Title: In vivo immobilization of D-hydantoinase in Escherichia coli
D-p-Hydroxyphenylglcine (D-HPG) is a precursor required for the synthesis of semi-synthetic antibiotics. This unnatural amino acid can be produced by a transformation reaction mediated by D-hydantoinase (D-HDT) and D-amidohydrolase. In this study, a method was developed to integrate production and immobilization of recombinant D-HDT in vivo. This was approached by first fusion of the gene encoding D-HDT with phaP (encoding phasin) of Ralstonia eutropha H16. The fusion gene was then expressed in the Escherichia coli strain that carried a heterologous synthetic pathway for polyhydroxyalkanoate (PHA). As a result, D-HDT was found to associate with isolated PHA granules. Further characterization illustrated that D-HDT immobilized on PHA exhibited the maximum activity at pH 9 and 60oC and had a half-life of 95 h at 40oC. Moreover, PHA-bound D-HDT could be reused for 8 times with the conversion yield exceeding 90%. Overall, it illustrates the feasibility of this approach to facilitate in vivo immobilization of enzymes in heterologous E. coli strain, which may open a new avenue of enzyme application in industry.
D-hydroxyphenylglycine (D-HPG)是人工半合成抗生素cephalosporin 的先驅物，D型氨基酸D-HPG可採用二段式酵素法D-hydantoinase (D-HDT)及N-carbamoyl -D- amino acid amidohydrolase (D-NCAase)來進行生產，本研究利用PHA顆粒為固定化載體，發展創新之單一步驟酵素生產和胞內固定化技術。我們選殖phaCAB operon，於大腸桿菌中表現來合成PHA 顆粒，並以D-HDT的生產為例，將D-hydantoinase基因和phasin融合，最後於生產PHA 顆粒之大腸桿菌中進行融合蛋白質表現。研究結果顯示，我們成功的利用胞內固定化系統將D-HDT固定於PHA 顆粒上，利用該固定化D-HDT進行酵素轉換之最佳操作條件為pH9.0及60oC，在40oC下的半衰期可達95小時，此外，固定化D-HDT可重複使用8次仍然保有90%的轉化率，此固定化酵素方法可提供一個具有高穩定性、胞內固定化和低成本之固定化程序，深具工業開發價值。
Chen, Shan Yu*, Chien, Y-W, Chao, Y-P. (2014) In vivo immobilization of D-hydantoinase in Escherichia coli. Journal of Bioscience and Bioengineering. 118: 78-81