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Trigonopsis variabilis D-amino acid oxidase accounts for 35% of Escherichia coli protein when added Dmethionine suppresses the toxic activity of the recombinant product. Permeabilized E. coli cells are reusable and stabilized enzyme preparations. The purified oxidase lacks the microheterogeneity of the natural enzyme. Oriented immobilization of a chimeric oxidase maintains 80% of the original activity in microparticle-bound enzymes.D-Amino acid oxidases (DAOs) (EC 1.4.3.3.) are well-studied and technologically useful flavoenzymes that catalyze the O 2 -dependent transformation of an ␣-amino acid substrate into the corresponding ␣-keto acid, H 2 O 2 , and NH 3 (for reviews, see references 11, 12, and 17). Economically, the most important use of DAO is the industrial conversion of cephalosporin C into glutaryl-7-aminocephalosporanic acid (2,10,14). DAO from the yeast Trigonopsis variabilis (TvDAO) has been a prime candidate for use in commercial applications because it displays good activity with cephalosporin C and is reasonably resistant to the oxidants (O 2 , H 2 O 2 ) occurring in the process (13). Current industrial enzyme production integrates relatively slow, multistage bioreactor cultivation of T. variabilis and several steps of downstream processing to obtain an immobilized biocatalyst deficient in catalase and esterase activities (8,14). The technical-grade oxidase obtained in this way is structurally microheterogeneous due to the partial oxidation of Cys108 into a cysteine sulfinic acid (16). Oxidatively modified TvDAO is catalytically only about one-quarter as efficient as the native enzyme (16). Production of a recombinant TvDAO potentially could eliminate many shortcomings of the established process, especially in a prokaryotic host with a reducing cellular environment that should prevent cysteine oxidation in TvDAO. Escherichia coli thus seems to be a logical candidate, but toxicity of the enzyme for this bacterium may have hampered efficient heterologous expression so far. TvDAO produced in E. coli has often been precipitated in inclusion bodies (3,6,7,17), or the soluble portion of it has displayed much lower specific activity than the natural enzyme, likely because a large fraction of the protein was recovered in the inactive apo form (1, 9). Thus, recombinant production of this important industrial biocatalyst in E. coli has not been established (17) and was therefore reexamined in this study, in which we focused on strain physiology and protein quality, as well as on the development of new oxidase preparations based on the recombinant enzyme.Expression vectors bearing the wild-type TvDAO gene and two novel genes encoding chimeric enzymes in which a 12-amino-acid peptide (Strep-Tag II) was fused in frame to the N terminus and the C terminus of TvDAO were constructed using standard methods (15). Further details and information about the oligonucleotide primers used are summarized in Table S.1 in the supplemental material. After sequencing, the final constructs pTvDAO, pTvDAOstrepN, and pTvDAOstr...

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Western diet is frequently low in essential metal ions. A common method for preventing metal ion deficiency is pharmacological supplementation, especially in a highly available form such as metalo-protein complexes. Accordingly, in this work, specific lactic acid bacteria were assessed for their ability to bind copper ions. Significant amounts of copper ions were bound, and the binding potential was found to be strain specific. Differences among the strains were evaluated with the Langmuir model for biosorption. Binding of copper was a fast process, strongly influenced by ionic strength, pH and biomass concentration. During the process, copper ions significantly reduced cell viability. Discharge of copper ions in a simulated gastrointestinal tract was examined; 85-90% of copper ions bound to LAB were discharged in the gastrointestinal model system.

This study deals with nonthermal food processing techniques and the results and findings present in this study are the root for further prospective studies. The food industry is looking for nonthermal methods that will enable food preservation, reduce deterioration of food compounds and structure and prolong food shelf life.