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Purification and characterization of an alkaline serine-protease produced by a new isolated Aspergillus clavatus ES1
Process Biochemistry 42 (2007) 791–797 www.elsevier.com/locate/procbio
Purification and characterization of an alkaline serine-protease produced by a new isolated Aspergillus clavatus ES1 Mohamed Hajji, Safia Kanoun, Moncef Nasri *, Ne´ji Gharsallah Laboratoire de Ge´nie Enzymatique et de Microbiologie, Ecole Nationale d’Inge´nieurs de Sfax, B.P. ‘‘W’’, 3038 Sfax, Tunisia Received 1 August 2006; received in revised form 12 January 2007; accepted 25 January 2007
Abstract An extracellular bleach stable protease from the fungus Aspergillus clavatus ES1, isolated from wastewater, was purified and characterized. The protease of ES1 strain was purified to homogeneity using acetone precipitation, Sephadex G-100 gel filtration and CM-Sepharose ion exchange chromatography, with a 7.5-fold increase in specific activity and 29% recovery. The molecular mass was estimated to be 32 kDa on SDS-PAGE. The optimum pH and temperature for the proteolytic activity were pH 8.5 and 50 8C, respectively. The enzyme was stable in the pH range of 7.0– 9.0. The protease was activated by divalent cations such as Ca2+ and Mg2+. The alkaline protease showed extreme stability towards non-ionic surfactants (5% Tween 80 and 5% Triton X-100). In addition, the enzyme was relatively stable towards oxidizing agents, retaining more than 71 and 53% of its initial activity after 1 h incubation in the presence of 1 and 2% (w/ v) sodium perborate, respectively. The N-terminal sequence of the first 15 amino acids of the purified alkaline protease of A. clavatus ES1 showed high similarity with other fungal alkaline proteases. The activity was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serineprotease. # 2007 Elsevier Ltd. All rights reserved. Keywords: Serine alkaline protease; Purification; Aspergillus clavatus; Bleaches; Detergents
1. Introduction Proteases are the most important industrial enzymes accounting for approximately 60% of the total industrial enzyme market [1]. They have diverse applications in a wide variety of industries, such as in detergent, food, pharmaceutical, leather, silk and recovery of silver from used X-ray films [2,3]. The industrial demand of proteolytic enzymes, with appropriate specificity and stability to pH, temperature and surfactants, continues to stimulate the search for new enzyme sources. Proteases with high activity and stability in high alkaline range and high temperatures are interesting for bioengineering and biotechnological applications. Their major application is in detergent industry because the pH of laundry detergents is generally in the range of 9.0–12.0. Alkaline proteases are used as cleaning additives in detergents to
* Corresponding author. Tel.: +216 96 501 698; fax: +216 74 275 595. E-mail addresses: [email protected], [email protected] (M. Nasri). 1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2007.01.011
facilitate the release of proteins. Proteases in detergent industries account for approximately 30% of the total world enzyme production [4]. Alkaline proteases are particularly important because they are both stable and active at high pH solutions and in the presence of surfactants and oxidizing agents [5]. They are produced by a wide range of microorganisms including bacteria, moulds, yeasts and also mammalian tissues. Most of the commercial alkaline proteases were isolated from Bacillus species [6]. A considerable number of fungal species are known to produce extracellular alkaline proteases such as Conidiobolus coronatus [7], Arthrobotrys olgospora [8], Trichoderma harzianum [9], Cephalosporium sp. KM388 [10] and A. fumigatus [11]. Isolation and screening of microorganisms from naturally occurring alkaline habitats or from alkaline wastewater was expected to provide new strains producing enzymes active and stable in highly alkaline conditions. This article deals with the purification and characterization of an alkaline protease produced by Aspergillus clavatus ES1 strain isolated from wastewater.
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2. Materials and methods 2.1. Materials Column chromatography materials, molecular markers for electrophoresis and casein were purchased from Sigma Chemicals Co. St. Louis, USA. All the microbiological media components were product of Bio-Rad, France. Other chemicals used were of analytical grade.
2.2. Methods 2.2.1. Microorganism A. clavatus ES1 used throughout this study was isolated from wastewater. The isolate was propagated on potato-dextrose-agar plates at 30 8C, and inocula were prepared from 7-day-old colonies by flooding with 10 ml of sterile distilled water and scraping off the agar plates. 2.2.2. PCR amplification and ITS of 18S rDNA gene sequencing Fungal DNA was extracted from freeze-dried mycelia according to RuizDuenas et al. [12]. PCR amplification of the internal transcribed spacer ‘ITS’ region of the ribosomal DNA was performed using primers ITS1 (50 TCCGTAGGTGAACCTGCGG-30 ) and ITS4 (50 -TCCTCCGCTTATTGATATGC-30 ) [13]. PCR product was cloned using pMOSBlue vector (Amersham). Nucleotide sequencing of the cloned PCR product was performed following the application of automated DNA sequencing method based on dideoxynucleotide chain termination method [14] using universal primers: U-19 50 -GTTTTCCCAGTCACGACG-30 and T7 50 -TAATACGACTCACTATAGGG-30 . The nucleotide sequence was analyzed with the GenBank database using BLAST program [15]. 2.2.3. Culture and growth conditions The medium used for isolation of protease producing strains was consisted of (g/l): peptone, 5.0; yeast extract, 3.0; skimmed milk 25% (v/v) and bacteriological agar, 12.0; pH was adjusted to 9.0. The medium used for protease production by ES1 strain was composed of (g/l): CaCl27H2O 0.4; KH2PO4 1.0; Na2HPO4 0.8; MgSO47H2O 0.5; ZnCl2 0.1; NaCl 0.3; whole sardinella (Sardinella aurita) fish flour 2.0 and wheat bran 10 (pH 6.0). Media were autoclaved at 120 8C for 20 min. Culture media were inoculated with 107 spores/ml and incubated on a rotatory shaker (150 rev/min) for 72 h at 30 8C, in 300 ml Erlenmeyer flasks with a working volume of 30 ml. The cultures were centrifuged to remove fungi mycelia and the supernatants were used for estimation of proteolytic activity. All experiments were carried out in duplicate and repeated at least twice. 2.2.4. Assay of protease activity Protease activity was measured by the method of Kembhavi et al. [16] using casein as a substrate. Half milliliter of the enzyme, suitably diluted, was mixed with 0.5 ml 100 mM Tris–HCl (pH 8.5) containing 1% casein, and incubated for 15 min at 50 8C. The reaction was stopped by addition of 0.5 ml trichloroacetic acid (20%; w/v). The mixture was allowed to stand at room temperature for 15 min and then centrifuged at 10,000 g for 15 min to remove the precipitate. The acid soluble material was estimated spectrophotometrically at 280 nm. A standard curve was generated using solutions of 0–50 mg/l tyrosine. One unit of protease activity was defined as the amount of enzyme required to liberate 1 mg of tyrosine per min under the experimental conditions used. 2.2.5. Protein determination Protein concentration was determined by the method of Bradford [17] using bovine serum albumin as a standard. 2.2.6. Protease purification 2.2.6.1. Acetone treatment. The culture supernatant containing the extracellular enzyme was first subjected to acetone precipitation. Acetone fractions of 0–40%, 40–60% and 60–80% (v/v) were collected by centrifugation at 10,000 g and the pellet obtained in each fraction was suspended in a minimal volume of 100 mM Tris–HCl, pH 8.0. 2.2.6.2. Sephadex G-100 gel filtration. The 60–80% (v/v) acetone fraction was subjected to gel filtration on a Sephadex G-100 column (3 cm 100 cm)
equilibrated with 25 mM Tris–HCl pH 8.0 containing 0.05% (v/v) Triton X100. Enzyme fractions of 5 ml were collected at a flow rate of 25 ml/h with the same buffer. Protein content (Abs 280 nm) and protease activity were measured. Fractions showing protease activities were pooled.
2.2.6.3. CM-Sepharose separation. The active fractions were applied to a CM-Sepharose column (3 cm 30 cm) equilibrated with 25 mM Tris–HCl buffer pH 8.0. After being washed with the same buffer, bound proteins were eluted with a linear gradient of sodium chloride in the range of 0– 0.5 M in the equilibrating buffer. Fractions (5 ml each) were collected at a flow rate of 120 ml/h. The fractions with high protease activity were pooled and stored at 20 8C for further analysis. All the purification steps were conducted at temperatures not exceeding 4 8C.
2.2.7. Polyacrylamide gel electrophoresis Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out for the determination of purity and molecular mass of the enzyme as described by Laemmli [18] using a 5% (w/v) stacking gel and a 15% (w/v) separating gel. The molecular mass of the enzyme was estimated using a low molecular mass calibration kit as markers consisting of bovine serum albumin (66 kDa), egg white ovalbumin (45 kDa), glyceraldehyde-3-P deshydrogenase (36 kDa), bovine carbonic anhydrase (29 kDa), bovine trypsinogen (24 kDa), soybean trypsin inhibitor (20.1 kDa) and bovine a-lactalbumin (14.2 kDa).
2.2.8. N-terminal amino acid sequence of the enzyme The purified enzyme was electrophoretically transferred from SDS-PAGE to a polyvinylidiene difluoride membrane (PVDF). The region containing the protease band on the PVDF was excised and the protein N-terminal amino acid sequence was determined by the Edman degradation method on an ABI Procise 494 protein sequencer (Applied Biosystems).
2.2.9. Effect of pH and temperature on the activity and stability of the enzyme The optimum pH of the purified enzyme was studied over a pH range of 6.0– 11.0. For the measurement of pH stability, the enzyme was kept at 4 8C for 1 h in different buffers at 100 mM and the residual proteolytic activity was determined under standard assay conditions. The following buffer systems were used: phosphate buffer, pH 6.0–7.5; Tris–HCl buffer, pH 8.0–8.5; glycine–NaOH buffer, pH 9.0–11.0. To analyse the effect of temperature, the activity was tested at different temperatures (40–70 8C) using casein as substrate for 15 min at pH 8.5. Thermal inactivation was examined by incubating the purified enzyme at 30, 40, 50 and 60 8C for 60 min. Aliquots were withdrawn at desired time intervals to test the remaining activity at pH 8.5 and 50 8C. The non-heated enzyme was considered as a control (100%).
2.2.10. Effect of metal ions, oxidizing agents and enzyme inhibitors The effect of various metal ions (5 mM) on enzyme activity was investigated using CaCl2, MnSO4, ZnSO4, CoSO4, CuSO4, BaCl2 and MgSO4. The effects of some surfactants (Triton X-100, Tween 80 and SDS) and oxidizing agents (H2O2 and sodium perborate) on enzyme stability were also studied by pre-incubating enzyme for 1 h at 4 8C. The residual activity was measured at pH 8.5 and 50 8C. The activity of the enzyme (without any surfactants) was considered as 100%. The effects of enzyme inhibitors on protease activity were studied using phenylmethylsulfonyl fluoride (PMSF), dithio-bis-nitrobenzoic acid (DTNB) and ethylene-diaminetetraacetic acid (EDTA). The purified enzyme was preincubated with inhibitors for 30 min at 4 8C and then the remaining enzyme activity was estimated using casein as a substrate.
2.2.11. Nucleotide sequence accession number The ITS nucleotide sequence of the 18S rDNA gene has been submitted to the GenBank database and assigned accession number EF151929.
M. Hajji et al. / Process Biochemistry 42 (2007) 791–797
Fig. 1. Phylogenetic tree of A. clavatus ES1 associated with the other members of the Aspergillus genus. Topology was inferred using the neighbour-joining method [19].
3. Results and discussion
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Fig. 2. Purification profile of alkaline protease from A. clavatus ES1 by gel filtration on Sephadex G-100 column. The concentrated enzyme preparation (60–80%) was applied to a 3 cm 100 cm column, equilibrated and eluted with 25 mM Tris–HCl buffer (pH 8.0) at a flow rate of 25 ml/h.
3.1. Identification of the microorganism and phylogenetic analysis Several fungi strains producing extracellular proteases were screened from different biotopes. Of these, isolate ES1 exhibiting a large zone of hydrolysis on skim milk agar plates was selected. In order to identify ES1 strain, the internal transcribed spacer region of 18S rDNA (860 pb) was amplified, cloned in pMOSBlue vector and sequenced. The ITS nucleotide sequence was analyzed with the GenBank database using BLAST program and showed 100 and 95% homology with ITS of A. clavatus and A. fumigatus strains, respectively. Multiple sequence alignment was performed with CLC Gene Workbench software version (2.0.2). The phylogenetic tree was constructed by using neighbour-joining method [19] (Fig. 1). Multiple alignment and phylogenetic tree showed that ES1 strain is A. clavatus. The strain was inoculated in conical flasks and incubated at 30 8C for 72 h. The cultures were then filtrated and centrifuged and the supernatant was used as the enzyme solution.
Fig. 3. Elution profile of A. clavatus ES1 protease from a CM-Sepharose column. The enzyme was eluted with a linear gradient of NaCl (0–0.5 M) in 25 mM Tris–HCl buffer (pH 8.0) at a flow rate of 120 ml/h.
The molecular mass of the ES1 serine protease was similar to those of Ophiostoma piceae 387N [20] and A. fumigatus [21] and was higher than serine protease from Trichoderma virens (29 kDa) [22]. Fungal serine proteases with molecular mass between 32 and 35 kDa were also reported [23–26].
3.2. Purification of ES1 protease The ES1 protease was purified by the three-step procedure described in Section 2. In the first step, the culture supernatant was precipitated with acetone. The 60–80% (v/v) acetone fraction showed higher specific activity (15,880 U/mg of protein) than 40–60% (v/v) acetone fraction (13,230 U/mg of protein). No activity was detected in the final supernatant and in 0–40% acetone fraction. The 60–80% (v/v) acetone fraction was then successively subjected to gel filtration on a Sephadex G-100 column and to ion exchange chromatography on CMSepharose column. The elution profiles of proteins and protease activities are shown in Figs. 2 and 3. The results of the purification procedure are summarized in Table 1. After the final purification step, the enzyme was purified 7.5-fold with a recovery of 29% and a specific activity of 37,600 U/mg of protein. The purified ES1 protease was homogenous on SDSPAGE and its molecular mass was estimated to be 32 kDa (Fig. 4).
Fig. 4. SDS-PAGE of the purified protease from A. clavatus ES1. Lane 1: purified ES1 protease; lane 2: molecular mass markers.
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Table 1 Summary of the purification of A. clavatus ES1 protease Purification steps
Total activity (U)
Total protein (mg)
Specific activity (U/mg)
Recovery (%)
Purification (fold)
Crude extract 40–60% acetone fraction 60–80% acetone fraction Sephadex G-100 CM-Sepharose
31800 13500 13500 12000 9400
6.4 1.02 0.85 0.56 0.25
4970 13230 15880 21430 37600
100 42 42 37 29
1 2.6 3.2 4.3 7.5
All operations were carried out at 4 8C. Only 60–80% acetone fraction was subjected to gel-filtration on Sephadex G-100.
3.3. N-terminal amino acid sequence of A. clavatus ES1 protease
3.4. Effect of pH and temperature on enzyme activity and stability
The N-terminal sequence of the first 15 amino acid residues of the purified alkaline protease was found to be A-L-T-T-Q-SG-A-P-W-G-L-G-S-I. The 15-N terminal amino acid sequence showed high similarity to those of serine proteases from fungi strains: A. fumigatus [21], T. virens [22], T. hamatum [26], A. viridunitans [27], O. piceae [28] and Beauvirea nivea [29] (Fig. 5). The highest homology was observed with the most popular subtilisin-like serine protease from A. fumigatus [21]. The ES1 protease differs from that of A. fumigatus with only one residue in the first 15 amino acids. The Lys-6 in A. fumigatus alkaline protease was replaced by Ser-6 in ES1 enzyme.
The effect of pH on protease activity was studied by using casein as a substrate at various pH values at 50 8C. The pHactivity profile of the purified ES1 protease is shown in Fig. 6. ES1 protease is active in the pH range of 6.0–11.0, with an optimum at pH 8.5. The relative activities at pH 9.0 and 10.0 were about 93 and 58%, respectively, of that at pH 8.5. These findings are in accordance with several earlier reports showing pH optima of 8.0–9.5 for fungi proteases such as A. fumigatus [21,23,24], A. parasiticus [30] and A. clavatus CCT2759 [25].
Fig. 5. Comparison of N-terminal amino acid sequence of the purified alkaline protease from A. clavatus ES1 with other enzymes from: A. fumigatus [21], T. virens [22], T. hamatum [26], A. viridunitans [27], O. piceae [28] and B. nivea [29].
Fig. 6. Effect of pH on enzyme activity. The pH profile was determined in different buffers of varying pH values at 50 8C. Standard deviations were 2.5% (based on three replicates).
Fig. 7. Effect of pH on protease stability. Enzyme samples were incubated in the respective buffers and percent residual activity was calculated. Standard deviations were 2.5% (based on three replicates).
Fig. 8. Effect of temperature on protease activity. The temperature was determined by assaying enzyme activity at various temperatures values using 100 mM Tris–HCl buffer at pH 8.5. Standard deviations were 2.5% (based on three replicates).
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Table 3 Effect of inhibitors on protease activity Inhibitors
Concentration (mM)
Residual activity (%)
None PMSF Dithionitrobenzoate EDTA
– 5 1 5 10 5
100 0 91 92 87 93
b-Mercaptoethanol
Enzyme activity measured in the absence of any inhibitor was taken as 100%. The remaining protease activity was measured after pre-incubation of enzyme with each inhibitor at 4 8C for 30 min.
Fig. 9. The temperature stability of ES1 protease. The enzyme was incubated at different temperatures and samples were withdrawn at 15 min intervals up to 60 min. Percent residual activity was calculated: 30 8C (&), 40 8C (&), 50 8C (~) and 60 8C (*). Standard deviations were 2.5% (based on three replicates).
similar to that of A. parasiticus protease [30] and was higher than that of A. clavatus CCT2759 [25] which showed a half-life of 18 min at 50 8C. 3.5. Effect of metal ions and enzyme inhibitors
However, our results differ from an A. clavatus alkaline protease reported by Ogundero and Osunlaja [31], which had a maximum protease activity at pH 7.8. The pH stability profile of the purified protease showed that the enzyme is highly stable at pH 8.0–9.0 (Fig. 7). The protease retained about 68 and 54% of its initial activity at pH 7.0 and 10.0, respectively. The effect of temperature on the activity of A. clavatus ES1 protease was examined at various temperatures. The ES1 enzyme was active between 40 and 70 8C with an optimum around 50 8C (Fig. 8). The relative activities at 40 and 60 8C were about 65 and 70%, respectively. The optimum temperature of A. clavatus ES1 protease was similar to that of O. piceae 387N serine protease [20] and was higher than those of A. clavatus CCT2759 [25], A. fumigatus TKU003 [11] and A. fumigatus CBS113.26 [24], which had optima temperatures between 37 and 42 8C. The thermal stability profile of the purified protease showed that the enzyme is completely stable at 30 8C. At 40 8C, the enzyme retained more than 65% of its initial activity after 1 h incubation, while, 50 and 10% of the maximal activity remained within 30 min of incubation at 50 and 60 8C, respectively (Fig. 9). The thermal stability of ES1 protease was Table 2 Effect of various metal ions (5 mM) on alkaline protease activity Metal ions
Relative activity (%)
None CaCl2 MgSO4 ZnCl2 CuSO4 MnSO4 BaCl2 CoSO4
100 124 108 16 62 56 77 0
The activity of the protease was determined by incubating the enzyme in the presence of various metal ions for 15 min at 50 8C and pH 8.5.
The effects of various metal ions (5 mM) on the activity of ES1 protease were studied at pH 8.5 and 50 8C by the addition of the respective cations to the reaction mixture (Table 2). The addition of CaCl2 and MgSO4 increased protease activity by 124 and 108% of the control, respectively. However, BaCl2, CuSO4 and ZnSO4 inhibited the enzyme activity by 23, 38 and 84%, respectively. The protease activity was completely inhibited by CoSO4. In order to determine the nature of the protease, enzyme activity was measured in the presence of different enzyme inhibitors (Table 3). The enzyme was strongly inhibited by the serine protease inhibitor (PMSF) indicating that the enzyme is a serine protease. Thiol reagent (DTNB) was practically without influence on the activity of the purified enzyme. The enzyme was slightly inhibited by the chelating agent EDTA (5 mM), with 8% of its original activity being lost, indicating the importance of Ca2+ in enzyme stabilization. These findings are in line with several earlier reports showing that active structure of serine proteases contains Ca2+ binding site(s) and the removal of Ca2+ from the strong binding site is associated to a significant reduction in thermal stability [32]. The high activity of ES1 protease in the presence of EDTA was very useful for application as detergent additive because chelating agents are components of most detergents. 3.6. Effect of oxidizing agents and surfactants on protease stability In order to be effective during washing, a good detergent protease must be compatible and stable with all commonly used detergent compounds such as surfactants, bleaches, oxidizing agents and other additives, which could be present in the formulation [2,5]. As shown in Table 4, the enzyme was highly stable in the presence of the non-ionic surfactants like Tween 80 and Triton X-100. In addition, the ES1 protease retained 72, 56 and 65% of its activity after incubation 1 h at 4 8C in the
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Table 4 Stability of ES1 alkaline protease in the presence of various surfactants and detergents Additives
Concentration
Residual activity (%)
None Tween 80 Triton X-100 SDS SDS H2 O2 Sodium perborate Sodium perborate
5% (v/v) 5% (v/v) 0.1% (w/v) 0.5% (w/v) 5% (v/v) 1% (w/v) 2% (w/v)
100 100 100 90 33 65 72 56
The enzyme was pre-incubated with surfactants and oxidizing agents for 1 h at 4 8C and the remaining activity was measured at pH 8.5 and 50 8C. The activity is expressed as a percentage of the activity level in the absence of additives.
presence of 1 and 2% (w/v) sodium perborate and 5% (v/v) hydrogen peroxide, respectively. This is a very important characteristic for its eventual use in detergent formulations. The strong anionic surfactant (SDS) at 0.1% (w/v) caused a moderate inhibition (10%). These findings are different to those of Tremacoldi et al. [25] who reported that the protease from A. clavatus CCT2759 was strongly inhibited by SDS, Tween 80 and carbonate ion. The stability of ES1 protease against SDS was lower than A. parasiticus protease which retained about 97% of its initial activity after 1 h incubation with 2% SDS at room temperature [30]. 4. Conclusion In the present study, an alkaline protease designated ES1 protease produced by a fungus A. clavatus ES1 was purified and characterized. The purification to homogeneity of the enzyme was achieved by acetone precipitation (60–80%), gel filtration through Sephadex G-100 and ion exchange chromatography on CM-Cellulose. After the final purification step, the enzyme was purified 7.5-fold with a specific activity of 37,600 U/mg and 29% recovery. The purified enzyme was homogenous on SDSPAGE and its molecular mass was estimated to be 32 kDa. The enzyme showed an optimum temperature at 50 8C and optimum pH of 8.5. The 15 N-terminal amino acid sequence of the ES1 protease revealed 93 and 86% homology with serine-proteases from A. fumigatus [21] and A. viridunitans, [27] respectively. The high proteolytic activity of ES1 protease at pH values of 8.0–9.5, at moderate temperature and stability towards various surfactants and bleaching agents suggest its suitability for inclusion in liquid detergent compositions. Acknowledgements We are grateful to Professor Sami Sayadi for his help in the ribosomal DNA sequencing and Professor Hafedh Mejdoub for his help in the amino acid sequencing. We also thank Mr. Zouhaier Bouallagui for proofreading this paper. This work was financed by the Ministe`re de la Recherche Scientifique, de la Technologie et du De´veloppement des Compe´tences, Tunisia.
References [1] Rao MB, Tankasale AM, Ghatge MS, Desphande VV. Molecular and biotechnological aspects of microbial proteases. Microbiol Mol Biol Rev 1998;62:597–634. [2] Gupta R, Beg QK, Lorenz P. Bacterial alkaline proteases: molecular approaches and industrial applications. Appl Microbiol Biotechnol 2002;59:15–32. [3] Kumar CG, Takagi H. Microbial alkaline proteases: from a bioindustrial viewpoint. Biotechnol Adv 1999;17:561–94. [4] Kalisz HM. Microbial proteinases. In: Fietcher A, editor. Advances in biochemical engineering/biotechnology (enzymes studies), 36. Berlin Heidelberg, New York: Springer; 1988. p. 3–61. [5] Gupta R, Gupta K, Saxena RK, Khan S. Bleach-stable, alkaline protease from Bacillus sp.. Biotechnol Lett 1999;21:135–8. [6] Maurer KH. Detergent proteases. Curr Opin Biotech 2004;15:330–4. [7] Phadatare SU, Deshpande VV, Srinivasan MC. High activity alkaline protease from Conidiobolus coronatus (NCL 86.8.20): enzyme production and compatibility with commercial detergents. Enzyme Microb Technol 1993;15:72–6. [8] Tunlid A, Rosen S, Ek B, Rask L. Purification and characterization of an extracellular serine protease from the nematode-trapping fungus Arthrobotrys oligospora. Microbiology 1994;140:1687–95. [9] Dunaevsky TE, Gruban TN, Beliokova GA, Belozersky MA. Enzymes secreted by filamentous fungi, regulation and purification of an extracellular protease of Trichoderma harzianum. Biochemistry (Mosc) 2000;65:723–7. [10] Tsuchiya K, Arai T, Seki K, Kimura T. Purification and some properties of alkaline proteinases from Cephalosporium sp. KM388. Agric Biol Chem 1987;51:2959–65. [11] Wang SL, Chen YH, Wang CL, Yen YH, Chern MK. Purification and characterization of a serine protease extracellularly produced by Aspergillus fumigatus in a shrimp and crab shell powder medium. Enzyme Microb Technol 2005;36:660–5. [12] Ruiz-Duenas FJ, Martinez MJ, Martinez AT. Molecular characterization of a novel peroxydase isolated from the ligninolytic fungus Pleurotus eryngii. Mol Microbiol 1999;31:223–35. [13] White TJ, Bruns T, Lee S, Taylor JW. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR protocols: a guide to methods and applications. New York: Academic press, Inc.; 1990. p. 315–22. [14] Sanger F, Niklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 1977;74:5463–7. [15] Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997;25: 3389–402. [16] Kembhavi AA, Kulkarni A, Pant AA. Salt-tolerant and thermostable alkaline protease from Bacillus subtilis NCIM No 64. Appl Biochem Biotechnol 1993;38:83–92. [17] Bradford M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 1976;72:248–54. [18] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680–5. [19] Saitou K, Nei M. A neighbour-joining method: new method for reconstructing phylogenetic trees. Mol Biol Evolution 1987;44: 406–25. [20] Abraham LD, Breuil C. Isolation and characterization of a subtilisin-like serine protease secreted by the sap-staining fungus Ophiostoma piceae. Enzyme Microb Technol 1996;18:133–40. [21] Reichard U, Buttner S, Eiffert H, Staib F, Ruchel R. Purification and characterization of an extracellular serine proteinase from Aspergillus fumigatus and its detection in tissue. J Med Microbiol 1990;33: 243–51. [22] Pozo MJ, Baek JM, Garcia JM, Kenerley CM. Functional analysis of tvsp1, a serine protease-encoding gene in the biocontrol agent Trichoderma virens. Fungal Genet Biol 2004;41:336–48.
M. Hajji et al. / Process Biochemistry 42 (2007) 791–797 [23] Monod M, Togni G, Rahalison L, Frenk E. Isolation and characterization of an extracellular alkaline protease of Aspergillus fumigatus. J Med Microbiol 1991;35:23–8. [24] Larcher G, Bouchara JP, Annaix V, Symoens F, Chabasse F, Tronchin G. Purification and characterization of a fibrinogenolytic serine proteinase from Aspergillus fumigatus culture filtrate. FEBS Lett 1992;308:65–9. [25] Tremacoldi CR, Monti R, Selistre-De-Arau´jo HS, Carmona EC. Purification and properties of an alkaline protease of Aspergillus clavatus. World J Microbiol Biotechnol 2007;23:295–9. [26] Steyaert JM, Stewart A, Ridgway HJ. Co-expression of two genes, chitinase (chit 42) and proteinase ( prb 1), implicated in mycoparasitism by Trichoderma Hamatum. Mycologica 2004;96:1245–52. [27] Katz ME, Dougall AM, Weeks K, Cheetham BF. Multiple genetically distinct groups revealed among clinical isolates identified as atypical Aspergillus fumigatus. J Clin Microbiol 2005;43:551–5.
797
[28] Hoffman B, Breuil C. Cloning and genetic analysis of subtilases in sap staining fungi. Curr Genet 2002;41:168–75. [29] Plucienniczak G, Marciniak-Rusek A, Pass-Dziegielewska L. The serine protease gene of cyclosporin-producing fungi Beauveria nivea. Institute of Biotechnology and Antibiotics, Staroscinska 5. Warsaw Poland 2002;502– 16. [30] Tunga R, Shrivastava B, Banerjee R. Purification and characterization of a protease from solid state cultures of Aspergillus parasiticus. Process Biochem 2003;38:1553–8. [31] Ogundero VW, Osunlaja SO. The purification and activities of an alkaline protease of Aspergillus clavatus from Nigerian poultry feeds. J Basic Microbiol 1986;26:241–8. [32] Vieille C, Zeikus JG. Hyperthermophilic enzymes: sources, uses and molecular mechanisms for thermostability. Microbiol Mol Biol Rev 2001;65:1–43.
Purification and characterization of an alkaline serine-protease produced by a new isolated Aspergillus clavatus ES1 Mohamed Hajji, Safia Kanoun, Moncef Nasri *, Ne´ji Gharsallah Laboratoire de Ge´nie Enzymatique et de Microbiologie, Ecole Nationale d’Inge´nieurs de Sfax, B.P. ‘‘W’’, 3038 Sfax, Tunisia Received 1 August 2006; received in revised form 12 January 2007; accepted 25 January 2007
Abstract An extracellular bleach stable protease from the fungus Aspergillus clavatus ES1, isolated from wastewater, was purified and characterized. The protease of ES1 strain was purified to homogeneity using acetone precipitation, Sephadex G-100 gel filtration and CM-Sepharose ion exchange chromatography, with a 7.5-fold increase in specific activity and 29% recovery. The molecular mass was estimated to be 32 kDa on SDS-PAGE. The optimum pH and temperature for the proteolytic activity were pH 8.5 and 50 8C, respectively. The enzyme was stable in the pH range of 7.0– 9.0. The protease was activated by divalent cations such as Ca2+ and Mg2+. The alkaline protease showed extreme stability towards non-ionic surfactants (5% Tween 80 and 5% Triton X-100). In addition, the enzyme was relatively stable towards oxidizing agents, retaining more than 71 and 53% of its initial activity after 1 h incubation in the presence of 1 and 2% (w/ v) sodium perborate, respectively. The N-terminal sequence of the first 15 amino acids of the purified alkaline protease of A. clavatus ES1 showed high similarity with other fungal alkaline proteases. The activity was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serineprotease. # 2007 Elsevier Ltd. All rights reserved. Keywords: Serine alkaline protease; Purification; Aspergillus clavatus; Bleaches; Detergents
1. Introduction Proteases are the most important industrial enzymes accounting for approximately 60% of the total industrial enzyme market [1]. They have diverse applications in a wide variety of industries, such as in detergent, food, pharmaceutical, leather, silk and recovery of silver from used X-ray films [2,3]. The industrial demand of proteolytic enzymes, with appropriate specificity and stability to pH, temperature and surfactants, continues to stimulate the search for new enzyme sources. Proteases with high activity and stability in high alkaline range and high temperatures are interesting for bioengineering and biotechnological applications. Their major application is in detergent industry because the pH of laundry detergents is generally in the range of 9.0–12.0. Alkaline proteases are used as cleaning additives in detergents to
* Corresponding author. Tel.: +216 96 501 698; fax: +216 74 275 595. E-mail addresses: [email protected], [email protected] (M. Nasri). 1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2007.01.011
facilitate the release of proteins. Proteases in detergent industries account for approximately 30% of the total world enzyme production [4]. Alkaline proteases are particularly important because they are both stable and active at high pH solutions and in the presence of surfactants and oxidizing agents [5]. They are produced by a wide range of microorganisms including bacteria, moulds, yeasts and also mammalian tissues. Most of the commercial alkaline proteases were isolated from Bacillus species [6]. A considerable number of fungal species are known to produce extracellular alkaline proteases such as Conidiobolus coronatus [7], Arthrobotrys olgospora [8], Trichoderma harzianum [9], Cephalosporium sp. KM388 [10] and A. fumigatus [11]. Isolation and screening of microorganisms from naturally occurring alkaline habitats or from alkaline wastewater was expected to provide new strains producing enzymes active and stable in highly alkaline conditions. This article deals with the purification and characterization of an alkaline protease produced by Aspergillus clavatus ES1 strain isolated from wastewater.
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2. Materials and methods 2.1. Materials Column chromatography materials, molecular markers for electrophoresis and casein were purchased from Sigma Chemicals Co. St. Louis, USA. All the microbiological media components were product of Bio-Rad, France. Other chemicals used were of analytical grade.
2.2. Methods 2.2.1. Microorganism A. clavatus ES1 used throughout this study was isolated from wastewater. The isolate was propagated on potato-dextrose-agar plates at 30 8C, and inocula were prepared from 7-day-old colonies by flooding with 10 ml of sterile distilled water and scraping off the agar plates. 2.2.2. PCR amplification and ITS of 18S rDNA gene sequencing Fungal DNA was extracted from freeze-dried mycelia according to RuizDuenas et al. [12]. PCR amplification of the internal transcribed spacer ‘ITS’ region of the ribosomal DNA was performed using primers ITS1 (50 TCCGTAGGTGAACCTGCGG-30 ) and ITS4 (50 -TCCTCCGCTTATTGATATGC-30 ) [13]. PCR product was cloned using pMOSBlue vector (Amersham). Nucleotide sequencing of the cloned PCR product was performed following the application of automated DNA sequencing method based on dideoxynucleotide chain termination method [14] using universal primers: U-19 50 -GTTTTCCCAGTCACGACG-30 and T7 50 -TAATACGACTCACTATAGGG-30 . The nucleotide sequence was analyzed with the GenBank database using BLAST program [15]. 2.2.3. Culture and growth conditions The medium used for isolation of protease producing strains was consisted of (g/l): peptone, 5.0; yeast extract, 3.0; skimmed milk 25% (v/v) and bacteriological agar, 12.0; pH was adjusted to 9.0. The medium used for protease production by ES1 strain was composed of (g/l): CaCl27H2O 0.4; KH2PO4 1.0; Na2HPO4 0.8; MgSO47H2O 0.5; ZnCl2 0.1; NaCl 0.3; whole sardinella (Sardinella aurita) fish flour 2.0 and wheat bran 10 (pH 6.0). Media were autoclaved at 120 8C for 20 min. Culture media were inoculated with 107 spores/ml and incubated on a rotatory shaker (150 rev/min) for 72 h at 30 8C, in 300 ml Erlenmeyer flasks with a working volume of 30 ml. The cultures were centrifuged to remove fungi mycelia and the supernatants were used for estimation of proteolytic activity. All experiments were carried out in duplicate and repeated at least twice. 2.2.4. Assay of protease activity Protease activity was measured by the method of Kembhavi et al. [16] using casein as a substrate. Half milliliter of the enzyme, suitably diluted, was mixed with 0.5 ml 100 mM Tris–HCl (pH 8.5) containing 1% casein, and incubated for 15 min at 50 8C. The reaction was stopped by addition of 0.5 ml trichloroacetic acid (20%; w/v). The mixture was allowed to stand at room temperature for 15 min and then centrifuged at 10,000 g for 15 min to remove the precipitate. The acid soluble material was estimated spectrophotometrically at 280 nm. A standard curve was generated using solutions of 0–50 mg/l tyrosine. One unit of protease activity was defined as the amount of enzyme required to liberate 1 mg of tyrosine per min under the experimental conditions used. 2.2.5. Protein determination Protein concentration was determined by the method of Bradford [17] using bovine serum albumin as a standard. 2.2.6. Protease purification 2.2.6.1. Acetone treatment. The culture supernatant containing the extracellular enzyme was first subjected to acetone precipitation. Acetone fractions of 0–40%, 40–60% and 60–80% (v/v) were collected by centrifugation at 10,000 g and the pellet obtained in each fraction was suspended in a minimal volume of 100 mM Tris–HCl, pH 8.0. 2.2.6.2. Sephadex G-100 gel filtration. The 60–80% (v/v) acetone fraction was subjected to gel filtration on a Sephadex G-100 column (3 cm 100 cm)
equilibrated with 25 mM Tris–HCl pH 8.0 containing 0.05% (v/v) Triton X100. Enzyme fractions of 5 ml were collected at a flow rate of 25 ml/h with the same buffer. Protein content (Abs 280 nm) and protease activity were measured. Fractions showing protease activities were pooled.
2.2.6.3. CM-Sepharose separation. The active fractions were applied to a CM-Sepharose column (3 cm 30 cm) equilibrated with 25 mM Tris–HCl buffer pH 8.0. After being washed with the same buffer, bound proteins were eluted with a linear gradient of sodium chloride in the range of 0– 0.5 M in the equilibrating buffer. Fractions (5 ml each) were collected at a flow rate of 120 ml/h. The fractions with high protease activity were pooled and stored at 20 8C for further analysis. All the purification steps were conducted at temperatures not exceeding 4 8C.
2.2.7. Polyacrylamide gel electrophoresis Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out for the determination of purity and molecular mass of the enzyme as described by Laemmli [18] using a 5% (w/v) stacking gel and a 15% (w/v) separating gel. The molecular mass of the enzyme was estimated using a low molecular mass calibration kit as markers consisting of bovine serum albumin (66 kDa), egg white ovalbumin (45 kDa), glyceraldehyde-3-P deshydrogenase (36 kDa), bovine carbonic anhydrase (29 kDa), bovine trypsinogen (24 kDa), soybean trypsin inhibitor (20.1 kDa) and bovine a-lactalbumin (14.2 kDa).
2.2.8. N-terminal amino acid sequence of the enzyme The purified enzyme was electrophoretically transferred from SDS-PAGE to a polyvinylidiene difluoride membrane (PVDF). The region containing the protease band on the PVDF was excised and the protein N-terminal amino acid sequence was determined by the Edman degradation method on an ABI Procise 494 protein sequencer (Applied Biosystems).
2.2.9. Effect of pH and temperature on the activity and stability of the enzyme The optimum pH of the purified enzyme was studied over a pH range of 6.0– 11.0. For the measurement of pH stability, the enzyme was kept at 4 8C for 1 h in different buffers at 100 mM and the residual proteolytic activity was determined under standard assay conditions. The following buffer systems were used: phosphate buffer, pH 6.0–7.5; Tris–HCl buffer, pH 8.0–8.5; glycine–NaOH buffer, pH 9.0–11.0. To analyse the effect of temperature, the activity was tested at different temperatures (40–70 8C) using casein as substrate for 15 min at pH 8.5. Thermal inactivation was examined by incubating the purified enzyme at 30, 40, 50 and 60 8C for 60 min. Aliquots were withdrawn at desired time intervals to test the remaining activity at pH 8.5 and 50 8C. The non-heated enzyme was considered as a control (100%).
2.2.10. Effect of metal ions, oxidizing agents and enzyme inhibitors The effect of various metal ions (5 mM) on enzyme activity was investigated using CaCl2, MnSO4, ZnSO4, CoSO4, CuSO4, BaCl2 and MgSO4. The effects of some surfactants (Triton X-100, Tween 80 and SDS) and oxidizing agents (H2O2 and sodium perborate) on enzyme stability were also studied by pre-incubating enzyme for 1 h at 4 8C. The residual activity was measured at pH 8.5 and 50 8C. The activity of the enzyme (without any surfactants) was considered as 100%. The effects of enzyme inhibitors on protease activity were studied using phenylmethylsulfonyl fluoride (PMSF), dithio-bis-nitrobenzoic acid (DTNB) and ethylene-diaminetetraacetic acid (EDTA). The purified enzyme was preincubated with inhibitors for 30 min at 4 8C and then the remaining enzyme activity was estimated using casein as a substrate.
2.2.11. Nucleotide sequence accession number The ITS nucleotide sequence of the 18S rDNA gene has been submitted to the GenBank database and assigned accession number EF151929.
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Fig. 1. Phylogenetic tree of A. clavatus ES1 associated with the other members of the Aspergillus genus. Topology was inferred using the neighbour-joining method [19].
3. Results and discussion
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Fig. 2. Purification profile of alkaline protease from A. clavatus ES1 by gel filtration on Sephadex G-100 column. The concentrated enzyme preparation (60–80%) was applied to a 3 cm 100 cm column, equilibrated and eluted with 25 mM Tris–HCl buffer (pH 8.0) at a flow rate of 25 ml/h.
3.1. Identification of the microorganism and phylogenetic analysis Several fungi strains producing extracellular proteases were screened from different biotopes. Of these, isolate ES1 exhibiting a large zone of hydrolysis on skim milk agar plates was selected. In order to identify ES1 strain, the internal transcribed spacer region of 18S rDNA (860 pb) was amplified, cloned in pMOSBlue vector and sequenced. The ITS nucleotide sequence was analyzed with the GenBank database using BLAST program and showed 100 and 95% homology with ITS of A. clavatus and A. fumigatus strains, respectively. Multiple sequence alignment was performed with CLC Gene Workbench software version (2.0.2). The phylogenetic tree was constructed by using neighbour-joining method [19] (Fig. 1). Multiple alignment and phylogenetic tree showed that ES1 strain is A. clavatus. The strain was inoculated in conical flasks and incubated at 30 8C for 72 h. The cultures were then filtrated and centrifuged and the supernatant was used as the enzyme solution.
Fig. 3. Elution profile of A. clavatus ES1 protease from a CM-Sepharose column. The enzyme was eluted with a linear gradient of NaCl (0–0.5 M) in 25 mM Tris–HCl buffer (pH 8.0) at a flow rate of 120 ml/h.
The molecular mass of the ES1 serine protease was similar to those of Ophiostoma piceae 387N [20] and A. fumigatus [21] and was higher than serine protease from Trichoderma virens (29 kDa) [22]. Fungal serine proteases with molecular mass between 32 and 35 kDa were also reported [23–26].
3.2. Purification of ES1 protease The ES1 protease was purified by the three-step procedure described in Section 2. In the first step, the culture supernatant was precipitated with acetone. The 60–80% (v/v) acetone fraction showed higher specific activity (15,880 U/mg of protein) than 40–60% (v/v) acetone fraction (13,230 U/mg of protein). No activity was detected in the final supernatant and in 0–40% acetone fraction. The 60–80% (v/v) acetone fraction was then successively subjected to gel filtration on a Sephadex G-100 column and to ion exchange chromatography on CMSepharose column. The elution profiles of proteins and protease activities are shown in Figs. 2 and 3. The results of the purification procedure are summarized in Table 1. After the final purification step, the enzyme was purified 7.5-fold with a recovery of 29% and a specific activity of 37,600 U/mg of protein. The purified ES1 protease was homogenous on SDSPAGE and its molecular mass was estimated to be 32 kDa (Fig. 4).
Fig. 4. SDS-PAGE of the purified protease from A. clavatus ES1. Lane 1: purified ES1 protease; lane 2: molecular mass markers.
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Table 1 Summary of the purification of A. clavatus ES1 protease Purification steps
Total activity (U)
Total protein (mg)
Specific activity (U/mg)
Recovery (%)
Purification (fold)
Crude extract 40–60% acetone fraction 60–80% acetone fraction Sephadex G-100 CM-Sepharose
31800 13500 13500 12000 9400
6.4 1.02 0.85 0.56 0.25
4970 13230 15880 21430 37600
100 42 42 37 29
1 2.6 3.2 4.3 7.5
All operations were carried out at 4 8C. Only 60–80% acetone fraction was subjected to gel-filtration on Sephadex G-100.
3.3. N-terminal amino acid sequence of A. clavatus ES1 protease
3.4. Effect of pH and temperature on enzyme activity and stability
The N-terminal sequence of the first 15 amino acid residues of the purified alkaline protease was found to be A-L-T-T-Q-SG-A-P-W-G-L-G-S-I. The 15-N terminal amino acid sequence showed high similarity to those of serine proteases from fungi strains: A. fumigatus [21], T. virens [22], T. hamatum [26], A. viridunitans [27], O. piceae [28] and Beauvirea nivea [29] (Fig. 5). The highest homology was observed with the most popular subtilisin-like serine protease from A. fumigatus [21]. The ES1 protease differs from that of A. fumigatus with only one residue in the first 15 amino acids. The Lys-6 in A. fumigatus alkaline protease was replaced by Ser-6 in ES1 enzyme.
The effect of pH on protease activity was studied by using casein as a substrate at various pH values at 50 8C. The pHactivity profile of the purified ES1 protease is shown in Fig. 6. ES1 protease is active in the pH range of 6.0–11.0, with an optimum at pH 8.5. The relative activities at pH 9.0 and 10.0 were about 93 and 58%, respectively, of that at pH 8.5. These findings are in accordance with several earlier reports showing pH optima of 8.0–9.5 for fungi proteases such as A. fumigatus [21,23,24], A. parasiticus [30] and A. clavatus CCT2759 [25].
Fig. 5. Comparison of N-terminal amino acid sequence of the purified alkaline protease from A. clavatus ES1 with other enzymes from: A. fumigatus [21], T. virens [22], T. hamatum [26], A. viridunitans [27], O. piceae [28] and B. nivea [29].
Fig. 6. Effect of pH on enzyme activity. The pH profile was determined in different buffers of varying pH values at 50 8C. Standard deviations were 2.5% (based on three replicates).
Fig. 7. Effect of pH on protease stability. Enzyme samples were incubated in the respective buffers and percent residual activity was calculated. Standard deviations were 2.5% (based on three replicates).
Fig. 8. Effect of temperature on protease activity. The temperature was determined by assaying enzyme activity at various temperatures values using 100 mM Tris–HCl buffer at pH 8.5. Standard deviations were 2.5% (based on three replicates).
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Table 3 Effect of inhibitors on protease activity Inhibitors
Concentration (mM)
Residual activity (%)
None PMSF Dithionitrobenzoate EDTA
– 5 1 5 10 5
100 0 91 92 87 93
b-Mercaptoethanol
Enzyme activity measured in the absence of any inhibitor was taken as 100%. The remaining protease activity was measured after pre-incubation of enzyme with each inhibitor at 4 8C for 30 min.
Fig. 9. The temperature stability of ES1 protease. The enzyme was incubated at different temperatures and samples were withdrawn at 15 min intervals up to 60 min. Percent residual activity was calculated: 30 8C (&), 40 8C (&), 50 8C (~) and 60 8C (*). Standard deviations were 2.5% (based on three replicates).
similar to that of A. parasiticus protease [30] and was higher than that of A. clavatus CCT2759 [25] which showed a half-life of 18 min at 50 8C. 3.5. Effect of metal ions and enzyme inhibitors
However, our results differ from an A. clavatus alkaline protease reported by Ogundero and Osunlaja [31], which had a maximum protease activity at pH 7.8. The pH stability profile of the purified protease showed that the enzyme is highly stable at pH 8.0–9.0 (Fig. 7). The protease retained about 68 and 54% of its initial activity at pH 7.0 and 10.0, respectively. The effect of temperature on the activity of A. clavatus ES1 protease was examined at various temperatures. The ES1 enzyme was active between 40 and 70 8C with an optimum around 50 8C (Fig. 8). The relative activities at 40 and 60 8C were about 65 and 70%, respectively. The optimum temperature of A. clavatus ES1 protease was similar to that of O. piceae 387N serine protease [20] and was higher than those of A. clavatus CCT2759 [25], A. fumigatus TKU003 [11] and A. fumigatus CBS113.26 [24], which had optima temperatures between 37 and 42 8C. The thermal stability profile of the purified protease showed that the enzyme is completely stable at 30 8C. At 40 8C, the enzyme retained more than 65% of its initial activity after 1 h incubation, while, 50 and 10% of the maximal activity remained within 30 min of incubation at 50 and 60 8C, respectively (Fig. 9). The thermal stability of ES1 protease was Table 2 Effect of various metal ions (5 mM) on alkaline protease activity Metal ions
Relative activity (%)
None CaCl2 MgSO4 ZnCl2 CuSO4 MnSO4 BaCl2 CoSO4
100 124 108 16 62 56 77 0
The activity of the protease was determined by incubating the enzyme in the presence of various metal ions for 15 min at 50 8C and pH 8.5.
The effects of various metal ions (5 mM) on the activity of ES1 protease were studied at pH 8.5 and 50 8C by the addition of the respective cations to the reaction mixture (Table 2). The addition of CaCl2 and MgSO4 increased protease activity by 124 and 108% of the control, respectively. However, BaCl2, CuSO4 and ZnSO4 inhibited the enzyme activity by 23, 38 and 84%, respectively. The protease activity was completely inhibited by CoSO4. In order to determine the nature of the protease, enzyme activity was measured in the presence of different enzyme inhibitors (Table 3). The enzyme was strongly inhibited by the serine protease inhibitor (PMSF) indicating that the enzyme is a serine protease. Thiol reagent (DTNB) was practically without influence on the activity of the purified enzyme. The enzyme was slightly inhibited by the chelating agent EDTA (5 mM), with 8% of its original activity being lost, indicating the importance of Ca2+ in enzyme stabilization. These findings are in line with several earlier reports showing that active structure of serine proteases contains Ca2+ binding site(s) and the removal of Ca2+ from the strong binding site is associated to a significant reduction in thermal stability [32]. The high activity of ES1 protease in the presence of EDTA was very useful for application as detergent additive because chelating agents are components of most detergents. 3.6. Effect of oxidizing agents and surfactants on protease stability In order to be effective during washing, a good detergent protease must be compatible and stable with all commonly used detergent compounds such as surfactants, bleaches, oxidizing agents and other additives, which could be present in the formulation [2,5]. As shown in Table 4, the enzyme was highly stable in the presence of the non-ionic surfactants like Tween 80 and Triton X-100. In addition, the ES1 protease retained 72, 56 and 65% of its activity after incubation 1 h at 4 8C in the
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Table 4 Stability of ES1 alkaline protease in the presence of various surfactants and detergents Additives
Concentration
Residual activity (%)
None Tween 80 Triton X-100 SDS SDS H2 O2 Sodium perborate Sodium perborate
5% (v/v) 5% (v/v) 0.1% (w/v) 0.5% (w/v) 5% (v/v) 1% (w/v) 2% (w/v)
100 100 100 90 33 65 72 56
The enzyme was pre-incubated with surfactants and oxidizing agents for 1 h at 4 8C and the remaining activity was measured at pH 8.5 and 50 8C. The activity is expressed as a percentage of the activity level in the absence of additives.
presence of 1 and 2% (w/v) sodium perborate and 5% (v/v) hydrogen peroxide, respectively. This is a very important characteristic for its eventual use in detergent formulations. The strong anionic surfactant (SDS) at 0.1% (w/v) caused a moderate inhibition (10%). These findings are different to those of Tremacoldi et al. [25] who reported that the protease from A. clavatus CCT2759 was strongly inhibited by SDS, Tween 80 and carbonate ion. The stability of ES1 protease against SDS was lower than A. parasiticus protease which retained about 97% of its initial activity after 1 h incubation with 2% SDS at room temperature [30]. 4. Conclusion In the present study, an alkaline protease designated ES1 protease produced by a fungus A. clavatus ES1 was purified and characterized. The purification to homogeneity of the enzyme was achieved by acetone precipitation (60–80%), gel filtration through Sephadex G-100 and ion exchange chromatography on CM-Cellulose. After the final purification step, the enzyme was purified 7.5-fold with a specific activity of 37,600 U/mg and 29% recovery. The purified enzyme was homogenous on SDSPAGE and its molecular mass was estimated to be 32 kDa. The enzyme showed an optimum temperature at 50 8C and optimum pH of 8.5. The 15 N-terminal amino acid sequence of the ES1 protease revealed 93 and 86% homology with serine-proteases from A. fumigatus [21] and A. viridunitans, [27] respectively. The high proteolytic activity of ES1 protease at pH values of 8.0–9.5, at moderate temperature and stability towards various surfactants and bleaching agents suggest its suitability for inclusion in liquid detergent compositions. Acknowledgements We are grateful to Professor Sami Sayadi for his help in the ribosomal DNA sequencing and Professor Hafedh Mejdoub for his help in the amino acid sequencing. We also thank Mr. Zouhaier Bouallagui for proofreading this paper. This work was financed by the Ministe`re de la Recherche Scientifique, de la Technologie et du De´veloppement des Compe´tences, Tunisia.
References [1] Rao MB, Tankasale AM, Ghatge MS, Desphande VV. Molecular and biotechnological aspects of microbial proteases. Microbiol Mol Biol Rev 1998;62:597–634. [2] Gupta R, Beg QK, Lorenz P. Bacterial alkaline proteases: molecular approaches and industrial applications. Appl Microbiol Biotechnol 2002;59:15–32. [3] Kumar CG, Takagi H. Microbial alkaline proteases: from a bioindustrial viewpoint. Biotechnol Adv 1999;17:561–94. [4] Kalisz HM. Microbial proteinases. In: Fietcher A, editor. Advances in biochemical engineering/biotechnology (enzymes studies), 36. Berlin Heidelberg, New York: Springer; 1988. p. 3–61. [5] Gupta R, Gupta K, Saxena RK, Khan S. Bleach-stable, alkaline protease from Bacillus sp.. Biotechnol Lett 1999;21:135–8. [6] Maurer KH. Detergent proteases. Curr Opin Biotech 2004;15:330–4. [7] Phadatare SU, Deshpande VV, Srinivasan MC. High activity alkaline protease from Conidiobolus coronatus (NCL 86.8.20): enzyme production and compatibility with commercial detergents. Enzyme Microb Technol 1993;15:72–6. [8] Tunlid A, Rosen S, Ek B, Rask L. Purification and characterization of an extracellular serine protease from the nematode-trapping fungus Arthrobotrys oligospora. Microbiology 1994;140:1687–95. [9] Dunaevsky TE, Gruban TN, Beliokova GA, Belozersky MA. Enzymes secreted by filamentous fungi, regulation and purification of an extracellular protease of Trichoderma harzianum. Biochemistry (Mosc) 2000;65:723–7. [10] Tsuchiya K, Arai T, Seki K, Kimura T. Purification and some properties of alkaline proteinases from Cephalosporium sp. KM388. Agric Biol Chem 1987;51:2959–65. [11] Wang SL, Chen YH, Wang CL, Yen YH, Chern MK. Purification and characterization of a serine protease extracellularly produced by Aspergillus fumigatus in a shrimp and crab shell powder medium. Enzyme Microb Technol 2005;36:660–5. [12] Ruiz-Duenas FJ, Martinez MJ, Martinez AT. Molecular characterization of a novel peroxydase isolated from the ligninolytic fungus Pleurotus eryngii. Mol Microbiol 1999;31:223–35. [13] White TJ, Bruns T, Lee S, Taylor JW. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR protocols: a guide to methods and applications. New York: Academic press, Inc.; 1990. p. 315–22. [14] Sanger F, Niklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 1977;74:5463–7. [15] Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997;25: 3389–402. [16] Kembhavi AA, Kulkarni A, Pant AA. Salt-tolerant and thermostable alkaline protease from Bacillus subtilis NCIM No 64. Appl Biochem Biotechnol 1993;38:83–92. [17] Bradford M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 1976;72:248–54. [18] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680–5. [19] Saitou K, Nei M. A neighbour-joining method: new method for reconstructing phylogenetic trees. Mol Biol Evolution 1987;44: 406–25. [20] Abraham LD, Breuil C. Isolation and characterization of a subtilisin-like serine protease secreted by the sap-staining fungus Ophiostoma piceae. Enzyme Microb Technol 1996;18:133–40. [21] Reichard U, Buttner S, Eiffert H, Staib F, Ruchel R. Purification and characterization of an extracellular serine proteinase from Aspergillus fumigatus and its detection in tissue. J Med Microbiol 1990;33: 243–51. [22] Pozo MJ, Baek JM, Garcia JM, Kenerley CM. Functional analysis of tvsp1, a serine protease-encoding gene in the biocontrol agent Trichoderma virens. Fungal Genet Biol 2004;41:336–48.
M. Hajji et al. / Process Biochemistry 42 (2007) 791–797 [23] Monod M, Togni G, Rahalison L, Frenk E. Isolation and characterization of an extracellular alkaline protease of Aspergillus fumigatus. J Med Microbiol 1991;35:23–8. [24] Larcher G, Bouchara JP, Annaix V, Symoens F, Chabasse F, Tronchin G. Purification and characterization of a fibrinogenolytic serine proteinase from Aspergillus fumigatus culture filtrate. FEBS Lett 1992;308:65–9. [25] Tremacoldi CR, Monti R, Selistre-De-Arau´jo HS, Carmona EC. Purification and properties of an alkaline protease of Aspergillus clavatus. World J Microbiol Biotechnol 2007;23:295–9. [26] Steyaert JM, Stewart A, Ridgway HJ. Co-expression of two genes, chitinase (chit 42) and proteinase ( prb 1), implicated in mycoparasitism by Trichoderma Hamatum. Mycologica 2004;96:1245–52. [27] Katz ME, Dougall AM, Weeks K, Cheetham BF. Multiple genetically distinct groups revealed among clinical isolates identified as atypical Aspergillus fumigatus. J Clin Microbiol 2005;43:551–5.
797
[28] Hoffman B, Breuil C. Cloning and genetic analysis of subtilases in sap staining fungi. Curr Genet 2002;41:168–75. [29] Plucienniczak G, Marciniak-Rusek A, Pass-Dziegielewska L. The serine protease gene of cyclosporin-producing fungi Beauveria nivea. Institute of Biotechnology and Antibiotics, Staroscinska 5. Warsaw Poland 2002;502– 16. [30] Tunga R, Shrivastava B, Banerjee R. Purification and characterization of a protease from solid state cultures of Aspergillus parasiticus. Process Biochem 2003;38:1553–8. [31] Ogundero VW, Osunlaja SO. The purification and activities of an alkaline protease of Aspergillus clavatus from Nigerian poultry feeds. J Basic Microbiol 1986;26:241–8. [32] Vieille C, Zeikus JG. Hyperthermophilic enzymes: sources, uses and molecular mechanisms for thermostability. Microbiol Mol Biol Rev 2001;65:1–43.