High Expression Of Reductase CgKR2in Pichia Pastoris And Its Applications

Ethyl (R)-2-hydroxy-4-phenylbutyrate ((R)-HPBE) is an important precursor for the synthesis of a variety of angiotensin converting enzyme (ACE) inhibitors, such as benazepril, enalapril and ramipril. Different technical processes have been reported for the preparation of (R)-HPBE, including both chemical and biological methods. Although chemical methods may need fewer procedures, they require high-pressure reactors, expensive metal catalysts, substrate pre-purification and lower ee value of the product (R)-HPBE. Biological methods, on the other hands, has attracted much more attention due to its significant advantages such as high conversion, eco-friendliness, and remarkable stereoselectivity(e.g.,>98%ee).A reducatse CgKR2was discovered previously by data mining in our lab. In this work, the CgKR.2gene was successfully inserted into Pichia pastoris, and in the screening for high copy numbers, two recombinants KM71/3.5k-CgKR2and GS115/9k-CgKR2showed the highest enzyme activity. The Km value of the purified reductase produced by Pichia pastoris/CgKR2towards OPBE did not change much as compared with that by E. coli/CgKR2.Then the higher cell density cultivation in the fermentor was carried out. As for KM71/3.5K-CgKR2, a methanol concentration of0.5%(v/v) showed a better result than using DO-stat method. The production of intracellularly expressed enzyme was154U/g for the wet cells, and the total enzyme production was160kU/2L, which corresponds to6.67g pure protein per liter of CgKR2fermentation broth. For the secreted expression, methanol feeding was controlled by DO-stat method. After induction, the CgKR2production in the supernatant reached43.7kU/L, corresponding to3.0g/L of CgKR2protein. No better result was obtained at lower induction temperatures.(R)-HPBE was produced by CgKR2wet cells or culture supernatant of P. pastoris/pCgKR.2via reduction of OPBE at1M in10mL aqueous phase system with>99%conversion and>97%ee, coupled with glucose dehydrogenase and glucose for NADPH regeneration. Then preparative-scale reactions were carried out, a0.5L reaction was introduced using KM71whole cells as catalysts, the space-time yield of (R)-HPBE production reached202.8g·LT-1·d-while a STY of216.4g·L-1·d-1(0.1L reaction) was achieved using culture supernatant, making P. pastoris/pCgKR2a competitive and promising biocatalyst for industrial use.