Enzyme-catalysed carbon-carbon bond formation: large-scale production of Escherichia coli transketolase.

ABSTRACT

Escherichia coli strain JM107/pQR700 possesses the vector pBGS18, a high copy number plasmid carrying kanamycin resistance, into which a 4.4 kb fragment containing the transketolase gene had been cloned. The bacterium was grown at 20 and 1000 1 scale for the production of transketolase. The specific growth rate was maintained at 0.15 h-1 until the bacterial concentration reached 20 g dry wt per litre at which point the culture was harvested. The clarified cell extract obtained after disruption of the bacteria in a high-pressure homogeniser contained about 230 U ml-1 of the enzyme, which represented about 40% of the total protein released. No further purification was done at large scale as the clarified cell extract could be used satisfactorily for biotransformations.