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

• Published in: Protein expression and purification 2020-11, Vol.175, p.105715-105715, Article 105715
• Main Authors: Khine, Aye Aye, Lu, Pei-Chieh, Ko, Tzu-Ping, Huang, Kai-Fa, Chen, Hao-Ping
• Format: Article
• Language: English
• Subjects: 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; Borneol; Borneol dehydrogenase; Camphor; Cloning, Molecular; Gene Expression; Pseudomonas; Pseudomonas - enzymology; Pseudomonas - genetics
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2020.105715

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. •Two borneol dehydrogenases (BDH) from Pseudomonas sp. TCU-HL1 were characterized.•Insoluble BDH1 was converted into a soluble form by adding glycerol in LB medium.•The kcat for (+)-borneol of soluble BDH1 is 34-fold higher than that of the refolded.•A new gene, bdh2, encoding a BDH isozyme, was identified in strain TCU-HL1.•Both BDH1 and BDH2 prefer to degrade (+)-borneol, rather than (−)-borneol.