The bioconversion of L-phenylalanine (L-Phe) to 2-phenylethanol (PEA) by the yeast Saccharomyces cerevisiae is limited by the toxicity of the product. PEA extraction by a separate organic phase in the fermenter is the ideal in situ product recovery (ISPR) technique to enhance productivity. Oleic acid was chosen as organic phase for two-phase fed-batch cultures, although it interfered to some extent with yeast viability. There was a synergistic inhibitory impact toward S. cerevisiae in the presence of PEA, and therefore a maximal PEA concentration in the aqueous phase of only 2.1 g/L was achieved, compared to 3.8 g/L for a normal fed-batch culture. However, the overall PEA concentration in the fermenter was increased to 12.6 g/L, because the PEA concentration in the oleic phase attained a value of 24 g/L. Thus, an average volumetric PEA production rate of 0.26 g L(-1) h(-1) and a maximal volumetric PEA production rate of 0.47 g L(-1) h(-1) were achieved in the two-phase fed-batch culture. As ethanol inhibition had to be avoided, the production rates were limited by the intrinsic oxidative capacity of S. cerevisiae. In addition, the high viscosity of the two-phase system lowered the k(l)a, and therefore also the productivity. Thus, if a specific ISPR technique is planned, it consequently has to be remembered that the productivity of this bioconversion process is also quickly limited by the k(l)a of the fermenter at high cell densities.
Phospholipases A2 (PLA2s) may be grouped into distinct families of proteins that catalyse the hydrolysis of the 2-acyl bond of phospholipids and perform a variety of biological functions. The best characterized are the small (relative molecular mass approximately 14,000) calcium-dependent, secretory enzymes of diverse origin, such as pancreatic and venom PLA2s. The structures and functions of several PLA2s are known. Recently, high-resolution crystal structures of complexes of secretory PLA2s with phosphonate phospholipid analogues have provided information about the detailed stereochemistry of transition-state binding, confirming the proposed catalytic mechanism of esterolysis. By contrast, studies on mammalian nonpancreatic secretory PLA2s (s-PLA2s) have only recently begun; s-PLA2s are scarce in normal cells and tissues but large amounts are found in association with local and systemic inflammatory processes and tissue injury in animals and man. Such s-PLAs have been purified from rabbit and rat inflammatory exudate, from synovial fluid from patients with rheumatoid arthritis and from human platelets. Cloning and sequencing shows that the primary structure of the human s-PLA2 has about 37% homology with that of bovine pancreatic PLA2 and 44% homology with that of Crotalus atrox PLA2. The human s-PLA2 is an unusually basic protein, yet contains most of the highly conserved amino-acid residues and sequences characteristic of the PLA2s sequenced so far. Here we report the refined, three-dimensional crystal structure at 2.2 A resolution of recombinant human rheumatoid arthritic synovial fluid PLA2. This may aid the development of potent and specific inhibitors of this enzyme using structure-based design.
A novel in situ product removal (ISPR) method that uses microcapsules to extract inhibitory products from the reaction suspension is introduced into fermentation technology. More specifically, L-phenylalanine (L-Phe) was transformed by Saccharomyces cerevisiae to 2-phenylethanol (PEA), which is inhibitory toward the yeast. In order to continuously remove PEA from the vicinity of the cells, the reaction suspension was brought into contact with capsules of 2.2-mm diameter that had a hydrophobic core of dibutyl sebacate and an alginate-based wall. This novel process combines the advantages of a normal in situ extraction process (fast mass transfer and simple process set-up) with the benefits of a membrane-based process (reduction of the solvent toxicity and avoidance of stable emulsions). In particular, the microbial cells are shielded from the phase toxicity of the organic solvent by a hydrogel layer surrounding the organic core. By placing the microcapsules into the fermenter, the final overall concentration of PEA in a fed-batch culture was increased from 3.8 to 5.6 g/L because a part of the inhibitory product dissolved in the dibutyl sebacate core. In another fermentation experiment, the capsules were placed in a fluidized bed that was connected via a loop to the fermenter. In addition, the fluidized bed was connected via a second loop to a back-extractor to regenerate the capsules. By alternating the extraction and back-extraction cycles, it was possible to limit the PEA concentration of the fed-batch culture in the fermenter to 2.4 g/L while producing important quantities of PEA that accumulated in an external reservoir.
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