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Abstract: The activity of permeabilized cells of Escherichia coli strains during the biotransformation of trimethylamonium compounds, such as crotonobetaine and d-carnitine, into l-carnitine was determined. Permeabilization of cells did not affect the biotransformation of d-carnitine but improved the yield of l-carnitine obtained when crotonobetaine was used as biotransformation substrate (by nearly two-fold). A higher degree of cell activity with respect to control (45% yield) was observed with organic solvents such as acetone, ethanol, isopropyl alcohol and toluene (65–70% yield), detergents such as Triton X-100 and Tween 20 (70–75% yield) and polyethylenimine (80–89% yield). However, maximum cell activity was attained with 2% of the detergents, Triton X-100 and Tween 20, and 0.1% of polyethylenimine, using a temperature of 37°C and 10–30min of treatment time, in which case a yield of 75–89% (100% increase) was obtained with E. coli O44 K74 and 85–94% (80% increase) with E. coli K38 T7-5KE32. Both detergents and polyethylenimine produced alterations in the outer membrane of treated cells since thin-sectioned bacteria were observed by transmision electron microscopy. No l-carnitine dehydratase or carnitine racemase enzyme activity was detected within the extracellular medium when detergents and polyethyleneimine were used. Furthermore, transport studies showed that trimethylammonium compound carriers were not affected by permeabilization, while the rate of l-carnitine transport increased by 20%. [Copyright &y& Elsevier]

Resting cells of a wild and a recombinant strain of Escherichia coli were used for d(+)-carnitine racemisation into l(−)-carnitine. A scheme of cell reuse and cell regeneration/biotransformation for enlarging the production process and reducing process costs was established. For the recombinant strain high levels of d(+)-carnitine racemase activity were obtained by growing the cells under anaerobic conditions at 41 °C. The biotransformation process depended on both cell and substrate concentrations. The addition of fumarate as an energy providing substrate during the cell resting state improved the biotransformation process at high substrate concentrations. In this way, the use of 500 mmol/l d(+)-carnitine and 200 mmol/l fumarate provided 28.2 and 36.3 g/l l(−)-carnitine (35% and 41% product yield and 3.50 and 1.45 g/l h l(−)-carnitine productivity) for the recombinant and wild strains, respectively. A storage study showed that resting cells retained 70% of their initial biotransformation capacity after 2 months storage at 4 °C. Resting cells could be reused in a biotransformation/regeneration system, cutting the overall process time by approximately 70 and 25% for the transformed and wild strain, respectively. [Copyright &y& Elsevier]

Membrane reactors with a Proteus sp. strain as the biocatalyst were used to attain continuous l-carnitine production from crotonobetaine. Hollow fiber cartridges with fibers of polysulfone provided adequate filtration rates and were reusable after long-term operation (consecutive runs of 140 h followed). Running under optimum medium composition, these systems enabled high cell densities (35–45 g dry weight/l) to be reached after 35–40 h at dilution rates ranging from 0.1 to 1.5 h−1, while cross-flow filtration units with flat-sheet membranes made of polyvinylidene difluoride (PVDF) showed lower cell concentrations (up to 25 g dry weight/l) and levels of l-carnitine production. The composition of the hollow fiber membranes probably improved filtration flow rates and definitely speed up the cleaning-up procedure because cake layers did not adhere tightly to the membranes. Furthermore, resting cell-cycles were maintained in continuous operation for 2–3 days with crotonobetaine and phosphate buffer as the only medium components, followed by cycles of cell re-activation, meaning that the system was stable for long periods of time (140–200 h). During resting cell-cycles, high cell decay rates were observed due to nutrient shortages. The hollow fiber system was able to produce high levels of l-carnitine during the growing/resting cycles with 35–50% conversion and productivities as high as 40.5 g/(l h). The optimum dilution rate was 1.0 h−1 as mass-transfer problems appeared at higher dilution rates because of high cell densities. The process described is presented as a way of recycling d-carnitine from the chemical industry, since, when dehydrated, this compound renders crotonobetaine. [Copyright &y& Elsevier]

A method for preparing dynamic membranes by the cross-flow filtration of two water-soluble polymers (gelatin and/or polyethyleneimine, PEI) on an α-alumina ceramic support, followed by covalent attachment of Candida antarctica lipase B (CALB) has been optimized. The obtained derivatives were tested for different immobilization parameters and selected as a function of the operational stability for the continuous hydrolysis of p-nitrophenyl palmitate (PNPP). The gelatin–PEI active membrane provided the best results (93% coupling efficiency, 4×10−3 U cm−2 activity and 22 h half-life time). This enzymatic membrane was also used as catalyst for the continuous kinetically controlled synthesis of butyl butyrate from vinyl butyrate and 1-butanol in three different organic solvents (hexane, acetone and acetonitrile) at low water content (<3% v/v). A clear enhancement in the operational stability of the enzymatic dynamic membranes was observed in these anhydrous media. The lipase-membrane derivative prepared with gelatin–PEI showed 28×10−3 U cm−2 synthetic activity and a 203 day half-life time in hexane media. [Copyright &y& Elsevier]