Characterization and gene cloning of a novel serine protease with nematicidal activity from Trichoderma pseudokoningii SMF2.
Trichoderma pseudokoningii SMF2 is a biocontrol fungus with inhibitory ability against phytopathogenic fungi. Here, a crude extract of strain SMF2 in a solid ferment exhibited strong nematicidal activity against Meloidogyne incognita, and a novel serine protease SprT with nematicidal activity was purified from the crude extract. Protease SprT has a molecular mass of 31 kDa, a pH optimum of 8.5, and a temperature optimum of 60-65 degrees C. It had good thermostability, and was stable in an alkaline environment. SprT could degrade bovine serum albumin, lysozyme, and gelatin, and its activity was enhanced by many metal ions. The cuticles of nematodes treated by protease SprT obviously crimpled. Purified protease SprT could kill juveniles of M. incognita and inhibit egg hatch, suggesting that it is involved in the nematicidal process of T. pseudokoningii SMF2. The full-length cDNA gene-encoding protease SprT was cloned by rapid amplification of cDNA ends. Sequence analysis showed that SprT is a monodomain subtilase containing 284 amino acid residues. It had higher identities and a closer relation to the nematicidal serine proteases (59-69%) from nematode parasitic fungi than to the serine proteases (<50%) from Trichoderma. Protease SprT represents the first well-characterized subtilase with nematicidal activity from Trichoderma.
Chen
Lei-Lei
LL
The State Key Laboratory of Microbial Technology, Shandong University, Jinan, China.
Liu
Li-Jun
LJ
Shi
Mei
M
Song
Xiao-Yan
XY
Zheng
Chang-Ying
CY
Chen
Xiu-Lan
XL
Zhang
Yu-Zhong
YZ
D000818:Animals; D000969:Antinematodal Agents / Q000302:isolation & purification / Q000494:pharmacology*; D003001:Cloning, Molecular; D020536:Enzyme Activators / Q000494:pharmacology; D004795:Enzyme Stability; D005780:Gelatin / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009113:Muramidase / Q000378:metabolism; D010802:Phylogeny; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology*; D012710:Serum Albumin, Bovine / Q000378:metabolism; D016019:Survival Analysis; D013696:Temperature; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics; D014431:Tylenchoidea / Q000187:drug effects*
D000969:Antinematodal Agents; D020536:Enzyme Activators; D008670:Metals; D012710:Serum Albumin, Bovine; D005780:Gelatin; D009113:Muramidase; D012697:Serine Endopeptidases