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Overexpression of epoxide hydrolase in Rhodococcus ruber with high robustness for the synthesis of chiral epichlorohydrin

Liang, Youxiang, Jiao, Song, Wang, Miaomiao, Yu, Huimin, Shen, Zhongyao
Process biochemistry 2019 v.79 pp. 49-56
Agrobacterium radiobacter, Escherichia coli, Rhodococcus ruber, biocatalysts, enantiomers, epichlorohydrins, epoxide hydrolase, epoxides, glycols, pH, proteins, thermal stability
Epoxide hydrolases (EHs) are attractive enzymes for producing enantiopure epoxides and diols, but do not display enough stability when lysates or Escherichia coli whole cells are used as biocatalysts. In this work, an organic-solvent tolerant strain Rhodococcus ruber THdAdN was utilized to overexpress an epoxide hydrolase from Agrobacterium radiobacter (ArEH), using E. coli BL21(DE3) as a control. The proportion of ArEH in all soluble proteins of R. ruber THdAdN(ArEH) reached 30.3%, which was comparable to that of E. coli BL21(DE3)(ArEH). Due to a higher cell density in flask cultivation, the maximum ArEH activity of R. ruber THdAdN(ArEH) toward epichlorohydrin (ECH) reached 5.4 U/mL, approximately 5-fold higher than that of E. coli BL21(DE3)(ArEH). More importantly, compared with E. coli BL21(DE3)(ArEH), ArEH in R. ruber THdAdN(ArEH) showed a 10-fold enhanced thermostability, better tolerance against alkali pH, and reduced substrate and product inhibition, which significantly improved its performance in the resolution of high concentration ECH. By substrate feeding, 98.5% ee (R)-ECH was obtained with a 35.5% yield from 512 mM racemic ECH hydrolyzed by R. ruber THdAdN(ArEH). Compared with using free enzyme as biocatalysts, utilization of the R. ruber cells harboring ArEH improved the final (R)-ECH concentration by 46%.