Fatty acid synthetase and acetyl CoA carboxylase mutants have been used to study several aspects of fatty acid biosynthesis in yeast: the contribution of the various enzymes of fatty acid biosynthesis and modification to the overall cellular fatty acid composition, the mechanism of fatty acyl chain elongation in yeast, the molecular structure and the reaction mechanism of the fatty acid synthetase complex and the genetic control of the biosynthesis of this multi-enzyme system. Genetic and biochemical evidence suggest an alpha6beta6 molecular structure of this complex, where alpha and beta are multifunctional proteins comprising, respectively, 3 and 5 of the various fatty acid synthetase component functions. The two subunits alpha and beta are synthesized on two different, unliked genes, fas 2 and fas 1. The biosynthesis of both is coordinated. The various component enzyme activities reside in distinct domains on the multifunctional chains. While most domains appear to be functionally independent, the three acyl transferases exhibit extensive mutual interactions. It is suggested that the biosynthesis of a multifunctional protein is favoured on the grounds of kinetics and regulation as compared with the formation of a complex of the corresponding individual enzymes.

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A competitive enzyme-linked immunosorbent assay (ELISA) was developed to quantify the amount of the preservative and potential allergen lysozyme in cheese using a commercially available monoclonal antibody against hen egg white lysozyme. The limit of detection for lysozyme in a cheese matrix amounted to 2.73 ng/mL, and the working range comprises 3.125-800 ng/mL. Intra- and interassay coefficients of variation were lower than 12%. Neither cross-reactivity with alpha-lactalbumin and human lysozyme nor unspecific interference with matrix components was observed. The recovery of lysozyme-spiked cheese ranged from 87.4 to 93.6% at four concentrations (50, 100, 200, and 400 mg/kg). The ELISA method was also compared to a high-performance liquid chromatography (HPLC) method, confirming the reliability and accuracy of the ELISA. A total of 21 commercially available cheese samples produced with and without lysozyme were analyzed with ELISA as well as HPLC. Both methods showed good agreement with a correlation index of R2=0.990.

1. By dissociation and subsequent reassociation of appropriate pairs of mutant fatty acid synthetases, hybrid multienzyme complexes were obtained whose overall fatty acid synthetase activities were restored to a considerable extent. The complementation thus achieved in vitro could be both intragenic and intergenic and was, in all cases studied, in agreement with the known complementation characteristics of fatty acid synthetase cr;l.c)2. Similarly, the method of reversible dissociation could be used to reactivate a wild-type fatty acid synthetase which had undergone considerable loss of activity due to prolonged storage. It is believed that only those subunits which have retained their native conformation are used in the reassociation of the complex.3. Specific component enzyme activities were determined with a variety of different mutant fatty acid synthetases and with several hybrid enzymes obtained from them by complementation in vivo or in vitro. The results indicate that the activities of most fatty acid synthetase functional domains are influenced by homologous and/or heterologous subunit interactions within the ct61jh oligomeric complex. Thus, mutational inactivation of any one of the active sites in subunit fl simultancously also alters the other four catalytic sites of this subunit. The lack of complementation occasionally observed between certain mutants of two different ,fas 1 complementation groups may be explained by this effect. Furthermore, alp interactions are indicated by the fact that enoylreductase-deficient mutant enzymes (B defect) always exhibit dramatically increased [Lketoacyl synthase ( x domain) activities.4. The incorporation of F M N in vitro into wild-type Fatty acid synthetase apo-enzyme from which the Ravin had been removed leads to a fully active enzyme complex only when the cofactor is present during the subunit assembly process. However, addition of FMN to a fully associated apo-enzyme restores only 50 of its original overall specific activity. If F M N was replaced by FAD in either of these experiments, reactivation occurred only to an extent of 50-70% of that observed with FMN.