Andrea Rodríguez-Martín, Raquel Acosta, Félix Núñez, Mª José Benitoa, Miguel A. Asensio*
Higiene y Seguridad Alimentaria, Facultad de Veterinaria. Universidad de Extremadura, Avda. de la Universidad, s/n. 10071- Cáceres, Spain.
a Nutrición y Bromatología, Escuela de Ingenierías Agrarias, Universidad de Extremadura, Carretera de
Cáceres s/n, 06071- Badajoz, Spain
* Corresponding author: Telephone (+34) 927 257 125; fax: (+34) 927 257 110. E-mail: [email protected]
ABSTRACT
The strain RP42C from Penicillium chrysogenum produces a small protein PgAFP that inhibits the growth of some toxigenic moulds. The gene encoding the precursor of this antifungal protein has been characterized. cDNA was prepared from isolated total RNA by RT-PCR. The cDNA fragment encoding the pgafp gene was obtained by PCR using degenerate primers based on two known amino acid sequences of the purified PgAFP. Full-length cDNA of pgafp was obtained by RACE-PCR. Comparison of nucleotide sequence from genomic fragment and cDNA of the gene, revealed the presence of a 279 bp coding region which is interrupted by two introns of 63 and 62 bp in length. The precursor of the antifungal protein consists of 92 amino acids and appears to be processed to the mature 58 amino acids PgAFP by two-step process. The deduced amino acid sequence of the preproprotein revealed 75 % identity to an antifungal protein predicted from the Aspergillus niger CBS 513.88 genome.
KEY WORDS: preproprotein, protective cultures, dry fermented foods. INTRODUCTION
The strain RP42C of the mould Penicillium chrysogenum secretes a small protein PgAFP that has been purified for its ability to inhibit toxigenic moulds isolated from dry-cured meat products (Acosta et al., 2009a,b). Several amino acid sequences of this protein were obtained by electrospray ionization mass spectrometry (Rodríguez-Martín et al., 2009) revealing a high homology with the antifungal protein Anafp (Lee et al., 1999). Anafp protein belongs to a new group of antimicrobial proteins named small, basic and cysteine-
rich antifungal proteins. The recent sequencing of the genome of different moulds has shown several genes that could encode proteins with common characteristics to this group. However, only four proteins have been reported: Anafp from Aspergillus niger (Lee et al., 1999), PAF from Penicillium chrysogenum (Marx et al., 1995), AFP from Aspergillus giganteus (Nakaya et al., 1990; Lacadena et al., 1995), and AcAFP from Aspergillus clavatus (Skouri-Gargouri and Gargouri, 2008). Only AFP and PAF have been genetically characterizated. In addition a gene homologous to paf gene has been described from Penicillium nalgiovense (Geisen, 2000).
The genes encoding PAF and AFP have open reading frames interrupted by two introns with conserved splice sites. They encode products of 92 and 94 amino acids, respectively, and these proteins are synthesized as a long precursor with a peptide signal and a presequence that are removed to obtain mature proteins.
Genetic characterization of the gene encoding PgAFP will allow to clon it in other microorganisms, to increase PgAFP production and to obtain protective cultures for future applications in foods, environment or medicine. In addition, the genetic sequence will give valuable information for a better characterization of the protein.
MATERIALS AND METHODS Microbial strain
P. chrysogenum RP42C was isolated from dry-cured ham, and belongs to the fungal collection of Food Hygiene, University of Extremadura.
DNA isolation
P. chrysogenum RP42C was grown in malt extract broth made with 2% (w/v) glucose, 2% (w/v) malt extract, and 0.1% (w/v) peptone, pH 4.5 at 25ºC under continuous shaking for 5 days. The mycelium was obtained by filtering the culture through Whatman paper nº2. Two grams of mycelium were broken with mortar and pestle after freezing by adding liquid nitrogen. Fungal lysis was performed by homogenizing the obtained powder in 4 ml 0.05 M Tris-HCl, pH 8; 0.005 M ethylenediamine tetraacetic acid (EDTA); 0.05 M NaCl (TES) buffer and 1% (w/v) sodium dodecyl sulfate (SDS). Then, 200 µl proteinase K (20 µg/µl) were added, the mix was incubated at 60ºC for 35 min, cooled on ice, and extracted with phenol-chloroform-isoamyl alcohol (25:24:1). The suspension was centrifuged at
3000×g for 2 min at 4 ºC. The upper phase containing DNA was transferred to a fresh tube and precipitated by adding 3 M sodium acetate pH 5.2 to a final concentration of 10% (v/v) and two volumes of cold ethanol. After centrifugation, the pellet was cleaned with 70% (v/v) ethanol, centrifuged again in the conditions above mentioned, resuspended in sterile water, and treated with 50 µl RNase (10 µg/µl) at 37ºC for 60 min. Finally, DNA was extrated with phenol-chloroform-isoamyl alcohol, precipitated again as indicated above, resuspended in water, and stored at -70ºC.
Quantity and quality of purified DNA was determined spectrophotometrically in a Biophotometer (Eppendorf AG, Hamburg, Germany).
Genomic DNA amplification
Polymerase chain reactions were performed with genomic DNA (100 ng) as template in 50 µl reaction mixtures containing 50 pmol each of forward and reverse primers, 0.5 mM each of the deoxyribonucleotide triphosphates (dNTPs), 0.1 vol of 10× PCR buffer (22.5 mM MgCl2, 500 mM Tris-HCl pH 9.2, 140 mM ammonium sulfate), 1.8 mM MgCl2 and 2.5
units pfu turbo polymerase (Stratagene, La Jolla, USA). The degenerated primers, pgafp- DPF1 and pgafp-DPR1A (Table 1), were designed from amino acid sequences previously obtained by mass spectrometry (Rodríguez-Martín et al., 2009). Three reactions were done: one with both forward and reverse primers, another only with forward, and another only with reverse primer.
DNA amplification was carried out in a Px2 thermocycler (Thermo Scientific, Cramlington, UK). The PCR program consisted of initial denaturation (94ºC for 5 min), 35
Primer names Amino acid sequences and designed primers*
pgafp-DPF1
L K H N T C T 5’-TN AAR CAY AAY ACN TGN AC-3’
pgafp-DPR1A
C G S A A N 3’-CR CCN AGN CGN CGN TT-5’ * R for purine, Y for pirimidine, N for A, C, T, or G.
cycles of denaturation (94ºC for 5 s), annealing (45-57ºC range for 5 s, gradient 12), and extension (72ºC for 1 s) steps, followed by a final extension at 72 ºC for 4 min. PCR products were electrophoresed on 2% (w/v) agarose gels with MetaPhor agarose (BMA, Rockland, ME, USA). Two different markers were used for agarose gels: DNA marker from Gibco (0.15-12kb bands) (Gibco, GrandIsland, NY, USA) and DNA size standard from Bio-Rad (0.05-2kb) (Bio-rad Laboratories, Madrid, Spain). PCR products detected only in the reactions with both reverse and forward primers were gel-purified and cloned into the pCR2.1-TOPO vector (Invitrogen, Barcelona, Spain). One Shot TOP10 Chemically Competent E. coli (Invitrogen) were transformed with that vector. To confirm the insert in the vector, digestions with restriction enzyme EcoRI or PCR with M13 forward and reverse primers (Invitrogen) were carried out. Finally, vectors were sequenced in the Institute of Biomedicine (CSIC, Valencia, Spain).
RNA isolation
P. chrysogenum RP42C was grown in malt extract broth as indicated for DNA isolation. Subsequently, mycelium was quickly frozen with liquid nitrogen and stored at -80ºC until RNA extraction. Mycelium was broken with mortar and pestle after adding liquid nitrogen. RNA extraction was performed with RNeasy Plant Mini Kit (Qiagen) following the manufacturer’s instructions. The final extracted RNA was resuspended in 40 µl of RNase- free deionized water.
Rapid amplification of cDNA ends-Polymerase Chain Reaction (RACE-PCR)
Amplification of 5’ and 3’ ends of pgafp gene was carried out using the SMART RACE cDNA Amplification Kit (Clontech Laboratories, Saint Germain en Laye, France).
cDNA synthesis by reverse transcriptase-polymerase chain reaction (RT-PCR). Two separate populations of cDNA were synthesized according to the amplification kit instructions: 5’-RACE-Ready cDNA and 3’-RACE-Ready cDNA. After incorporating the SMART sequence into both the 5’- and 3’-RACE-Ready cDNA populations, RACE PCR reactions were performed using the Universal Primer A Mix (UPM), in conjunction with distinct gene-specific primers.
Gene-specific primers design. A gene-specific primer (GSP) was designed for amplifying the 3’- end (3’-RACE GSP: 5’-GGGTGGAAAGAACCATGTAGTCAATTGCG-3’) from
the sequence previously obtained by PCR with degenerate primers pgafp-DPF1 and pgafp- DPR1A. Amplification of the 5’- end was carried out designing a gene-specific primer (5’- RACE GSP: 5’-AACTGGGGTCTGGCAGTCAACCCTC-3’) from the 3’-end also previously obtained.
Polymerase chain reactions were performed with 5’- or 3’-RACE-Ready cDNAs (50 ng) as template in 50 µl reaction mixtures containing 1 mM of each of the dNTPs (Roche,
Madrid, Spain), 0.5 µM of the 5’- or 3’-RACE GSP primers, 5 µl of Universal Primer A Mix (10×), 0.1 vol of 10× PCR buffer (10 mM Tris-HCl, pH 8.8, 50 mM KCl, 0.1 % Triton X-100), 4 mM MgCl2 and 2.5 units pfu turbo polymerase (Stratagene). The PCR
program for obtaining the 3’-end consisted of 35 cycles of denaturation (94ºC for 30s), annealing (68ºC for 30s), and extension (72ºC for 3 min).
The PCR program for 5’-end amplification was: 5 cycles of 2 steps (94ºC for 30 s and 72ºC for 3 min), 5 cycles of 3 steps (94ºC for 30 s, 70ºC for 30 s and 72ºC for 3 min) and 27 cycles of 3 steps (94ºC for 30 s, 68ºC for 30 s and 72ºC for 3 min). Finally, 5’- and 3’- RACE-PCR products were gel-purified and cloned into pCR2.1-TOPO vectors, which were used to transform One Shot TOP10 Chemically Competent E. coli (Invitrogen) before purifying and sequencing the plasmid.
Digestions with restriction enzyme Eco RI (Roche) or PCR with M13 forward primer (Invitrogen) were performed to confirm the insert in the vector.
Full-length cDNA amplification. The full cDNA fragment of the gene pgafp was obtained by PCR with specific primers (Fwd-PrePro pgafp: 5’-ATGCAGATCACCAGCATTGCC- 3’ and Rev-PrePro/Mature pgafp: 5’-TCAAACTGGGGTCTGGCAGTC-3’) designed from 5’ and 3’ ends. PCR reaction mixtures at a final volume of 50 µl contained 5µl of 5’- RACE-Ready cDNA, 0.5 µM each of forward and reverse primers, 0.5 mM each of the dNTPs, 0.1 vol of 10× PCR buffer (10 mM Tris-HCl, pH 8.8, 50 mM KCl, 0.1% Triton X- 100), 4 mM MgCl2 and 2.5 units pfu turbo polymerase (Stratagene). The PCR program
consisted of 35 cycles of denaturation (94ºC for 30 s), annealing (65ºC for 30 s), and extension (72ºC for 3 min). PCR products were gel-purified and cloned into pCR2.1- TOPO vectors, which were used to transform One Shot TOP10 Chemically Competent E. coli (Invitrogen) before purifying and sequencing the plasmid. Digestions with restriction enzyme Eco RI (Roche) or PCR with M13 forward primer (Invitrogen) were performed to confirm if the insert was in the vector.
Amplification of the whole pgafp gene
The gene sequence was amplified by PCR using Fwd-PrePro pgafp and Rev- PrePro/Mature pgafp primers. PCR mixtures were composed by 100 ng of genomic DNA, 0.5 µM each of forward and reverse primers, 0.5 mM each of the dNTPs, 0.1 vol of 10× PCR buffer (10 mM Tris-HCl, pH 8.8, 50 mM KCl, 0.1% Triton X-100), 4 mM MgCl2,
and 2.5 units pfu turbo polymerase (Stratagene) and deionized water until a final volume of 50µl. The PCR program consisted of 30 cycles of denaturation (94ºC for 30 s), annealing (65ºC for 30 s), and extension (72ºC for 3 min). PCR products were cloned in pCR2.1- TOPO vector that later was used to transform cells of One Shot TOP10 Chemically Competent E. coli. The plasmid was purified and sequenced. Introns sequences were confirmed by GenScan software.
RESULTS
Partial amplification of pgafp gene using degenerated primers
The pgafp gene partial amplification was done using the two degenerate primers, pgafp- DPF1 and pgafp-DPR2A (Table 1). After different reactions, the optimal annealing temperature was 45.1ºC. PCR products run in agarose gel showed one small band of around 75 bp that was amplified only in reactions with both the forward and the reverse primers (Figure 1). This band was selected for cloning and sequencing.
Figure 1. Products obtained by PCR using DPF1 primer (F), DPF1+DPR2A primers (F+R), and DPR2A (R) primer. M: DNA standards. The arrow indicates the selected band for cloning and sequencing.
After sequencing, the fragment found was 70 bp length (5’-tgaagcataatacgtgcacatacctaaag ggtggaaagaaccatgtagtcaattgcggttctgccgctaa-3’), and the translated sequence (KHNTCTYL KGGKNHVVNCGSAA) was compared in Basic Local Alignment Search Tool (BLAST) showing common sequences with Anafp from Aspergillus niger.
Sequence of pgafp gene by RACE-PCR
Both 3’ and 5’ -ends of the gene pgafp were obtained by amplification from cDNA. mRNA was extracted from a 5 days-old culture of P. chrysogenum RP42C and was used to obtain the two different populations of 5’- and 3’-RACE-Ready cDNA by RT-PCR. The gene specific primer 3’-RACE GSP (5’-GGGTGGAAAGAACCATGTAGTCAATTGCG-3’), designed from the known pgafp sequence of genomic DNA, allowed to amplify the 3’-end by RACE-PCR. The resulting product of about 450 bp included 121 bp of the 3’-end of pgafp (Figure 2). Amplification of the 5’-end was carried out with a new gene specific primer, 5’-RACE GSP (5’-AACTGGGGTCTGGCAGTCAACCCTC-3’), designed from 3’-end sequence. The obtained band, of approximately 440 bp, included the full pgafp cDNA gene.
Amplifications of complete gene in genomic DNA and cDNA were performed by designing two new primers Fwd-full pgafp (5’-ATGCAGATCACCAGCATTGCC-3’) and Rev-full pgafp (5’-TCAAACTGGGGTCTGGCAGTC-3’). The sequence of genomic DNA was 404 bp, whereas the amplified cDNA was 279 bp (Figure 3). The difference between
B) A)
Figure 2. cDNA fragments of 5’- and 3’-RACE PCR (A and B, respectively). M: DNA standards.
the sequences revealed the existence of two introns of 63 and 62 bp (Figure 4), absent in the cDNA, which were confirmed by GenScan software.
The open reading frame was translated to protein by ExPASy proteomics server at the Swiss Institute of Bioinformatics (http://www.expasy.org). Analysis of the signal peptidase
Figure 3. pgafp gene amplification of the with genomic DNA (1) and cDNA (2). M: DNA standards. 5’-atgcagatcaccagcattgccattgtcttcttcgccgcaatgggtgcggttgctaacccc M Q I T S I A I V F F A A M G A V A N P atcgcgagggagtcggacgatcttgatgcccgagacgtacagcttagtaaattcggagga I A R E S D D L D A R D V Q L S K F G G gtaagttcttcttacaagacgtctatatagaaatagcactaacctttctgaaccacttta << Intron 1 caggaatgcagcttgaaacacaacacgtgcacatacctaaagggtggaaagaaccatgta >> E C S L K H N T C T Y L K G G K N H V gtcaattgcggttcggccgccaacaagaaggtaggttccgattcgattcggggccaattg V N C G S A A N K K << Intron 2 atttgttcttatcatttaatcttcatctacagtgcaagtctgatcgccaccactgtgaat >> C K S D R H H C E acgatgagcaccacaagagggttgactgccagaccccagtttga-3’ Y D E H H K R V D C Q T P V -
Figure 4. Full-length DNA sequence of pgafp gene. The open reading frame has been translated to amino acids. Consensus splice sequences and internal marker of fungal introns are underlined.
cleavage site predicted by the program SignalP (Nielsen et al., 1997) suggested that the pre-sequence consisted of the first 18 amino acids (MQITSIAIVFFAAMGAVA).
DISCUSSION
P. chrysogenum RP42C produces a high amount of the PgAFP in the first week of incubation (Acosta, 2006). Thus, to obtain appropriate cDNA populations, a 5 days-old culture was used to make sure that the mRNA from gene encoding PgAFP was actively being produced. The RACE-PCR was effective to obtain pgafp gene. Thus, the designed primers and obtained cDNA populations were adequate to amplify the 3’ and 5’ –ends of pgafp. When the complete sequence was obtained, a difference between the pgafp gene from genomic DNA (404 bp) and from cDNA (279 bp) could be observed (Figure 3). Thus, the existence of two introns of 63 and 62 bp in the gene, which are absent in the cDNA, was confirmed by GenScan software. The small size of these introns compared to those of mammalian introns is a typical feature of fungal genes (Gurr et al., 1987). The 5’- and 3’-ends of the two introns are quite similar (Figure 4) and coincide with the consensus splice sequences for fungal introns: 5’-splice donor site GTNNGT (N= A, G, T or C) and the consensus 3’-splice acceptor site DYAG (D= A, G or T; Y= C or T). In addition, the first intron has the internal CTAAC sequence, very common in fungal introns (Wiesner et al., 1988).
The pgafp open reading frame encodes a 92 amino acid-long precursor of the PgAFP. The first 18 amino acids correspond with a secretion signal sequence according to the SignalP program (Nielsen et al., 1997). The next residues (19-34) were not found in the amino acid sequence obtained from the purified protein (Rodríguez-Martín et al., 2009). These 16 amino acids constitute a prosequence that would be removed before or during the release of mature PgAFP. Prosequences play an important role by preventing protein activity before secretion. Only after the prosequence is cleaved off, the mature protein adopts its correct tertiary structure and gains its final activity. Antifungal proteins produced as preproproteins, such as PAF and AFP (Nakaya et al., 1990; Marx et al., 1995), have been described from other moulds.
The deduced mature PgAFP has 58 amino acid residues. It shows a high percentage (27.6 %) of basic amino acids. The isoelectric point was estimated to be 8.8 using the Compute pI tool from Expasy (Gasteiger et al., 2005). The isoelectric point of PgAFP estimated by isoelectrofocusing in previous studies (Acosta et al., 2009b) was 9.2, higher than the
predicted value with Compute pI tool. However, the prediction can be not exact for highly basic and small proteins (Gasteiger et al., 2005). A pI of 9.2 results in a net positive charge under physiological conditions, which is a common characteristic among antifungal proteins from moulds.
The accessibility of the whole genome sequence of an increasing number of microorganisms facilitates the search for genes encoding potential antifungal proteins. Three of these genes, those from Aspergillus niger, Neosartorya fischeri, and Gibberella zeae, show partial homology with PgAFP protein. The sequences of the hypothetical proteins from these genes as well as other reported antifungal proteins are aligned in Figure 5. The protein showing the highest homology (75%) with the precursor of PgAFP was the hypothetical protein from A. niger. Despite both PAF and PgAFP precursors are produced by Penicillium chrysogenum, they only share a similarity of 43%.
There are six cysteines conserved in all the similar proteins (Figure 5). Those residues would play an important role on stabilizing of the tertiary structure of PgAFP by formation of disulfide bridges (Nakaya et al. 1990; Marx et al., 1995; Lee et al., 1999).
PgAFP ---MQITSIAIVFFAAMGAVANPIARE---SDDLDARD--VQLSKFGGECSLKHNTCTYL
Anafp ---LSKYGGECSLEHNTCTYR
A.niger ---MQLTSIAIILFAAMGAIANPIAAE---ADNLVARE--AELSKYGGECSVEHNTCTYL
N.fischeri ---MQITKISLFLFVGIGVVASPIHAE---SDGLNARAVNAADLEYKGECFTKDNTCKYK
G.zeae ---MQFSTIIPLFVAAMGVVATPVNSP---AQELDARGNLFPRLEYWGKCTKAENRCKYK
PAF ---MQITTVALFLFAAMGGVATPIESV---SNDLDARAEAGVLAKYTGKCTKSKNECKYK AFP MKFVSLASLGFALVAALGAVATPVEADSLTAGGLDARDESAVLATYNGKCYKKDNICKYK
A.clavatus MKVVSLASLGFALVAALGVVASPVDADSLAAGGLDARDESAVQATYDGKCYKKDNICKYK
PgAFP K-GGKNHVVNCGSAANKKCKSDRHHCEYDEHHKRVDCQTPV
Anafp K-DGKNHVVSCPSAANLRCKTDRHHCRYDDHHKTVDCQTPV
A.niger K-GGKDHIVSCPSAANLRCKTERHHCEYDEHHKTVDCQTPV
N.fischeri ID-GKTYLAKCPSAANTKCEKDGNKCTYDSYNRKVKCDFRH
G.zeae NDKGKDVLQNCPKFDNKKCTKDGNSCKWDSASKALTCY---
PAF NDAGKDTFIKCPKFDNKKCTKDNNKCTVDTYNNAVDCD--- AFP AQSGKTAICKCYV---KKCPRDGAKCEFDSYKGKCYC----
A.clavatus AQSGKTAICKCYV---KVCPRDGAKCEFDSYKGKCYC----
Figure 5. Alignment of PgAFP amino acid sequences (Rodríguez-Martín et al., 2009) with other antifungal proteins. Accession numbers in Genbank are XP_001391221 (hypothetical protein from Aspergillus niger CBS 513.88), XP_001262586 (hypothetical protein from
Neosartorya fischeri NRRL 181), XP_384921 (hypothetical protein from Gibberella zeae PH-
1), AAA92718 (PAF from P. chrysogenum), P17737 (AFP from A. giganteus) and XP_001267787 (hypothetical protein from Aspergillus clavatus). Common amino acids are denoted by shaded background letters.
In previous studies PgAFP showed a molecular weight of 6,494 Da by mass spectrometry (Rodríguez-Martín et al., 2009). Translating the genetic sequence according to Gasteiger et al. (2005) the predicted mass of the mature protein was 6,500 Da, very close to the weight determined by mass spectrometry. In previous works, PgAFP protein was tested for having glycosylations by treatments with deglycosilases and chemical compounds, and no linked- oligosaccharides were found. The minimal difference found between predicted and experimental molecular weights confirms that this protein is not glycosylated.
Recently, the genome of P. chrysogenum Wisconsin 54-1255 has been sequenced (Van den Berg et al., 2008). A putative protein can be found in BLAST (accession number CAP80456) that differs from the precursor of PgAFP in only three amino acids. However, the production of the protein by this mould has not been reported.
In conclusion, the gene enconding the antifungal protein PgAFP has been sequenced. Such as similar proteins, PgAFP is synthesized as a long precursor with a peptide signal and a presequence that are removed to obtain mature proteins. The characterization of pgafp gene will permit its cloning for overproduction or heterologous expression of the protein in other microorganisms.
ACKNOWLEDGEMENTS
This work was supported by the Spanish Ministry of Education and Science (AGL2004- 06546-ALI) and FEDER. Andrea Rodríguez and Raquel Acosta were the recipients of FPI grants from the Spanish Ministry of Education and Science. Authors would like to thank Dr. Susan Liddell (Division of Animal Sciences) and Prof. Ian Connerton (Division of Food Sciences) for their support during Andrea Rodriguez stay’s at Nottingham University.
REFERENCES
Acosta, R. Doctoral Thesis: “Selección de Penicillium productores de péptidos antifúngicos para su utilización en productos cárnicos madurados”. University of Extremadura. Spain. 2006.
Acosta, R., Rodríguez-Martín, A., Martín, A., Núñez, F., and Asensio, M.A. Selection of antifungal protein-producing molds from dry-cured meat products. Int. J. Food Microbiol. Submitted (2009a).
Acosta, R., Rodríguez-Martín, A., Bermúdez, E., Núñez, F., and Asensio, M.A. Partial characterization and antifungal activity of a protein from Penicillium chrysogenum. Food Microbiol. Submitted (2009b).
Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M.R., Appel, R.D., and Bairoch, A. Protein Identification and Analysis Tools on the ExPASy Server;
(In) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press. Full text - Copyright Humana Press (2005).
Geisen, R. P. nalgiovense carries a gene which is homologous to the paf gene of P.
chrysogenum which codes for an antifungal peptide. Int. J. Food Microbiol. 62: 95-101 (2000).
Gurr, S.J., Uncles, S.E., and Kinghorn, J.R. The structure and organization of nuclear genes of filamentous fungi. In: Kinghorn, J.R. (Ed.). Gene structure in Eukaryotic
Microbes. IRL Press, Oxford, pp. 93-193 (1987).
Lacadena, J., Martínez del Pozo, A., Gasset, M., Patiño, B., Campos-Olivas, R., Vázquez, C., Martínez-Ruiz, A., Mancheño, J.M., Oñaderra, M., Gavilanes, J.G. Characterization of the antifungal protein secreted by the mould Aspergillus giganteus. Arch. Biochem. Biophys. 20: 273-281 (1995).
Lee, D.G., Shin, S.Y., Maeng, C-Y., Jin, Z.Z., Kim, K.L. and Hahm, K-S. Isolation and characterization of a novel antifungal peptide from Aspergillus niger. Biochem. Biophy.