Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (9): 207-214.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1450

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Gene Cloning of an Aldehyde Dehydrogenase from Bursaphelenchus xylophilus and Biochemical Characterization

LI Wen-shuo(), WANG Lin-song, DU Gui-cai, GUO Qun-qun, ZHANG Ting-ting, YANG Hong LI Rong-gui   

  1. College of Life Sciences,Qingdao University,Qingdao 266071
  • Received:2021-11-21 Online:2022-09-26 Published:2022-10-11

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