Journal of Biotechnology 45 ( 19%) 173- 179 ,O”INAL 0, Biotechnolo Enzyme-catalysed carbon-carbon bond formation: Large-scale production of Escherichia coli transketolase Gordon R. Hobbs, Robin K. Mitra, Rashmi P. Chauhan, John M. Woodley, Malcolm D. Lilly * zyxwvutsrqponmlkjihgfedcbaZYXWVUTSR The Advanced Cenrre for Biochemical Engineering, Department of Chemical and Biochemical Engineering, University College London, Torringron Pluce, London W CIE 7JE, UK Received 25 July 1995; revised 21 October 1995; accepted 24 October 1995 Abstract zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Escherichiu coli strain JM 107/pQR700 possesses the vector pBGS 18, 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- ’ 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-’ 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. Keywords: Escherichia coli; Transketolase; Large scale; Bioprocess; Recovery 1. Introduction The industrial use of enzymes for the synthesis of chemicals is receiving increased attention as their potential for novel chemistry is more widely recog- nised. However, much less has been published on the production of microorganisms and enzymes for use as catalysts in these biotransformations. Previously we have described the large scale production and isolation of cholesterol oxidase from Nocurdia rhodochrous (Buckland et al., 1974) and chloroper- oxidase from Culduriomyces fumago (Smith et al., * Corresponding author. 1987). More recently developments in recombinant DNA technology allow improvements in enzyme production in a variety of host organisms giving the biochemical engineer a greater choice of catalyst options (Woodley and Lilly, 1994). In many cases this is leading to the development of processes which previously were economically non-viable. Stereospecific carbon-carbon bond synthesis is an important reaction in synthetic organic chemistry and may be catalysed by several enzymes, including transketolase (EC 2.2.1 .I 1. This enzyme in vivo catalyses the transfer of a two carbon ketol group between phosphorylated sugars in the presence of thiamine pyrophosphate and magnesium (II) ions. However, the enzyme will also act on non-phos- phorylated compounds. When P-hydroxypyruvate is 0168-1656/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved SD/ 0168-1656(95)00165-4