Cloning, characterization, expression and antifungal activity of an alkaline serine protease of Aureobasidium pullulans PL5 involved in the biological control of postharvest pathogens.
An alkaline protease gene was amplified from genomic DNA and cDNA of the antagonistic yeast-like fungus Aureobasidium pullulans PL5, a biocontrol agent effective against Monilinia laxa on stone fruit and Botrytis cinerea and Penicillium expansum on pome fruits. An open reading frame of 1248 bp encoding a 415-amino acid (aa) protein with a calculated molecular weight (M(r)) of 42.9 kDa and an isoelectric point (pI) of 4.5 was characterized. The cDNAALP5 gene had an 18-amino acid signal peptide, one N-gylcosylation, one histidine active site, and one serine active site. The ALP5 gene with a M(r) of 1351 bp contained two introns. One intron was of 54 bp, while the other was of 50 bp. Protein BLAST and phylogenetic tree analysis of the deduced amino sequences from the cDNAALP5 gene showed that the encoded protein had 100% homology to a protease enzyme (ALP2) of a sea strain of A. pullulans, suggesting that the protein ALP5 was an alkaline serine protease. Expression of ALP5 in Escherichia coli BL21 (DE3), followed by identification with Western-blotting, purification with Ni-NTA and analysis of enzymatic activity, yielded an homogeneous recombinant ALP5 which hydrolysed the substrate casein and inhibited the mycelial growth of the pathogens. At its optimal pH of 10.0 and reaction temperature of 50°C, the recombinant protease exhibited the highest activity towards the substrate casein, though the highest stability was at lower temperatures and pH between 7.0 and 9.0. This study provided the direct evidence that extracellular proteases secreted by the antagonist A. pullulans PL5 played a role in the biocontrol activities against some postharvest pathogens of apple and peach.
Centre of Competence for the Innovation in the Agro-environmental Sector, University of Torino, via L. da Vinci 44, I-10095 Grugliasco (TO), Italy.
Dianpeng
D
Zhang
Davide
D
Spadaro
Silvia
S
Valente
Angelo
A
Garibaldi
Maria Lodovica
ML
Gullino
D000595:Amino Acid Sequence; D000818:Animals; D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D061046:Biological Control Agents; D020171:Botrytis / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D019976:Cloning, Organism; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005638:Fruit / Q000382:microbiology*; D006868:Hydrolysis; D007438:Introns; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010407:Penicillium / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D017385:Sequence Homology; D012694:Serine / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D057057:Serine Proteases
D001426:Bacterial Proteins; D061046:Biological Control Agents; D018076:DNA, Complementary; D012694:Serine; D010450:Endopeptidases; D057057:Serine Proteases; D012697:Serine Endopeptidases; C087822:alkaline protease