Shrestha, Sunil; (1994) A comparison of the solvent stability of three gram negative organisms. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

A growing interest lies in the use of biocatalysts in industry to carry out stereo- and regio-specific conversions. Many of these reactions involve poorly water-soluble reactants and/or products. This poses problems in attaining high substrate and product concentrations in the reactor, and two-liquid phase systems can be employed in such cases. The main disadvantage of this approach is destabilisation of the biocatalyst by the organic solvent. Protection against solvent is afforded by the outer membrane in Gram negative cells. Naphthalene hydroxylation by Gram negative organisms was studied to compare solvent stability in an aqueous saturated buffer system, and a two-liquid phase system. The naphthalene dioxygenase gene, nahA, was isolated and inserted into plasmid pMMB66EH. The new plasmid construct was used to transform E. coli JM107 and P. aeruginosa strains PAC1R and PAC610. P. putida UV4 which expresses toluene dioxygenase activity from chromosomally encoded genes, was used to complete the array of microorganisms studied. Exposure to solvent, both in two-liquid phase systems and in solvent saturated buffer, showed that differences in cell outer membrane composition affected the degree to which cells retained stability. Solvent hydrophobicity and presence of an aqueous:organic phase interface, were also important in determining the rate at which solvent damage occurred. Loss of stability was shown to be a time dependent phenomenon, with sustained exposure leading to increased loss of stability, even with very hydrophobic solvents. PAC1R(pSS2) was unable to convert naphthalene at the same rate as PAC610(pSS2). This difference is thought to be due to differences in the extent of interactions between components of their outer membranes affecting the rate of uptake of the substrate. Partial permeabilisation by solvents increased the activity observed in PAC1R(pSS2). Activity retention shown by UV4 was lower than that of JM107(pSS2) in hexane saturated buffer for the first 4hours of biotransformation, but greater over the final 21hour period. UV4 also retained a greater level of activity than JM107(pSS2) over the entire biotransformation in tetradecane saturated buffer. PAC1R(pSS2) activity appeared to be stimulated by the presence of saturating levels of hexane and tetradecane over the first 4hours, but declined with continued exposure. This stimulated level of activity was still significantly lower than that of JM107(pSS2). Pentanol saturated buffer caused immediate and complete loss of activity in all cells.

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