Cloning, expression, identification and characterization of borneol dehydrogenase isozymes in Pseudomonas sp. TCU-HL1

Protein Expr Purif. 2020 Nov:175:105715. doi: 10.1016/j.pep.2020.105715. Epub 2020 Jul 29.

Abstract

Borneol is a bicyclic plant monoterpene. It can be degraded by soil microorganisms through the conversion of borneol dehydrogenase (BDH) and a known camphor degradation pathway. Recombinant BDH from Pseudomonas sp. TCU-HL1 was produced in the form of inclusion body. The refolded BDH1 tends to precipitate. Insoluble recombinant BDH1 was converted into a soluble form by adding glycerol in LB medium. The kcat and kcat/Km values of soluble form BDH1 for (+)-borneol turned out to be about 34-fold and 45-fold higher, respectively, than those of the refolded enzyme. On the other hand, a gene knockout mutant, TCU-HL1Δbdh, was constructed to investigate the possible presence of a second copy of the bdh gene in TCU-HL1 genome. A new gene, bdh2, encoding a BDH isozyme, was identified, and the recombinant BDH2 protein was produced in a soluble form. Both bdh1 and bdh2 genes are expressed in the crude extract of wild type TCU-HL1, as shown by RT-qPCR results. Both BDH isozymes prefer to degrade (+)-borneol, rather than (-)-borneol, probably because (+)-camphor is the main form present in nature.

Keywords: Borneol; Borneol dehydrogenase; Camphor; Pseudomonas.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alcohol Oxidoreductases* / biosynthesis
  • Alcohol Oxidoreductases* / chemistry
  • Alcohol Oxidoreductases* / genetics
  • Alcohol Oxidoreductases* / isolation & purification
  • Bacterial Proteins* / biosynthesis
  • Bacterial Proteins* / chemistry
  • Bacterial Proteins* / genetics
  • Bacterial Proteins* / isolation & purification
  • Cloning, Molecular*
  • Gene Expression*
  • Pseudomonas* / enzymology
  • Pseudomonas* / genetics

Substances

  • Bacterial Proteins
  • Alcohol Oxidoreductases
  • borneol dehydrogenase