Purification and properties of an insecticidal metalloprotease produced by Photorhabdus luminescens strain 0805-P5G, the entomopathogenic nematode symbiont.
A total of 13 Photorhabdus luminescens strains were screened for proteolytic activity. The P. luminescens strain 0805-P5G had the highest activity on both skim milk and gelatin plates. The protease was purified to electrophoretical homogeneity by using a two-step column chromatographic procedure. It had a molecular weight of 51.8 kDa, as determined by MALDI-TOF mass spectrometry. The optimum pH, temperature, as well as pH and thermal stabilities were 8, 60 °C, 5-10, and 14-60 °C, respectively. It was completely inhibited by EDTA and 1,10-phenanthroline. Bioassay of the purified protease against Galleria mellonella by injection showed high insecticidal activity. The protease also showed high oral toxicity to the diamondback moth (Plutella xylostella) of a Taiwan field-collected strain, but low toxicity to an American strain. To our knowledge, this is the first report to demonstrate that the purified protease of P. luminescens has direct toxicity to P. xylostella and biopesticide potentiality.
Institute of Biotechnology and Bioinformatics, Asia University, Wufeng, Taichung 413, Taiwan. mhmeng@dragon.nchu.edu.tw.
Yu-Tzu
YT
Chang
Chienyan
C
Hsieh
Li-Ching
LC
Wu
Hebron C
HC
Chang
Suey-Sheng
SS
Kao
Menghsiao
M
Meng
Feng-Chia
FC
Hsieh
D000595:Amino Acid Sequence; D000818:Animals; D004275:DNA, Ribosomal / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007306:Insecticides / Q000737:chemistry / Q000302:isolation & purification* / Q000633:toxicity; D007814:Larva / Q000187:drug effects; D013058:Mass Spectrometry; D045726:Metalloproteases / Q000737:chemistry / Q000302:isolation & purification* / Q000633:toxicity; D008969:Molecular Sequence Data; D009036:Moths / Q000187:drug effects; D009348:Nematoda / Q000382:microbiology*; D020637:Photorhabdus / Q000201:enzymology*; D010802:Phylogeny; D011480:Protease Inhibitors / Q000494:pharmacology; D013559:Symbiosis; D013696:Temperature
D004275:DNA, Ribosomal; D007306:Insecticides; D011480:Protease Inhibitors; D045726:Metalloproteases