Folia Microbiol. 54 (2), 105–109 (2009)
http://www.biomed.cas.cz/mbu/folia/
PepJ Is a New Extracellular Proteinase of Aspergillus nidulans T. EMRIa, M. SZILÁGYIa, K. LÁSZLÓa, M. M-HAMVASb, I. PÓCSIa aDepartment of Microbial Biotechnology and Cell Biology, bDepartment of Botany, Faculty of Science and Technology,
University of Debrecen, 4032 Debrecen, Hungary fax +36 52 512 925 e-mail
[email protected] Received 18 July 2008 Revised version 6 January 2009
ABSTRACT. Under carbon starvation, Aspergillus nidulans released a metallo-proteinase with activities comparable to those of PrtA, the major extracellular serine proteinase of the fungus. The relative molar mass of the enzyme was 19 kDa as determined with both denaturing and renaturing SDS PAGE, while its isoelectric point and pH and temperature optima were 8.6, 5.5 and 65 °C, respectively. The enzyme was stable at pH 3.5–10.5 and was still active at 95 °C in the presence of azocasein substrate. MALDI-TOF MS analysis demonstrated that the proteinase was encoded by the pepJ gene (locus ID AN7962.3), and showed high similarity to deuterolysin from Aspergillus oryzae. The size of the mature enzyme, its EDTA sensitivity and heat stability also supported the view that A. nidulans PepJ is a deuterolysin-type metallo-proteinase.
Abbreviations EP(s)
extracellular proteinase(s)
SDS-PAGE
sodium dodecylsulfate-polyacrylamide gel electrophoresis
Proteinases are industrial enzymes widely used for, e.g., milk clotting, meat tenderization, beer clarifying, hair removing in leather industry, manufacture of biological washing powders and contact lens cleaning fluid, medicine (e.g., to remove gastrointestinal parasites and dead skin from burnt patients) or in laboratory work (e.g., protein sequencing, processing of recombinant fusion proteins) (Rawlings et al. 2007). Not surprisingly, proteinases account for ≈60 % of total enzyme sales. In the fermentation industry, fungal proteinases are not only important as valuable products but they can destroy other valuable protein products as well and influence the morphology of the cultures via affecting autolytic cell wall degradation (Emri et al. 2008). Although up to six different groups of EPs have so far been identified in the aspergilli (Katz et al. 2000), there are only a few pieces of information available on the EPs of A. nidulans. The best-known EP of A. nidulans is PrtA, an alkaline serine proteinase. The regulation of the prtA gene has been published in detail (Katz et al. 2000, 2006, 2008), the PrtA gene product being purified and characterized (Peña-Montes et al. 2008). Moreover, the transcription of the pepJ gene putatively encoding a metallo-proteinase was demonstrated by Northern blot analysis during nitrogen starvation or in the presence of 1 % (W/V) bovine serum albumine; PepJ is likely an extracellular metallo-proteinase (Katz et al. 2008). In the case of the prtB gene, which encodes a putative aspartate proteinase, there are RT-PCR experiments suggesting that the gene is actively transcribed (vanKuyk et al. 2000; Molnár et al. 2006). Here we present data on the purification and characterization of an EP from carbon starving A. nidulans cultures, which is released at activities comparable to those of PrtA, the major extracellular serine proteinase of this fungus (Katz et al. 2008). We also demonstrate that this proteinase is a deuterolysin-type metallo-proteinase and is encoded by the pepJ gene (locus ID AN7962.3). MATERIALS AND METHODS Organism and growth conditions. Aspergillus nidulans MK191 (yA1 pabaA1 argB2 [argB]) and MK189 (yA1 pabaA1 argB2 prtA::argB) strains were a kind gift of M. Katz (University of New England, Armidale, Australia). Strains were grown in shake flasks (500 mL) containing 100 mL minimal-nitrate medium, pH 6.5 (Barratt et al. 1965) supplemented with 0.5 % (W/V) yeast extract. Culture media were inoculated with 5 × 107 spores and incubated (200 rpm, 20 h, 37 °C). Mycelia from 20-h-old cultures were washed and transferred into glucose-free minimal medium containing 200 g/L 4-amino-benzoic acid and were further cultivated (200 rpm, 1 d, 37 °C).
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SDS-PAGE and zymogram analysis of EPs. SDS-PAGE was performed on 12.5 % gels (LeBlanc and Cochrane 1987) using Page RulerTM Prestained Protein Ladder (Fermentas) as molar mass (M) standards; protein bands were visualized by Coomassie Blue staining (Laemmli 1970). Zymogram analysis was carried out on 7.5 or 10 % (W/V) SDS-polyacrylamide gels containing 400 ppm (W/V) gelatin as proteinase substrate (Schlereth et al. 2000). To allow proteolytic enzymes to renaturate, SDS was removed from the gels by two 10-min washes in 20 % (V/V) 2-propanol dissolved in phosphate buffer (pH 5.5; M-Hamvas et al. 2003). For M standards, the Low Molecular Weight Calibration Kit of Pharmacia was used. Gelatin degradation (visible as clear bands on a dark background after Coomassie staining) revealed the sites of active proteinases. Purification and MALDI-TOF MS analysis of EP. Protein content of the mycelium-free fermentation broth (obtained by filtration of 1-d-old washed cultures on sintered glass) was precipitated with 90 % diammonium sulfate at 4 °C for 1 h, and dialyzed overnight at 4 °C against 75 mmol/L Tris–acetic acid buffer (pH 9.3). Chromatofocusing was carried out on a Polybuffer Exchanger 94 (Pharmacia Biotech, Sweden) column between pH 9 and 6 (Binod et al. 2005). The pH of the active protein peak was determined and regarded as an isoelectric point of the enzyme. The active protein peak was run on 12.5 % SDS-PAGE and MALDI-TOF MS analysis of the band was done according to Pusztahelyi et al. (2007). Proteinase activity was measured in 0.2 mol/L Na2HPO4–0.1 mol/L citric acid buffer (pH 6.5) using azocaseine as substrate in a final concentration of 12.5 mg/mL. Samples and enzyme-free buffer (control) were incubated with the substrate for 30 min at 37 °C, then the reaction was stopped with 4 volumes of 5 % (V/W) trichloroacetic acid. After centrifugation (10 min, 5000 g, 4 °C), the supernatant was treated with 1 volume of 0.5 mol/L NaOH and the absorbance at 440 nm was measured (Tomarelli et al. 1949). One unit of enzyme activity was defined as the amount of enzyme resulting in the increase of absorbance of 10–3 per min. Determination of pH optimum, pH stability, temperature optimum, heat stability and EDTA sensitivity of the purified proteinase. Because of the low solubility of azocaseine at acidic pH, the pH dependence of enzyme activity was determined with bovine serum albumin as substrate (final concentration 12.5 mg/mL) at pH 3.5–7.5 (0.2 mol/L Na2HPO4–0.1 mol/L citric acid buffer) and at pH 8.5–10.5 (0.2 mol/L NaOH– 0.2 mol/L Gly buffer). Samples and enzyme-free buffer (control) were incubated (30 min, 37 °C) with the substrate solution. After stopping the reaction with 4 volumes of 5 % (W/V) trichloroacetic acid, samples were centrifuged (10 min, 5000 g, 4 °C) and the absorbance at 280 nm was read. For determining the pH stability of the enzyme, samples were pre-incubated at the appropriate pH (1 d, 37 °C) and enzyme activity was determined as described above. Temperature dependence of the enzyme activity was determined by adjusting the incubation temperature to 5–95 °C. To study the heat and EDTA stability of the enzyme, samples were pre-incubated at the selected temperatures or with 10 mmol/L Na2-EDTA (pH 6.5; 30 min, 37 °C) and enzyme activity was determined as described above. RESULTS AND DISCUSSION The zymogram of EPs occurring in carbon-depleted A. nidulans MK191 revealed two major proteinases with 19 and 35 kDa M values (Fig. 1). Since the zymogram of the DprtA gene-deletion mutant MK189 contained only one band at 19 kDa, the 35 kDa proteinase was considered to be the PrtA proteinase, which agrees with Peña-Montes et al. (2008). The widths of the two bands and the total EP activity produced by the control (MK191) and DprtA mutant strains (223 ± 17 and 104 ± 10 U/mL, respectively) indicated that the activities of PrtA and the other proteinase were comparable in carbon-starving cultures. When 7.5 % polyacrylamide gels were used, further proteinase bands
Fig. 1. Zymogram analysis of EPs produced by carbon-starving A. nidulans MK189 (A; control strain) and MK191 (B; DprtA deletion mutant) strains; M – molar-mass markers.
NEW EXTRACELLULAR PROTEINASE PepJ OF A. nidulans 107
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were visualized in both strains with lower mobility than that of PrtA but these bands were weak (and not observable on 10 % gel; data not shown). It is worth mentioning that Katz et al. (2008), using zymogram analysis with nondenaturing PAGE and milk agarose overlay for detection, detected 6 separate bands including those representing PrtA and a metallo-proteinase. The 19 kDa proteinase was purified by diammonium-sulfate precipitation and chromatofocusing to homogeneity tested by Coomassie staining (Table I, Fig. 2). The collected samples contained again only one proteinase with M of 19 kDa determined by denaturing SDS-PAGE (Fig. 2), indicating that the protein conTable I. Purification of PepJ from carbon starving Aspergillus nidulans cultures Purification step Crude extract (NH4)2SO4 precipitation Chromatofocusing
Protein µg
Specific activity U/mg protein
Yield %
8740 6670 252
37 8 124
100 17 10
Fig. 2. Purification and purity check of PepJ. A: Chromatofocusing of PepJ after (NH4)2SO4 precipitation and dialysis; closed squares – protein content (A280), open squares – proteinase activity (A440), circles – pH; B: Result of denaturing SDS-PAGE of fraction no. 12 (possessing the highest proteinase activity); M – molar-mass markers.
sisted of one subunit. MALDI-TOF MS analysis of the purified enzyme revealed that this proteinase was encoded by the AN7962.3 locus (pepJ; Katz et al. 2008; Broad Institute http://www.broad.mit.edu/ annotation/genome/aspergillus_group/MultiHome.html ). PepJ is a deuterolysin-type metalloproteinase; the expected molar mass of the protein (calculated from the sequence of the pepJ gene) was twice higher (37 kDa) than that found for the purified enzyme (19 kDa). Substantial posttranslational processing was also recorded for Aspergillus oryzae deuterolysin (neutral proteinase II; encoded by AO 090 010 000 493), where the 353 amino-acid long precursor protein consists of a 175-residue prepro region and the 177-residue processed enzyme with M = 19 kDa (Tatsumi et al. 1991). The transcribed penicillolysin from Penicillium citrinum also consists of a 155-residue propeptide segment and the 177-residue proteinases with M = 18 kDa (Matsumoto et al. 1994). The precursor protein encoded by the pepJ gene showed high similarity to the A. oryzae deuterolysin precursor protein (NPII; locus ID: P46076; 61 % identity and 76 % positive residues) and to the P. citrinum penicillolysin precursor protein (PlnC; locus ID: P47189; 61 % identity and 76 % positive residues) (NCBI databases http://www.ncbi.nlm.nih.gov/; blastp tool). Moreover, EDTA efficiently inactiva-
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ted the purified A. nidulans PepJ (47 % inactivation by exposure to 10 mmol/L Na2-EDTA for 30 min), which also supports its metallo-proteinase nature. Considering the enzymological characteristics of PepJ, its isoelectric point was alkaline (pI 8.6; Fig. 2) similarly to penicillolysin (pI 9.6; Yamaguchi et al. 1993). It is worth noting that the pI of the alkaline serine proteinase PrtA of A. nidulans is acidic (4.5; Peña-Montes et al. 2008). Although the pH optimum of PepJ was at pH 5.5, it showed still significant activity up to pH 10.5 (Fig. 3), which is important in terms of the physiology of carbon-starving cultures. In the absence of easily metabolizable carbon source, filamentous fungi degrade and catabolize intracellular protein and peptide reserves as well as proteins released into the culture media during autolysis; these processes result in the liberation of ammonia and, consequently, in the alkalinization of culture media (Emri et al. 2004). The enzyme also showed significant pH stability (3.5– 10.5) at 37 °C (Fig. 3).
Fig. 3. Enzymological characterization of purified PepJ (proteinase activity, %); mean ± SD calculated from quadruplicates, SD being always
