Department of Biochemistry, Tzu Chi University, Hualien, 97004, Taiwan; Institute of Medical Sciences, School of Medicine, Tzu Chi University, Hualien, 97004, Taiwan; Department of Biochemistry, University of Medicine 1, Yangon, Myanmar.
Department of Biochemistry, Tzu Chi University, Hualien, 97004, Taiwan.
Protein Expr Purif. 2020 Nov;175:105715. doi: 10.1016/j.pep.2020.105715. Epub 2020 Jul 29.
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 k and k/K 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.
龙脑是一种双环植物单萜。它可以通过土壤微生物中的龙脑脱氢酶（BDH）和已知的樟脑降解途径转化而被降解。来自假单胞菌 sp. TCU-HL1 的重组 BDH 以包涵体的形式产生。复性的 BDH1 容易沉淀。通过在 LB 培养基中添加甘油，可将不溶性重组 BDH1 转化为可溶性形式。可溶性形式 BDH1 的 (+)-龙脑的 k 和 k/K 值分别比复性酶高约 34 倍和 45 倍。另一方面，构建了基因敲除突变体 TCU-HL1Δbdh，以研究 TCU-HL1 基因组中是否存在第二个 bdh 基因的副本。鉴定出一个新基因 bdh2，它编码一种 BDH 同工酶，并以可溶性形式产生重组 BDH2 蛋白。如 RT-qPCR 结果所示，bdh1 和 bdh2 基因都在野生型 TCU-HL1 的粗提物中表达。两种 BDH 同工酶都优先降解 (+)-龙脑，而不是 (-)-龙脑，这可能是因为 (+)-樟脑是自然界中主要存在的形式。
