Candida cloacae oxidation of long-chain fatty acids to dioic acids.

Candida cloacae cells oxidize long-chain fatty acids to their corresponding dicarboxylic acids (dioic acids) at rates dependent on their chain length and degree of saturation. This is despite the well-known toxicity of the fatty acids. Among the saturated substrates, the oxidation is limited to lauric acid (C12). The addition of pristane (5% v/v), which acts as an inert carrier for the poorly water-soluble substrate, boosts the oxidation of lauric acid to a rate that is comparable to that of dodecane. When dissolved in pristane, myristic (C14) and palmitic (C16) acids are effective carbon sources for C. cloacae, but dioic acid production is very low. Media glucose concentration and pH also influence cell growth and productivity. After the glucose is depleted, oxidation is optimal at a low pH. A two-phase (pristane/water) reaction was tested in a 2-l stirred tank bioreactor in which growth and oxidation were separated. A 50% w/w conversion of lauric acid (10 g/l) to dodecanedioic acid was achieved. The bioreactor also alleviated poor mass transfer characteristics experienced in shake flasks.

A systematic approach to the large-scale production of protein crystals.

Crystallization has recently emerged as a suitable process for the manufacture of biocatalysts in the form of cross-linked enzyme crystals (CLECs) or for the recovery of proteins from fermentation broths. In both instances it is essential to define conditions which control crystal size and habit, and that yield a reliable recovery of the active protein. Experiments to define the crystallization conditions usually depend on a factorial design (either incomplete or sparse matrix) or reverse screening techniques. In this work, we describe a simple procedure that allows the effect of three factors, for example protein concentration, precipitant concentration and pH, to be varied simultaneously and smoothly over a wide range. The results are mapped onto a simple triangular diagram where a 'window of crystallization' is immediately apparent, and that conveniently describes variations either in the crystal features, such as their yield, size, and habit, or in the recovery of biological activity. The approach is illustrated with two enzymes, yeast alcohol dehydrogenase (ADH I) and Candida rugosa lipase. For ADH the formation of two crystal habits (rod and hexagonal) could be controlled as a function of pH (6.5-10) and temperature (4-25 degrees C). At pH 7, in 10 to 16% w/v polyethylene glycol (PEG) 4000, only rod-shaped crystals formed whereas at pH 8, in 10 to 14% w/v PEG, only hexagonal crystals existed. For both enzymes, catalyst recovery was greatest at high crystallization agent concentrations and low protein concentration. For ADH, the greatest activity recovery was 87% whereas for the lipase crystals, by using 45% v/v 2-methyl-2,4-pentanediol (MPD) as the crystallization agent, a crystal recovery of 250 crystals per µl was obtained. For the lipase system, the use of crystal seeding was also shown to increase the crystal recovery by up to a factor of four. From the crystallization windows, the original conditions based on literature precedent (35% v/v MPD, 1 mM CaCl(2), 1.8 mg protein/ml) were altered (47.5% v/v MPD, 2 mM CaCl(2), 3 mg protein/ml). This led to an improved recovery of the lipase under conditions that scale reliably from 0.5 ml to 500 ml with no change in size, shape or recovery of the crystals themselves. Finally, these crystals were crosslinked with 5% v/v glutaraldehyde and mass and activity balances were calculated for the entire process of CLEC production. Up to 35% of the lipase activity present in the crude solid was finally recovered in the lipase CLECs after propan-2-ol fractionation, crystallization, and crosslinking.