Isolation And Heterologous Expression Of Trans-epoxysuccinate Hydrolase

Meso-tartaric acid is mainly applied in salt industry and membrane electrolysis industry, which was the hydrolysis product of trans-epoxysuccinate catalyzed by trans-epoxysuccinate hydrolase (TESH). Until now, the research on biotransformation of meso-tartaric acid was mainly focused on the screening of the wild-type TESH-producing strain and the optimizing of the fermentation conditions. Since the reported TESH showed low activity and low stability, it is necessary to find a strain which can produce the enzyme with high activity and stability.In this study, a novel trans-epoxysuccinate hydrolase (TESH) producing strain for production of meso-tartaric acid was isolated. Based on the morphological and biochemical properties along with 16S rDNA sequence analysis, the isolate was identified as Pseudomonas koreensis (named BK-9). TESH from BK-9 was purified into a homogeneous state by combination of ammonium sulfate precipitation, DEAE-Sepharose chromatography and Phenyl-Sepharose chromatography. Its accurate molecular mass was estimated to be 32.25 kDa by MALDI-TOF-MS analysis. TESH remained stable at temperature up to 40 ? and in a broad pH range of 3.5-11. It's metal-independent and the Km and Vmax for trans-epoxysuccinate were 8.22 ± 0.2 mM and 2.5 ±0.1 mM/min, respectively.According to sequencing results of N-terminal and C-terminal of TESH, the encoded gene of TESH was successfully cloned from Pseudomonas koreensis BK-9. What's more, we successfully constructed an engineering strain E.coli-pET-15b(+)-TESH that was able to over express TESH. After fermentation in a 5 L fermentor, TESH activity of E.coli-pET-15b(+)-TESH reached 35542 U/g, which showed high potential for commercial production of meso-tartaric acid.