African Journal of Biotechnology Vol. 8 (18), pp. 4582-4589, 15 September, 2009 Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2009 Academic Journals Full Length Research Paper Extracellular acid protease from Aspergillus niger I1: purification and characterization Rayda Siala#, Alya Sellami-Kamoun#, Mohamed Hajji, Ines Abid, Neji Gharsallah and Moncef Nasri* Laboratoire de Génie Enzymatique et de Microbiologie, Ecole Nationale d'Ingénieurs de Sfax, B.P. (W) 3038 Sfax, Tunisia. Accepted 30 April, 2009 A new strain of Aspergillus niger producing acid protease was isolated and identified by universal primers NL1 and NL4. The acid protease from A. niger I1 was purified to homogeneity by ultrafiltration using a 10-KDa cut-off membrane, gel filtration on Sephadex G-75 and ion exchange chromatography on CM-Sephadex C-50, with a 3.55-fold increase in specific activity and 56% recovery. The molecular weight of the protease was estimated to be 50 kDa on SDS-PAGE and gel filtration, which is higher than those from other A. niger strains. Carbohydrate content of the purified protease, determined by the chemical anthrone method, was calculated to be 16%. The Km and Vmax for caseinolytic activity of the purified enzyme were found to be 1.02 mM and 2.2 µmol/min, respectively. The enzyme was optimally active at 60°C and pH 3.0. The most metal ions tested had no significant effect on protease activity. The enzyme activity was inhibited by pepstatin A, suggesting that the purified enzyme is an aspartic protease. Key words: Acid protease, Aspergillus niger, purification, aspergillopepsin, glycosylation. INTRODUCTION Proteases constitute one of the most important groups of (Ishibashi et al., 1988). Acid proteases have been industrial enzymes accounting for about 60% of the total isolated and characterized from mammals, plants, industrial market (Rao et al., 1998). They have diverse bacteria and fungi (Wu and Hang, 1998). A considerable applications in a wide variety of industries, such as in number of Aspergilli species are known to produce detergent, food, pharmaceutical, leather, silk and for extracellular acid proteases such as Aspergillus niger recovery of silver from used X-ray films (Ward, 1983; (O’Donnell et al., 2001), A. oryzae (Tsujita and Endo, Gupta et al., 2002). The industrial demand for highly 1978), A. awamori (Moralejo et al., 2002), A. fumigatus active preparations of proteolytic enzymes with (Reichard et al., 1995) and A. saitoi (Tello-Solis and appropriate specificity and stability to extreme pH and Hernandez-Arana, 1995). These enzymes are predo- temperature continues to stimulate the search for new minantly extracellular, isolated in active form from the enzyme sources. culture medium and some of them are available in Proteases with high activity and stability in acid pH commercial scale (Rao et al., 1998). Several fungal acid range have important industrial applications, especially in proteases called aspergillopepsin have been purified and the food processing industry, such as dairy industry as characterized from Aspergillus strains (Ishishima, 2004; milk clotting agents for the manufacture of cheese Percin et al., 2009). Their molecular weight have been (Sumantha et al., 2006) or to improve food flavours reported to range from 30 to 40 kDa and showed maximal activity at pH 3.0 to 4.0 (Rao et al., 1998). A. niger is known to produce five different endopeptidases (Van den Hombergh et al., 1997), two carboxypeptidases *Corresponding author. E-mail: [email protected] or (Dal Degan et al., 1992) and one aminopeptidase [email protected]. Tel: +216 74 274 088. Fax: +216 74 275 (Basten et al., 2001). The two major extracellular acid 595. proteases are called proctases A and B. Proctase A (20 kDa), a non-pepsin type protease is not inhibited by #These authors contribute equally to this article pepstatin (Takahashi et al., 1991), whereas proctase B or Siala et al. 4583 aspergillopepsin I (35 kDa) which is inhibited by pepstatin Protein content belonging to the A1 family of proteases (Inoue et al., The protein content of individual fractions, obtained after different 1995; 1996). steps of chromatography, was monitored by measuring the absor- The isolation and screening of microorganisms from bance at 280 nm. Quantitative estimation of protein was determined naturally occurring acid habitats are expected to provide by the method of Bradford (Bradford, 1976) using bovine serum new strains producing enzymes active and stable in acid albumin as a standard. conditions. This paper deals with the purification and characterization of an acid protease produced by A. niger Purification of acid protease I1 strain newly isolated from olive oil mill wastewater. Ultrafiltration: The crude supernatant (190 ml) was concentrated by ultrafiltration with a 10-kDa molecular weight cut-off membrane MATERIALS AND METHODS (MSI Miscron Separations inc., Webstboro, MA, USA) in an Amicon Chemicals Model 8050 stirred ultrafiltration cell (Amicon corp., Danvers, USA). The pressure of the cell was kept at 75 psi by nitrogen gas. Casein, pepstatin A, ethylenediaminetetraacetic acid (EDTA), glycine, trichloroacetic acid (TCA), molecular mass markers were purchased from Sigma Chemical Co. (St. Louis MO, USA). Sodium Sephadex G-75 filtration dodecyl sulphate (SDS), acrylamide, ammonium per-sulphate, tetramethylethylenediamine and Coomassie brilliant blue R250 The concentrated extract was then subjected to gel filtration on a were from Bio-Rad Laboratories (France). Supports of chromato- Sephadex G-75 column (3 cm x 90 cm) previously equilibrated with graphy used for protease purification: Sephadex G-75 and CM- 25 mM glycine-HCl buffer pH 3.0. Fractions of 4.0 ml were collected Sephadex C-50 were from Pharmacia (Uppsala, Sweden). All other at a flow rate of 25 ml/h after elution with the same buffer. Protein reagents were of analytical grade. content (abs. at 280 nm) and protease activity in each fraction were measured. Fractions showing protease activities were pooled. Microorganism and primers CM-Sephadex C-50 separation The strain used throughout this study was isolated from olive oil mill wastewater. The isolate was identified as A. niger with universal The active fractions were applied to a CM-Sephadex C-50 column primers NL1 (5’GCATATCAATAAGCGGAGGAAAAG) and NL4 (3 cm x 30 cm) equilibrated with 25 mM glycine-HCl buffer, pH 3.0. (5’GGTCCGTGTTTCAAGACGG). The strain was maintained on After being washed with the same buffer, bound proteins were potato-dextrose-agar plates at 30°C. Spores for inoculum were eluted with a linear gradient of sodium chloride in the range of 0-0.5 prepared from 7 days-old colonies by flooding with 10 ml of sterile M in the equilibrating buffer. Fractions (6.0 ml each) were collected distilled water and scraping off the agar plates. at a flow rate of 96 ml/h, and analyzed for protease activity and protein concentration. Active fractions were pooled and stored at Culture and growth conditions 4°C for further analysis. All the purification steps were carried out at temperature not exceeding 4°C. The medium used for protease production by A. niger I1 strain was composed of (g/l): CaCl2 7.H2O 0.4, KH2PO4 7.0, Na2HPO4 2.5, MgSO4 7. H2O 0.5, ZnCl2 0.1, NaCl 0.3, fish (Sardinella aurita) flour Polyacrylamide gel electrophoresis 5, Hull grain of wheat 10; pH 6.0. Media were autoclaved at 120°C for 20 min. Cultures were inoculated with 107 spores/ml and Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS- incubated in a rotatory shaker at 150 rpm for 72 h at 30°C, in 300 PAGE) at 12% was carried out to determine the purity and ml Erlenmeyer flasks with a working volume of 50 ml. The cultures molecular weight of the enzyme, as described by Laemmli (1970). were centrifuged at 10,000 rpm for 10 min at 4°C to remove fungi The molecular weight of the enzyme was estimated using a low- mycelia and supernatants were used as the enzyme solution. All molecular weight calibration kit as markers consisting of bovine experiments were carried out in duplicate and repeated at least serum albumin (66 kDa), egg white ovalbumin (45 kDa), twice. glyceraldehyde-3-P deshydrogenase (36 kDa), bovine carbonic anhydrase (29 kDa), bovine trypsinogen (24 kDa), soybean trypsin inhibitor (20.1 kDa) and bovine -lactalbumin (14.2 kDa). Protein Assay of protease activity bands were visualized after staining with Coomassie brilliant blue R-250. Protease activity was measured by the method of Kembhavi et al. (1993), using casein as a substrate. Half milliliter of the enzyme, suitably diluted, was mixed with 0.5 ml of 100 mM glycine-HCl (pH Detection of proteolytic activity on polyacrylamide gels 3.0) containing 1% casein, and incubated for 5 min at 60°C. The reaction was stopped by addition of 0.5 ml trichloroacetic acid (20%. Zymography was performed in conjunction with SDS-PAGE w/v). The mixture was allowed to stand at room temperature for 15 according to the method described by Garcia-Carreno et al. (1993) min and then centrifuged at 10,000 rpm for 15 min and the with slight modification. The sample was not heated before loading precipitate was removed. The absorbance was measured at 280 in the gel. After electrophoresis, the gel was submerged in 100 mM nm using a UV spectrophotometer. A standard curve was glycine−HCl buffer (pH 3.0) containing 2.5% Triton X-100 for 30 min generated using solutions of 0-50 mg/l tyrosine. One unit of at 4°C, with constant agitation to remove SDS. Triton X-100 was protease activity was defined as the amount of enzyme required to then removed by washing the gel three times with 100 mM liberate 1 µg of tyrosine in one minute under the experimental glycine−HCl buffer (pH 3.0).