<i>Lactobacillus plantarum</i> (<i>Lb. plantarum</i>) NRRL B4496 and <i>Lactococcus lactis</i> (<i>Lc. lactis</i>) NRRL B1821 prolidase genes were isolated, cloned, and sequenced. The sequence-confirmed genes were subcloned into the expression systems. The recombinant prolidases from the pKK223-3 systems were purified through ammonium sulphate precipitation and anion-exchange column chromatography. Recombinant <i>Lb. plantarum prolidase</i>, however, demonstrated a loss of activity during the purification. The following characterization work was performed on purified recombinant <i>Lc. lactis prolidase</i>. <p>The mass spectroscopic result and the molecular modelling suggested a 80 kDa homodimer with two metal cations at the catalytic centre of the prolidase. The optimum temperature was 50 ºC and showed more than 50% activities between 40 and 55 ºC. The enzyme was most stable at 30 ºC and withstood 20 min of heat-treatment up to 60 ºC, however, lost activity over 70 ºC. Circular dichroism indicated a denaturation temperature of 67 ºC. The optimum pH was 6.5 for hydrolyzing Leu-Pro and the enzyme did not display any activity below pH 5.5 nor above pH 7 with this peptide. However, Phe-Pro was hydrolyzed the fastest at pH 7 and Arg-Pro had a maximum rate at pH 9. This metallopeptidase exhibited a broad range of metal cation preference, hydrolyzing Leu-Pro with Mn++, Co++, Zn++, Ca++, and Mg++. Further kinetic analysis showed unusual allostery of the enzyme (Hill coefficient: 1.3). The unique substrate intakes onGlu-Pro and tripeptides were observed while Val-Pro was not hydrolyzed. The molecular modelling of this prolidase suggested a difference in the substrate specificity resulting from a loop structure, L33 to R40, near the substrate binding site.

Current industry chilling practices with and without the application of 2 percent L-lactic acid were compared for their effectiveness at reducing levels of Salmonella, Yersinia enterocolitica, Campylobacter coli, and common indicator organisms used in industry (aerobic plate count APC, Escherichia coli, and coliforms) on pork variety meats. Pork livers, hearts, intestines, and stomachs were either inoculated individually with 1 of the 3 pathogens or not inoculated and subjected to 1 of 5 treatments: 1 (water wash + lactic acid spray + freeze), 2 (freeze), 3 (water wash + lactic acid spray + chill + freeze), 4 (chill + freeze), and 5 (water wash + freeze). Samples were analyzed between treatment steps and after 2 months, 4 months, and 6 months of frozen storage. Results of effects of the steps within treatments showed that reductions in levels of pathogens after the water wash and lactic acid spray were significantly different (P<0.05) across variety meats. Treatment of variety meats with water wash and lactic acid before chilling resulted in >/= 0.5 log CFU/sample (P<0.05) reductions when compared to chilling alone. Regardless of treatments, reductions in levels of Salmonella and Y. enterocolitica of 0.6-1.3 log CFU/sample were observed after freezing (0 degrees C) overnight. Freezing reduced C. coli by >/= 2.2 log CFU/sample regardless of previous treatment. Throughout 6 months of frozen storage, reductions were observed in levels of all microorganisms equal to or greater than 1.3 log CFU/sample. The greatest reductions were observed on samples treated with lactic acid (Treatments 1 and 3) (1.3-5.0 log CFU/sample) while the smallest reductions were reported for samples without any spray treatment (Treatments 2 and 4) (0.7-4.5 log CFU/sample). Large reductions were observed in levels of C. coli (2.9-5.0 log CFU/sample) for all treatments. The results of this study suggest that, while the application of a water wash followed by freezing reduced levels of pathogens by approximately 1 log CFU/sample, the application of lactic acid before chilling and freezing variety meats results in significantly larger (P<0.05) reductions in microorganisms. Results also show that aerobic plate counts, E. coli, and coliforms follow similar trends to the pathogens.

Thesis (M.A.)--University of Missouri-Columbia, 2003. / Typescript. Includes bibliographical references (leaves 75-79). Also available on the Internet.

Thesis (M.A.)--University of Missouri-Columbia, 2003. / Typescript. Includes bibliographical references (leaves 75-79). Also available on the Internet.

No description available.

Abstract available in PDF file. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1977.

No description available.

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / Enzyme concentrates are available for use in commercial wineries to aid in wine processing, or to enhance wine quality. However, pectolytic enzyme remains the sole product routinely used in most wineries. One disadvantage of some of the products currently available commercially is they contain enzymes sourced from microorganisms not usually associated with grape juice or wine, typically fungi such as Aspergillus species. As a result, enzymes are inefficient catalysts under the harsh oenological conditions. In addition, some products contain secondary, and potentially undesirable, contaminant enzyme activities. Clearly there is the potential to develop enzyme preparations specifically for use in grape juice and wine. A potential source of such enzymes are the lactic acid bacteria (LAB), the organisms more commonly associated with the conduct of the malolactic fermentation (MLF) during vinification. In this study, the production of cell-associated enzymes with potential oenological applications by LAB was investigated. A screening of 50 LAB isolates for the production of lipases, esterases, tannases, and polysaccharide-degrading enzymes revealed wine LAB can produce enzymes of oenological importance. In general, activity towards polysaccharide substrates was more frequent among the lactobacilli and pediococci strains. Lipase activity was observed in three lactobacilli, and all strains were found to have tannase activity. Similarly, all strains displayed some esterase activity, although the activity was markedly stronger among the Oenococcus oeni. On the basis of the initial screen, a more detailed characterisation of the esterase activity of selected LAB isolates was conducted. Esterase activity was examined across a range of pH, temperature, and ethanol concentrations - all important oenological parameters. In addition, substrate specificity was determined using six ester substrates. In general, activity was maximal at pH values close to 6.0, and temperatures close to 40°C, although exceptions were observed with some strains. Increases in ethanol concentration resulted in lower activity for most lactobacilli and pediococci, but stimulated the esterase activity of all O. oeni. Work conducted with dairy LAB isolates has suggested esterases may be capable of both hydrolysing and synthesising esters. In the wine industry, the results of some volatile-profiling studies tend to support this theory, with concentrations of esters being reported to both increase and decrease during MLF. Malolactic fermentation trials were conducted in wine with six strains of O. oeni and GCMS was used to quantify particular esters before and after MLF. Some esters were found to increase in concentration during MLF, while others were found to decrease. These findings suggest LAB esterases are in fact capable of both synthesising and hydrolysing ester substrates in wine. To further dissect the esterase make-up of selected LAB strains, attempts to clone and heterologously express three structural genes for these enzymes were made. Three putative esterase genes were identified in O. oeni and cloned. Sequencing was completed and alignment with published esterase sequences used to reveal theoretical proteins of the O. oeni genes with high homology with those from other organisms. Of note, key features, such as active site motifs, were conserved in each O. oeni sequence. Expression of the recombinant proteins in E. coli resulted in higher esterase activity in one of the clones compared to the host. These results indicate that the open reading frame of one esterase gene in 0. oeni has been identified. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1297212 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2007

Thesis (Master)--İzmir Institute of Technology, İzmir, 2005. / Includes bibliographical references (leaves. 56-62).

Thesis (M.S.)--University of Wisconsin--Madison, 1968. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.