Characterization of an extracellular lipase from the biocontrol fungus, Nomuraea rileyi MJ, and its toxicity toward Spodoptera litura.
An extracellular lipase from Nomuraea rileyi MJ was purified 23.9-fold with 1.69% yield by ammonium sulfate precipitation followed by Sephacryl S-100 HR column chromatography. By mass spectrometry and SDS-polyacrylamide gel electrophoresis, the molecular weight of the homogenous lipase was 81kDa. The N-terminal sequence was determined as LeuSerValGluGlnThrLysLeuSerLysLeuAlaTyrAsnAsp and it showed no homology to sequences of known lipases. The optimum pH and temperature for activity were 8.0 and 35°C, respectively. The enzyme was stable in the pH range 7.0-9.0 and at 15-35°C for 1h. Higher activity was observed in the presence of surfactants, Na(+), NH(4)(+) ions, NaN(3) and ethylenediaminetetraacetic acid (EDTA), while Co(2+) and Cu(2+) ions, cysteine and dithiothreitol (DTT) strongly inhibited activity. The purified lipase hydrolyzed both synthetic and natural triglycerides with maximum activity for trilaurin and coconut oil, respectively. It also hydrolyzed esters of p-nitrophenol (pNP) with highest activity for p-nitrophenyl caprate (pNPCA). The purified lipase was found to promote N. rileyi spore germination in vitro in that germination reached 98% in conidial suspensions containing purified lipase at 2.75 U. Moreover, it enhanced toxicity of N. rileyi toward Spodoptera litura larvae with mortality via topical application reaching 63.3% at 4-10days post-treatment which calculated to be 2.7 times higher than the mortality obtained using conidial suspensions alone.
Supakdamrongkul
Piyaporn
P
Department of Microbiology, Faculty of Pharmacy, Mahidol University, 447 Sri-Ayudhaya Rd., Phayathai, Ratchathewi, Bangkok, Thailand.
Bhumiratana
Amaret
A
Wiwat
Chanpen
C
D000595:Amino Acid Sequence; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D045604:Extracellular Fluid / Q000737:chemistry / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005658:Fungi / Q000201:enzymology* / Q000235:genetics; D007525:Isoelectric Focusing; D008049:Lipase / Q000378:metabolism*; D008969:Molecular Sequence Data; D010572:Pest Control, Biological / Q000379:methods*; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera / Q000382:microbiology*; D013379:Substrate Specificity
D005656:Fungal Proteins; D008049:Lipase