A plug-and-play system for enzyme production at commercially viable levels in fed-batch cultures of Escherichia coli BL21 (DE3)

Commercial exploitation of enzymes in biotransformation necessitates a robust method for enzyme production that yields high enzyme titer. Nitrilases are a family of hydrolases that can transform nitriles to enantiopure carboxylic acids, which are important pharmaceutical intermediates. Here, we report a fed-batch method that uses a defined medium and involves growth under carbon limiting conditions using DO-stat feeding approach combined with an optimized post-induction strategy, yielding high cell densities and maximum levels of active and soluble enzyme. This strategy affords strict control of nutrient feeding and growth rates, and ensures sustained protein synthesis over a longer period. The method was optimized for highest titer of nitrilase reported so far (247 kU/l) using recombinant E. coli expressing the Alcaligenes sp. ECU0401 nitrilase. The fed-batch protocol presented here can also be employed as template to produce a wide variety of enzymes with minimal modification, as demonstrated for alcohol dehydrogenase and formate dehydrogenase.