Cyclohexanone monooxygenase was produced from Acinetobacter calcoaceticus grown on a medium containing both glutamate (30 g l −1 ) and cyclohexanol (1 g l −1 ). Productivity was increased to 650 U l −1 , an order of magnitude greater than previous production methods, thereby enhancing the potential commercial utility of this enzyme.

Transketolase from Escherichia coli used for the asymmetric synthesis of carbon–carbon bonds has been immobilised on two commercial supports (Eupergit-C and Amberlite XAD-7). Stabilisation against oxidative affects could only be achieved by the addition of reducing agents. However, immobilisation did have a significant effect on stabilising the enzyme activity against the denaturing effects of the aldehyde acceptor required for the reaction. Using the transketolase catalysed synthesis of l-erythrulose from glycolaldehyde and β-hydroxypyruvate an improvement of 80 and 100 fold was found in the half-life of Amberlite XAD-7 and Eupergit-C immobilised preparations, respectively. Such an improvement may avoid the need for reactant feeding strategies in subsequent reactor designs.

Biochemical engineering science is the fundamental research into all aspects of the interactions between engineering and other disciplines necessary to underpin the development of industrial-scale biologically-based processes. Its importance to European industry, justifying its incorporation into the Framework IV Programme, and the need to maintain a strong activity within Europe in this exciting field are emphasised. Some challenges and opportunities are discussed. The need for an integrated multidisciplinary and rational approach providing industry with strategies for process selection, design and operations based on generic principles and guidelines is highlighted.

Annals of the New York Academy of SciencesVolume 799, Issue 1 p. 434-445 Carbon-Carbon Bond Synthesis Reactor Design and Operation for Transketolase-catalyzed Biotransformationsa JOHN M. WOODLEY, JOHN M. WOODLEY Advanced Center for Biochemical Engineering Department of Chemical and Biochemical Engineering University College London London WC1E 7JE, United KingdomSearch for more papers by this authorROBIN K. MITRA, ROBIN K. MITRA Advanced Center for Biochemical Engineering Department of Chemical and Biochemical Engineering University College London London WC1E 7JE, United KingdomSearch for more papers by this authorMALCOLM D. LILLY, MALCOLM D. LILLY Advanced Center for Biochemical Engineering Department of Chemical and Biochemical Engineering University College London London WC1E 7JE, United KingdomSearch for more papers by this author JOHN M. WOODLEY, JOHN M. WOODLEY Advanced Center for Biochemical Engineering Department of Chemical and Biochemical Engineering University College London London WC1E 7JE, United KingdomSearch for more papers by this authorROBIN K. MITRA, ROBIN K. MITRA Advanced Center for Biochemical Engineering Department of Chemical and Biochemical Engineering University College London London WC1E 7JE, United KingdomSearch for more papers by this authorMALCOLM D. LILLY, MALCOLM D. LILLY Advanced Center for Biochemical Engineering Department of Chemical and Biochemical Engineering University College London London WC1E 7JE, United KingdomSearch for more papers by this author First published: October 1996 https://doi.org/10.1111/j.1749-6632.1996.tb33238.xCitations: 13 a This research was supported by the Biotechnology and Biological Sciences Research Council and the participating departments at University College London, the University of Edinburgh, and the University of Exeter. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume799, Issue1Enzyme Engineering XIIIOctober 1996Pages 434-445 RelatedInformation

The effect of water-miscible cosolvents on biotransformations of poorly water-soluble substrates by immobilized cells was investigated, using Δ1-dehydrogenation of hydrocortisone by Arthrobacter simplex as a model. Criteria for solvent selection on the basis of retention of enzymic activity were postulated and tested. Diols were considered to be the most suitable group of solvents. Substrate solubility increased tenfold in 30% (v/v) ethylene glycol, but reaction rates were significantly slower in such solutions. This was mainly caused by a decrease of oxygen solubility in the presence of the cosolvent and conformational changes imposed on the intracellular enzyme by cosolvent molecules penetrating the cell. The inhibition could be eliminated by the addition of an artificial electron acceptor, phenazine methosulphate (PMS). Reaction rates faster than those for substrate suspensions (no cosolvent added) could thus be achieved. Immobilization of Arthrobacter simplex in cross-linked polyacrylamide hydrazide gave high retentions of activity. PMS exhibited toxic effects on the entrapped cells, leading to reduced activity after extended use.

The disruption of Pseudomonas putida cells capable of n-alkane assimilation was investigated by enzymic lysis and mechanical disruption in a high pressure-homogeniser, with a view to the isolation of alkane hydroxylase activity. Examination of the conditions for enzymic lysis showed that disruption with lysozyme/EDTA could be replaced effectively with lysozyme alone in phosphate buffer, pH 8.0 (I=0.05). This allowed inclusion of DNase during the lysis procedure for high bacterial concentrations and gave improved cell disruption. Mechanical disruption resulted in the solubilisation of alkane hydroxylase activity. In contrast enzymic lysis allowed the isolation of an insoluble fraction containing alkane hydroxylase activity, and although some solubilisation of the enzyme system did occur much of the activity was retained in the insoluble fraction. This fraction also contained a high level of n-alkane or diethoxymethane inducible, NAD-independent alcohol dehydrogenase activity.

The induction of alkane hydroxylase activity was investigated in two strains of Pseudomonas putida with a view to the production of primary alcohols. n-Nonanol production rates (16.0 μmol/g dry wt/h) with an alcohol dehydrogenase negative mutant P. putida PpS173 were considerably lower than might be expected from the growth of a wild type on n-alkane. Production of cells by fed-batch culture on n-nonane, with a specific alkane hydroxylase activity of 3.9 mmol/g/h, was considered most suitable for isolation of the alkane hydroxylase.

A mutant of E. coli constitutive for β-galactosidase has been grown in batch culture with the carbon source, glycerol, fed at various fixed rates to the culture. High feeding rates where growth was only slightly restricted gave final enzyme levels similar to those obtained in cultures where all the glycerol was added initially. Low feeding rates resulted in breakdown of the β-galactosidase formed and gave reduced final levels of the enzyme.