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Overexpression of Lactobacillus casei D-Hydroxyisocaproic Acid Dehydrogenase in Cheddar Cheese. Jeffery R. Broadbent, 2004.Metabolism of aromatic amino acids by lactic acid bacteria is an important source of off-flavor compounds in Cheddar cheese . Previous work has shown that  -keto acids produced from Trp, Tyr, and Phe by aminotransferase enzymes are chemically labile and may degrade spontaneously into a variety of off-flavor compounds . However, dairy lactobacilli can convert unstable
-keto acids to more-stable
-hydroxy acids via the action of
-keto acid dehydrogenases such as D-hydroxyisocaproic acid dehydrogenase . To further characterize the role of this enzyme in cheese flavor, the Lactobacillus casei D-hydroxyisocaproic acid dehydrogenase gene was cloned into the high-copy-number vector pTRKH2 and transformed into L . casei ATCC 334 . Enzyme assays confirmed that
-keto acid dehydrogenase activity was significantly higher in pTRKH2:dhic transformants than in wild-type cells . Reduced-fat Cheddar cheeses were made with Lactococcus lactis starter only, starter plus L . casei ATCC 334, and starter plus L . casei ATCC 334 transformed with pTRKH2:dhic . After 3 months of aging, the cheese chemistry and flavor attributes were evaluated instrumentally by gas chromatography-mass spectrometry and by descriptive sensory analysis . The culture system used significantly affected the concentrations of various ketones, aldehydes, alcohols, and esters and one sulfur compound in cheese . Results further indicated that enhanced expression of D-hydroxyisocaproic acid dehydrogenase suppressed spontaneous degradation of
-keto acids, but sensory work indicated that this effect retarded cheese flavor development .
Identification of an Actinobacillus pleuropneumoniae Consensus Promoter Structure. Scott M. Doree, 2003. Recombination Activity of a Distinctive Integron-Gene Cassette System Associated with Pseudomonas stutzeri Populations in Soil. Andrew J. Holmes, 2003.Class 1 integrons have strongly influenced the evolution of multiple antibiotic resistance . Diverse integrons have recently been detected directly in a range of natural environments . In order to characterize the properties of these environmental integrons, we sought to isolate organisms containing integrons from soils, which resulted in the isolation of Pseudomonas stutzeri strain Q . Further isolation efforts targeted at this species resulted in recovery of two other strains (P and BAM) . 16S rRNA sequences and chromosome mapping showed that these three strains are very closely related clonal variants in a single genomovar of P . stutzeri . Only strains Q and BAM were found to contain an integron and an associated gene cassette array . The intI and attI components of these strains showed 99 and 90% identity, respectively . The structure of these integrons and their associated gene cassettes was similar to that reported previously for other integron classes . The two integrons contained nonoverlapping sets of cassette-associated genes . In contrast, many of the cassette-associated recombination sites in the two integrons were similar and were considered to constitute a distinct subfamily consisting of 59-base element (59-be) recombination sites (the Pseudomonas subfamily) . The recombination activity of P . stutzeri integron components was tested in cointegrate assays . IntIPstQ was shown to catalyze site-specific recombination between its cognate attI site and 59-be sites from antibiotic resistance gene cassettes . While IntIPstQ did not efficiently mediate recombination between members of the Pseudomonas 59-be subfamily and other 59-be types, the former sites were functional when they were tested with IntI1 . We concluded that integrons present in P . stutzeri possess recombination activity and represent a hot spot for genomic diversity in this species . Involvement of Two Cytosolic Enzymes and a Novel Intermediate, 5'-Oxoaverantin, in the Pathway from 5'-Hydroxyaverantin to Averufin in Aflatoxin Biosynthesis. Emi Sakuno, 2003.During aflatoxin biosynthesis, 5'-hydroxyaverantin (HAVN) is converted to averufin (AVR) . Although we had previously suggested that this occurs in one enzymatic step, we demonstrate here that this conversion is composed of two enzymatic steps by showing that the two enzyme activities in the cytosol fraction of Aspergillus parasiticus were clearly separated by Mono Q column chromatography . An enzyme, HAVN dehydrogenase, catalyzes the first reaction from HAVN to a novel intermediate, another new enzyme catalyzes the next reaction from the intermediate to AVR, and the intermediate is a novel substance, 5'-oxoaverantin (OAVN), which was determined by physicochemical methods . We also purified both of the enzymes, HAVN dehydrogenase and OAVN cyclase, from the cytosol fraction of A . parasiticus by using ammonium sulfate fractionation and successive chromatographic steps . The HAVN dehydrogenase is a homodimer composed of 28-kDa subunits, and it requires NAD, but not NADP, as a cofactor for its activity . Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis of tryptic peptides of the purified HAVN dehydrogenase revealed that this enzyme coincides with a protein deduced from the adhA gene in the aflatoxin gene cluster of A . parasiticus . Also, the OAVN cyclase enzyme is a homodimer composed of 79-kDa subunits which does not require any cofactor for its activity . Further characterizations of both enzymes were performed .
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