Cloning and overexpression of Pochonia chlamydosporia chitinase gene pcchi44, a potential virulence factor in infection against nematodes
The fungus Pochonia chlamydosporia is a biocontrol agent with commercial potential for root-knot and cyst nematodes. In this study, a gene (designated pcchi44) encoding an extracellular endochitinase was isolated for the first time from P. chlamydosporia using degenerate primers and a genome walking technique. The 2385-bp pcchi44 sequence is interrupted by three introns and encodes a 424-amino acid polypeptide. The cDNA sequence of pcchi44 was amplified via reverse transcription polymerase chain reaction and overexpressed in Escherichia coli BL21. The recombinant chitinase PCCHI44 was purified as a protein of ca. 44 kDa with an optimal activity at pH 6.0 and 50 °C. The Vmax and Km value of PCCHI44 using 4-methylumbelliferyl-β-d-N,N′,N″-triacetylchitotrioside as substrate were 8.37 × 10−3 μM s−1 and 9.65 μM, respectively. The hydrolytic activity of PCCHI44 could be completely inhibited by acetazolamide. The chitinase PCCHI44 could damage eggs of both the root-knot nematode Meloidogyne incognita and the insect Bombyx mori.
Journal: Process Biochemistry - Volume 45, Issue 5, May 2010, Pages 810–814