Abstract:

Pine wood nematode（PWN）,Bursaphelenchus xylophilus,is a causal pathogen of pine wilt disease（PWD）that is devastating to dozens of pine species. An gene aldh of aldehyde dehydrogenase from PWNs was successfully cloned through RT-PCR,its length was 1 353 bp and encoded a protein containing 451 amino acids residues. Prior studies demonstrated a nematicide fomepizole resulted in the up-regulation of aldh expression. Sequences alignment showed that the conserved catalytic position was Leu residue,which was different to Cys residue in most other aldehyde dehydrogenases,and its adjacent residue at C-terminal was His rather than the common nonpolar amino acid residue such as Leu. The aldh-encoding aldehyde dehydrogenase was over expressed in E. coli BL21（DE3）transformed with pET-15b-aldh via IPTG induction,and the recombinant aldehyde dehydrogenase was purified by Ni-NTA affinity chromatography. The Km of the aldehyde dehydrogenase with formaldehye as substrate was 27.87 mmol/L,the optimal pH value and temperature was 7.5 and 25℃,respectively. The enzyme activities were enhanced by Fe³⁺and Ni²⁺,while inhibited by Ca²⁺,Mn²⁺,Na⁺ and K⁺. The recombinant aldehyde dehydrogenase showed different catalytic activities towards 5 aldehydes,and vanillin was found to be its optimal substrate in the 5 tested aldehydes. This study lays a foundation for investigating the roles of aldehyde dehydrogenase in PWD and offers a clue to explore aldehyde dehydrogenases utilized in industry.

Key words: Bursaphelenchus xylophilus, aldehyde dehydrogenase, purification, properties