Biocontrol activity of an alkaline serine protease from Aureobasidium pullulans expressed in Pichia pastoris against four postharvest pathogens on apple.
The yeast-like fungus Aureobasidium pullulans PL5 is a microbial antagonist against postharvest pathogens of fruits. The strain is able to produce hydrolases, including glucanases, chitinases and proteases. The alkaline serine protease gene ALP5 from A. pullulans was cloned, inserted into the vector pPIC9 to construct pPIC9/ALP5, and then expressed in Pichia pastoris strain KM71. ALP5 had a molecular mass of 42.9kDa after 5days growth with 1% methanol induction at 28°C. The recombinant protease expressed in P. pastoris showed its highest activity under alkaline conditions (at pH10) and a temperature of 50°C. The antifungal activity of the recombinant protease was investigated against Penicillium expansum, Botrytis cinerea, Monilinia fructicola and Alternaria alternata in vitro and on apple. The recombinant protease reduced significantly the spore germination and the germ tube length of the tested pathogens in PDB medium. The highest level of protease efficacy was observed against M. fructicola and B. cinerea, whereas a lower efficacy was observed against P. expansum and A. alternata indicating a possible effect of the pathogen cell wall composition on the proteolytic activity of the recombinant protease. The presence of protease was able to cause the swelling of the hyphae of B. cinerea, under an optical microscope. The recombinant protease expressed in P. pastoris was more active against the pathogens in vitro than the same enzyme expressed in E. coli in previous studies. The efficacy of ALP5 was also evaluated against the pathogens in vivo on cv Golden Delicious apples. The protease was more efficient in controlling M. fructicola, B. cinerea and P. expansum than A. alternata. However, the extent of the activity was dependent on the enzyme concentration and the length of fruit storage. This study demonstrated the capacity of the alkaline serine protease to keep its enzymatic activity for some days in the unfavorable environment of the fruit wounds. The alkaline serine protease could be developed as a postharvest treatment with antimicrobial activity for fruit undergoing a short storage period.
Centre of Competence for the Innovation in the Agro-environmental Sector - AGROINNOVA, University of Torino, via L. da Vinci 44, I-10095 Grugliasco, TO, Italy.
Houda
H
Banani
Centre of Competence for the Innovation in the Agro-environmental Sector - AGROINNOVA, University of Torino, via L. da Vinci 44, I-10095 Grugliasco, TO, Italy; DiSAFA - Dept. Agricultural, Forestry and Food Sciences, University of Torino, via L. da Vinci 44, I-10095 Grugliasco, TO, Italy. Electronic address: davide.spadaro@unito.it.
Davide
D
Spadaro
Centre of Competence for the Innovation in the Agro-environmental Sector - AGROINNOVA, University of Torino, via L. da Vinci 44, I-10095 Grugliasco, TO, Italy.
Dianpeng
D
Zhang
Centre of Competence for the Innovation in the Agro-environmental Sector - AGROINNOVA, University of Torino, via L. da Vinci 44, I-10095 Grugliasco, TO, Italy.
Slavica
S
Matic
Centre of Competence for the Innovation in the Agro-environmental Sector - AGROINNOVA, University of Torino, via L. da Vinci 44, I-10095 Grugliasco, TO, Italy.
Angelo
A
Garibaldi
Centre of Competence for the Innovation in the Agro-environmental Sector - AGROINNOVA, University of Torino, via L. da Vinci 44, I-10095 Grugliasco, TO, Italy; DiSAFA - Dept. Agricultural, Forestry and Food Sciences, University of Torino, via L. da Vinci 44, I-10095 Grugliasco, TO, Italy.
Maria Lodovica
ML
Gullino
D000935:Antifungal Agents / Q000494:pharmacology; D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics; D005511:Food Handling; D005516:Food Microbiology / Q000379:methods*; D025301:Hyphae / Q000187:drug effects; D027845:Malus / Q000382:microbiology*; D003904:Mitosporic Fungi / Q000187:drug effects*; D010572:Pest Control, Biological / Q000592:standards*; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000378:metabolism / Q000494:pharmacology*; D013172:Spores, Fungal / Q000187:drug effects
D000935:Antifungal Agents; D011994:Recombinant Proteins; D012697:Serine Endopeptidases
Alternaria alternata; Botrytis cinerea; Monilinia fructicola; Penicillium expansum; Postharvest; Recombinant expression