Wilkinson, Diane; (1997) Organic solvent tolerance of gram negative bacteria for biocatalysis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Microorganisms have widespread application in biocatalysis due to their ability to perform complex chemical reactions, forming chiral compounds of use in chemical synthesis. Many of these chemical conversions involve hydrophobic compounds with low aqueous solubility. One method used to improve these processes has been the addition of organic solvent to the reaction. Solvent may be added in low (subsaturation) concentrations or at higher concentrations when a second organic phase is formed. However solvents have been shown to be toxic to the biocatalyst. Their toxicity has been related to the solvent log P value, the critical membrane concentration and interactions with membrane bound proteins. Toxicity can be split into inherent molecular toxicity and phase toxicity caused by the presence of a liquid-liquid interface. This work examined the molecular toxicity of ethanol and the phase toxicity of a hexane-phosphate buffer interface to naphthalene dioxygenase activity, used as a representative test system. The naphthalene dioxygenase nah A gene encoded on pSS2 was successfully transfered and expressed in Pseudomonas aeruginosa PAC610 and Pseudomonas putida PpG277. The enzyme activity of these strains together with Escherichia coli JM107 pSS2 and P. aeruginosa PACIR pSS2 was investigated by the production of naphthalene dihydrodiol. After induction in the early exponential growth phase optimal enzyme activity occured during mid-late exponential phase. Large differences in the maximal specific enzyme activity were noted between bacterial strains (P. putida PpG277 > E. coli JM107 > P. aeruginosa PACIR = P. aeruginosa PAC610). P. putida PpG277 was shown to contain other dioxygenase activity and non-specific activity against the dihydrodiol product. Addition of up to 4% (w/v) ethanol to an aqueous naphthalene dioxygenase biotransformation increased the aqueous saturation concentrations of naphthalene by 38% from 0.42 mgL-1 to 0.58 mgL-1 and the naphthalene dihydrodiol by 34.5% from 18 mgL-1 to 25 mgL-1 at 4% (w/v). Comparing the specific naphthalene dioxygenase activity using ethanol and glucose as carbon sources indicated that under all conditions used, higher specific activity was achieved using glucose. The ratios of specific activity (ethanol/glucose) were shown to decrease with increasing carbon concentration for Pseudomonas spp, conversely E. coli biotransformations showed a sharp increase in this ratio at the highest carbon concentration. The primary disadvantage with these single phase biotransformations was shown to be the toxic nature of naphthalene dihydrodiol. Naphthalene dihydrodiol was found to be toxic above 0.05mgL-1. Liquid-liquid interfacial effects were determined using a Lewis cell reactor. In this system the reactor was operated with two defined liquid phases and a known interfacial area. The mass transfer in these Lewis cells was characterised using specific interfacial areas (interfacial area/aqueous volume) between 24.29-39.48 m-1 and agitation rates between 100-300 rpm using a top solvent phase of either hexane, cyclohexane, methylcyclohexane or toluene. Alteration of the aqueous volume or agitation rate of the aqueous phase controlled the solvent mass transfer enabling solvent exposure at a constant mass transfer rate with different specific interfacial areas. Using this experimental design, adsorption of E. coli or crude porcine lipase to a hexane-liquid interface reduced the interfacial tension of a hexane-phosphate buffer interface from 43 mNm-1 to 22mNm-1 and decreased the mass transfer rate by 50%. The aqueous saturation concentration increased by 7% due to absorption of solvent by cells and interaction of solvent with proteins. Interfacial effects due to the direct contact of the biocatalyst with the interface were seen within the first 30 minutes after this time the molecular effects of hexane became apparent. Strategies that protect biocatalysts from this contact, such as immobilisation and membrane bioreactors are thought to inhibit this interfacial damage.

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