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Aim: Purification and characterization of an aminotransferase (AT) specific for the degradation of branched-chain amino acids from Lactobacillus paracasei subsp. paracasei CHCC 2115. Methods and Results:The purification protocol consisted of anion exchange chromatography, affinity chromatography and hydrophobic interaction chromatography. The enzyme was found to exist as a monomer with a molecular mass of 40-50 kDa. The AT converted isoleucine, leucine and valine at a similar rate with a-ketoglutarate as the amino group acceptor; minor activity was shown for methionine. The enzyme had pH and temperature optima of 7AE3 and 43°C, respectively, and activity was detected at the pH and salt conditions found in cheese (pH 5AE2, 4% NaCl). Hg 2+ completely inhibited the enzyme, and the inhibition pattern was similar to that for pyridoxal-5¢-phosphate-dependent enzymes, when studying the effect of other metal ions, thiol-and carbonylbinding agents. The N-terminal sequence of the enzyme was SVNIDWNNLGFDYMQLPYRYVAHXKDGVXD, and had at the amino acid level, 60 and 53% identity to a branched-chain amino acid AT of Lact. plantarum and Lactococcus lactis, respectively. Conclusions: The results suggest that Lact. paracasei subsp. paracasei CHCC 2115 may contribute to development of flavour in cheese. Significance and Impact of the Study: The findings of this work contribute to the knowledge of transamination performed by cheese-related bacteria, and in the understanding and control of amino acid catabolism and the production of aroma compounds.

Data are presented which indicate that the repression of pur gene expression seen after the addition of preformed purines to cultures of Salmonella typhimurium is the consequence of the presence or the formation of the purine bases, hypoxanthine and guanine. This conclusion is based on the following observations. First, it was impossible to find a correlation between the size of any individual purine nucleotide pool and the level of the first four enzymes in the de novo biosynthetic pathway. Second, adenine plus guanosine served as a perfect source of purine nucleotides, but their presence caused no repression of pur gene expression if the cells lacked purine nucleoside phosphorylase activity. This enzyme is needed to convert adenine and guanosine to hypoxanthine and guanine, but not for their conversion to nucleotides. Third, addition of guanine to a strain lacking guanine phosphoribosyltransferase (gpt) resulted in a repression of the level of the purine de novo biosynthetic enzymes, a reduction of the growth rate, and a fall in the pools of ATP and GTP. Addition of hypoxanthine to a strain lacking hypoxanthine phosphoribosyltransferase (hpt) had a similar, although weaker, effect. If the cells lacked both hypoxanthine and guanine phosphoribosyltransferases (hpt gpt), their basal level of the purine de novo biosynthetic enzymes was repressed in minimal medium. Such cells grow slower than wild-type cells and excrete purines, probably due to the inability to salvage endogenously formed hypoxanthine and guanine.

Aminotransferase (AT) activity against 18 amino acids was studied in ten strains of three species of Lactobacillus. A method for permeabilisation of cells was developed using toluene and ethanol combined with mechanical treatment. It was found that the AT activities in the washed permeabilised cells (W-PC) corresponded well to that in cell-free extracts (CFE). The AT specificity pattern was species as well as strain dependant. Strains of Lb. helveticus had high specificity for aromatic amino acids (ArAA) and lower activity against branched-chain amino acids (BcAA) and Asp, while strains of Lb. paracasei subsp. paracasei degraded BcAA and Asp, but had a lower and variable specificity against ArAA. One of the Lb. paracasei strains was characterised by having very high AT activity against all three BcAA (Ile, Leu, Val) compared with any of the other Lb. paracasei strains tested. Strains of Lb. danicus, which is a newly discovered Lactobacillus species isolated from cheese, had up to about 20 times higher AT activity against Leu than Lb. paracasei and Lb. helveticus. The permeabilised cells of Lb. danicus had also considerably higher AT activity against ArAA than Lb. paracasei and Lb. helveticus strains, and also higher AT activity against Asp. All Lactobacillus strains tested had AT activity against Met, but at a much lower rate than against other amino acids. Results of this study also demonstrated a chemical reaction between α-ketoglutaric acid and Asp that was catalysed by pyridoxal-5-phosphate without any AT present.

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An automated method (FastChrom) for baseline correction, peak detection and assignment (grouping) of similar peaks across samples has been developed. The method has been tested both on artificial data and a dataset obtained from gas chromatograph analysis of wine samples. As part of the automated approach, a new method for baseline estimation has been developed and compared with other methods. FastChrom has been shown to perform at least as well as conventional software. However, compared to other approaches, FastChrom finds more peaks in the chromatograms and not only those with retention times defined by the user. FastChrom is fast and easy to use and offers the possibility of applying a retention time index which facilitates the identification of peaks and the comparison between experiments.

The chromosomal locations of the genes purG and purI on the Escherichia coli linkage map are the opposites of those of Salmonella typhimurium. By methods which permit the identification of lesions in any of the five early enzymes of the purine de novo pathway, the gene-enzyme relationships of the purG and purI loci have been reevaluated in these two organisms. The results demonstrate that the relative locations of the genes encoding the two enzymes (phosphoribosylformylglycinamidine synthetase and phosphoribosylaminoimidazole synthetase) are similar in the two organisms. The gene products have been correctly determined in S. typhimurium. The gene products currently listed for the loci in E. coli are incorrect. The E. coli purG locus is equivalent to the S. typhimurium purI locus, and the E. coli purI locus is equivalent to the S. typhimurium purG locus.