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Cloning, expression, identification and characterization of borneol dehydrogenase isozymes in Pseudomonas sp. TCU-HL1.
Protein Expression and Purification ( IF 1.4 ) Pub Date : 2020-07-29 , DOI: 10.1016/j.pep.2020.105715
Aye Aye Khine,Pei-Chieh Lu,Tzu-Ping Ko,Kai-Fa Huang,Hao-Ping Chen

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.



中文翻译:

假单胞菌中冰片脱氢酶同工酶的克隆,表达,鉴定和鉴定 TCU-HL1。

冰片是双环植物单萜。土壤微生物可通过冰片脱氢酶(BDH)的转化和已知的樟脑降解途径将其降解。来自假单胞菌sp。的重组BDH 。TCU-HL1以包涵体形式产生。重新折叠的BDH1倾向于沉淀。通过在LB培养基中添加甘油将不溶性重组BDH1转化为可溶形式。结果表明,(+)-冰片的可溶形式BDH1的k catk cat / K m值分别比重新折叠的酶高约34倍和45倍。另一方面,基因敲除突变体TCU- HL1Δbdh被构建以研究TCU-HL1基因组中bdh基因第二个拷贝的可能存在。鉴定了编码BDH同工酶的新基因bdh2,并以可溶形式生产了重组BDH2蛋白。既BDH1bdh2基因在野生型TCU-HL1的粗提取物中表达,如图通过RT-qPCR结果。两种BDH同工酶都倾向于降解(+)-冰片,而不是(-)-冰片,这可能是因为(+)-樟脑是自然界中的主要形式。

更新日期:2020-08-04
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