title: Heterologous expression of an aspartic protease gene from biocontrol fungus Trichoderma asperellum in Pichia pastoris.
abstract: Trichoderma asperellum parasitizes a large variety of phytopathogenic fungi. The mycoparasitic activity of T. asperellum depends on the secretion of complex mixtures of hydrolytic enzymes able to degrade the host cell wall and proteases which are a group of enzymes capable of degrading proteins from host. In this study, a full-length cDNA clone of aspartic protease gene, TaAsp, from T. asperellum was obtained and sequenced. The 1,185 bp long cDNA sequence was predicted to encode a 395 amino acid polypeptide with molecular mass of 42.3 kDa. The cDNA of TaAsp was inserted into the pPIC9K vector and transformed into yeast Pichia pastoris GS115 for heterologous expression. A clearly visible band with molecular mass about 42 kDa in the SDS-PAGE gel indicated that the transformant harboring the gene TaAsp had been successfully translated in P. pastoris and produced a recombinant protein. Enzyme characterization test showed that the optimum fermentation time for P. pastoris GS115 transformant was 72 h. Enzyme activity of the recombinant aspartic proteinase remained relatively stable at 25-60 °C and pH 3.0-9.0, which indicated its good prospect of application in biocontrol. The optimal pH value and temperature of the enzyme activity were pH 4.0 and 40 °C, and under this condition, with casein as the substrate, the recombinant protease activity was 18.5 U mL(-1). In order to evaluate antagonistic activity of the recombinant protease against pathogenic fungi, five pathogenic fungi, Fusarium oxysporum, Alternaria alternata, Cytospora chrysosperma, Sclerotinia sclerotiorum and Rhizoctonia solani, were applied to the test of in vitro inhibition of their mycelial growth by culture supernatant of P. pastoris GS115 transformant.
authors.affiliation: School of Life Science and Engineering, Harbin Institute of Technology, P.O. 437, No 92, West Da-Zhi Street, Harbin, 150001, Heilongjiang, China.
authors.forename: Xiaoxue
authors.initials: X
authors.lastname: Yang
authors.forename: Hua
authors.initials: H
authors.lastname: Cong
authors.forename: Jinzhu
authors.initials: J
authors.lastname: Song
authors.forename: Junzheng
authors.initials: J
authors.lastname: Zhang
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology*; D057055:Aspartic Acid Proteases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D061046:Biological Control Agents; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005285:Fermentation; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D005658:Fungi / Q000187:drug effects* / Q000254:growth & development; D015870:Gene Expression; D008213:Lymphocyte Activation; D008958:Models, Molecular; D025282:Mycelium / Q000187:drug effects / Q000254:growth & development; D010843:Pichia / Q000235:genetics*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis; D017385:Sequence Homology; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics*
chemical_list: D000935:Antifungal Agents; D061046:Biological Control Agents; D005656:Fungal Proteins; D011994:Recombinant Proteins; D057055:Aspartic Acid Proteases