease of B. licheniformis BA17 can be proposed as a convenient addi-tive for commercial detergents.

Effect of amino acid inhibitors and metal ions on alkaline protease of Bacillus licheniformis BA17The effects of metal ions on alkaline protease of B. licheniformis B17 are given in Table 3. Among the metal ions tested at 30 °C, enzyme activity increased in the presence of Cu+2, Mg+2, K+1 and

141312111098

100

80

60

40

20

0

100

80

60

40

20

0

pH

Rel

ativ

e ac

tivity

(%

)

Res

idua

l act

ivit

y (%

)

FIG. 3 - A: The temperature profile of the alkaline protease from Bacillus licheniformis BA17 in the absence (m) and presence (●) of 5 mM Ca+2 ions. B: Inactivation kinetics of B. licheniformis BA17 alkaline protease at different temperatures.

FIG. 4 - The pH stability (●) and the pH profile (m) of the alkaline protease from Bacillus licheniformis BA17.

A B

9080706050403020100

100

80

60

40

20

0

Temperature (°C)

Rel

ativ

e ac

tivity

(%

)

0 mM Ca+2

5 mM Ca+2

300250200150100500

0

-0,5

-1

-1,5

-2

-2,5

-3

Time (min)

Ln

(Eý/

E0)

40°C 50°C 60°C

88 S ÖZTÜRK et al.

Mn+2 ions, despite the reports on strong inhibitor effects of Cu+2, Mg+2 and Mn+2 ions on alkaline proteases of some Bacillus sp. (Singh et al., 2001; Beg and Gupta, 2003; Gupta et al., 2005; Setyorini et al., 2006). While 6 and 27% activity losses were observed in the presence of Co+2 and Fe+2 ions, Ca+2 did not show any inhibitory effects on enzyme activity, similar to reports (Beg and Gupta, 2003; Patel et al., 2006) where no (or only a slight) inhibitory effect of Ca+2 ion on enzyme activities have been detected. Residual activity of BA17 alkaline protease in the presence of amino acid inhibitors are given in Table 4. Among the active site directed irreversible inhibitors studied, PMSF at 1 and 5 mM con-centrations caused complete loss of the alkaline protease activity within 2 h at 30 °C. This enzyme is a serine alkaline protease, since PMSF caused complete loss of alkaline protease activity. Similarly, the majority of the alkaline proteases of Bacillus sp. are completely inhibited by the serine protease inhibitor PMSF

(Singh et al., 2001; Beg and Gupta, 2003; Joo et al., 2003; Kazan et al., 2005; Dodia et al., 2008). EDTA, 1 mM and 5 mM, reduced the protease activity of B. licheniformis BA17 to 39 and 19%, respectively. Such unusual inhibition of bacterial protease by both EDTA and PMSF was also obtained for B. cereus MTCC 6840 alkaline protease (Joshi et al., 2007). Since the Cu+2, Mg+2 and Mn+2 ions cause an increase in the activity of BA17, it can be concluded that the enzyme could be a metal-activated enzyme and it has a serine residue at the active site.

Effect of surface active agents on alkaline protease activityAs given in Table 5, almost 30% of enzyme activity loss was observed in the presence of 5% SDS (w/v). Triton X-100 at 1% (v/v) concentration did not effect the enzyme activity. However, in the presence of 1% Tween 20 (v/v), 23.5% increase in acti-vity was found. In order to be an effective detergent additive under washing conditions; the alkaline proteases should be stable against the effects of various surface active agents available in detergent composition. The alkaline protease of B. licheniformis BA17 showed considerable stability towards Triton X-100, Tween 20 and SDS, and therefore this also makes it a good candidate as an additive for commercial detergents. Hadj-Ali et al. (2007) report-ed similar results for B. licheniformis NH1 alkaline protease. NH1 alkaline protease was highly stable in the presence of non-ionic surfactants as Tween-20 and Triton X-100. They also reported that 5% SDS caused 20% loss of NH1 protease activity.

Substrate specificity of alkaline proteaseThe substrate specificity of alkaline protease was investigated using various synthetic peptide peptidyl-pNAs (Table 6). Among the substrates studied, the highest specificity was observed for

TABLE 5 - Effect of surface-active agents on Bacillus licheniformis BA17 alkaline protease activity without incubation and after 2.5 h incubation

Surface-active agent Residual activity (%)* Residual activity (%)**

Free enzyme (FE) 100

FE+5% SDS (w/v) 70 74

FE+1% Tween 20 (v/v) 124 111

FE+1% TritonX-100 (v/v) 98 96

* Without incubation; ** 2.5 h incubation.

TABLE 3 - Effect of metal ions (5 mM) on Bacillus licheniformis BA17 alkaline protease

Ion Relative activity (%)Free enzyme (FE) 100

FE+Co+2 94

FE+Cu+2 123

FE+Mg+2 104

FE+Fe+2 73

FE+Mn+2 106

FE+Ca+2 100

FE+K+1 112

TABLE 4 - Effect of amino acid inhibitors on Bacillus licheniformis BA17 alkaline protease

Active site inhibitor Concentration (mM) Residual activity (%)

Phenyl methyl sulphonyl fluoride (PMSF) 1 3

5 0

Ethylene diamine tetra acetic acid (EDTA) 1 39

5 19

Diethyl pyrocarbonate (DEPC) 10 100

Iodoacetemide 10 100

N-α-p tosyl-L-lysine chloro-methyl ketone (TLCK) 10 51

Ethylacetimidate 10 98

Phenylglyoxal 10 98

Iodoacetic acid 10 95

N-Ethyl maleimidate 10 100

Ann. Microbiol., 59 (1), 83-90 (2009) 89

N-Suc-Ala-Ala-Pro-Pne-pNA which was in accordance with the reports for alkaline protease from halotolerant B. clausii I-52 (Joo and Chang, 2005).The enzyme had a relatively low specificity for N-α-Benzol-Leu-Arg-pNA, N-Suc-Gly-Gly-Phe-pNA and had no specificity for L-leu pNA. The B. licheniformis alkaline protease hydrolysed native proteinacious substrates as skim-milk, haemoglobin, gelatine and BSA. The enzyme had a higher specificity for skim milk and casein (data not shown). Km and Vmax values for hydrolysis of casein were found to be 0.209 mg/ml and 5.5 µmol tyrosine/ml/min, respectively. Considering its high activity and stability at high tempera-tures, pH and in the presence of surfactants, B. licheniformis BA17 alkaline serine protease may find potential application in laundry detergents. In addition, BA17 alkaline protease was active in the absence of Ca2+. Since detergents used in domes-tic and industrial washing processes are alkaline and contain sequestering agents to remove Ca2+ (which adversely affects the water hardness and foam characteristics), B. licheniformis BA17 alkaline protease would be of biotechnological interest, particu-larly as a detergent additive.

AcknowledgementThis research was supported by The Scientific and Technical Research Council of Turkey (TUBITAK-TBAG) by the Project No. TBAG 2321-(103T069) and Marmara University Research Foundation (BAPKO) by the project number BSE - 073/1311.

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TABLE 6 - Substrate specificity of Bacillus licheniformis BA17 alkaline protease toward synthetic substrates

Synthetic substrate Relative activity (%)

N-Suc-Ala-Ala-Pro-Leu-pNA 63.4

L-Leu-pNA 0

N-Suc-Ala-Ala-Pro-Phe-pNA 100

N-Suc-Gly-Gly-Phe-pNA 0.13

N-α-Benzol-Leu-Arg-pNA 0.6

90 S ÖZTÜRK et al.

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